Prof Patrik Rorsman FRS FMedSci

Research Area: Cell and Molecular Biology
Scientific Themes: Diabetes, Endocrinology & Metabolism
Keywords: diabetes, calcium, exocytosis, insulin, glucagon and somatostatin
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Insufficient insulin secretion represents an important facet of diabetes. Insulin is secreted from the ß-cells of the pancreatic islets when the blood glucose concentration rises above the normal ~5 mM. Precise knowledge about the cellular control and kinetics of insulin secretion is essential because type-2 diabetes involves the complete loss of rapid insulin secretion and a substantial reduction of sustained secretion. The loss of insulin secretion in diabetes is accompanied by defects in the release of the other islet hormones. For example, the regulation of glucagon release shows several abnormalities that exacerbate the metabolic consequences of insulin deficiency and type-2 diabetes is therefore best described as a multihormonal disorder.


The aim of our research is to explain how changes in the plasma glucose concentration via islet cell electrical activity and increases in the cytoplasmic Ca2+-concentration regulate exocytotic release of insulin as well as glucagon and somatostatin. Our work involves a combination of techniques to study secretion at the molecular, cellular and systemic levels. This requires sophisticated methodology to record the minute electrical currents flowing across biological membranes and secretion in individual cells at millisecond resolution. We also use optical techniques that allow us to monitor the movements of single secretory granules within the cell prior and during secretion.

These studies will promote our understanding of the fundamental processes that control insulin secretion under physiological conditions and determine the defects associated with clinical diabetes. Ultimately, our studies will allow the development of new diabetes therapies by identifying novel drug targets.

Name Department Institution Country
Prof Frances M Ashcroft Department of Physiology The University of Oxford United Kingdom
Vinnakota KC, Cha CY, Rorsman P, Balaban RS, La Gerche A, Wade-Martins R, Beard DA, Jeneson JA. 2016. Improving the physiological realism of experimental models. Interface Focus, 6 (2), pp. 20150076. | Show Abstract | Read more

The Virtual Physiological Human (VPH) project aims to develop integrative, explanatory and predictive computational models (C-Models) as numerical investigational tools to study disease, identify and design effective therapies and provide an in silico platform for drug screening. Ultimately, these models rely on the analysis and integration of experimental data. As such, the success of VPH depends on the availability of physiologically realistic experimental models (E-Models) of human organ function that can be parametrized to test the numerical models. Here, the current state of suitable E-models, ranging from in vitro non-human cell organelles to in vivo human organ systems, is discussed. Specifically, challenges and recent progress in improving the physiological realism of E-models that may benefit the VPH project are highlighted and discussed using examples from the field of research on cardiovascular disease, musculoskeletal disorders, diabetes and Parkinson's disease.

Briant L, Salehi A, Vergari E, Zhang Q, Rorsman P. 2016. Glucagon secretion from pancreatic α-cells. Ups J Med Sci, 121 (2), pp. 113-119. | Show Abstract | Read more

Type 2 diabetes involves a ménage à trois of impaired glucose regulation of pancreatic hormone release: in addition to impaired glucose-induced insulin secretion, the release of the hyperglycaemic hormone glucagon becomes dysregulated; these last-mentioned defects exacerbate the metabolic consequences of hypoinsulinaemia and are compounded further by hypersecretion of somatostatin (which inhibits both insulin and glucagon secretion). Glucagon secretion has been proposed to be regulated by either intrinsic or paracrine mechanisms, but their relative significance and the conditions under which they operate are debated. Importantly, the paracrine and intrinsic modes of regulation are not mutually exclusive; they could operate in parallel to control glucagon secretion. Here we have applied mathematical modelling of α-cell electrical activity as a novel means of dissecting the processes that underlie metabolic regulation of glucagon secretion. Our analyses indicate that basal hypersecretion of somatostatin and/or increased activity of somatostatin receptors may explain the loss of adequate counter-regulation under hypoglycaemic conditions, as well as the physiologically inappropriate stimulation of glucagon secretion during hyperglycaemia seen in diabetic patients. We therefore advocate studying the interaction of the paracrine and intrinsic mechanisms; unifying these processes may give a more complete picture of the regulation of glucagon secretion from α-cells than studying the individual parts.

Collins SC, Do HW, Hastoy B, Hugill A, Adam J, Chibalina MV, Galvanovskis J, Godazgar M et al. 2016. Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion. Diabetes, 65 (7), pp. 1952-1961. | Show Abstract | Read more

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.

Tarasov AI, Rorsman P. 2016. Dramatis Personae in β-Cell Mass Regulation: Enter SerpinB1. Cell Metab, 23 (1), pp. 8-10. | Show Abstract | Read more

How is β-cell mass adjusted to changes in the functional insulin requirements? The answer to this question is central to the understanding of the causes and (potentially) the therapy of type 2 diabetes. In this issue of Cell Metabolism, El Ouaamari et al. (2016) report that increased production of the protease inhibitor SerpinB1 in the liver links insulin resistance to stimulation of β-cell proliferation.

Szabat M, Modi H, Ramracheya R, Girbinger V, Chan F, Lee JT, Piske M, Kamal S et al. 2015. High-content screening identifies a role for Na(+) channels in insulin production. R Soc Open Sci, 2 (12), pp. 150306. | Show Abstract | Read more

Insulin production is the central feature of functionally mature and differentiated pancreatic β-cells. Reduced insulin transcription and dedifferentiation have been implicated in type 2 diabetes, making drugs that could reverse these processes potentially useful. We have previously established ratiometric live-cell imaging tools to identify factors that increase insulin promoter activity and promote β-cell differentiation. Here, we present a single vector imaging tool with eGFP and mRFP, driven by the Pdx1 and Ins1 promoters, respectively, targeted to the nucleus to enhance identification of individual cells in a high-throughput manner. Using this new approach, we screened 1120 off-patent drugs for factors that regulate Ins1 and Pdx1 promoter activity in MIN6 β-cells. We identified a number of compounds that positively modulate Ins1 promoter activity, including several drugs known to modulate ion channels. Carbamazepine was selected for extended follow-up, as our previous screen also identified this use-dependent sodium channel inhibitor as a positive modulator of β-cell survival. Indeed, carbamazepine increased Ins1 and Ins2 mRNA in primary mouse islets at lower doses than were required to protect β-cells. We validated the role of sodium channels in insulin production by examining Nav1.7 (Scn9a) knockout mice and remarkably islets from these animals had dramatically elevated insulin content relative to wild-type controls. Collectively, our experiments provide a starting point for additional studies aimed to identify drugs and molecular pathways that control insulin production and β-cell differentiation status. In particular, our unbiased screen identified a novel role for a β-cell sodium channel gene in insulin production.

Cane MC, Parrington J, Rorsman P, Galione A, Rutter GA. 2016. The two pore channel TPC2 is dispensable in pancreatic β-cells for normal Ca²⁺ dynamics and insulin secretion. Cell Calcium, 59 (1), pp. 32-40. | Show Abstract | Read more

Ca(2+) signals are central to the stimulation of insulin secretion from pancreatic β-cells by glucose and other agents, including glucagon-like peptide-1 (GLP-1). Whilst Ca(2+) influx through voltage-gated Ca(2+) channels on the plasma membrane is a key trigger for glucose-stimulated secretion, mobilisation of Ca(2+) from acidic stores has been implicated in the control of more localised Ca(2+) changes and membrane potential. Nicotinic acid adenine dinucleotide phosphate (NAADP), generated in β-cells in response to high glucose, is a potent mobiliser of these stores, and has been proposed to act through two pore channels (TPC1 and TPC2, murine gene names Tpcn1 and Tpcn2). Whilst the role of TPC1 in the control of Ca(2+) mobilisation and insulin secretion was recently confirmed, conflicting data exist for TPC2. Here, we used the selective and efficient deleter strain, Ins1Cre to achieve β-cell selective deletion of the Tpcn2 gene in mice. βTpcn2 KO mice displayed normal intraperitoneal and oral glucose tolerance, and glucose-stimulated Ca(2+) dynamics and insulin secretion from islets were similarly normal. GLP-1-induced Ca(2+) increases involved an increase in oscillation frequency from 4.35 to 4.84 per minute (p=0.04) at 8mM glucose, and this increase was unaffected by the absence of Tpcn2. The current data thus indicate that TPC2 is not absolutely required for normal glucose- or incretin-stimulated insulin secretion from the β-cell. Our findings suggest that TPC1, whose expression tended to increase in Tpcn2 null islets, might be sufficient to support normal Ca(2+) dynamics in response to stimulation by nutrients or incretins.

Wu B, Wei S, Petersen N, Ali Y, Wang X, Bacaj T, Rorsman P, Hong W, Südhof TC, Han W. 2015. Synaptotagmin-7 phosphorylation mediates GLP-1-dependent potentiation of insulin secretion from β-cells. Proc Natl Acad Sci U S A, 112 (32), pp. 9996-10001. | Show Abstract | Read more

Glucose stimulates insulin secretion from β-cells by increasing intracellular Ca(2+). Ca(2+) then binds to synaptotagmin-7 as a major Ca(2+) sensor for exocytosis, triggering secretory granule fusion and insulin secretion. In type-2 diabetes, insulin secretion is impaired; this impairment is ameliorated by glucagon-like peptide-1 (GLP-1) or by GLP-1 receptor agonists, which improve glucose homeostasis. However, the mechanism by which GLP-1 receptor agonists boost insulin secretion remains unclear. Here, we report that GLP-1 stimulates protein kinase A (PKA)-dependent phosphorylation of synaptotagmin-7 at serine-103, which enhances glucose- and Ca(2+)-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca(2+)-triggered exocytosis, whereas a phospho-inactive synaptotagmin-7 mutant disrupts GLP-1 potentiation of insulin secretion. Our findings thus suggest that synaptotagmin-7 is directly activated by GLP-1 signaling and may serve as a drug target for boosting insulin secretion. Moreover, our data reveal, to our knowledge, the first physiological modulation of Ca(2+)-triggered exocytosis by direct phosphorylation of a synaptotagmin.

Arredouani A, Ruas M, Collins SC, Parkesh R, Clough F, Pillinger T, Coltart G, Rietdorf K et al. 2015. Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP) and Endolysosomal Two-pore Channels Modulate Membrane Excitability and Stimulus-Secretion Coupling in Mouse Pancreatic β Cells. J Biol Chem, 290 (35), pp. 21376-21392. | Show Abstract | Read more

Pancreatic β cells are electrically excitable and respond to elevated glucose concentrations with bursts of Ca(2+) action potentials due to the activation of voltage-dependent Ca(2+) channels (VDCCs), which leads to the exocytosis of insulin granules. We have examined the possible role of nicotinic acid adenine dinucleotide phosphate (NAADP)-mediated Ca(2+) release from intracellular stores during stimulus-secretion coupling in primary mouse pancreatic β cells. NAADP-regulated Ca(2+) release channels, likely two-pore channels (TPCs), have recently been shown to be a major mechanism for mobilizing Ca(2+) from the endolysosomal system, resulting in localized Ca(2+) signals. We show here that NAADP-mediated Ca(2+) release from endolysosomal Ca(2+) stores activates inward membrane currents and depolarizes the β cell to the threshold for VDCC activation and thereby contributes to glucose-evoked depolarization of the membrane potential during stimulus-response coupling. Selective pharmacological inhibition of NAADP-evoked Ca(2+) release or genetic ablation of endolysosomal TPC1 or TPC2 channels attenuates glucose- and sulfonylurea-induced membrane currents, depolarization, cytoplasmic Ca(2+) signals, and insulin secretion. Our findings implicate NAADP-evoked Ca(2+) release from acidic Ca(2+) storage organelles in stimulus-secretion coupling in β cells.

Chandra V, Albagli-Curiel O, Hastoy B, Piccand J, Randriamampita C, Vaillant E, Cavé H, Busiah K et al. 2014. RFX6 Regulates Insulin Secretion by Modulating Ca<sup>2+</sup> Homeostasis in Human β Cells Cell Reports, 9 (6), pp. 2206-2218. | Show Abstract | Read more

© 2014 The Authors.Development and function of pancreatic β cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse β cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human β cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human β cell line EndoC-βH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca2+-channel genes resulting in the reduction in L-type Ca2+-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G) that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca2+-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes.

Rorsman P, Zhang Q. 2014. Matthias Braun, 23 July 1966-16 November 2013 Diabetologia, 57 (12), pp. 2431-2432. | Read more

Gheni G, Ogura M, Iwasaki M, Yokoi N, Minami K, Nakayama Y, Harada K, Hastoy B et al. 2014. Glutamate acts as a key signal linking glucose metabolism to incretin/cAMP action to amplify insulin secretion. Cell Rep, 9 (2), pp. 661-673. | Show Abstract | Read more

Incretins, hormones released by the gut after meal ingestion, are essential for maintaining systemic glucose homeostasis by stimulating insulin secretion. The effect of incretins on insulin secretion occurs only at elevated glucose concentrations and is mediated by cAMP signaling, but the mechanism linking glucose metabolism and cAMP action in insulin secretion is unknown. We show here, using a metabolomics-based approach, that cytosolic glutamate derived from the malate-aspartate shuttle upon glucose stimulation underlies the stimulatory effect of incretins and that glutamate uptake into insulin granules mediated by cAMP/PKA signaling amplifies insulin release. Glutamate production is diminished in an incretin-unresponsive, insulin-secreting β cell line and pancreatic islets of animal models of human diabetes and obesity. Conversely, a membrane-permeable glutamate precursor restores amplification of insulin secretion in these models. Thus, cytosolic glutamate represents the elusive link between glucose metabolism and cAMP action in incretin-induced insulin secretion.

Rorsman P, Zhang Q. 2014. Matthias Braun, 23 July 1966-16 November 2013. Diabetologia, 57 (12), pp. 2431-2432. | Read more

Brereton MF, Iberl M, Shimomura K, Zhang Q, Proks P, Adriaenssens AA, Spiliotis II, Dace W et al. 2014. Reversible changes in pancreatic islet structure and function produced by elevated blood glucose DIABETOLOGIA, 57 pp. S93-S93.

Tarasov AI, Galvanovskis J, Leenders F, Rorsman P. 2014. Biphasic nature of glucose-induced insulin secretion is independent on energy metabolism DIABETOLOGIA, 57 pp. S178-S179.

Zhang Q, Salehi A, Ramracheya R, Rorsman P. 2014. Elevation of cAMP promotes an Epac2 dependent, L-type Ca2+-channel coupled, Ca2+-induced Ca2+ release in mouse alpha cells DIABETOLOGIA, 57 pp. S253-S253.

Zhang Q, Chibalina MV, Bengtsson M, Groschner LN, Ramracheya R, Rorsman NJ, Leiss V, Nassar MA et al. 2014. Na+ current properties in islet α- and β-cells reflect cell-specific Scn3a and Scn9a expression. J Physiol, 592 (21), pp. 4677-4696. | Show Abstract | Read more

Mouse pancreatic β- and α-cells are equipped with voltage-gated Na(+) currents that inactivate over widely different membrane potentials (half-maximal inactivation (V0.5) at -100 mV and -50 mV in β- and α-cells, respectively). Single-cell PCR analyses show that both α- and β-cells have Nav1.3 (Scn3) and Nav1.7 (Scn9a) α subunits, but their relative proportions differ: β-cells principally express Nav1.7 and α-cells Nav1.3. In α-cells, genetically ablating Scn3a reduces the Na(+) current by 80%. In β-cells, knockout of Scn9a lowers the Na(+) current by >85%, unveiling a small Scn3a-dependent component. Glucagon and insulin secretion are inhibited in Scn3a(-/-) islets but unaffected in Scn9a-deficient islets. Thus, Nav1.3 is the functionally important Na(+) channel α subunit in both α- and β-cells because Nav1.7 is largely inactive at physiological membrane potentials due to its unusually negative voltage dependence of inactivation. Interestingly, the Nav1.7 sequence in brain and islets is identical and yet the V0.5 for inactivation is >30 mV more negative in β-cells. This may indicate the presence of an intracellular factor that modulates the voltage dependence of inactivation.

Chandra V, Albagli-Curiel O, Hastoy B, Piccand J, Randriamampita C, Vaillant E, Cavé H, Busiah K et al. 2014. RFX6 regulates insulin secretion by modulating Ca2+ homeostasis in human β cells. Cell Rep, 9 (6), pp. 2206-2218. | Show Abstract | Read more

Development and function of pancreatic β cells involve the regulated activity of specific transcription factors. RFX6 is a transcription factor essential for mouse β cell differentiation that is mutated in monogenic forms of neonatal diabetes. However, the expression and functional roles of RFX6 in human β cells, especially in pathophysiological conditions, are poorly explored. We demonstrate the presence of RFX6 in adult human pancreatic endocrine cells. Using the recently developed human β cell line EndoC-βH2, we show that RFX6 regulates insulin gene transcription, insulin content, and secretion. Knockdown of RFX6 causes downregulation of Ca(2+)-channel genes resulting in the reduction in L-type Ca(2+)-channel activity that leads to suppression of depolarization-evoked insulin exocytosis. We also describe a previously unreported homozygous missense RFX6 mutation (p.V506G) that is associated with neonatal diabetes, which lacks the capacity to activate the insulin promoter and to increase Ca(2+)-channel expression. Our data therefore provide insights for understanding certain forms of neonatal diabetes.

Rorsman P, Ramracheya R, Rorsman NJ, Zhang Q. 2014. ATP-regulated potassium channels and voltage-gated calcium channels in pancreatic alpha and beta cells: similar functions but reciprocal effects on secretion. Diabetologia, 57 (9), pp. 1749-1761. | Show Abstract | Read more

Closure of ATP-regulated K(+) channels (K(ATP) channels) plays a central role in glucose-stimulated insulin secretion in beta cells. K(ATP) channels are also highly expressed in glucagon-producing alpha cells, where their function remains unresolved. Under hypoglycaemic conditions, K(ATP) channels are open in alpha cells but their activity is low and only ~1% of that in beta cells. Like beta cells, alpha cells respond to hyperglycaemia with K(ATP) channel closure, membrane depolarisation and stimulation of action potential firing. Yet, hyperglycaemia reciprocally regulates glucagon (inhibition) and insulin secretion (stimulation). Here we discuss how this conundrum can be resolved and how reduced K(ATP) channel activity, via membrane depolarisation, paradoxically reduces alpha cell Ca(2+) entry and glucagon exocytosis. Finally, we consider whether the glucagon secretory defects associated with diabetes can be attributed to impaired K(ATP) channel regulation and discuss the potential for remedial pharmacological intervention using sulfonylureas.

Latreille M, Hausser J, Stützer I, Zhang Q, Hastoy B, Gargani S, Kerr-Conte J, Pattou F et al. 2014. MicroRNA-7a regulates pancreatic β cell function. J Clin Invest, 124 (6), pp. 2722-2735. | Show Abstract | Read more

Dysfunctional microRNA (miRNA) networks contribute to inappropriate responses following pathological stress and are the underlying cause of several disease conditions. In pancreatic β cells, miRNAs have been largely unstudied and little is known about how specific miRNAs regulate glucose-stimulated insulin secretion (GSIS) or impact the adaptation of β cell function to metabolic stress. In this study, we determined that miR-7 is a negative regulator of GSIS in β cells. Using Mir7a2 deficient mice, we revealed that miR-7a2 regulates β cell function by directly regulating genes that control late stages of insulin granule fusion with the plasma membrane and ternary SNARE complex activity. Transgenic mice overexpressing miR-7a in β cells developed diabetes due to impaired insulin secretion and β cell dedifferentiation. Interestingly, perturbation of miR-7a expression in β cells did not affect proliferation and apoptosis, indicating that miR-7 is dispensable for the maintenance of endocrine β cell mass. Furthermore, we found that miR-7a levels are decreased in obese/diabetic mouse models and human islets from obese and moderately diabetic individuals with compensated β cell function. Our results reveal an interconnecting miR-7 genomic circuit that regulates insulin granule exocytosis in pancreatic β cells and support a role for miR-7 in the adaptation of pancreatic β cell function in obesity and type 2 diabetes.

Brereton MF, Iberl M, Shimomura K, Zhang Q, Adriaenssens AE, Proks P, Spiliotis II, Dace W et al. 2014. Reversible changes in pancreatic islet structure and function produced by elevated blood glucose. Nat Commun, 5 pp. 4639. | Show Abstract | Read more

Diabetes is characterized by hyperglycaemia due to impaired insulin secretion and aberrant glucagon secretion resulting from changes in pancreatic islet cell function and/or mass. The extent to which hyperglycaemia per se underlies these alterations remains poorly understood. Here we show that β-cell-specific expression of a human activating KATP channel mutation in adult mice leads to rapid diabetes and marked alterations in islet morphology, ultrastructure and gene expression. Chronic hyperglycaemia is associated with a dramatic reduction in insulin-positive cells and an increase in glucagon-positive cells in islets, without alterations in cell turnover. Furthermore, some β-cells begin expressing glucagon, whilst retaining many β-cell characteristics. Hyperglycaemia, rather than KATP channel activation, underlies these changes, as they are prevented by insulin therapy and fully reversed by sulphonylureas. Our data suggest that many changes in islet structure and function associated with diabetes are attributable to hyperglycaemia alone and are reversed when blood glucose is normalized.

Rorsman P, Ramracheya R, Rorsman NJG, Zhang Q. 2014. ATP-regulated potassium channels and voltage-gated calcium channels in pancreatic alpha and beta cells: Similar functions but reciprocal effects on secretion Diabetologia, 57 (9), pp. 1749-1761. | Show Abstract | Read more

Closure of ATP-regulated K+ channels (KATP channels) plays a central role in glucose-stimulated insulin secretion in beta cells. KATP channels are also highly expressed in glucagon-producing alpha cells, where their function remains unresolved. Under hypoglycaemic conditions, KATP channels are open in alpha cells but their activity is low and only ~1% of that in beta cells. Like beta cells, alpha cells respond to hyperglycaemia with KATP channel closure, membrane depolarisation and stimulation of action potential firing. Yet, hyperglycaemia reciprocally regulates glucagon (inhibition) and insulin secretion (stimulation). Here we discuss how this conundrum can be resolved and how reduced KATP channel activity, via membrane depolarisation, paradoxically reduces alpha cell Ca2+ entry and glucagon exocytosis. Finally, we consider whether the glucagon secretory defects associated with diabetes can be attributed to impaired KATP channel regulation and discuss the potential for remedial pharmacological intervention using sulfonylureas. © 2014 Springer-Verlag Berlin Heidelberg.

Zhang Q, Ramracheya R, Lahmann C, Tarasov A, Bengtsson M, Braha O, Braun M, Brereton M et al. 2013. Role of KATP channels in glucose-regulated glucagon secretion and impaired counterregulation in type 2 diabetes. Cell Metab, 18 (6), pp. 871-882. | Show Abstract | Read more

Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na(+) channels involved in action potential firing that, via reduced action potential height and Ca(2+) entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.

Soni A, Amisten S, Rorsman P, Salehi A. 2013. GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion. Biochem Biophys Res Commun, 441 (3), pp. 643-648. | Show Abstract | Read more

GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12-16 week old mice strongly (2.5-fold) increased basal (1 mmol/l) and moderately (40%) potentiated glucose-(20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Down-regulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and β-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type 2 diabetes.

Cited:

36

Scopus

Ashcroft FM, Rorsman P. 2013. KATP channels and islet hormone secretion: New insights and controversies Nature Reviews Endocrinology, 9 (11), pp. 660-669. | Show Abstract | Read more

ATP-sensitive potassium channels (K ATP channels) link cell metabolism to electrical activity by controlling the cell membrane potential. They participate in many physiological processes but have a particularly important role in systemic glucose homeostasis by regulating hormone secretion from pancreatic islet cells. Glucose-induced closure of K ATP channels is crucial for insulin secretion. Emerging data suggest that K ATP channels also play a key part in glucagon secretion, although precisely how they do so remains controversial. This Review highlights the role of K ATP channels in insulin and glucagon secretion. We discuss how K ATP channels might contribute not only to the initiation of insulin release but also to the graded stimulation of insulin secretion that occurs with increasing glucose concentrations. The various hypotheses concerning the role of K ATP channels in glucagon release are also reviewed. Furthermore, we illustrate how mutations in K ATP channel genes can cause hyposecretion or hypersecretion of insulin, as in neonatal diabetes mellitus and congenital hyperinsulinism, and how defective metabolic regulation of the channel may underlie the hypoinsulinaemia and the hyperglucagonaemia that characterize type 2 diabetes mellitus. Finally, we outline how sulphonylureas, which inhibit K ATP channels, stimulate insulin secretion in patients with neonatal diabetes mellitus or type 2 diabetes mellitus, and suggest their potential use to target the glucagon secretory defects found in diabetes mellitus. © 2013 Macmillan Publishers Limited. All rights reserved.

Cited:

32

Scopus

Amisten S, Salehi A, Rorsman P, Jones PM, Persaud SJ. 2013. An atlas and functional analysis of G-protein coupled receptors in human islets of Langerhans Pharmacology and Therapeutics, 139 (3), pp. 359-391. | Show Abstract | Read more

G-protein coupled receptors (GPCRs) regulate hormone secretion from islets of Langerhans, and recently developed therapies for type-2 diabetes target islet GLP-1 receptors. However, the total number of GPCRs expressed by human islets, as well as their function and interactions with drugs, is poorly understood. In this review we have constructed an atlas of all GPCRs expressed by human islets: the 'islet GPCRome'. We have used this atlas to describe how islet GPCRs interact with their endogenous ligands, regulate islet hormone secretion, and interact with drugs known to target GPCRs, with a focus on drug/receptor interactions that may affect insulin secretion. The islet GPCRome consists of 293 GPCRs, a majority of which have unknown effects on insulin, glucagon and somatostatin secretion. The islet GPCRs are activated by 271 different endogenous ligands, at least 131 of which are present in islet cells. A large signalling redundancy was also found, with 119 ligands activating more than one islet receptor. Islet GPCRs are also the targets of a large number of clinically used drugs, and based on their coupling characteristics and effects on receptor signalling we identified 107 drugs predicted to stimulate and 184 drugs predicted to inhibit insulin secretion. The islet GPCRome highlights knowledge gaps in the current understanding of islet GPCR function, and identifies GPCR/ligand/drug interactions that might affect insulin secretion, which are important for understanding the metabolic side effects of drugs. This approach may aid in the design of new safer therapeutic agents with fewer detrimental effects on islet hormone secretion. © 2013 Elsevier Inc.

McCarthy MI, Rorsman P, Gloyn AL. 2013. TCF7L2 and diabetes: a tale of two tissues, and of two species. Cell Metab, 17 (2), pp. 157-159. | Show Abstract | Read more

Human genetics is revealing ever more variants that influence propensity to common diseases, but progress in translating these discoveries into the biological mechanisms responsible for predisposition continues to lag behind. A recent paper in Cell (Boj et al., 2012) using rodent models to examine how diabetes-associated variants near TCF7L2 perturb metabolic regulation provides surprising results.

Soni A, Amisten S, Rorsman P, Salehi A. 2013. GPRC5B a putative glutamate-receptor candidate is negative modulator of insulin secretion. Biochem Biophys Res Commun, 441 (3), pp. 643-648. | Show Abstract | Read more

GPRC5B is an orphan receptor belonging to the group C family of G protein-coupled receptors (GPCRs). GPRC5B is abundantly expressed in both human and mouse pancreatic islets, and both GPRC5B mRNA and protein are up-regulated 2.5-fold in islets from organ donors with type 2 diabetes. Expression of Gprc5b is 50% lower in islets isolated from newborn (<3 weeks) than in adult (>36 weeks) mice. Lentiviral shRNA-mediated down-regulation of Gprc5b in intact islets from 12 to 16 week-old mice strongly (2.5-fold) increased basal (1 mmol/l) and moderately (40%) potentiated glucose (20 mmol/l) stimulated insulin secretion and also enhanced the potentiating effect of glutamate on insulin secretion. Downregulation of Gprc5b protected murine insulin-secreting clonal MIN6 cells against cytokine-induced apoptosis. We propose that increased expression of GPRC5B contributes to the reduced insulin secretion and b-cell viability observed in type-2 diabetes. Thus, pharmacological targeting of GPRC5B might provide a novel means therapy for the treatment and prevention of type-2 diabetes.

Braun M, Ramracheya R, Rorsman P. 2012. Autocrine regulation of insulin secretion. Diabetes Obes Metab, 14 Suppl 3 (SUPPL.3), pp. 143-151. | Show Abstract | Read more

Impaired insulin secretion from pancreatic β-cells is a major factor in the pathogenesis of type 2 diabetes. The main regulator of insulin secretion is the plasma glucose concentration. Insulin secretion is modified by other nutrients, circulating hormones and the autonomic nervous system, as well as local paracrine and autocrine signals. Autocrine signalling involves diffusible molecules that bind to receptors on the same cell from which they have been released. The first transmitter to be implicated in the autocrine regulation of β-cell function was insulin itself. The importance of autocrine insulin signalling is underscored by the finding that mice lacking insulin receptors in β-cells are glucose intolerant. In addition to insulin, β-cells secrete a variety of additional substances, including peptides (e.g. amylin, chromogranin A and B and their cleavage products), neurotransmitters (ATP and γ-aminobutyric acid) and ions (e.g. zinc). Here we review the autocrine effects of substances secreted from β-cells, with a focus on acute effects in stimulus-secretion coupling, present some novel data and discuss the general significance of autocrine signals for the regulation of insulin secretion.

Rorsman P, Braun M. 2013. Regulation of insulin secretion in human pancreatic islets. Annu Rev Physiol, 75 (1), pp. 155-179. | Show Abstract | Read more

Pancreatic β cells secrete insulin, the body's only hormone capable of lowering plasma glucose levels. Impaired or insufficient insulin secretion results in diabetes mellitus. The β cell is electrically excitable; in response to an elevation of glucose, it depolarizes and starts generating action potentials. The electrophysiology of mouse β cells and the cell's role in insulin secretion have been extensively investigated. More recently, similar studies have been performed on human β cells. These studies have revealed numerous and important differences between human and rodent β cells. Here we discuss the properties of human pancreatic β cells: their glucose sensing, the ion channel complement underlying glucose-induced electrical activity that culminates in exocytotic release of insulin, the cellular control of exocytosis, and the modulation of insulin secretion by circulating hormones and locally released neurotransmitters. Finally, we consider the pathophysiology of insulin secretion and the interactions between genetics and environmental factors that may explain the current diabetes epidemic.

Kailey B, van de Bunt M, Cheley S, Johnson PR, MacDonald PE, Gloyn AL, Rorsman P, Braun M. 2012. SSTR2 is the functionally dominant somatostatin receptor in human pancreatic β- and α-cells. Am J Physiol Endocrinol Metab, 303 (9), pp. E1107-E1116. | Show Abstract | Read more

Somatostatin-14 (SST) inhibits insulin and glucagon secretion by activating G protein-coupled somatostatin receptors (SSTRs), of which five isoforms exist (SSTR1-5). In mice, the effects on pancreatic β-cells are mediated by SSTR5, whereas α-cells express SSTR2. In both cell types, SSTR activation results in membrane hyperpolarization and suppression of exocytosis. Here, we examined the mechanisms by which SST inhibits secretion from human β- and α-cells and the SSTR isoforms mediating these effects. Quantitative PCR revealed high expression of SSTR2, with lower levels of SSTR1, SSTR3, and SSTR5, in human islets. Immunohistochemistry showed expression of SSTR2 in both β- and α-cells. SST application hyperpolarized human β-cells and inhibited action potential firing. The membrane hyperpolarization was unaffected by tolbutamide but antagonized by tertiapin-Q, a blocker of G protein-gated inwardly rectifying K⁺ channels (GIRK). The effect of SST was mimicked by an SSTR2-selective agonist, whereas a SSTR5 agonist was marginally effective. SST strongly (>70%) reduced depolarization-evoked exocytosis in both β- and α-cells. A slightly weaker inhibition was observed in both cell types after SSTR2 activation. SSTR3- and SSTR1-selective agonists moderately reduced the exocytotic responses in β- and α-cells, respectively, whereas SSTR4- and SSTR5-specific agonists were ineffective. SST also reduced voltage-gated P/Q-type Ca²⁺ currents in β-cells, but normalization of Ca²⁺ influx to control levels by prolonged depolarizations only partially restored exocytosis. We conclude that SST inhibits secretion from both human β- and α-cells by activating GIRK and suppressing electrical activity, reducing P/Q-type Ca²⁺ currents, and directly inhibiting exocytosis. These effects are mediated predominantly by SSTR2 in both cell types.

Salehi A, Gunnerud U, Muhammed SJ, Ostman E, Holst JJ, Björck I, Rorsman P. 2012. The insulinogenic effect of whey protein is partially mediated by a direct effect of amino acids and GIP on β-cells. Nutr Metab (Lond), 9 (1), pp. 48. | Show Abstract | Read more

BACKGROUND: Whey protein increases postprandial serum insulin levels. This has been associated with increased serum levels of leucine, isoleucine, valine, lysine, threonine and the incretin hormone glucose-dependent insulinotropic polypeptide (GIP). We have examined the effects of these putative mediators of whey's action on insulin secretion from isolated mouse Langerhans islets. METHODS: Mouse pancreatic islets were incubated with serum drawn from healthy individuals after ingestion of carbohydrate equivalent meals of whey protein (whey serum), or white wheat bread (control serum). In addition the effect of individual amino acid combinations on insulin secretion was also tested. Furthermore, the stimulatory effects of whey serum on insulin secretion was tested in vitro in the absence and presence of a GIP receptor antagonist ((Pro(3))GIP[mPEG]). RESULTS: Postprandial amino acids, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) responses were higher after whey compared to white wheat bread. A stimulatory effect on insulin release from isolated islets was observed with serum after whey obtained at 15 min (+87%, P < 0.05) and 30 min (+139%, P < 0.05) postprandially, compared with control serum. The combination of isoleucine, leucine, valine, lysine and threonine exerted strong stimulatory effect on insulin secretion (+270%, P < 0.05), which was further augmented by GIP (+558% compared to that produced by glucose, P < 0.05). The stimulatory action of whey on insulin secretion was reduced by the GIP-receptor antagonist (Pro(3))GIP[mPEG]) at both 15 and 30 min (-56% and -59%, P < 0.05). CONCLUSIONS: Compared with white wheat bread meal, whey causes an increase of postprandial insulin, plasma amino acids, GIP and GLP-1 responses. The in vitro data suggest that whey protein exerts its insulinogenic effect by preferential elevation of the plasma concentrations of certain amino acids, GIP and GLP-1.

Rosengren AH, Braun M, Mahdi T, Andersson SA, Travers ME, Shigeto M, Zhang E, Almgren P et al. 2012. Reduced insulin exocytosis in human pancreatic β-cells with gene variants linked to type 2 diabetes. Diabetes, 61 (7), pp. 1726-1733. | Show Abstract | Read more

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca(2+) sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

Ashcroft FM, Rorsman P. 2012. Diabetes mellitus and the β cell: the last ten years. Cell, 148 (6), pp. 1160-1171. | Show Abstract | Read more

Diabetes is a major global problem. During the past decade, the genetic basis of various monogenic forms of the disease, and their underlying molecular mechanisms, have been elucidated. Many genes that increase type 2 diabetes (T2DM) risk have also been identified, but how they do so remains enigmatic. Nevertheless, defective insulin secretion emerges as the main culprit in both monogenic and polygenic diabetes, with environmental and lifestyle factors, via obesity, accounting for the current dramatic increase in T2DM. There also have been significant advances in therapy, particularly for some monogenic disorders. We review here what ails the β cell and how its function may be restored.

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Rorsman P, Braun M, Zhang Q. 2012. Regulation of calcium in pancreatic α- and β-cells in health and disease Cell Calcium, 51 (3-4), pp. 300-308. | Show Abstract | Read more

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca 2+-influx via voltage-gated Ca 2+-channels, elevation of [Ca 2+] i and initiation of Ca 2+-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca 2+-channel present in α- and β-cells and the modulation of hormone secretion by Ca 2+-dependent and -independent processes. We also consider how subtle changes in Ca 2+-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes. © 2011 Elsevier Ltd.

Rorsman P, Braun M, Zhang Q. 2012. Regulation of calcium in pancreatic α- and β-cells in health and disease. Cell Calcium, 51 (3-4), pp. 300-308. | Show Abstract | Read more

The glucoregulatory hormones insulin and glucagon are released from the β- and α-cells of the pancreatic islets. In both cell types, secretion is secondary to firing of action potentials, Ca(2+)-influx via voltage-gated Ca(2+)-channels, elevation of [Ca(2+)](i) and initiation of Ca(2+)-dependent exocytosis. Here we discuss the mechanisms that underlie the reciprocal regulation of insulin and glucagon secretion by changes in plasma glucose, the roles played by different types of voltage-gated Ca(2+)-channel present in α- and β-cells and the modulation of hormone secretion by Ca(2+)-dependent and -independent processes. We also consider how subtle changes in Ca(2+)-signalling may have profound impact on β-cell performance and increase risk of developing type-2 diabetes.

Yashiro H, Tsujihata Y, Takeuchi K, Hazama M, Johnson PR, Rorsman P. 2012. The effects of TAK-875, a selective G protein-coupled receptor 40/free fatty acid 1 agonist, on insulin and glucagon secretion in isolated rat and human islets. J Pharmacol Exp Ther, 340 (2), pp. 483-489. | Show Abstract | Read more

G protein-coupled receptor 40 (GPR40)/free fatty acid 1 (FFA1) is a G protein-coupled receptor involved in free fatty acid-induced insulin secretion. To analyze the effect of our novel GPR40/FFA1-selective agonist, [(3S)-6-({2',6'-dimethyl-4'-[3-(methylsulfonyl)propoxy]biphenyl-3-yl}methoxy)-2,3-dihydro-1-benzofuran-3-yl]acetic acid hemi-hydrate (TAK-875), on insulin and glucagon secretion, we performed hormone secretion assays and measured intracellular Ca²⁺ concentration ([Ca²⁺](i)) in both human and rat islets. Insulin and glucagon secretion were measured in static and dynamic conditions by using groups of isolated rat and human pancreatic islets. [Ca²⁺](i) was recorded by using confocal microscopy. GPR40/FFA1 expression was measured by quantitative polymerase chain reaction. In both human and rat islets, TAK-875 enhanced glucose-induced insulin secretion in a glucose-dependent manner. The stimulatory effect of TAK-875 was similar to that produced by glucagon-like peptide-1 and correlated with the elevation of β-cell [Ca²⁺](i). TAK-875 was without effect on glucagon secretion at both 1 and 16 mM glucose in human islets. These data indicate that GPR40/FFA1 influences mainly insulin secretion in a glucose-dependent manner. The β-cell-specific action of TAK-875 in human islets may represent a therapeutically useful feature that allows plasma glucose control without compromising counter-regulation of glucagon secretion, thus minimizing the risk of hypoglycemia.

Walker JN, Ramracheya R, Zhang Q, Johnson PR, Braun M, Rorsman P. 2011. Regulation of glucagon secretion by glucose: paracrine, intrinsic or both? Diabetes Obes Metab, 13 Suppl 1 (SUPPL. 1), pp. 95-105. | Show Abstract | Read more

Glucagon secretion is regulated by glucose but the mechanisms involved remain hotly debated. Both intrinsic (within the α-cell itself) and paracrine (mediated by factors released β- and/or δ-cells) have been postulated. Glucagon secretion is maximally suppressed by glucose concentrations that do not affect insulin and somatostatin secretion, a finding that highlights the significance of intrinsic regulation of glucagon secretion. Experiments on islets from mice lacking functional ATP-sensitive potassium channels (K(ATP)-channels) indicate that these channels are critical to the α-cell's capacity to sense changes in extracellular glucose. Here, we review recent data on the intrinsic and paracrine regulation of glucagon secretion in human pancreatic islets. We propose that glucose-induced closure of the K(ATP)-channels, via membrane depolarization, culminates in reduced electrical activity and glucagon secretion by voltage-dependent inactivation of the ion channels involved in action potential firing. We further demonstrate that glucagon secretion measured in islets isolated from donors with type-2 diabetes is reduced at low glucose and that glucose stimulates rather than inhibits secretion in these islets. We finally discuss the relative significance of paracrine and intrinsic regulation in the fed and fasted states and propose a unifying model for the regulation of glucagon secretion that incorporates both modes of control.

Rorsman P, Eliasson L, Kanno T, Zhang Q, Gopel S. 2011. Electrophysiology of pancreatic β-cells in intact mouse islets of Langerhans. Prog Biophys Mol Biol, 107 (2), pp. 224-235. | Show Abstract | Read more

When exposed to intermediate glucose concentrations (6-16 mol/l), pancreatic β-cells in intact islets generate bursts of action potentials (superimposed on depolarised plateaux) separated by repolarised electrically silent intervals. First described more than 40 years ago, these oscillations have continued to intrigue β-cell electrophysiologists. To date, most studies of β-cell ion channels have been performed on isolated cells maintained in tissue culture (that do not burst). Here we will review the electrophysiological properties of β-cells in intact, freshly isolated, mouse pancreatic islets. We will consider the role of ATP-regulated K⁺-channels (K(ATP)-channels), small-conductance Ca²⁺-activated K⁺-channels and voltage-gated Ca²⁺-channels in the generation of the bursts. Our data indicate that K(ATP)-channels not only constitute the glucose-regulated resting conductance in the β-cell but also provide a variable K⁺-conductance that influence the duration of the bursts of action potentials and the silent intervals. We show that inactivation of the voltage-gated Ca²⁺-current is negligible at voltages corresponding to the plateau potential and consequently unlikely to play a major role in the termination of the burst. Finally, we propose a model for glucose-induced β-cell electrical activity based on observations made in intact pancreatic islets.

Galvanovskis J, Braun M, Rorsman P. 2011. Exocytosis from pancreatic β-cells: mathematical modelling of the exit of low-molecular-weight granule content. Interface Focus, 1 (1), pp. 143-152. | Show Abstract | Read more

Pancreatic β-cells use Ca(2+)-dependent exocytosis of large dense core vesicles to release insulin. Exocytosis in β-cells has been studied biochemically, biophysically and optically. We have previously developed a biophysical method to monitor release of endogenous intragranular constituents that are co-released with insulin. This technique involves the expression of ionotropic membrane receptors in the β-cell plasma membrane and enables measurements of exocytosis of individual vesicles with sub-millisecond resolution. Like carbon fibre amperometry, this method allows fine details of the release process, like the expansion of the fusion pore (the narrow connection between the granule lumen and the extracellular space), to be monitored. Here, we discuss experimental data obtained with this method within the framework of a simple mathematical model that describes the release of low-molecular constituents during exocytosis of the insulin granules. Our findings suggest that the fusion pore functions as a molecular sieve, allowing differential release of low- and high-molecular-weight granule constituents.

Hoppa MB, Jones E, Karanauskaite J, Ramracheya R, Braun M, Collins SC, Zhang Q, Clark A et al. 2011. Multivesicular exocytosis in rat pancreatic beta cells Diabetologia, pp. 1-12.

Walker JN, Johnson PRV, Shigeto M, Hughes SJ, Clark A, Rorsman P. 2011. Glucose-responsive beta cells in islets isolated from a patient with long-standing type 1 diabetes mellitus Diabetologia, 54 (1), pp. 200-202. | Read more

MacDonald PE, Rorsman P. 2011. Per-arnt-sim (PAS) domain kinase (PASK) as a regulator of glucagon secretion Diabetologia, 54 (4), pp. 719-721. | Show Abstract | Read more

The physiological and pathophysiological regulation of glucagon secretion from pancreatic alpha cells remains a hotly debated topic. The mechanism(s) contributing to the glucose sensitivity of glucagon release and its impaired regulation in diabetes remain unclear. A paper in the current issue of Diabetologia by da Silva Xavier and colleagues (doi: 10.1007/s00125-010-2010-7 ) provides intriguing new insight into a metabolic sensing pathway mediated by the per-arnt-sim (PAS) domain kinase (PASK) that may contribute to both the paracrine and the intrinsic glucose regulation of alpha cells. Importantly, the authors show that PASK is decreased in islets from patients with type 2 diabetes, providing a potential mechanism for impaired suppression of glucagon by hyperglycaemia in this disease. Much work remains to be done to determine the exact role and mechanism of PASK in alpha and beta cells. Nevertheless, the present work introduces a new player in the metabolic regulation of glucagon secretion. © 2011 Springer-Verlag.

Akhmedov D, Braun M, Mataki C, Park KS, Pozzan T, Schoonjans K, Rorsman P, Wollheim CB, Wiederkehr A. 2010. Mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in INS-1E clonal beta cells. FASEB J, 24 (11), pp. 4613-4626. | Show Abstract | Read more

Glucose-evoked mitochondrial signals augment ATP synthesis in the pancreatic β cell. This activation of energy metabolism increases the cytosolic ATP/ADP ratio, which stimulates plasma membrane electrical activity and insulin granule exocytosis. We have recently demonstrated that matrix pH increases during nutrient stimulation of the pancreatic β cell. Here, we have tested whether mitochondrial matrix pH controls oxidative phosphorylation and metabolism-secretion coupling in the rat β-cell line INS-1E. Acidification of the mitochondrial matrix pH by nigericin blunted nutrient-dependent respiratory and ATP responses (continuously monitored in intact cells). Using electrophysiology and single cell imaging, we find that the associated defects in energy metabolism suppress glucose-stimulated plasma membrane electrical activity and cytosolic calcium transients. The same parameters were unaffected after direct stimulation of electrical activity with tolbutamide, which bypasses mitochondrial function. Furthermore, lowered matrix pH strongly inhibited sustained, but not first-phase, insulin secretion. Our results demonstrate that the matrix pH exerts a control function on oxidative phosphorylation in intact cells and that this mode of regulation is of physiological relevance for the generation of downstream signals leading to insulin granule exocytosis. We propose that matrix pH serves a novel signaling role in sustained cell activation.

Braun M, Rorsman P. 2010. The glucagon-producing alpha cell: an electrophysiologically exceptional cell. Diabetologia, 53 (9), pp. 1827-1830. | Show Abstract | Read more

Activation of potassium channels normally serves to reduce cellular activity but recent data indicate that the glucagon-secreting alpha cells are different in this respect and that inhibition of voltage-gated potassium channels results in a paradoxical inhibition of glucagon secretion. Here we discuss these findings and attempt to provide a model for the regulation of glucagon secretion that incorporates these observations.

Ramracheya R, Ward C, Shigeto M, Walker JN, Amisten S, Zhang Q, Johnson PR, Rorsman P, Braun M. 2010. Membrane potential-dependent inactivation of voltage-gated ion channels in alpha-cells inhibits glucagon secretion from human islets. Diabetes, 59 (9), pp. 2198-2208. | Show Abstract | Read more

OBJECTIVE: To document the properties of the voltage-gated ion channels in human pancreatic alpha-cells and their role in glucagon release. RESEARCH DESIGN AND METHODS: Glucagon release was measured from intact islets. [Ca(2+)](i) was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated alpha-cells identified by immunocytochemistry. RESULT: Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl(2). Glucose remained inhibitory in the presence of ZnCl(2) and after blockade of type-2 somatostatin receptors. Human alpha-cells are electrically active at 1 mmol/l glucose. Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude. Human alpha-cells contain heteropodatoxin-sensitive A-type K(+)-channels, stromatoxin-sensitive delayed rectifying K(+)-channels, tetrodotoxin-sensitive Na(+)-currents, and low-threshold T-type, isradipine-sensitive L-type, and omega-agatoxin-sensitive P/Q-type Ca(2+)-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, omega-agatoxin, and isradipine. The [Ca(2+)](i) oscillations depend principally on Ca(2+)-influx via L-type Ca(2+)-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV. Blocking P/Q-type Ca(2+)-currents abolished depolarization-evoked exocytosis. CONCLUSIONS: Human alpha-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

De Marinis YZ, Salehi A, Ward CE, Zhang Q, Abdulkader F, Bengtsson M, Braha O, Braun M et al. 2010. GLP-1 inhibits and adrenaline stimulates glucagon release by differential modulation of N- and L-type Ca2+ channel-dependent exocytosis. Cell Metab, 11 (6), pp. 543-553. | Show Abstract | Read more

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).

Collins SC, Hoppa MB, Walker JN, Amisten S, Abdulkader F, Bengtsson M, Fearnside J, Ramracheya R et al. 2010. Progression of diet-induced diabetes in C57BL6J mice involves functional dissociation of Ca2(+) channels from secretory vesicles. Diabetes, 59 (5), pp. 1192-1201. | Show Abstract | Read more

OBJECTIVE: The aim of the study was to elucidate the cellular mechanism underlying the suppression of glucose-induced insulin secretion in mice fed a high-fat diet (HFD) for 15 weeks. RESEARCH DESIGN AND METHODS: C57BL6J mice were fed a HFD or a normal diet (ND) for 3 or 15 weeks. Plasma insulin and glucose levels in vivo were assessed by intraperitoneal glucose tolerance test. Insulin secretion in vitro was studied using static incubations and a perfused pancreas preparation. Membrane currents, electrical activity, and exocytosis were examined by patch-clamp technique measurements. Intracellular calcium concentration ([Ca(2+)](i)) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca(2+)](i). The functional data were complemented by analyses of histology and gene transcription. RESULTS: After 15 weeks, but not 3 weeks, mice on HFD exhibited hyperglycemia and hypoinsulinemia. Pancreatic islet content and beta-cell area increased 2- and 1.5-fold, respectively. These changes correlated with a 20-50% reduction of glucose-induced insulin secretion (normalized to insulin content). The latter effect was not associated with impaired electrical activity or [Ca(2+)](i) signaling. Single-cell capacitance and TIRFM measurements of exocytosis revealed a selective suppression (>70%) of exocytosis elicited by short (50 ms) depolarization, whereas the responses to longer depolarizations were (500 ms) less affected. The loss of rapid exocytosis correlated with dispersion of Ca(2+) entry in HFD beta-cells. No changes in gene transcription of key exocytotic protein were observed. CONCLUSIONS: HFD results in reduced insulin secretion by causing the functional dissociation of voltage-gated Ca(2+) entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes.

Obermüller S, Calegari F, King A, Lindqvist A, Lundquist I, Salehi A, Francolini M, Rosa P, Rorsman P, Huttner WB, Barg S. 2010. Defective secretion of islet hormones in chromogranin-B deficient mice. PLoS One, 5 (1), pp. e8936. | Show Abstract | Read more

Granins are major constituents of dense-core secretory granules in neuroendocrine cells, but their function is still a matter of debate. Work in cell lines has suggested that the most abundant and ubiquitously expressed granins, chromogranin A and B (CgA and CgB), are involved in granulogenesis and protein sorting. Here we report the generation and characterization of mice lacking chromogranin B (CgB-ko), which were viable and fertile. Unlike neuroendocrine tissues, pancreatic islets of these animals lacked compensatory changes in other granins and were therefore analyzed in detail. Stimulated secretion of insulin, glucagon and somatostatin was reduced in CgB-ko islets, in parallel with somewhat impaired glucose clearance and reduced insulin release, but normal insulin sensitivity in vivo. CgB-ko islets lacked specifically the rapid initial phase of stimulated secretion, had elevated basal insulin release, and stored and released twice as much proinsulin as wildtype (wt) islets. Stimulated release of glucagon and somatostatin was reduced as well. Surprisingly, biogenesis, morphology and function of insulin granules were normal, and no differences were found with regard to beta-cell stimulus-secretion coupling. We conclude that CgB is not required for normal insulin granule biogenesis or maintenance in vivo, but is essential for adequate secretion of islet hormones. Consequentially CgB-ko animals display some, but not all, hallmarks of human type-2 diabetes. However, the molecular mechanisms underlying this defect remain to be determined.

Jeyabalan J, Nesbit MA, Galvanovskis J, Callaghan R, Rorsman P, Thakker RV. 2010. SEDLIN forms homodimers: characterisation of SEDLIN mutations and their interactions with transcription factors MBP1, PITX1 and SF1. PLoS One, 5 (5), pp. e10646. | Show Abstract | Read more

BACKGROUND: SEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1). SEDLIN mutations cause X-linked spondyloepiphyseal dysplasia tarda (SEDT). METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of 4 missense (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and the most C-terminal nonsense (Gln131Stop) SEDT-associated mutations on interactions with MBP1, PITX1 and SF1 by expression in COS7 cells. Wild-type SEDLIN was present in the cytoplasm and nucleus and interacted with MBP1, PITX1 and SF1; the SEDLIN mutations did not alter these subcellular localizations or the interactions. However, SEDLIN was found to homodimerize, and the formation of dimers between wild-type and mutant SEDLIN would mask a loss in these interactions. A mammalian SEDLIN null cell-line is not available, and the interactions between SEDLIN and the transcription factors were therefore investigated in yeast, which does not endogenously express SEDLIN. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with MBP1, PITX1 and SF1. Three-dimensional modelling studies of SEDLIN revealed that Asp47 resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of SEDLIN and thereby disrupt protein-protein interactions. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate that SEDLIN is present in the nucleus, forms homodimers and that SEDT-associated mutations cause a loss of interaction with the transcription factors MBP1, PITX1 and SF1.

De Marinis YZ, Zhang E, Amisten S, Taneera J, Renström E, Rorsman P, Eliasson L. 2010. Enhancement of glucagon secretion in mouse and human pancreatic alpha cells by protein kinase C (PKC) involves intracellular trafficking of PKCalpha and PKCdelta. Diabetologia, 53 (4), pp. 717-729. | Show Abstract | Read more

AIMS/HYPOTHESIS: Protein kinase C (PKC) regulates exocytosis in various secretory cells. Here we studied intracellular translocation of the PKC isoenzymes PKCalpha and PKCdelta, and investigated how activation of PKC influences glucagon secretion in mouse and human pancreatic alpha cells. METHODS: Glucagon release from intact islets was measured in static incubations, and the amounts released were determined by RIA. Exocytosis was monitored as increases in membrane capacitance using the patch-clamp technique. The expression of genes encoding PKC isoforms was analysed by real-time PCR. Intracellular PKC distribution was assessed by confocal microscopy. RESULTS: The PKC activator phorbol 12-myristate 13-acetate (PMA) stimulated glucagon secretion from mouse and human islets about fivefold (p < 0.01). This stimulation was abolished by the PKC inhibitor bisindolylmaleimide (BIM). Whereas PMA potentiated exocytosis more than threefold (p < 0.001), BIM inhibited alpha cell exocytosis by 60% (p < 0.05). In mouse islets, the PKC isoenzymes, PKCalpha and PKCbeta1, were highly abundant, while in human islets PKCeta, PKCepsilon and PKCzeta were the dominant variants. PMA stimulation of human alpha cells correlated with the translocation of PKCalpha and PKCdelta from the cytosol to the cell periphery. In the mouse alpha cells, PKCdelta was similarly affected by PMA, whereas PKCalpha was already present at the cell membrane in the absence of PMA. This association of PKCalpha in alpha cells was principally dependent on Ca(2+) influx through the L-type Ca(2+) channel. CONCLUSIONS/INTERPRETATION: PKC activation augments glucagon secretion in mouse and human alpha cells. This effect involves translocation of PKCalpha and PKCdelta to the plasma membrane, culminating in increased Ca(2+)-dependent exocytosis. In addition, we demonstrated that PKCalpha translocation and exocytosis exhibit differential Ca(2+) channel dependence.

Hoppa MB, Collins S, Ramracheya R, Hodson L, Amisten S, Zhang Q, Johnson P, Ashcroft FM, Rorsman P. 2009. Chronic palmitate exposure inhibits insulin secretion by dissociation of Ca(2+) channels from secretory granules. Cell Metab, 10 (6), pp. 455-465. | Show Abstract | Read more

Long-term (72 hr) exposure of pancreatic islets to palmitate inhibited glucose-induced insulin secretion by >50% with first- and second-phase secretion being equally suppressed. This inhibition correlated with the selective impairment of exocytosis evoked by brief (action potential-like) depolarizations, whereas that evoked by long ( approximately 250 ms) stimuli was unaffected. Under normal conditions, Ca(2+) influx elicited by brief membrane depolarizations increases [Ca(2+)](i) to high levels within discrete microdomains and triggers the exocytosis of closely associated insulin granules. We found that these domains of localized Ca(2+) entry become dispersed by long-term (72 hr), but not by acute (2 hr), exposure to palmitate. Importantly, the release competence of the granules was not affected by palmitate. Thus, the location rather than the magnitude of the Ca(2+) increase determines its capacity to evoke exocytosis. In both mouse and human islets, the palmitate-induced secretion defect was reversed when the beta cell action potential was pharmacologically prolonged.

Reed AA, Loh NY, Terryn S, Lippiat JD, Partridge C, Galvanovskis J, Williams SE, Jouret F et al. 2010. CLC-5 and KIF3B interact to facilitate CLC-5 plasma membrane expression, endocytosis, and microtubular transport: relevance to pathophysiology of Dent's disease. Am J Physiol Renal Physiol, 298 (2), pp. F365-F380. | Show Abstract | Read more

Renal tubular reabsorption is important for extracellular fluid homeostasis and much of this occurs via the receptor-mediated endocytic pathway. This pathway is disrupted in Dent's disease, an X-linked renal tubular disorder that is characterized by low-molecular-weight proteinuria, hypercalciuria, nephrolithiasis, and renal failure. Dent's disease is due to mutations of CLC-5, a chloride/proton antiporter, expressed in endosomes and apical membranes of renal tubules. Loss of CLC-5 function alters receptor-mediated endocytosis and trafficking of megalin and cubilin, although the underlying mechanisms remain to be elucidated. Here, we report that CLC-5 interacts with kinesin family member 3B (KIF3B), a heterotrimeric motor protein that facilitates fast anterograde translocation of membranous organelles. Using yeast two-hybrid, glutathione-S-transferase pull-down and coimmunoprecipitation assays, the COOH terminus of CLC-5 and the coiled-coil and globular domains of KIF3B were shown to interact. This was confirmed in vivo by endogenous coimmunoprecipitation of CLC-5 and KIF3B and codistribution with endosomal markers in mouse kidney fractions. Confocal live cell imaging in kidney cells further demonstrated association of CLC-5 and KIF3B, and transport of CLC-5-containing vesicles along KIF3B microtubules. KIF3B overexpression and underexpression, using siRNA, had reciprocal effects on whole cell chloride current amplitudes, CLC-5 cell surface expression, and endocytosis of albumin and transferrin. Clcn5(Y/-) mouse kidneys and isolated proximal tubular polarized cells showed increased KIF3B expression, whose effects on albumin endocytosis were dependent on CLC-5 expression. Thus, the CLC-5 and KIF3B interaction is important for CLC-5 plasma membrane expression and for facilitating endocytosis and microtubular transport in the kidney.

Rosengren AH, Jokubka R, Tojjar D, Granhall C, Hansson O, Li DQ, Nagaraj V, Reinbothe TM et al. 2010. Overexpression of alpha2A-adrenergic receptors contributes to type 2 diabetes. Science, 327 (5962), pp. 217-220. | Show Abstract | Read more

Several common genetic variations have been associated with type 2 diabetes, but the exact disease mechanisms are still poorly elucidated. Using congenic strains from the diabetic Goto-Kakizaki rat, we identified a 1.4-megabase genomic locus that was linked to impaired insulin granule docking at the plasma membrane and reduced beta cell exocytosis. In this locus, Adra2a, encoding the alpha2A-adrenergic receptor [alpha(2A)AR], was significantly overexpressed. Alpha(2A)AR mediates adrenergic suppression of insulin secretion. Pharmacological receptor antagonism, silencing of receptor expression, or blockade of downstream effectors rescued insulin secretion in congenic islets. Furthermore, we identified a single-nucleotide polymorphism in the human ADRA2A gene for which risk allele carriers exhibited overexpression of alpha(2A)AR, reduced insulin secretion, and increased type 2 diabetes risk. Human pancreatic islets from risk allele carriers exhibited reduced granule docking and secreted less insulin in response to glucose; both effects were counteracted by pharmacological alpha(2A)AR antagonists.

Li DQ, Jing X, Salehi A, Collins SC, Hoppa MB, Rosengren AH, Zhang E, Lundquist I et al. 2009. Suppression of sulfonylurea- and glucose-induced insulin secretion in vitro and in vivo in mice lacking the chloride transport protein ClC-3. Cell Metab, 10 (4), pp. 309-315. | Show Abstract | Read more

Priming of insulin secretory granules for release requires intragranular acidification and depends on vesicular Cl(-)-fluxes, but the identity of the chloride transporter/ion channel involved is unknown. We tested the hypothesis that the chloride transport protein ClC-3 fulfills these actions in pancreatic beta cells. In ClC-3(-/-) mice, insulin secretion evoked by membrane depolarization (high extracellular K(+), sulfonylureas), or glucose was >60% reduced compared to WT animals. This effect was mirrored by a approximately 80% reduction in depolarization-evoked beta cell exocytosis (monitored as increases in cell capacitance) in single ClC-3(-/-) beta cells, as well as a 44% reduction in proton transport across the granule membrane. ClC-3 expression in the insulin granule was demonstrated by immunoblotting, immunostaining, and negative immuno-EM in a high-purification fraction of large dense-core vesicles (LDCVs) obtained by phogrin-EGFP labeling. The data establish the importance of granular Cl(-) fluxes in granule priming and provide direct evidence for the involvement of ClC-3 in the process.

Gilon P, Rorsman P. 2009. NALCN: a regulated leak channel. EMBO Rep, 10 (9), pp. 963-964. | Read more

Williams SE, Reed AA, Galvanovskis J, Antignac C, Goodship T, Karet FE, Kotanko P, Lhotta K et al. 2009. Uromodulin mutations causing familial juvenile hyperuricaemic nephropathy lead to protein maturation defects and retention in the endoplasmic reticulum. Hum Mol Genet, 18 (16), pp. 2963-2974. | Show Abstract | Read more

Familial juvenile hyperuricaemic nephropathy (FJHN), an autosomal dominant disorder, is caused by mutations in the UMOD gene, which encodes Uromodulin, a glycosylphosphatidylinositol-anchored protein that is expressed in the thick ascending limb of the loop of Henle and excreted in the urine. Uromodulin contains three epidermal growth factor (EGF)-like domains, a cysteine-rich region which includes a domain of eight cysteines and a zona pellucida (ZP) domain. Over 90% of UMOD mutations are missense, and 62% alter a cysteine residue, implicating a role for protein misfolding in the disease. We investigated 20 northern European FJHN probands for UMOD mutations. Wild-type and mutant Uromodulins were functionally studied by expression in HeLa cells and by the use of western blot analysis and confocal microscopy. Six different UMOD missense mutations (Cys32Trp, Arg185Gly, Asp196Asn, Cys217Trp, Cys223Arg and Gly488Arg) were identified. Patients with UMOD mutations were phenotypically similar to those without UMOD mutations. The mutant Uromodulins had significantly delayed maturation, retention in the endoplasmic reticulum (ER) and reduced expression at the plasma membrane. However, Gly488Arg, which is the only mutation we identified in the ZP domain, was found to be associated with milder in vitro abnormalities and to be the only mutant Uromodulin detected in conditioned medium from transfected cells, indicating that the severity of the mutant phenotypes may depend on their location within the protein. Thus, FJHN-causing Uromodulin mutants are retained in the ER, with impaired intracellular maturation and trafficking, thereby indicating mechanisms whereby Uromodulin mutants may cause the phenotype of FJHN.

Braun M, Ramracheya R, Amisten S, Bengtsson M, Moritoh Y, Zhang Q, Johnson PR, Rorsman P. 2009. Somatostatin release, electrical activity, membrane currents and exocytosis in human pancreatic delta cells. Diabetologia, 52 (8), pp. 1566-1578. | Show Abstract | Read more

AIMS/HYPOTHESIS: The aim of this study was to characterise electrical activity, ion channels, exocytosis and somatostatin release in human delta cells/pancreatic islets. METHODS: Glucose-stimulated somatostatin release was measured from intact human islets. Membrane potential, currents and changes in membrane capacitance (reflecting exocytosis) were recorded from individual human delta cells identified by immunocytochemistry. RESULTS: Somatostatin secretion from human islets was stimulated by glucose and tolbutamide and inhibited by diazoxide. Human delta cells generated bursting or sporadic electrical activity, which was enhanced by tolbutamide but unaffected by glucose. Delta cells contained a tolbutamide-insensitive, Ba(2+)-sensitive inwardly rectifying K(+) current and two types of voltage-gated K(+) currents, sensitive to tetraethylammonium/stromatoxin (delayed rectifying, Kv2.1/2.2) and 4-aminopyridine (A current). Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents contributed to the action potential upstroke but TTX had no effect on somatostatin release. Delta cells are equipped with Ca(2+) channels blocked by isradipine (L), omega-agatoxin (P/Q) and NNC 55-0396 (T). Blockade of any of these channels interferes with delta cell electrical activity and abolishes glucose-stimulated somatostatin release. Capacitance measurements revealed a slow component of depolarisation-evoked exocytosis sensitive to omega-agatoxin. CONCLUSIONS/INTERPRETATION: Action potential firing in delta cells is modulated by ATP-sensitive K(+)-channel activity. The membrane potential is stabilised by Ba(2+)-sensitive inwardly rectifying K(+) channels. Voltage-gated L- and T-type Ca(2+) channels are required for electrical activity, whereas Na(+) currents and P/Q-type Ca(2+) channels contribute to (but are not necessary for) the upstroke of the action potential. Action potential repolarisation is mediated by A-type and Kv2.1/2.2 K(+) channels. Exocytosis is tightly linked to Ca(2+)-influx via P/Q-type Ca(2+) channels. Glucose stimulation of somatostatin secretion involves both K(ATP) channel-dependent and -independent processes.

Olofsson CS, Håkansson J, Salehi A, Bengtsson M, Galvanovskis J, Partridge C, SörhedeWinzell M, Xian X et al. 2009. Impaired insulin exocytosis in neural cell adhesion molecule-/- mice due to defective reorganization of the submembrane F-actin network. Endocrinology, 150 (7), pp. 3067-3075. | Show Abstract | Read more

The neural cell adhesion molecule (NCAM) is required for cell type segregation during pancreatic islet organogenesis. We have investigated the functional consequences of ablating NCAM on pancreatic beta-cell function. In vivo, NCAM(-/-) mice exhibit impaired glucose tolerance and basal hyperinsulinemia. Insulin secretion from isolated NCAM(-/-) islets is enhanced at glucose concentrations below 15 mM but inhibited at higher concentrations. Glucagon secretion from pancreatic alpha-cells evoked by low glucose was also severely impaired in NCAM(-/-) islets. The diminution of insulin secretion is not attributable to defective glucose metabolism or glucose sensing (documented as glucose-induced changes in intracellular Ca(2+) and K(ATP)-channel activity). Resting K(ATP) conductance was lower in NCAM(-/-) beta-cells than wild-type cells, and this difference was abolished when F-actin was disrupted by cytochalasin D (1 muM). In wild-type beta-cells, the submembrane actin network disassembles within 10 min during glucose stimulation (30 mM), an effect not seen in NCAM(-/-) beta-cells. Cytochalasin D eliminated this difference and normalized insulin and glucagon secretion in NCAM(-/-) islets. Capacitance measurements of exocytosis indicate that replenishment of the readily releasable granule pool is suppressed in NCAM(-/-) alpha- and beta-cells. Our data suggest that remodeling of the submembrane actin network is critical to normal glucose regulation of both insulin and glucagon secretion.

Karanauskaite J, Hoppa MB, Braun M, Galvanovskis J, Rorsman P. 2009. Quantal ATP release in rat beta-cells by exocytosis of insulin-containing LDCVs. Pflugers Arch, 458 (2), pp. 389-401. | Show Abstract | Read more

Quantal release of adenosine triphosphate (ATP) was monitored in rat pancreatic beta-cells expressing P2X(2) receptors. Stimulation of exocytosis evoked rapidly activating and deactivating ATP-dependent transient inward currents (TICs). The unitary charge (q) of the events recorded at 0.2 microM [Ca(2+)](i) averaged 4.3 pC. The distribution of the 3 square root q of these events could be described by a single Gaussian. The rise times averaged approximately 5 ms over a wide range of TIC amplitudes. In beta-cells preloaded with 5-hydroxytryptamine (5-HT; accumulating in insulin granules), ATP was coreleased with 5-HT during >90% of the release events. Following step elevation of [Ca(2+)](i) to approximately 5 microM by photo release of caged Ca(2+), an increase in membrane capacitance was observed after 33 ms, whereas ATP release first became detectable after 43 ms. The step increase in [Ca(2+)](i) produced an initial large TIC followed by a series of smaller events that echoed the changes in membrane capacitance (DeltaC(m)). Mathematical modeling suggests that the large initial TIC reflects the superimposition of many unitary events. Exocytosis, measured as DeltaC(m) or TICs, was complete within 2 s after elevation of [Ca(2+)](i) with no sign of endocytosis masking the capacitance increase. The relationship between total charge (Q) and DeltaC(m) was linear with a slope of approximately 1.2 pC/fF. The latter value predicts a capacitance increase of 3.6 fF for the observed mean value of q, close to that expected for exocytosis of individual insulin granules. Our results indicate that measurements of ATP release and DeltaC(m) principally (> or =85-95%) report exocytosis of insulin granules.

Poy MN, Hausser J, Trajkovski M, Braun M, Collins S, Rorsman P, Zavolan M, Stoffel M. 2009. miR-375 maintains normal pancreatic alpha- and beta-cell mass. Proc Natl Acad Sci U S A, 106 (14), pp. 5813-5818. | Show Abstract | Read more

Altered growth and development of the endocrine pancreas is a frequent cause of the hyperglycemia associated with diabetes. Here we show that microRNA-375 (miR-375), which is highly expressed in pancreatic islets, is required for normal glucose homeostasis. Mice lacking miR-375 (375KO) are hyperglycemic, exhibit increased total pancreatic alpha-cell numbers, fasting and fed plasma glucagon levels, and increased gluconeogenesis and hepatic glucose output. Furthermore, pancreatic beta-cell mass is decreased in 375KO mice as a result of impaired proliferation. In contrast, pancreatic islets of obese mice (ob/ob), a model of increased beta-cell mass, exhibit increased expression of miR-375. Genetic deletion of miR-375 from these animals (375/ob) profoundly diminished the proliferative capacity of the endocrine pancreas and resulted in a severely diabetic state. Bioinformatic analysis of transcript data from 375KO islets revealed that miR-375 regulates a cluster of genes controlling cellular growth and proliferation. These data provide evidence that miR-375 is essential for normal glucose homeostasis, alpha- and beta-cell turnover, and adaptive beta-cell expansion in response to increasing insulin demand in insulin resistance.

Gloyn AL, Braun M, Rorsman P. 2009. Type 2 diabetes susceptibility gene TCF7L2 and its role in beta-cell function. Diabetes, 58 (4), pp. 800-802. | Read more

Gromada J, Duttaroy A, Rorsman P. 2009. The insulin receptor talks to glucagon? Cell Metab, 9 (4), pp. 303-305. | Show Abstract | Read more

Type 2 diabetes (T2DM) is not only a disorder of impaired insulin secretion but also glucagon oversecretion. However, the link between the two remains unclear. Is it possible that the latter is a consequence of the former? In this issue, Kawamori et al. (2009) have addressed this question by generating alpha cell-specific insulin receptor knockout mice.

Hanna ST, Pigeau GM, Galvanovskis J, Clark A, Rorsman P, MacDonald PE. 2009. Kiss-and-run exocytosis and fusion pores of secretory vesicles in human beta-cells. Pflugers Arch, 457 (6), pp. 1343-1350. | Show Abstract | Read more

Exocytosis of secretory vesicles results in the release of insulin from pancreatic beta-cells, although little is known about this process in humans. We examined the exocytosis of single secretory vesicles and their associated fusion pores in human beta-cells by cell-attached capacitance and conductance measurement. Unitary capacitance steps were observed, consistent with the exocytosis of single secretory vesicles. These were often coincident with increases in patch conductance representing the presence of a stable fusion pore. In some events, the fusion pore closed, mediating kiss-and-run, which contributed 20% of the exocytotic events. The cAMP-raising agent forskolin (5 microM) doubled the relative contribution of kiss-and-run. This effect was confirmed visually in MIN6 cells expressing a fluorescent granule probe. Thus, we demonstrate the unitary capacitance steps and fusion pores during single vesicle exocytosis in human beta-cells. Furthermore, these secretory vesicles can undergo rapid recycling by kiss-and-run, and this process is up-regulated by cAMP.

Gustavsson N, Wei SH, Hoang DN, Lao Y, Zhang Q, Radda GK, Rorsman P, Südhof TC, Han W. 2009. Synaptotagmin-7 is a principal Ca2+ sensor for Ca2+ -induced glucagon exocytosis in pancreas. J Physiol, 587 (Pt 6), pp. 1169-1178. | Show Abstract | Read more

Hormones such as glucagon are secreted by Ca(2+)-induced exocytosis of large dense-core vesicles, but the mechanisms involved have only been partially elucidated. Studies of pancreatic beta-cells secreting insulin revealed that synaptotagmin-7 alone is not sufficient to mediate Ca(2+)-dependent insulin granule exocytosis, and studies of chromaffin cells secreting neuropeptides and catecholamines showed that synaptotagmin-1 and -7 collaborate as Ca(2+) sensors for exocytosis, and that both are equally involved. As no other peptide secretion was analysed, it remains unclear whether synaptotagmins generally act as Ca(2+) sensors in large dense-core vesicle exocytosis in endocrine cells, and if so, whether synaptotagmin-7 always functions with a partner in that role. In particular, far less is known about the mechanisms underlying Ca(2+)-triggered glucagon release from alpha-cells than insulin secretion from beta-cells, even though insulin and glucagon together regulate blood glucose levels. To address these issues, we analysed the role of synaptotagmins in Ca(2+)-triggered glucagon exocytosis. Surprisingly, we find that deletion of a single synaptotagmin isoform, synaptotagmin-7, nearly abolished Ca(2+)-triggered glucagon secretion. Moreover, single-cell capacitance measurements confirmed that pancreatic alpha-cells lacking synaptotagmin-7 exhibited little Ca(2+)-induced exocytosis, whereas all other physiological and morphological parameters of the alpha-cells were normal. Our data thus identify synaptotagmin-7 as a principal Ca(2+) sensor for glucagon secretion, and support the notion that synaptotagmins perform a universal but selective function as individually acting Ca(2+) sensors in neurotransmitter, neuropeptide, and hormone secretion.

Mårtensson UE, Salehi SA, Windahl S, Gomez MF, Swärd K, Daszkiewicz-Nilsson J, Wendt A, Andersson N et al. 2009. Deletion of the G protein-coupled receptor 30 impairs glucose tolerance, reduces bone growth, increases blood pressure, and eliminates estradiol-stimulated insulin release in female mice. Endocrinology, 150 (2), pp. 687-698. | Show Abstract | Read more

In vitro studies suggest that the G protein-coupled receptor (GPR) 30 is a functional estrogen receptor. However, the physiological role of GPR30 in vivo is unknown, and it remains to be determined whether GPR30 is an estrogen receptor also in vivo. To this end, we studied the effects of disrupting the GPR30 gene in female and male mice. Female GPR30((-/-)) mice had hyperglycemia and impaired glucose tolerance, reduced body growth, increased blood pressure, and reduced serum IGF-I levels. The reduced growth correlated with a proportional decrease in skeletal development. The elevated blood pressure was associated with an increased vascular resistance manifested as an increased media to lumen ratio of the resistance arteries. The hyperglycemia and impaired glucose tolerance in vivo were associated with decreased insulin expression and release in vivo and in vitro in isolated pancreatic islets. GPR30 is expressed in islets, and GPR30 deletion abolished estradiol-stimulated insulin release both in vivo in ovariectomized adult mice and in vitro in isolated islets. Our findings show that GPR30 is important for several metabolic functions in female mice, including estradiol-stimulated insulin release.

Sumara G, Formentini I, Collins S, Sumara I, Windak R, Bodenmiller B, Ramracheya R, Caille D et al. 2009. Regulation of PKD by the MAPK p38delta in insulin secretion and glucose homeostasis. Cell, 136 (2), pp. 235-248. | Show Abstract | Read more

Dysfunction and loss of insulin-producing pancreatic beta cells represent hallmarks of diabetes mellitus. Here, we show that mice lacking the mitogen-activated protein kinase (MAPK) p38delta display improved glucose tolerance due to enhanced insulin secretion from pancreatic beta cells. Deletion of p38delta results in pronounced activation of protein kinase D (PKD), the latter of which we have identified as a pivotal regulator of stimulated insulin exocytosis. p38delta catalyzes an inhibitory phosphorylation of PKD1, thereby attenuating stimulated insulin secretion. In addition, p38delta null mice are protected against high-fat-feeding-induced insulin resistance and oxidative stress-mediated beta cell failure. Inhibition of PKD1 reverses enhanced insulin secretion from p38delta-deficient islets and glucose tolerance in p38delta null mice as well as their susceptibility to oxidative stress. In conclusion, the p38delta-PKD pathway integrates regulation of the insulin secretory capacity and survival of pancreatic beta cells, pointing to a pivotal role for this pathway in the development of overt diabetes mellitus.

Braun M, Ramracheya R, Johnson PR, Rorsman P. 2009. Exocytotic properties of human pancreatic beta-cells. Ann N Y Acad Sci, 1152 (1), pp. 187-193. | Show Abstract | Read more

Pancreatic beta-cells secrete insulin in response to elevated blood glucose via Ca(2+)-dependent fusion of secretory granules with the plasma membrane (regulated exocytosis). While exocytosis has been extensively investigated in rodent beta-cells, studies on human beta-cells are scarce. We have characterized the exocytotic properties of human beta-cells by insulin release measurements, carbon fiber amperometry, and capacitance measurements using the patch-clamp technique. Voltage-clamp depolarizations evoked capacitance increases in single beta-cells in a time- and voltage-dependent manner. The capacitance responses as well as insulin release from intact islets were strongly amplified by elevation of intracellular cAMP levels. Exocytosis was more dependent on Ca(2+) influx through P/Q-type than L-type Ca(2+) channels, reflecting the relative contribution of these channels to the total Ca(2+) current. Exocytosis (as monitored by capacitance or amperometric measurements) decreased during repetitive stimulation as a result of inactivation of Ca(2+) channels as well as depletion of a readily releasable pool of granules. These results reveal both similarities and differences between human and rodent beta-cells.

Girard CA, Wunderlich FT, Shimomura K, Collins S, Kaizik S, Proks P, Abdulkader F, Clark A et al. 2009. Expression of an activating mutation in the gene encoding the KATP channel subunit Kir6.2 in mouse pancreatic beta cells recapitulates neonatal diabetes. J Clin Invest, 119 (1), pp. 80-90. | Show Abstract | Read more

Neonatal diabetes is a rare monogenic form of diabetes that usually presents within the first six months of life. It is commonly caused by gain-of-function mutations in the genes encoding the Kir6.2 and SUR1 subunits of the plasmalemmal ATP-sensitive K+ (KATP) channel. To better understand this disease, we generated a mouse expressing a Kir6.2 mutation (V59M) that causes neonatal diabetes in humans and we used Cre-lox technology to express the mutation specifically in pancreatic beta cells. These beta-V59M mice developed severe diabetes soon after birth, and by 5 weeks of age, blood glucose levels were markedly increased and insulin was undetectable. Islets isolated from beta-V59M mice secreted substantially less insulin and showed a smaller increase in intracellular calcium in response to glucose. This was due to a reduced sensitivity of KATP channels in pancreatic beta cells to inhibition by ATP or glucose. In contrast, the sulfonylurea tolbutamide, a specific blocker of KATP channels, closed KATP channels, elevated intracellular calcium levels, and stimulated insulin release in beta-V59M beta cells, indicating that events downstream of KATP channel closure remained intact. Expression of the V59M Kir6.2 mutation in pancreatic beta cells alone is thus sufficient to recapitulate the neonatal diabetes observed in humans. beta-V59M islets also displayed a reduced percentage of beta cells, abnormal morphology, lower insulin content, and decreased expression of Kir6.2, SUR1, and insulin mRNA. All these changes are expected to contribute to the diabetes of beta-V59M mice. Their cause requires further investigation.

Zehetner J, Danzer C, Collins S, Eckhardt K, Gerber PA, Ballschmieter P, Galvanovskis J, Shimomura K et al. 2008. PVHL is a regulator of glucose metabolism and insulin secretion in pancreatic beta cells. Genes Dev, 22 (22), pp. 3135-3146. | Show Abstract | Read more

Insulin secretion from pancreatic beta cells is stimulated by glucose metabolism. However, the relative importance of metabolizing glucose via mitochondrial oxidative phosphorylation versus glycolysis for insulin secretion remains unclear. von Hippel-Lindau (VHL) tumor suppressor protein, pVHL, negatively regulates hypoxia-inducible factor HIF1alpha, a transcription factor implicated in promoting a glycolytic form of metabolism. Here we report a central role for the pVHL-HIF1alpha pathway in the control of beta-cell glucose utilization, insulin secretion, and glucose homeostasis. Conditional inactivation of Vhlh in beta cells promoted a diversion of glucose away from mitochondria into lactate production, causing cells to produce high levels of glycolytically derived ATP and to secrete elevated levels of insulin at low glucose concentrations. Vhlh-deficient mice exhibited diminished glucose-stimulated changes in cytoplasmic Ca(2+) concentration, electrical activity, and insulin secretion, which culminate in impaired systemic glucose tolerance. Importantly, combined deletion of Vhlh and Hif1alpha rescued these phenotypes, implying that they are the result of HIF1alpha activation. Together, these results identify pVHL and HIF1alpha as key regulators of insulin secretion from pancreatic beta cells. They further suggest that changes in the metabolic strategy of glucose metabolism in beta cells have profound effects on whole-body glucose homeostasis.

Zhang Q, Galvanovskis J, Abdulkader F, Partridge CJ, Göpel SO, Eliasson L, Rorsman P. 2008. Cell coupling in mouse pancreatic beta-cells measured in intact islets of Langerhans. Philos Trans A Math Phys Eng Sci, 366 (1880), pp. 3503-3523. | Show Abstract | Read more

The perforated whole-cell configuration of the patch-clamp technique was applied to functionally identified beta-cells in intact mouse pancreatic islets to study the extent of cell coupling between adjacent beta-cells. Using a combination of current- and voltage-clamp recordings, the total gap junctional conductance between beta-cells in an islet was estimated to be 1.22 nS. The analysis of the current waveforms in a voltage-clamped cell (due to the firing of an action potential in a neighbouring cell) suggested that the gap junctional conductance between a pair of beta-cells was 0.17 nS. Subthreshold voltage-clamp depolarization (to -55 mV) gave rise to a slow capacitive current indicative of coupling between beta-cells, but not in non-beta-cells, with a time constant of 13.5 ms and a total charge movement of 0.2 pC. Our data suggest that a superficial beta-cell in an islet is in electrical contact with six to seven other beta-cells. No evidence for dye coupling was obtained when cells were dialysed with Lucifer yellow even when electrical coupling was apparent. The correction of the measured resting conductance for the contribution of the gap junctional conductance indicated that the whole-cell KATP channel conductance (GK,ATP) falls from approximately 2.5 nS in the absence of glucose to 0.1 nS at 15 mM glucose with an estimated IC50 of approximately 4mM. Theoretical considerations indicate that the coupling between beta-cells within the islet is sufficient to allow propagation of [Ca2+]i waves to spread with a speed of approximately 80 microms-1, similar to that observed experimentally in confocal [Ca2+]i imaging.

Rorsman P, Salehi SA, Abdulkader F, Braun M, MacDonald PE. 2008. K(ATP)-channels and glucose-regulated glucagon secretion. Trends Endocrinol Metab, 19 (8), pp. 277-284. | Show Abstract | Read more

Glucagon, secreted by the alpha-cells of the pancreatic islets, is the most important glucose-increasing hormone of the body. The precise regulation of glucagon release remains incompletely defined but has been proposed to involve release of inhibitory factors from neighbouring beta-cells (paracrine control). However, the observation that glucose can regulate glucagon secretion under conditions when insulin secretion does not occur argues that the alpha-cell is also equipped with its own intrinsic (exerted within the alpha-cell itself) glucose sensing. Here we consider the possible mechanisms involved with a focus on ATP-regulated K(+)-channels and changes in alpha-cell membrane potential.

Collins SC, Salehi A, Eliasson L, Olofsson CS, Rorsman P. 2008. Long-term exposure of mouse pancreatic islets to oleate or palmitate results in reduced glucose-induced somatostatin and oversecretion of glucagon. Diabetologia, 51 (9), pp. 1689-1693. | Show Abstract | Read more

AIMS/HYPOTHESIS: Long-term exposure to NEFAs leads to inhibition of glucose-induced insulin secretion. We tested whether the release of somatostatin and glucagon, the two other major islet hormones, is also affected. METHODS: Mouse pancreatic islets were cultured for 72 h at 4.5 or 15 mmol/l glucose with or without 0.5 mmol/l oleate or palmitate. The release of glucagon and somatostatin during subsequent 1 h incubations at 1 or 20 mmol/l glucose as well as the islet content of the two hormones were determined. Lipid-induced changes in islet cell ultrastructure were assessed by electron microscopy. RESULTS: Culture at 15 mmol/l glucose increased islet glucagon content by approximately 50% relative to that observed following culture at 4.5 mmol/l glucose. Inclusion of oleate or palmitate reduced islet glucagon content by 25% (at 4.5 mmol/l glucose) to 50% (at 15 mmol/l glucose). Long-term exposure to the NEFA increased glucagon secretion at 1 mmol/l glucose by 50% (when islets had been cultured at 15 mmol/l glucose) to 100% (with 4.5 mmol/l glucose in the culture medium) and abolished the inhibitory effect of 20 mmol/l glucose on glucagon secretion. Somatostatin content was unaffected by glucose and lipids, but glucose-induced somatostatin secretion was reduced by approximately 50% following long-term exposure to either of the NEFA, regardless of whether the culture medium contained 4.5 or 15 mmol/l glucose. Ultrastructural evidence of lipid deposition was seen in <10% of non-beta cells but in >80% of the beta cells. CONCLUSIONS/INTERPRETATION: Long-term exposure to high glucose and/or NEFA affects the release of somatostatin and glucagon. The effects on glucagon secretion are very pronounced and in type 2 diabetes in vivo may aggravate the hyperglycaemic effects due to lack of insulin.

Eliasson L, Abdulkader F, Braun M, Galvanovskis J, Hoppa MB, Rorsman P. 2008. Novel aspects of the molecular mechanisms controlling insulin secretion. J Physiol, 586 (14), pp. 3313-3324. | Show Abstract | Read more

Pancreatic beta-cells secrete insulin by Ca(2+)-dependent exocytosis of secretory granules. beta-cell exocytosis involves SNARE (soluble NSF-attachment protein receptor) proteins similar to those controlling neurotransmitter release and depends on the close association of L-type Ca(2+) channels and granules. In most cases, the secretory granules fuse individually but there is ultrastructural and biophysical evidence of multivesicular exocytosis. Estimates of the secretory rate in beta-cells in intact islets indicate a release rate of approximately 15 granules per beta-cell per second, 100-fold higher than that observed in biochemical assays. Single-vesicle capacitance measurements reveal that the diameter of the fusion pore connecting the granule lumen with the exterior is approximately 1.4 nm. This is considerably smaller than the size of insulin and membrane fusion is therefore not obligatorily associated with release of the cargo, a feature that may contribute to the different rates of secretion detected by the biochemical and biophysical measurements. However, small molecules like ATP and GABA, which are stored together with insulin in the granules, are small enough to be released via the narrow fusion pore, which accordingly functions as a molecular sieve. We finally consider the possibility that defective fusion pore expansion accounts for the decrease in insulin secretion observed in pathophysiological states including long-term exposure to lipids.

Galvanovskis J, Rorsman P, Söderberg B. 2008. Probability of Exocytosis in Pancreatic β-Cells: Dependence on Ca2+ Sensing Latency Times, Ca2+ Channel Kinetic Parameters, and Channel Clustering pp. 299-311. | Show Abstract | Read more

The fusion of secretory vesicles and granules with the cell membrane prior to the release of their content into the extracellular space requires a transient increase of free Ca2+ concentration in the vicinity of the fusion site. Usually there is a short temporal delay in the onset of the actual fusion of membranes with reference to the rising free Ca2+ levels. This delay is described as a latency time of the Ca2+-sensing system of the secretory machinery and has been observed in several cell types, including pancreatic β-cells. The presence of a delay time of a finite length inherent to the secretory machinery of the cell has an essential effect on the probability for a certain granule to fuse with the cell membrane and to release its contents into the extracellular space during the action potential. We investigate here, theoretically and by numerical simulations, the extent of this influence and its dependence on the parameters of Ca2+ channels, channel clustering, the Ca2+-sensing system, and the length of depolarizing pulses.We use a linear probabilistic model for a random opening and closing of channels that yields an explicit expression for the Laplace transforms of the waiting time distributions for an event that at least one channel is open during the latency time. This allows one in principle to calculate the probability that a vesicle will fuse with the cell membrane during the action potential. We compare our theoretical results with numerical simulatio © 2008 Wiley-VCH Verlag GmbH & Co. KGaA.

Braun M, Ramracheya R, Bengtsson M, Zhang Q, Karanauskaite J, Partridge C, Johnson PR, Rorsman P. 2008. Voltage-gated ion channels in human pancreatic beta-cells: electrophysiological characterization and role in insulin secretion. Diabetes, 57 (6), pp. 1618-1628. | Show Abstract | Read more

OBJECTIVE: To characterize the voltage-gated ion channels in human beta-cells from nondiabetic donors and their role in glucose-stimulated insulin release. RESEARCH DESIGN AND METHODS: Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated beta-cells. The ion channel complement was determined by quantitative PCR. RESULTS: Human beta-cells express two types of voltage-gated K(+) currents that flow through delayed rectifying (K(V)2.1/2.2) and large-conductance Ca(2+)-activated K(+) (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K(V)2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents (Na(V)1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na(+) currents with TTX reduced glucose-stimulated (6-20 mmol/l) insulin secretion by 55-70%. Human beta-cells are equipped with L- (Ca(V)1.3), P/Q- (Ca(V)2.1), and T- (Ca(V)3.2), but not N- or R-type Ca(2+) channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca(2+) channels reduced glucose-induced (6 mmol/l) secretion by 60-70%. Membrane potential recordings suggest that L- and T-type Ca(2+) channels participate in action potential generation. Blockade of P/Q-type Ca(2+) channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca(2+) channels only had a minor effect. CONCLUSIONS: Voltage-gated T-type and L-type Ca(2+) channels as well as Na(+) channels participate in glucose-stimulated electrical activity and insulin secretion. Ca(2+)-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca(2+) influx through P/Q-type Ca(2+) channels.

Speidel D, Salehi A, Obermueller S, Lundquist I, Brose N, Renström E, Rorsman P. 2008. CAPS1 and CAPS2 regulate stability and recruitment of insulin granules in mouse pancreatic beta cells. Cell Metab, 7 (1), pp. 57-67. | Show Abstract | Read more

CAPS1 and CAPS2 regulate dense-core vesicle release of transmitters and hormones in neuroendocrine cells, but their precise roles in the secretory process remain enigmatic. Here we show that CAPS2(-/-) and CAPS1(+/-);CAPS2(-/-) mice, despite having increased insulin sensitivity, are glucose intolerant and that this effect is attributable to a marked reduction of glucose-induced insulin secretion. This correlates with diminished Ca(2+)-dependent exocytosis, a reduction in the size of the morphologically docked pool, a decrease in the readily releasable pool of secretory vesicles, slowed granule priming, and suppression of second-phase (but not first-phase) insulin secretion. In beta cells of CAPS1(+/-);CAPS2(-/-) mice, the lowered insulin content and granule numbers were associated with an increase in lysosome numbers and lysosomal enzyme activity. We conclude that although CAPS proteins are not required for Ca(2+)-dependent exocytosis to proceed, they exert a modulatory effect on insulin granule priming, exocytosis, and stability.

Bengtsson M, Hemberg M, Rorsman P, Ståhlberg A. 2008. Quantification of mRNA in single cells and modelling of RT-qPCR induced noise. BMC Mol Biol, 9 (1), pp. 63. | Show Abstract | Read more

BACKGROUND: Gene expression has a strong stochastic element resulting in highly variable mRNA levels between individual cells, even in a seemingly homogeneous cell population. Access to fundamental information about cellular mechanisms, such as correlated gene expression, motivates measurements of multiple genes in individual cells. Quantitative reverse transcription PCR (RT-qPCR) is the most accessible method which provides sufficiently accurate measurements of mRNA in single cells. RESULTS: Low concentration of guanidine thiocyanate was used to fully lyse single pancreatic beta-cells followed by RT-qPCR without the need for purification. The accuracy of the measurements was determined by a quantitative noise-model of the reverse transcription and PCR. The noise is insignificant for initial copy numbers >100 while at lower copy numbers the noise intrinsic of the PCR increases sharply, eventually obscuring quantitative measurements. Importantly, the model allows us to determine the RT efficiency without using artificial RNA as a standard. The experimental setup was applied on single endocrine cells, where the technical and biological noise levels were determined. CONCLUSION: Noise in single-cell RT-qPCR is insignificant compared to biological cell-to-cell variation in mRNA levels for medium and high abundance transcripts. To minimize the technical noise in single-cell RT-qPCR, the mRNA should be analyzed with a single RT reaction, and a single qPCR reaction per gene.

Wallis RH, Collins SC, Kaisaki PJ, Argoud K, Wilder SP, Wallace KJ, Ria M, Ktorza A, Rorsman P, Bihoreau MT, Gauguier D. 2008. Pathophysiological, genetic and gene expression features of a novel rodent model of the cardio-metabolic syndrome. PLoS One, 3 (8), pp. e2962. | Show Abstract | Read more

BACKGROUND: Complex etiology and pathogenesis of pathophysiological components of the cardio-metabolic syndrome have been demonstrated in humans and animal models. METHODOLOGY/PRINCIPAL FINDINGS: We have generated extensive physiological, genetic and genome-wide gene expression profiles in a congenic strain of the spontaneously diabetic Goto-Kakizaki (GK) rat containing a large region (110 cM, 170 Mb) of rat chromosome 1 (RNO1), which covers diabetes and obesity quantitative trait loci (QTL), introgressed onto the genetic background of the normoglycaemic Brown Norway (BN) strain. This novel disease model, which by the length of the congenic region closely mirrors the situation of a chromosome substitution strain, exhibits a wide range of abnormalities directly relevant to components of the cardio-metabolic syndrome and diabetes complications, including hyperglycaemia, hyperinsulinaemia, enhanced insulin secretion both in vivo and in vitro, insulin resistance, hypertriglyceridemia and altered pancreatic and renal histological structures. Gene transcription data in kidney, liver, skeletal muscle and white adipose tissue indicate that a disproportionately high number (43-83%) of genes differentially expressed between congenic and BN rats map to the GK genomic interval targeted in the congenic strain, which represents less than 5% of the total length of the rat genome. Genotype analysis of single nucleotide polymorphisms (SNPs) in strains genetically related to the GK highlights clusters of conserved and strain-specific variants in RNO1 that can assist the identification of naturally occurring variants isolated in diabetic and hypertensive strains when different phenotype selection procedures were applied. CONCLUSIONS: Our results emphasize the importance of rat congenic models for defining the impact of genetic variants in well-characterised QTL regions on in vivo pathophysiological features and cis-/trans- regulation of gene expression. The congenic strain reported here provides a novel and sustainable model for investigating the pathogenesis and genetic basis of risks factors for the cardio-metabolic syndrome.

Gerken T, Girard CA, Tung YC, Webby CJ, Saudek V, Hewitson KS, Yeo GS, McDonough MA et al. 2007. The obesity-associated FTO gene encodes a 2-oxoglutarate-dependent nucleic acid demethylase. Science, 318 (5855), pp. 1469-1472. | Show Abstract | Read more

Variants in the FTO (fat mass and obesity associated) gene are associated with increased body mass index in humans. Here, we show by bioinformatics analysis that FTO shares sequence motifs with Fe(II)- and 2-oxoglutarate-dependent oxygenases. We find that recombinant murine Fto catalyzes the Fe(II)- and 2OG-dependent demethylation of 3-methylthymine in single-stranded DNA, with concomitant production of succinate, formaldehyde, and carbon dioxide. Consistent with a potential role in nucleic acid demethylation, Fto localizes to the nucleus in transfected cells. Studies of wild-type mice indicate that Fto messenger RNA (mRNA) is most abundant in the brain, particularly in hypothalamic nuclei governing energy balance, and that Fto mRNA levels in the arcuate nucleus are regulated by feeding and fasting. Studies can now be directed toward determining the physiologically relevant FTO substrate and how nucleic acid methylation status is linked to increased fat mass.

Olofsson CS, Collins S, Bengtsson M, Eliasson L, Salehi A, Shimomura K, Tarasov A, Holm C, Ashcroft F, Rorsman P. 2007. Long-term exposure to glucose and lipids inhibits glucose-induced insulin secretion downstream of granule fusion with plasma membrane. Diabetes, 56 (7), pp. 1888-1897. | Show Abstract | Read more

Mouse beta-cells cultured at 15 mmol/l glucose for 72 h had reduced ATP-sensitive K+ (K(ATP)) channel activity (-30%), increased voltage-gated Ca2+ currents, higher intracellular free Ca2+ concentration ([Ca2+]i; +160%), more exocytosis (monitored by capacitance measurements, +100%), and greater insulin content (+230%) than those cultured at 4.5 mmol/l glucose. However, they released 20% less insulin when challenged with 20 mmol/l glucose. Glucose-induced (20 mmol/l) insulin secretion was reduced by 60-90% in islets cocultured at 4.5 or 15 mmol/l glucose and either oleate or palmitate (0.5 mmol/l). Free fatty acid (FFA)-induced inhibition of secretion was not associated with any major changes in [Ca2+]i or islet ATP content. Palmitate stimulated exocytosis by twofold or more but reduced K+-induced secretion by up to 60%. Basal (1 mmol/l glucose) K(ATP) channel activity was 40% lower in islets cultured at 4.5 mmol/l glucose plus palmitate and 60% lower in islets cultured at 15 mmol/l glucose plus either of the FFAs. Insulin content decreased by 75% in islets exposed to FFAs in the presence of high (15 mmol/l), but not low (4.5 mmol/l), glucose concentrations, but the number of secretory granules was unchanged. FFA-induced inhibition of insulin secretion was not associated with increased transcript levels of the apoptosis markers Bax (BclII-associated X protein) and caspase-3. We conclude that glucose and FFAs reduce insulin secretion by interference with the exit of insulin via the fusion pore.

Nielsen LB, Ploug KB, Swift P, Ørskov C, Jansen-Olesen I, Chiarelli F, Holst JJ, Hougaard P et al. 2007. Co-localisation of the Kir6.2/SUR1 channel complex with glucagon-like peptide-1 and glucose-dependent insulinotrophic polypeptide expression in human ileal cells and implications for glycaemic control in new onset type 1 diabetes. Eur J Endocrinol, 156 (6), pp. 663-671. | Show Abstract | Read more

OBJECTIVE: The ATP-dependent K+-channel (K(ATP)) is critical for glucose sensing and normal glucagon and insulin secretion from pancreatic endocrine alpha- and beta-cells. Gastrointestinal endocrine L- and K-cells are also glucose-sensing cells secreting glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotrophic polypeptide (GIP) respectively. The aims of this study were to 1) investigate the expression and co-localisation of the K(ATP) channel subunits, Kir6.2 and SUR1, in human L- and K-cells and 2) investigate if a common hyperactive variant of the Kir6.2 subunit, Glu23Lys, exerts a functional impact on glucose-sensing tissues in vivo that may affect the overall glycaemic control in children with new-onset type 1 diabetes. DESIGN AND METHODS: Western blot and immunohistochemical analyses were performed for expression and co-localisation studies. Meal-stimulated C-peptide test was carried out in 257 children at 1, 6 and 12 months after diagnosis. Genotyping for the Glu23Lys variant was by PCR-restriction fragment length polymorphism. RESULTS: Kir6.2 and SUR1 co-localise with GLP-1 in L-cells and with GIP in K-cells in human ileum tissue. Children with type 1 diabetes carrying the hyperactive Glu23Lys variant had higher HbA1C at diagnosis (coefficient = 0.61%, P = 0.02) and 1 month after initial insulin therapy (coefficient = 0.30%, P = 0.05), but later disappeared. However, when adjusting HbA1C for the given dose of exogenous insulin, the dose-adjusted HbA1C remained higher throughout the 12 month study period (coefficient = 0.42%, P = 0.03). CONCLUSIONS: Kir6.2 and SUR1 co-localise in the gastrointestinal endocrine L- and K-cells. The hyperactive Glu23Lys variant of the K(ATP) channel subunit Kir6.2 may cause defective glucose sensing in several tissues and impaired glycaemic control in children with type 1 diabetes.

MacDonald PE, De Marinis YZ, Ramracheya R, Salehi A, Ma X, Johnson PR, Cox R, Eliasson L, Rorsman P. 2007. A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans. PLoS Biol, 5 (6), pp. e143. | Show Abstract | Read more

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

MacDonald PE, Rorsman P. 2007. The ins and outs of secretion from pancreatic beta-cells: control of single-vesicle exo- and endocytosis. Physiology (Bethesda), 22 (2), pp. 113-121. | Show Abstract | Read more

Exocytosis of insulin-containing secretory vesicles in pancreatic beta-cells is crucial to maintenance of plasma glucose levels. They fuse with the plasma membrane in a regulated manner to release their contents and are subsequently recaptured either intact or through conventional clathrin-mediated endocytosis. Here, we discuss these mechanisms in beta-cells at the single-vesicle level.

Zhang Q, Bengtsson M, Partridge C, Salehi A, Braun M, Cox R, Eliasson L, Johnson PR et al. 2007. R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion. Nat Cell Biol, 9 (4), pp. 453-460. | Show Abstract | Read more

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.

Braun M, Wendt A, Karanauskaite J, Galvanovskis J, Clark A, MacDonald PE, Rorsman P. 2007. Corelease and differential exit via the fusion pore of GABA, serotonin, and ATP from LDCV in rat pancreatic beta cells. J Gen Physiol, 129 (3), pp. 221-231. | Show Abstract | Read more

The release of gamma-aminobutyric acid (GABA) and ATP from rat beta cells was monitored using an electrophysiological assay based on overexpression GABA(A) or P2X2 receptor ion channels. Exocytosis of LDCVs, detected by carbon fiber amperometry of serotonin, correlated strongly (approximately 80%) with ATP release. The increase in membrane capacitance per ATP release event was 3.4 fF, close to the expected capacitance of an individual LDCV with a diameter of 0.3 microm. ATP and GABA were coreleased with serotonin with the same probability. Immunogold electron microscopy revealed that approximately 15% of the LDCVs contain GABA. Prespike "pedestals," reflecting exit of granule constituents via the fusion pore, were less frequently observed for ATP than for serotonin or GABA and the relative amplitude (amplitude of foot compared to spike) was smaller: in some cases the ATP-dependent pedestal was missing entirely. An inward tonic current, not dependent on glucose and inhibited by the GABA(A) receptor antagonist SR95531, was observed in beta cells in clusters of islet cells. Noise analysis indicated that it was due to the activity of individual channels with a conductance of 30 pS, the same as expected for individual GABA(A) Cl- channels with the ionic gradients used. We conclude that (a) LDCVs accumulate ATP and serotonin; (b) regulated release of GABA can be accounted for by exocytosis of a subset of insulin-containing LDCVs; (c) the fusion pore of LDCVs exhibits selectivity and compounds are differentially released depending on their chemical properties (including size); and (d) a glucose-independent nonvesicular form of GABA release exists in beta cells.

Salehi A, Eliasson L, Ma X, Rorsman P, Håkanson R, Lundquist I. 2007. Secretory and electrophysiological characteristics of insulin cells from gastrectomized mice: evidence for the existence of insulinotropic agents in the stomach. Regul Pept, 139 (1-3), pp. 31-38. | Show Abstract | Read more

Mice were subjected to gastrectomy (GX) or sham operation (controls). Four to six weeks later the pancreatic islets were isolated and analysed for cAMP or alternatively incubated in a Krebs-Ringer based medium in an effort to study insulin secretion and cAMP accumulation in response to glucose or the adenylate cyclase activator forskolin. Freshly isolated islets from GX mice had higher cAMP content than islets from control mice, a difference that persisted after incubation for 1 h at a glucose concentration of 4 mmol/l. Addition of forskolin to this medium induced much greater cAMP and insulin responses in islets from GX mice than in islets from control mice. In contrast, the insulin response to high glucose (16.7 mmol/l) was much weaker in GX islets than in control islets. Glucose-induced insulin release was associated with a 2-fold rise in the cAMP content in control islets. Surprisingly no rise in cAMP was noted in GX islets incubated at high glucose. Capacitance measurements conducted on isolated insulin cells from GX mice revealed a much lower exocytotic response to a single 500 ms depolarisation (from -70 mV to zero) than in control insulin cells. Addition of cAMP to the cytosol enhanced the exocytotic response in insulin cells from control mice but not from GX mice. The depolarisation-triggered inward Ca(2+) current in insulin cells from GX mice did not differ from that in control mice, and hence the reduced exocytotic response following GX cannot be ascribed to a decreased Ca(2+) influx. Experiments involving a train of ten 500 ms depolarisations revealed that the exocytotic response was prominent in control insulin cells but modest in GX insulin cells. It seems that cAMP is capable of eliciting insulin release from insulin cells of GX mice only when cAMP is generated in a specific microdomain conceivably through the intervention of membrane-associated adenylate cyclases that can be activated by forskolin. The GX-evoked impairment of depolarisation-induced exocytosis and glucose-stimulated insulin release may reflect the lack of a gastric agent that serves to maintain an appropriate insulin response to glucose and an appropriate exocytotic response to depolarisation by raising cAMP in a special glucose-sensitive compartment possibly regulated by a soluble adenylate cyclase.

Jeans AF, Oliver PL, Johnson R, Capogna M, Vikman J, Molnár Z, Babbs A, Partridge CJ et al. 2007. A dominant mutation in Snap25 causes impaired vesicle trafficking, sensorimotor gating, and ataxia in the blind-drunk mouse. Proc Natl Acad Sci U S A, 104 (7), pp. 2431-2436. | Show Abstract | Read more

The neuronal soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex is essential for synaptic vesicle exocytosis, but its study has been limited by the neonatal lethality of murine SNARE knockouts. Here, we describe a viable mouse line carrying a mutation in the b-isoform of neuronal SNARE synaptosomal-associated protein of 25 kDa (SNAP-25). The causative I67T missense mutation results in increased binding affinities within the SNARE complex, impaired exocytotic vesicle recycling and granule exocytosis in pancreatic beta-cells, and a reduction in the amplitude of evoked cortical excitatory postsynaptic potentials. The mice also display ataxia and impaired sensorimotor gating, a phenotype which has been associated with psychiatric disorders in humans. These studies therefore provide insights into the role of the SNARE complex in both diabetes and psychiatric disease.

Cited:

63

Scopus

MacDonald PE, De Marinis YZ, Ramracheya R, Salehi A, Ma X, Johnson PRV, Cox R, Eliasson L, Rorsman P. 2007. A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans. PLoS biology, 5 (6), | Show Abstract | Read more

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

MacDonald PE, Braun M, Galvanovskis J, Rorsman P. 2006. Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells. Cell Metab, 4 (4), pp. 283-290. | Show Abstract | Read more

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.

Vikman J, Ma X, Hockerman GH, Rorsman P, Eliasson L. 2006. Antibody inhibition of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 reduces rapid exocytosis in insulin-secreting cells. J Mol Endocrinol, 36 (3), pp. 503-515. | Show Abstract | Read more

SNARE-proteins (soluble NSF-attachment protein receptor) are important for Ca(2+)-dependent exocytosis. We have used capacitance measurements and confocal imaging to dissect the role of synaptosomal protein of 25 kDa (SNAP-25) and syntaxin 1 in rapid exocytosis in insulin-secreting pancreatic beta-cells. Following immunoneutralization of syntaxin 1 and SNAP-25, exocytosis was strongly reduced and associated with a marked reduction in the size of the readily releasable pool (RRP) by 65% and 86% in the presence of the anti-SNAP-25 and anti-syntaxin 1 antibodies respectively. The size of the immediately releasable pool (IRP), a subset of RRP in close association with the voltage-dependent Ca(2+)-channels, was reduced to an equal extent. The reduction in IRP correlated with slowed release kinetics and the time constant (tau) increased from a control value of 16 to 36 ms and 51 ms after inclusion of anti-SNAP-25 and anti-syntaxin 1 antibodies respectively in the pipette solution. We further show that SNAP-25 and syntaxin 1 aggregate in clusters along the plasma membrane. The size of these clusters was estimated to be approximately 300 nm and every beta-cell contained approximately 400 SNAP-25/syntaxin 1 clusters. Whereas the inhibitory action of the anti-syntaxin 1 antibody on exocytosis could be attributed almost entirely to suppression of the voltage-dependent Ca(2+)-current (-40%), the effect of the anti-SNAP-25 antibody was not mediated by decreased Ca(2+)-entry and is more likely due to a direct interference with the exocytotic machinery. Our data are consistent with the concept that both syntaxin 1 and SNAP-25 are required for rapid exocytosis in beta-cells.

Taneera J, Rosengren A, Renstrom E, Nygren JM, Serup P, Rorsman P, Jacobsen SE. 2006. Failure of transplanted bone marrow cells to adopt a pancreatic beta-cell fate. Diabetes, 55 (2), pp. 290-296. | Show Abstract | Read more

Recent studies in normal mice have suggested that transplanted bone marrow cells can transdifferentiate into pancreatic beta-cells at relatively high efficiency. Herein, adopting the same and alternative approaches to deliver and fate map-transplanted bone marrow cells in the pancreas of normal as well as diabetic mice, we further investigated the potential of bone marrow transplantation as an alternative approach for beta-cell replacement. In contrast to previous studies, transplanted bone marrow cells expressing green fluorescence protein (GFP) under the control of the mouse insulin promoter failed to express GFP in the pancreas of normal as well as diabetic mice. Although bone marrow cells expressing GFP under the ubiquitously expressed beta-actin promoter efficiently engrafted the pancreas of normal and hyperglycemic mice, virtually all expressed CD45 and Mac-1/Gr-1, demonstrating that they adopt a hematopoietic rather than beta-cell fate, a finding further substantiated by the complete absence of GFP(+) cells expressing insulin and the beta-cell transcription factors pancreatic duodenal homeobox factor-1 and homeodomain protein. Thus, transplanted bone marrow cells demonstrated little, if any, capacity to adopt a beta-cell fate.

MacDonald PE, Rorsman P. 2006. Oscillations, intercellular coupling, and insulin secretion in pancreatic beta cells. PLoS Biol, 4 (2), pp. e49. | Read more

MacDonald PE, Rorsman P. 2006. Oscillations, intercellular coupling, and insulin secretion in pancreatic beta cells. PLoS biology, 4 (2), | Read more

MacDonald PE, Eliasson L, Rorsman P. 2005. Calcium increases endocytotic vesicle size and accelerates membrane fission in insulin-secreting INS-1 cells. J Cell Sci, 118 (Pt 24), pp. 5911-5920. | Show Abstract | Read more

In many cells, endocytotic membrane retrieval is accelerated by Ca2+. The effect of Ca2+ on single endocytotic vesicles and fission pore kinetics was examined by measuring capacitance and conductance changes in small membrane patches of insulin-secreting INS-1 cells. In intact cells, elevation of Ca2+ by glucose stimulation induced a 1.8-fold increase in membrane internalisation. This surprisingly resulted from an increased unitary capacitance of endocytotic vesicles whereas the frequency of endocytosis was unaltered. This effect of glucose was prevented by inhibition of L- or R-type Ca2+ channels. Extracellular (pipette) Ca2+ was found to regulate endocytotic vesicle capacitance in a bimodal manner. Vesicle capacitance was increased at intermediate Ca2+ (2.6 mM), but not at high Ca2+ (10 mM). Similar results were obtained upon direct application of 100 nM and 0.5 mM Ca2+ to the intracellular surface of inside-out excised membrane patches, and in these experiments the increase in vesicle capacitance was prevented by the calcineurin inhibitor deltamethrin. Endocytotic fission pore kinetics were accelerated by Ca2+ in both the intact cells and isolated membrane patches; however, the effect in this case was neither bimodal nor deltamethrin sensitive. Membrane retrieval can therefore be upregulated by a Ca2+-dependent increase in endocytotic vesicle size and acceleration of membrane fission in insulin-secreting INS-1 cells.

Bengtsson M, Ståhlberg A, Rorsman P, Kubista M. 2005. Gene expression profiling in single cells from the pancreatic islets of Langerhans reveals lognormal distribution of mRNA levels. Genome Res, 15 (10), pp. 1388-1392. | Show Abstract | Read more

The transcriptional machinery in individual cells is controlled by a relatively small number of molecules, which may result in stochastic behavior in gene activity. Because of technical limitations in current collection and recording methods, most gene expression measurements are carried out on populations of cells and therefore reflect average mRNA levels. The variability of the transcript levels between different cells remains undefined, although it may have profound effects on cellular activities. Here we have measured gene expression levels of the five genes ActB, Ins1, Ins2, Abcc8, and Kcnj11 in individual cells from mouse pancreatic islets. Whereas Ins1 and Ins2 expression show a strong cell-cell correlation, this is not the case for the other genes. We further found that the transcript levels of the different genes are lognormally distributed. Hence, the geometric mean of expression levels provides a better estimate of gene activity of the typical cell than does the arithmetic mean measured on a cell population.

Obermüller S, Lindqvist A, Karanauskaite J, Galvanovskis J, Rorsman P, Barg S. 2005. Selective nucleotide-release from dense-core granules in insulin-secreting cells. J Cell Sci, 118 (Pt 18), pp. 4271-4282. | Show Abstract | Read more

Secretory granules of insulin-secreting cells are used to store and release peptide hormones as well as low-molecular-weight compounds such as nucleotides. Here we have compared the rate of exocytosis with the time courses of nucleotide and peptide release by a combination of capacitance measurements, electrophysiological detection of ATP release and single-granule imaging. We demonstrate that the release of nucleotides and peptides is delayed by approximately 0.1 and approximately 2 seconds with respect to membrane fusion, respectively. We further show that in up to 70% of the cases exocytosis does not result in significant release of the peptide cargo, likely because of a mechanism that leads to premature closure of the fusion pore. Release of nucleotides and protons occurred regardless of whether peptides were secreted or not. These observations suggest that insulin-secreting cells are able to use the same secretory vesicles to release small molecules either alone or together with the peptide hormone.

MacDonald PE, Obermüller S, Vikman J, Galvanovskis J, Rorsman P, Eliasson L. 2005. Regulated exocytosis and kiss-and-run of synaptic-like microvesicles in INS-1 and primary rat beta-cells. Diabetes, 54 (3), pp. 736-743. | Show Abstract | Read more

We have applied cell-attached capacitance measurements to investigate whether synaptic-like microvesicles (SLMVs) undergo regulated exocytosis in insulinoma and primary pancreatic beta-cells. SLMV and large dense-core vesicle (LDCV) exocytosis was increased 1.6- and 2.4-fold upon stimulation with 10 mmol/l glucose in INS-1 cells. Exocytosis of both types of vesicles was coupled to Ca(2+) entry through l-type channels. Thirty percent of SLMV exocytosis in INS-1 and rat beta-cells was associated with transient capacitance increases consistent with kiss-and-run. Elevation of intracellular cAMP (5 micromol/l forskolin) increased SLMV exocytosis 1.6-fold and lengthened the duration of kiss-and-run events in rat beta-cells. Experiments using isolated inside-out patches of INS-1 cells revealed that the readily releasable pool (RRP) of SLMVs preferentially undergoes kiss-and-run exocytosis (67%), is proportionally larger than the LDCV RRP, and is depleted more quickly upon Ca(2+) stimulation. We conclude that SLMVs undergo glucose-regulated exocytosis and are capable of high turnover. Following kiss-and-run exocytosis, the SLMV RRP may be reloaded with gamma-aminobutyric acid and undergo several cycles of exo- and endocytosis. Our observations support a role for beta-cell SLMVs in a synaptic-like function of rapid intra-islet signaling.

Jing X, Li DQ, Olofsson CS, Salehi A, Surve VV, Caballero J, Ivarsson R, Lundquist I et al. 2005. CaV2.3 calcium channels control second-phase insulin release. J Clin Invest, 115 (1), pp. 146-154. | Show Abstract | Read more

Concerted activation of different voltage-gated Ca( (2+) ) channel isoforms may determine the kinetics of insulin release from pancreatic islets. Here we have elucidated the role of R-type Ca(V)2.3 channels in that process. A 20% reduction in glucose-evoked insulin secretion was observed in Ca(V)2.3-knockout (Ca(V)2.3(-/-)) islets, close to the 17% inhibition by the R-type blocker SNX482 but much less than the 77% inhibition produced by the L-type Ca(2+) channel antagonist isradipine. Dynamic insulin-release measurements revealed that genetic or pharmacological Ca(V)2.3 ablation strongly suppressed second-phase secretion, whereas first-phase secretion was unaffected, a result also observed in vivo. Suppression of the second phase coincided with an 18% reduction in oscillatory Ca(2+) signaling and a 25% reduction in granule recruitment after completion of the initial exocytotic burst in single Ca(V)2.3(-/-) beta cells. Ca(V)2.3 ablation also impaired glucose-mediated suppression of glucagon secretion in isolated islets (27% versus 58% in WT), an effect associated with coexpression of insulin and glucagon in a fraction of the islet cells in the Ca(V)2.3(-/-) mouse. We propose a specific role for Ca(V)2.3 Ca(2+) channels in second-phase insulin release, that of mediating the Ca(2+) entry needed for replenishment of the releasable pool of granules as well as islet cell differentiation.

Ma X, Zhang Y, Gromada J, Sewing S, Berggren PO, Buschard K, Salehi A, Vikman J, Rorsman P, Eliasson L. 2005. Glucagon stimulates exocytosis in mouse and rat pancreatic alpha-cells by binding to glucagon receptors. Mol Endocrinol, 19 (1), pp. 198-212. | Show Abstract | Read more

Glucagon, secreted by the pancreatic alpha-cells, stimulates insulin secretion from neighboring beta-cells by cAMP- and protein kinase A (PKA)-dependent mechanisms, but it is not known whether glucagon also modulates its own secretion. We have addressed this issue by combining recordings of membrane capacitance (to monitor exocytosis) in individual alpha-cells with biochemical assays of glucagon secretion and cAMP content in intact pancreatic islets, as well as analyses of glucagon receptor expression in pure alpha-cell fractions by RT-PCR. Glucagon stimulated cAMP generation and exocytosis dose dependently with an EC50 of 1.6-1.7 nm. The stimulation of both parameters plateaued at concentrations beyond 10 nm of glucagon where a more than 3-fold enhancement was observed. The actions of glucagon were unaffected by the GLP-1 receptor antagonist exendin-(9-39) but abolished by des-His1-[Glu9]-glucagon-amide, a specific blocker of the glucagon receptor. The effects of glucagon on alpha-cell exocytosis were mimicked by forskolin and the stimulatory actions of glucagon and forskolin on exocytosis were both reproduced by intracellular application of 0.1 mm cAMP. cAMP-potentiated exocytosis involved both PKA-dependent and -independent (resistant to Rp-cAMPS, an Rp-isomer of cAMP) mechanisms. The presence of the cAMP-binding protein cAMP-guanidine nucleotide exchange factor II in alpha-cells was documented by a combination of immunocytochemistry and RT-PCR and 8-(4-chloro-phenylthio)-2'-O-methyl-cAMP, a cAMP-guanidine nucleotide exchange factor II-selective agonist, mimicked the effect of cAMP and augmented rapid exocytosis in a PKA-independent manner. We conclude that glucagon released from the alpha-cells, in addition to its well-documented systemic effects and paracrine actions within the islet, also represents an autocrine regulator of alpha-cell function.

Rorsman P. 2005. Insulin secretion: Function and therapy of pancreatic beta-cells in diabetes British Journal of Diabetes and Vascular Disease, 5 (4), pp. 187-191. | Show Abstract

Insulin is secreted from the beta-cells of the pancreatic islets in response to an elevation of blood glucose concentration. This review describes a current view of the metabolic control of insulin secretion and the molecular mechanisms involved, including the role played by the beta-cell to ensure correct release of insulin as a result of electrical signals. It then considers what goes wrong in type 2 diabetes, a disease resulting from insufficient insulin secretion. It focuses on the influence of genetics exploring the theory of a genetic predisposition to type 2 diabetes, as well as the roles played by age and obesity. Finally, the mode of action of the hypoglycaemic sulphonylureas is discussed and the potential implications for the beta-cell associated with a sulphonylurea-based therapy.

Barg S, Rorsman P. 2004. Insulin secretion: a high-affinity Ca2+ sensor after all? J Gen Physiol, 124 (6), pp. 623-625. | Read more

Gromada J, Brock B, Schmitz O, Rorsman P. 2004. Glucagon-like peptide-1: regulation of insulin secretion and therapeutic potential. Basic Clin Pharmacol Toxicol, 95 (6), pp. 252-262. | Show Abstract | Read more

Glucagon-like peptide-1 (GLP-1) is an intestinally derived insulinotropic hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes. One of several important effects of GLP-1 is on nutrient-induced pancreatic hormone release and is mediated by binding to a specific G-protein coupled receptor resulting in the activation of adenylate cyclase and an increase in cAMP generation. In the beta-cell, cAMP binds and modulates activities of both protein kinase A and cAMP-regulated guanine nucleotide exchange factor II, thereby enhancing glucose-dependent insulin secretion. The stimulatory action of GLP-1 on insulin secretion involves interaction with a plethora of signal transduction processes including ion channel activity, intracellular Ca(2+) handling and exocytosis of the insulin-containing granules. In this review we focus principally on recent advances in our understanding on the cellular mechanisms proposed to underlie GLP-1's insulinotropic effect and attempt to incorporate this knowledge into a working model for the control of insulin secretion. Lastly, this review discusses the applicability of GLP-1 as a therapeutic agent for the treatment of type 2 diabetes.

Poy MN, Eliasson L, Krutzfeldt J, Kuwajima S, Ma X, Macdonald PE, Pfeffer S, Tuschl T, Rajewsky N, Rorsman P, Stoffel M. 2004. A pancreatic islet-specific microRNA regulates insulin secretion. Nature, 432 (7014), pp. 226-230. | Show Abstract | Read more

MicroRNAs (miRNAs) constitute a growing class of non-coding RNAs that are thought to regulate gene expression by translational repression. Several miRNAs in animals exhibit tissue-specific or developmental-stage-specific expression, indicating that they could play important roles in many biological processes. To study the role of miRNAs in pancreatic endocrine cells we cloned and identified a novel, evolutionarily conserved and islet-specific miRNA (miR-375). Here we show that overexpression of miR-375 suppressed glucose-induced insulin secretion, and conversely, inhibition of endogenous miR-375 function enhanced insulin secretion. The mechanism by which secretion is modified by miR-375 is independent of changes in glucose metabolism or intracellular Ca2+-signalling but correlated with a direct effect on insulin exocytosis. Myotrophin (Mtpn) was predicted to be and validated as a target of miR-375. Inhibition of Mtpn by small interfering (si)RNA mimicked the effects of miR-375 on glucose-stimulated insulin secretion and exocytosis. Thus, miR-375 is a regulator of insulin secretion and may thereby constitute a novel pharmacological target for the treatment of diabetes.

Gromada J, Rorsman P. 2004. New insights into the regulation of glucagon secretion by glucagon-like peptide-1. Horm Metab Res, 36 (11-12), pp. 822-829. | Show Abstract | Read more

Glucagon-like peptide-1 (GLP-1) is a potent incretin hormone currently under investigation for use as a novel therapeutic agent in the treatment of type 2 diabetes. One of several therapeutically important biological actions of GLP-1 in type 2 diabetic subjects is ability to induce strong suppression of glucagon secretion. The glucagonostatic action of GLP-1 results from its interaction with a specific G-protein coupled receptor resulting in the activation of adenylate cyclase and an increase in cAMP generation. In the pancreatic alpha-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion-channel activity and exocytosis of the glucagon-containing granules. In this short review, we will focus on recent advances in our understanding on the cellular mechanisms proposed to underlie the glucagonotropic action of GLP-1 and attempt to incorporate this knowledge into a working model for the control of glucagon secretion. Studies on the effects of GLP-1 on glucagon secretion are relevant to the pathogenesis of type 2 diabetes due to the likely contribution of hyperglucagonemia to impaired glucose tolerance in type 2 diabetes.

Olofsson CS, Salehi A, Göpel SO, Holm C, Rorsman P. 2004. Palmitate stimulation of glucagon secretion in mouse pancreatic alpha-cells results from activation of L-type calcium channels and elevation of cytoplasmic calcium. Diabetes, 53 (11), pp. 2836-2843. | Show Abstract | Read more

We have investigated the short-term effects of the saturated free fatty acid (FFA) palmitate on pancreatic alpha-cells. Palmitate (0.5 or 1 mmol/l bound to fatty acid-free albumin) stimulated glucagon secretion from intact mouse islets 1.5- to 2-fold when added in the presence of 1-15 mmol/l glucose. Palmitate remained stimulatory in islets depolarized with 30 mmol/l extracellular K(+) or exposed to forskolin, but it did not remain stimulatory after treatment with isradipine or triacsin C. The stimulatory action of palmitate on secretion correlated with a 3.5-fold elevation of intracellular free Ca(2+) when applied in the presence of 15 mmol/l glucose, a 40% stimulation of exocytosis (measured as increases in cell capacitance), and a 25% increase in whole-cell Ca(2+) current. The latter effect was abolished by isradipine, suggesting that palmitate selectively modulates l-type Ca(2+) channels. The effect of palmitate on exocytosis was not mediated by palmitoyl-CoA, and intracellular application of this FFA metabolite decreased rather than enhanced Ca(2+)-induced exocytosis. The stimulatory effects of palmitate on glucagon secretion were paralleled by a approximately 50% inhibition of somatostatin release. We conclude that palmitate increases alpha-cell exocytosis principally by enhanced Ca(2+) entry via l-type Ca(2+) channels and, possibly, relief from paracrine inhibition by somatostatin released by neighboring delta-cells.

Braun M, Wendt A, Buschard K, Salehi A, Sewing S, Gromada J, Rorsman P. 2004. GABAB receptor activation inhibits exocytosis in rat pancreatic beta-cells by G-protein-dependent activation of calcineurin. J Physiol, 559 (Pt 2), pp. 397-409. | Show Abstract | Read more

We have investigated the regulation of hormone secretion from rat pancreatic islets by the GABAB receptors (GABABRs). Inclusion of the specific GABABR antagonist CGP 55845 in the extracellular medium increased glucose-stimulated insulin secretion 1.6-fold but did not affect the release of glucagon and somatostatin. Conversely, addition of the GABABR agonist baclofen inhibited glucose-stimulated insulin secretion by approximately 60%. Using RT-PCR, transcription of GABABR1a-c,f and GABABR2 subunits was detected in beta-cells. Measurements of membrane currents and cell capacitance were applied to single beta-cells to investigate the mechanisms by which GABABR activation inhibits insulin secretion. In perforated-patch measurements, baclofen inhibited exocytosis elicited by 500-ms voltage-clamp depolarizations to 0 mV by < or = 80% and voltage-gated Ca2+ entry by only approximately 30%. Both effects were concentration-dependent with IC50 values of approximately 2 microm. The inhibitory action of baclofen was abolished in the presence of CGP 55845. The ability of baclofen to suppress exocytosis was prevented by pre-treatment with pertussis toxin and by inclusion of GDPbetaS in the intracellular medium, and became irreversible in the presence of GTPgammaS as expected for a process involving inhibitory G-proteins (Gi/o-proteins). The inhibitory effect of baclofen resulted from activation of the serine/threonine protein phosphatase calcineurin and pre-treatment with cyclosporin A or intracellular application of calcineurin autoinhibitory peptide abolished the effect. Addition of baclofen had no effect on [Ca2+]i and electrical activity in glucose-stimulated beta-cells. These data indicate that GABA released from beta-cells functions as an autocrine inhibitor of insulin secretion in pancreatic islets and that the effect is principally due to direct suppression of exocytosis.

Fex M, Olofsson CS, Fransson U, Bacos K, Lindvall H, Sörhede-Winzell M, Rorsman P, Holm C, Mulder H. 2004. Hormone-sensitive lipase deficiency in mouse islets abolishes neutral cholesterol ester hydrolase activity but leaves lipolysis, acylglycerides, fat oxidation, and insulin secretion intact. Endocrinology, 145 (8), pp. 3746-3753. | Show Abstract | Read more

Lipids are thought to serve as coupling factors in insulin secretion. Hormone-sensitive lipase (HSL) is expressed in pancreatic beta-cells and could potentially regulate insulin secretion via mobilization of stored triglycerides. Here, we examined the impact of HSL deficiency on fuel metabolism and insulin secretion in mouse islets. Lack of HSL resulted in abrogation of neutral cholesterol ester hydrolase activity, whereas diglyceride lipase activity remained intact. Although glucose stimulates lipolysis in rat islets, elevation of glucose with or without addition of cAMP failed to increase lipolysis in mouse islets regardless of genotype, as indicated by release of glycerol from islets. Storage of lipids, assayed as total acylglycerides, was unaltered in HSL null islets, and oxidation of fatty acids or glucose was not different. The intracellular rise in Ca(2+) triggered by glucose and its subsequent oscillations was unaffected in HSL null islets. Accordingly, insulin secretion in static incubations of islets, in response to fuel- and nonfuel secretagogues, was in no instance significantly different between wild-type and HSL null mice. The lacking impact of HSL deficiency on insulin secretion may be attributed to the failure of insulin secretagogues to stimulate lipolysis. Consequently, a regulatory function of lipid mobilization in insulin secretion in the mouse appears unlikely.

Kanno T, Ma X, Barg S, Eliasson L, Galvanovskis J, Göpel S, Larsson M, Renström E, Rorsman P. 2004. Large dense-core vesicle exocytosis in pancreatic beta-cells monitored by capacitance measurements. Methods, 33 (4), pp. 302-311. | Show Abstract | Read more

This article discusses the currently used methodologies for monitoring exocytosis as changes in cell capacitance. Details are given on composition of solutions, experimental protocols, and how the observed responses can be interpreted physiologically. The concepts are illustrated by examples from our own work on insulin-releasing pancreatic beta-cells. Finally, we consider the feasibility of applying capacitance measurements to endocrine cells in intact pancreatic islets, where the cells are electrically coupled to each other.

Hansson K, Ma X, Eliasson L, Czerwiec E, Furie B, Furie BC, Rorsman P, Stenflo J. 2004. The first gamma-carboxyglutamic acid-containing contryphan. A selective L-type calcium ion channel blocker isolated from the venom of Conus marmoreus. J Biol Chem, 279 (31), pp. 32453-32463. | Show Abstract | Read more

Contryphans constitute a group of conopeptides that are known to contain an unusual density of post-translational modifications including tryptophan bromination, amidation of the C-terminal residue, leucine, and tryptophan isomerization, and proline hydroxylation. Here we report the identification and characterization of a new member of this family, glacontryphan-M from the venom of Conus marmoreus. This is the first known example of a contryphan peptide carrying glutamyl residues that have been post-translationally carboxylated to gamma-carboxyglutamyl (Gla) residues. The amino acid sequence of glacontryphan-M was determined using automated Edman degradation and electrospray ionization mass spectrometry. The amino acid sequence of the peptide is: Asn-Gla-Ser-Gla-Cys-Pro-D-Trp-His-Pro-Trp-Cys. As with most other contryphans, glacontryphan-M is amidated at the C terminus and maintains the five-residue intercysteine loop. The occurrence of a D-tryptophan residue was confirmed by chemical synthesis and HPLC elution profiles. Using fluorescence spectroscopy we demonstrated that the Gla-containing peptide binds calcium with a K(D) of 0.63 mM. Cloning of the full-length cDNA encoding glacontryphan-M revealed that the primary translation product carries an N-terminal signal/propeptide sequence that is homologous to earlier reported contryphan signal/propeptide sequences up to 10 amino acids preceding the toxin region. Electrophysiological experiments, carried out on mouse pancreatic B-cells, showed that glacontryphan-M blocks L-type voltage-gated calcium ion channel activity in a calcium-dependent manner. Glacontryphan-M is the first contryphan reported to modulate the activity of L-type calcium ion channels.

Olofsson CS, Salehi A, Holm C, Rorsman P. 2004. Palmitate increases L-type Ca2+ currents and the size of the readily releasable granule pool in mouse pancreatic beta-cells. J Physiol, 557 (Pt 3), pp. 935-948. | Show Abstract | Read more

We have investigated the in vitro effects of the saturated free fatty acid palmitate on mouse pancreatic beta-cells by a combination of electrophysiological recordings, intracellular Ca(2+) ([Ca(2+)](i)) microfluorimetry and insulin release measurements. Addition of palmitate (1 mm, bound to fatty acid-free albumin) to intact islets exposed to 15 mm glucose increased the [Ca(2+)](i) by approximately 30% and insulin secretion 2-fold. Palmitate remained capable of increasing [Ca(2+)](i) and insulin release in the presence of tolbutamide and in islets depolarized by high K(+) in combination with diazoxide, indicating that the stimulation occurs independently of closure of ATP-regulated K(+) channels (K(ATP) channels). Palmitate (0.5 mm) augmented exocytosis (measured as an increase in cell capacitance) in single beta-cells and increased the size of the readily releasable pool (RRP) of granules 2-fold. Whole-cell peak Ca(2+) currents rose by approximately 25% following addition of 0.5 mm palmitate, an effect that was abolished in the presence of 10 microm isradipine indicating that the free fatty acid specifically acts on L-type Ca(2+) channels. The actions of palmitate on exocytosis and Ca(2+) currents were not mimicked by intracellular application of palmitoyl-CoA. We conclude that palmitate increases insulin secretion by a K(ATP) channel-independent mechanism exerted at the level of exocytosis and that involves both augmentation of L-type Ca(2+) currents and an increased size of the RRP.

Cited:

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Sinnegger-Brauns MJ, Hetzenauer A, Huber IG, Renstrom E, Wietzorrek G, Berjukov S, Cavalli M, Walter D et al. 2004. Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca2+ channels JOURNAL OF CLINICAL INVESTIGATION, 113 (10), pp. 1430-1439. | Show Abstract | Read more

Cav1.2 and Cav1.3 L-type Ca2+ channels (LTCCs) are believed to underlie Ca2+ currents in brain, pancreatic β cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause card iovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Cav1.2 α1 subunits (Ca v1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Cav1.2 from those of Cav1.3 and other LTCCs. DHP effects on pancreatic β cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Cav1.2DHP-/- mice, which rules out a direct role of Cav1.3 for these physiological processes. Using Cav1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Cav1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.

Göpel S, Zhang Q, Eliasson L, Ma XS, Galvanovskis J, Kanno T, Salehi A, Rorsman P. 2004. Capacitance measurements of exocytosis in mouse pancreatic alpha-, beta- and delta-cells within intact islets of Langerhans. J Physiol, 556 (Pt 3), pp. 711-726. | Show Abstract | Read more

Capacitance measurements of exocytosis were applied to functionally identified alpha-, beta- and delta-cells in intact mouse pancreatic islets. The maximum rate of capacitance increase in beta-cells during a depolarization to 0 mV was equivalent to 14 granules s(-1), <5% of that observed in isolated beta-cells. Beta-cell secretion exhibited bell-shaped voltage dependence and peaked at +20 mV. At physiological membrane potentials (up to approximately -20 mV) the maximum rate of release was approximately 4 granules s(-1). Both exocytosis (measured by capacitance measurements) and insulin release (detected by radioimmunoassay) were strongly inhibited by the L-type Ca(2+) channel blocker nifedipine (25 microm) but only marginally (<20%) affected by the R-type Ca(2+) channel blocker SNX482 (100 nm). Exocytosis in the glucagon-producing alpha-cells peaked at +20 mV. The capacitance increases elicited by pulses to 0 mV exhibited biphasic kinetics and consisted of an initial transient (150 granules s(-1)) and a sustained late component (30 granules s(-1)). Whereas addition of the N-type Ca(2+) channel blocker omega-conotoxin GVIA (0.1 microm) inhibited glucagon secretion measured in the presence of 1 mm glucose to the same extent as an elevation of glucose to 20 mm, the L-type Ca(2+) channel blocker nifedipine (25 microm) had no effect. Thus, glucagon release during hyperglycaemic conditions depends principally on Ca(2+)-influx through N-type rather than L-type Ca(2+) channels. Exocytosis in the somatostatin-secreting delta-cells likewise exhibited two kinetically separable phases of capacitance increase and consisted of an early rapid (600 granules s(-1)) component followed by a sustained slower (60 granules s(-1)) component. We conclude that (1) capacitance measurements in intact pancreatic islets are feasible; (2) exocytosis measured in beta-cells in situ is significantly slower than that of isolated cells; and (3) the different types of islet cells exhibit distinct exocytotic features.

Ashcroft FM, Rorsman P. 2004. Molecular defects in insulin secretion in type-2 diabetes. Rev Endocr Metab Disord, 5 (2), pp. 135-142. | Read more

Sinnegger-Brauns MJ, Hetzenauer A, Huber IG, Renström E, Wietzorrek G, Berjukov S, Cavalli M, Walter D et al. 2004. Isoform-specific regulation of mood behavior and pancreatic beta cell and cardiovascular function by L-type Ca 2+ channels. J Clin Invest, 113 (10), pp. 1430-1439. | Show Abstract | Read more

Ca(v)1.2 and Ca(v)1.3 L-type Ca(2+) channels (LTCCs) are believed to underlie Ca(2+) currents in brain, pancreatic beta cells, and the cardiovascular system. In the CNS, neuronal LTCCs control excitation-transcription coupling and neuronal plasticity. However, the pharmacotherapeutic implications of CNS LTCC modulation are difficult to study because LTCC modulators cause cardiovascular (activators and blockers) and neurotoxic (activators) effects. We selectively eliminated high dihydropyridine (DHP) sensitivity from Ca(v)1.2 alpha 1 subunits (Ca(v)1.2DHP-/-) without affecting function and expression. This allowed separation of the DHP effects of Ca(v)1.2 from those of Ca(v)1.3 and other LTCCs. DHP effects on pancreatic beta cell LTCC currents, insulin secretion, cardiac inotropy, and arterial smooth muscle contractility were lost in Ca(v)1.2DHP-/- mice, which rules out a direct role of Ca(v)1.3 for these physiological processes. Using Ca(v)1.2DHP-/- mice, we established DHPs as mood-modifying agents: LTCC activator-induced neurotoxicity was abolished and disclosed a depression-like behavioral effect without affecting spontaneous locomotor activity. LTCC activator BayK 8644 (BayK) activated only a specific set of brain areas. In the ventral striatum, BayK-induced release of glutamate and 5-HT, but not dopamine and noradrenaline, was abolished. This animal model provides a useful tool to elucidate whether Ca(v)1.3-selective channel modulation represents a novel pharmacological approach to modify CNS function without major peripheral effects.

Wendt A, Birnir B, Buschard K, Gromada J, Salehi A, Sewing S, Rorsman P, Braun M. 2004. Glucose inhibition of glucagon secretion from rat alpha-cells is mediated by GABA released from neighboring beta-cells. Diabetes, 53 (4), pp. 1038-1045. | Show Abstract | Read more

gamma-Aminobutyric acid (GABA) has been proposed to function as a paracrine signaling molecule in islets of Langerhans. We have shown that rat beta-cells release GABA by Ca(2+)-dependent exocytosis of synaptic-like microvesicles. Here we demonstrate that GABA thus released can diffuse over sufficient distances within the islet interstitium to activate GABA(A) receptors in neighboring cells. Confocal immunocytochemistry revealed the presence of GABA(A) receptors in glucagon-secreting alpha-cells but not in beta- and delta-cells. RT-PCR analysis detected transcripts of alpha(1) and alpha(4) as well as beta(1-3) GABA(A) receptor subunits in purified alpha-cells but not in beta-cells. In whole-cell voltage-clamp recordings, exogenous application of GABA activated Cl(-) currents in alpha-cells. The GABA(A) receptor antagonist SR95531 was used to investigate the effects of endogenous GABA (released from beta-cells) on pancreatic islet hormone secretion. The antagonist increased glucagon secretion at 1 mmol/l glucose twofold and completely abolished the inhibitory action of 20 mmol/l glucose on glucagon release. Basal and glucose-stimulated secretion of insulin and somatostatin were unaffected by SR95531. The L-type Ca(2+) channel blocker isradipine evoked a paradoxical stimulation of glucagon secretion. This effect was not observed in the presence of SR95531, and we therefore conclude that isradipine stimulates glucagon secretion by inhibition of GABA release.

Ashcroft F, Rorsman P. 2004. Type 2 diabetes mellitus: not quite exciting enough? Hum Mol Genet, 13 Spec No 1 (90001), pp. R21-R31. | Show Abstract | Read more

Type 2 diabetes mellitus is a serious metabolic disease that afflicts around 5% of the population in Western societies and over 150 million people worldwide. It is characterized by elevation of the blood glucose concentration, usually presents in middle age, and is exacerbated by obesity. Both genetic and environmental factors contribute to the disease but in the vast majority of cases the aetiology is still not understood. Here we present a novel hypothesis for the aetiology of type 2 diabetes. We postulate that the electrical activity of the insulin-secreting beta-cells of the pancreas acts to integrate the genetic and environmental factors that predispose to disease risk. Our hypothesis is supported by a substantial amount of data gathered from a range of different disciplines and makes predictions that can be tested experimentally both in vitro and in man.

Braun M, Wendt A, Birnir B, Broman J, Eliasson L, Galvanovskis J, Gromada J, Mulder H, Rorsman P. 2004. Regulated exocytosis of GABA-containing synaptic-like microvesicles in pancreatic beta-cells. J Gen Physiol, 123 (3), pp. 191-204. | Show Abstract | Read more

We have explored whether gamma-aminobutyric acid (GABA) is released by regulated exocytosis of GABA-containing synaptic-like microvesicles (SLMVs) in insulin-releasing rat pancreatic beta-cells. To this end, beta-cells were engineered to express GABA(A)-receptor Cl(-)-channels at high density using adenoviral infection. Electron microscopy indicated that the average diameter of the SLMVs is 90 nm, that every beta-cell contains approximately 3,500 such vesicles, and that insulin-containing large dense core vesicles exclude GABA. Quantal release of GABA, seen as rapidly activating and deactivating Cl(-)-currents, was observed during membrane depolarizations from -70 mV to voltages beyond -40 mV or when Ca(2+) was dialysed into the cell interior. Depolarization-evoked GABA release was suppressed when Ca(2+) entry was inhibited using Cd(2+). Analysis of the kinetics of GABA release revealed that GABA-containing vesicles can be divided into a readily releasable pool and a reserve pool. Simultaneous measurements of GABA release and cell capacitance indicated that exocytosis of SLMVs contributes approximately 1% of the capacitance signal. Mathematical analysis of the release events suggests that every SLMV contains 0.36 amol of GABA. We conclude that there are two parallel pathways of exocytosis in pancreatic beta-cells and that release of GABA may accordingly be temporally and spatially separated from insulin secretion. This provides a basis for paracrine GABAergic signaling within the islet.

Schulla V, Renström E, Feil R, Feil S, Franklin I, Gjinovci A, Jing XJ, Laux D et al. 2003. Impaired insulin secretion and glucose tolerance in beta cell-selective Ca(v)1.2 Ca2+ channel null mice. EMBO J, 22 (15), pp. 3844-3854. | Show Abstract | Read more

Insulin is secreted from pancreatic beta cells in response to an elevation of cytoplasmic Ca(2+) resulting from enhanced Ca(2+) influx through voltage-gated Ca(2+) channels. Mouse beta cells express several types of Ca(2+) channel (L-, R- and possibly P/Q-type). beta cell-selective ablation of the gene encoding the L-type Ca(2+) channel subtype Ca(v)1.2 (betaCa(v)1.2(-/-) mouse) decreased the whole-cell Ca(2+) current by only approximately 45%, but almost abolished first-phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca(2+) handling and glucose-induced electrical activity. However, high-resolution capacitance measurements of exocytosis in single beta cells revealed that the loss of first-phase insulin secretion in the betaCa(v)1.2(-/-) mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca(v)1.2 channel. Thus, the conduit of Ca(2+) entry determines the ability of the cation to elicit secretion.

Rorsman P, Renström E. 2003. Insulin granule dynamics in pancreatic beta cells. Diabetologia, 46 (8), pp. 1029-1045. | Show Abstract | Read more

Glucose-induced insulin secretion in response to a step increase in blood glucose concentrations follows a biphasic time course consisting of a rapid and transient first phase followed by a slowly developing and sustained second phase. Because Type 2 diabetes involves defects of insulin secretion, manifested as a loss of first phase and a reduction of second phase, it is important to understand the cellular mechanisms underlying biphasic insulin secretion. Insulin release involves the packaging of insulin in small (diameter approximately 0.3 micro m) secretory granules, the trafficking of these granules to the plasma membrane, the exocytotic fusion of the granules with the plasma membrane and eventually the retrieval of the secreted membranes by endocytosis. Until recently, studies on insulin secretion have been confined to the appearance of insulin in the extracellular space and the cellular events preceding exocytosis have been inaccessible to more detailed analysis. Evidence from a variety of secretory tissues, including pancreatic islet cells suggests, however, that the secretory granules can be functionally divided into distinct pools that are distinguished by their release competence and/or proximity to the plasma membrane. The introduction of fluorescent proteins that can be targeted to the secretory granules, in combination with the advent of new techniques that allow real-time imaging of granule trafficking in living cells (granule dynamics), has led to an explosion of our knowledge of the pre-exocytotic and post-exocytotic processes in the beta cell. Here we discuss these observations in relation to previous functional and ultra-structural data as well as the secretory defects of Type 2 diabetes.

Olsen HL, Hoy M, Zhang W, Bertorello AM, Bokvist K, Capito K, Efanov AM, Meister B et al. 2003. Phosphatidylinositol 4-kinase serves as a metabolic sensor and regulates priming of secretory granules in pancreatic beta cells. Proc Natl Acad Sci U S A, 100 (9), pp. 5187-5192. | Show Abstract | Read more

Insulin secretion is controlled by the beta cell's metabolic state, and the ability of the secretory granules to undergo exocytosis increases during glucose stimulation in a membrane potential-independent fashion. Here, we demonstrate that exocytosis of insulin-containing secretory granules depends on phosphatidylinositol 4-kinase (PI 4-kinase) activity and that inhibition of this enzyme suppresses glucose-stimulated insulin secretion. Intracellular application of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] stimulated exocytosis by promoting the priming of secretory granules for release and increasing the number of granules residing in a readily releasable pool. Reducing the cytoplasmic ADP concentration in a way mimicking the effects of glucose stimulation activated PI 4-kinase and increased exocytosis whereas changes of the ATP concentration in the physiological range had little effect. The PI(4,5)P(2)-binding protein Ca(2+)-dependent activator protein for secretion (CAPS) is present in beta cells, and neutralization of the protein abolished both Ca(2+)- and PI(4,5)P(2)-induced exocytosis. We conclude that ADP-induced changes in PI 4-kinase activity, via generation of PI(4,5)P(2), represents a metabolic sensor in the beta cell by virtue of its capacity to regulate the release competence of the secretory granules.

Høy M, Olsen HL, Andersen HS, Bokvist K, Buschard K, Hansen J, Jacobsen P, Petersen JS, Rorsman P, Gromada J. 2003. Imidazoline NNC77-0074 stimulates insulin secretion and inhibits glucagon release by control of Ca(2+)-dependent exocytosis in pancreatic alpha- and beta-cells. Eur J Pharmacol, 466 (1-2), pp. 213-221. | Show Abstract | Read more

We have investigated the effects of the novel imidazoline compound (+)-2-(2-(4,5-dihydro-1H-imidazol-2-yl)-thiopene-2-yl-ethyl)-pyridine (NNC77-0074) on stimulus-secretion coupling in isolated pancreatic alpha- and beta-cells. NNC77-0074 stimulated glucose-dependent insulin secretion in intact mouse pancreatic islets. No effect was observed at </=2.5 mM glucose and maximal stimulation occurred at 10-15 mM glucose. NNC77-0074 produced a concentration-dependent stimulation of insulin secretion. Half-maximal (EC(50)) stimulation was observed at 24 microM and at maximally stimulatory concentrations insulin release was doubled. The stimulatory action of NNC77-0074 on insulin secretion was not associated with membrane depolarisation or a change in the activity of ATP-sensitive K(+) channels. Using capacitance measurements, we found that NNC77-0074 stimulated depolarisation-induced exocytosis 2.6-fold without affecting the whole-cell Ca(2+) current when applied via the extracellular medium. The concentration dependence of the stimulatory action was determined by intracellular application of NNC77-0074 through the recording pipette. NNC77-0074 stimulated exocytosis half-maximal at 44 nM and at maximally stimulatory concentrations the rate of exocytosis was increased twofold. NNC77-0074 stimulated depolarised-induced insulin secretion from islets exposed to diazoxide and high external KCl (EC(50)=0.45 microM). The stimulatory action of NNC77-0074 was dependent on protein kinase C activity. NNC77-0074 potently inhibited glucagon secretion from rat islets (EC(50)=11 nM). This was not associated with a change in spontaneous electrical activity and ATP-sensitive K(+) channel activity but resulted from a reduction of the rate of Ca(2+)-dependent exocytosis in single rat alpha-cells (EC(50)=9 nM). Inhibition of exocytosis by NNC77-0074 was pertussis toxin-sensitive and mediated by activation of the protein phosphatase calcineurin. In rat somatotrophs, PC12 cells and mouse cortical neurons NNC77-0074 did not stimulate Ca(2+)-evoked exocytosis, whereas the other imidazoline compounds phentolamine and efaroxan produced 2.5-fold stimulation of exocytosis. Our data suggest that the imidazoline compound NNC77-0074 constitutes a novel class of antidiabetic compounds that stimulates glucose-dependent insulin release while inhibiting glucagon secretion. These actions are exclusively exerted by modulation of exocytosis of the insulin- and glucagon-containing granules.

Eliasson L, Ma X, Renström E, Barg S, Berggren PO, Galvanovskis J, Gromada J, Jing X et al. 2003. SUR1 regulates PKA-independent cAMP-induced granule priming in mouse pancreatic B-cells. J Gen Physiol, 121 (3), pp. 181-197. | Show Abstract | Read more

Measurements of membrane capacitance were applied to dissect the cellular mechanisms underlying PKA-dependent and -independent stimulation of insulin secretion by cyclic AMP. Whereas the PKA-independent (Rp-cAMPS-insensitive) component correlated with a rapid increase in membrane capacitance of approximately 80 fF that plateaued within approximately 200 ms, the PKA-dependent component became prominent during depolarizations >450 ms. The PKA-dependent and -independent components of cAMP-stimulated exocytosis differed with regard to cAMP concentration dependence; the K(d) values were 6 and 29 micro M for the PKA-dependent and -independent mechanisms, respectively. The ability of cAMP to elicit exocytosis independently of PKA activation was mimicked by the selective cAMP-GEFII agonist 8CPT-2Me-cAMP. Moreover, treatment of B-cells with antisense oligodeoxynucleotides against cAMP-GEFII resulted in partial (50%) suppression of PKA-independent exocytosis. Surprisingly, B-cells in islets isolated from SUR1-deficient mice (SUR1(-/-) mice) lacked the PKA-independent component of exocytosis. Measurements of insulin release in response to GLP-1 stimulation in isolated islets from SUR1(-/-) mice confirmed the complete loss of the PKA-independent component. This was not attributable to a reduced capacity of GLP-1 to elevate intracellular cAMP but instead associated with the inability of cAMP to stimulate influx of Cl(-) into the granules, a step important for granule priming. We conclude that the role of SUR1 in the B cell extends beyond being a subunit of the plasma membrane K(ATP)-channel and that it also plays an unexpected but important role in the cAMP-dependent regulation of Ca(2+)-induced exocytosis.

Kanno T, Rorsman P, Göpel SO. 2002. Glucose-dependent regulation of rhythmic action potential firing in pancreatic beta-cells by K(ATP)-channel modulation. J Physiol, 545 (Pt 2), pp. 501-507. | Show Abstract | Read more

The regulation of a K(+) current activating during oscillatory electrical activity (I(K,slow)) in an insulin-releasing beta-cell was studied by applying the perforated patch whole-cell technique to intact mouse pancreatic islets. The resting whole-cell conductance in the presence of 10 mM glucose amounted to 1.3 nS, which rose by 50 % during a series of 26 simulated action potentials. Application of the K(ATP)-channel blocker tolbutamide produced uninterrupted action potential firing and reduced I(K,slow) by approximately 50 %. Increasing glucose from 15 to 30 mM, which likewise converted oscillatory electrical activity into continuous action potential firing, reduced I(K,slow) by approximately 30 % whilst not affecting the resting conductance. Action potential firing may culminate in opening of K(ATP) channels by activation of ATP-dependent Ca(2+) pumping as suggested by the observation that the sarco-endoplasmic reticulum Ca(2+)-ATPase (SERCA) inhibitor thapsigargin (4 microM) inhibited I(K,slow) by 25 % and abolished bursting electrical activity. We conclude that oscillatory glucose-induced electrical activity in the beta-cell involves the opening of K(ATP)-channel activity and that these channels, in addition to constituting the glucose-regulated K(+) conductance, also play a role in the graded response to supra-threshold glucose concentrations.

Gromada J, Bokvist K, Høy M, Olsen HL, Lindström P, Hansen BS, Gotfredsen CF, Rorsman P, Thomsen MK. 2002. Nateglinide, but not repaglinide, stimulates growth hormone release in rat pituitary cells by inhibition of K channels and stimulation of cyclic AMP-dependent exocytosis. Eur J Endocrinol, 147 (1), pp. 133-142. | Show Abstract | Read more

OBJECTIVE: GH causes insulin resistance, impairs glycemic control and increases the risk of vascular diabetic complications. Sulphonylureas stimulate GH secretion and this study was undertaken to investigate the possible stimulatory effect of repaglinide and nateglinide, two novel oral glucose regulators, on critical steps of the stimulus-secretion coupling in single rat somatotrophs. METHODS: Patch-clamp techniques were used to record whole-cell ATP-sensitive K(+) (K(ATP)) and delayed outward K(+) currents, membrane potential and Ca(2+)-dependent exocytosis. GH release was measured from perifused rat somatotrophs. RESULTS: Both nateglinide and repaglinide dose-dependently suppressed K(ATP) channel activity with half-maximal inhibition being observed at 413 nM and 13 nM respectively. Both compounds induced action potential firing in the somatotrophs irrespective of whether GH-releasing hormone was present or not. The stimulation of electrical activity by nateglinide, but not repaglinide, was associated with an increased mean duration of the action potentials. The latter effect correlated with a reduction of the delayed outward K(+) current, which accounts for action potential repolarization. The latter effect had a K(d) of 19 microM but was limited to 38% inhibition. When applied at concentrations similar to those required to block K(ATP) channels, nateglinide in addition potentiated Ca(2+)-evoked exocytosis 3.3-fold (K(d)=3 microM) and stimulated GH release 4.5-fold. The latter effect was not shared by repaglinide. The stimulation of exocytosis by nateglinide was mimicked by cAMP and antagonized by the protein kinase A inhibitor Rp-cAMPS. CONCLUSION: Nateglinide stimulates GH release by inhibition of plasma membrane K(+) channels, elevation of cytoplasmic cAMP levels and stimulation of Ca(2+)-dependent exocytosis. By contrast, the effect of repaglinide was confined to inhibition of the K(ATP) channels.

Olofsson CS, Göpel SO, Barg S, Galvanovskis J, Ma X, Salehi A, Rorsman P, Eliasson L. 2002. Fast insulin secretion reflects exocytosis of docked granules in mouse pancreatic B-cells. Pflugers Arch, 444 (1-2), pp. 43-51. | Show Abstract | Read more

A readily releasable pool (RRP) of granules has been proposed to underlie the first phase of insulin secretion. In the present study we combined electron microscopy, insulin secretion measurements and recordings of cell capacitance in an attempt to define this pool ultrastructurally. Mouse pancreatic B-cells contain approximately 9,000 granules, of which 7% are docked below the plasma membrane. The number of docked granules was reduced by 30% (200 granules) during 10 min stimulation with high K+. This stimulus depolarized the cell to -10 mV, elevated cytosolic [Ca2+] ([Ca2+](i)) from a basal concentration of 130 nM to a peak of 1.3 microM and released 0.5 ng insulin/islet, corresponding to 200-300 granules/cell. The Ca2+ transient decayed towards the prestimulatory concentration within approximately 200 s, presumably reflecting Ca2+ channel inactivation. Renewed stimulation with high K+ failed to stimulate insulin secretion when applied in the absence of glucose. The size of the RRP, derived from the insulin measurements, is similar to that estimated from the increase in cell capacitance elicited by photolytic release of caged Ca2+. We propose that the RRP represents a subset of the docked pool of granules and that replenishment of RRP can be accounted for largely by chemical modification of granules already in place or situated close to the plasma membrane.

Kanno T, Gopel SO, Rorsman P, Wakui M. 2002. Cellular function in multicellular system for hormone-secretion: electrophysiological aspect of studies on alpha-, beta- and delta-cells of the pancreatic islet. Neurosci Res, 42 (2), pp. 79-90. | Show Abstract | Read more

We review a new method to explore the cellular functions in multicellular system by application of the perforated patch-clamp technique to intact pancreatic islet of Langerhans. Using this approach, the integrity of the islet is preserved and intercellular communication via gap junctions and paracrine processes are maintained. By using low-resistance patch electrodes, rapid current responses can be monitored under voltage-clamp control. We have applied this methodology to answer questions not resolved by patch-clamp experiments on isolated single insulin-secreting beta-cells. First, the role of a K(+)-current dependent on Ca(2+)-influx for the termination of burst of action potentials in beta-cells could be documented. Neither the current, nor the bursting pattern of electrical activity is preserved in isolated beta-cells. Second, the conductance of gap junctions (approximately 1 nS) between beta-cells was determined. Third, electrical properties of glucagon-producing alpha- and somatostatin-secreting delta-cells and the different mechanisms for glucose-sensing in these cells could be explored. The findings emanating from these experiments may have implications for neuroscience research such as the mechanism of oscillatory electrical activity in general and processes involved in the glucose-sensing in some neurons, which response to changes of blood glucose concentration.

Barg S, Olofsson CS, Schriever-Abeln J, Wendt A, Gebre-Medhin S, Renström E, Rorsman P. 2002. Delay between fusion pore opening and peptide release from large dense-core vesicles in neuroendocrine cells. Neuron, 33 (2), pp. 287-299. | Show Abstract | Read more

Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.

Barg S, Ma X, Eliasson L, Galvanovskis J, Göpel SO, Obermüller S, Platzer J, Renström E et al. 2001. Fast exocytosis with few Ca(2+) channels in insulin-secreting mouse pancreatic B cells. Biophys J, 81 (6), pp. 3308-3323. | Show Abstract | Read more

The association of L-type Ca(2+) channels to the secretory granules and its functional significance to secretion was investigated in mouse pancreatic B cells. Nonstationary fluctuation analysis showed that the B cell is equipped with <500 alpha1(C) L-type Ca(2+) channels, corresponding to a Ca(2+) channel density of 0.9 channels per microm(2). Analysis of the kinetics of exocytosis during voltage-clamp depolarizations revealed an early component that reached a peak rate of 1.1 pFs(-1) (approximately 650 granules/s) 25 ms after onset of the pulse and is completed within approximately 100 ms. This component represents a subset of approximately 60 granules situated in the immediate vicinity of the L-type Ca(2+) channels, corresponding to approximately 10% of the readily releasable pool of granules. Experiments involving photorelease of caged Ca(2+) revealed that the rate of exocytosis was half-maximal at a cytoplasmic Ca(2+) concentration of 17 microM, and concentrations >25 microM are required to attain the rate of exocytosis observed during voltage-clamp depolarizations. The rapid component of exocytosis was not affected by inclusion of millimolar concentrations of the Ca(2+) buffer EGTA but abolished by addition of exogenous L(C753-893), the 140 amino acids of the cytoplasmic loop connecting the 2(nd) and 3(rd) transmembrane region of the alpha1(C) L-type Ca(2+) channel, which has been proposed to tether the Ca(2+) channels to the secretory granules. In keeping with the idea that secretion is determined by Ca(2+) influx through individual Ca(2+) channels, exocytosis triggered by brief (15 ms) depolarizations was enhanced 2.5-fold by the Ca(2+) channel agonist BayK8644 and 3.5-fold by elevating extracellular Ca(2+) from 2.6 to 10 mM. Recordings of single Ca(2+) channel activity revealed that patches predominantly contained no channels or many active channels. We propose that several Ca(2+) channels associate with a single granule thus forming a functional unit. This arrangement is important in a cell with few Ca(2+) channels as it ensures maximum usage of the Ca(2+) entering the cell while minimizing the influence of stochastic variations of the Ca(2+) channel activity.

Gromada J, Høy M, Buschard K, Salehi A, Rorsman P. 2001. Somatostatin inhibits exocytosis in rat pancreatic alpha-cells by G(i2)-dependent activation of calcineurin and depriming of secretory granules. J Physiol, 535 (Pt 2), pp. 519-532. | Show Abstract | Read more

1. Measurements of cell capacitance were used to investigate the molecular mechanisms by which somatostatin inhibits Ca(2+)-induced exocytosis in single rat glucagon-secreting pancreatic alpha-cells. 2. Somatostatin decreased the exocytotic responses elicited by voltage-clamp depolarisations by 80 % in the presence of cyclic AMP-elevating agents such as isoprenaline and forskolin. Inhibition was time dependent and half-maximal within 22 s. 3. The inhibitory action of somatostatin was concentration dependent with an IC(50) of 68 nM and prevented by pretreatment of the cells with pertussis toxin. The latter effect was mimicked by intracellular dialysis with specific antibodies to G(i1/2) and by antisense oligonucleotides against G proteins of the subtype G(i2). 4. Somatostatin lacked inhibitory action when applied in the absence of forskolin or in the presence of the L-type Ca(2+) channel blocker nifedipine. The size of the omega-conotoxin-sensitive and forskolin-independent component of exocytosis was limited to 60 fF. By contrast, somatostatin abolished L-type Ca(2+) channel-dependent exocytosis in alpha-cells exposed to forskolin. The magnitude of the latter pool amounted to 230 fF. 5. The inhibitory effect of somatostatin on exocytosis was mediated by activation of the serine/threonine protein phosphatase calcineurin and was prevented by pretreatment with cyclosporin A and deltamethrin or intracellularly applied calcineurin autoinhibitory peptide. Experiments using the stable ATP analogue AMP-PCP indicate that somatostatin acts by depriming of granules. 6. We propose that somatostatin receptors associate with L-type Ca(2+) channels and couple to G(i2) proteins leading to a localised activation of calcineurin and depriming of secretory granules situated close to the L-type Ca(2+) channels.

Barg S, Huang P, Eliasson L, Nelson DJ, Obermüller S, Rorsman P, Thévenod F, Renström E. 2001. Priming of insulin granules for exocytosis by granular Cl(-) uptake and acidification. J Cell Sci, 114 (Pt 11), pp. 2145-2154. | Show Abstract

ATP-dependent priming of the secretory granules precedes Ca(2+)-regulated neuroendocrine secretion, but the exact nature of this reaction is not fully established in all secretory cell types. We have further investigated this reaction in the insulin-secreting pancreatic B-cell and demonstrate that granular acidification driven by a V-type H(+)-ATPase in the granular membrane is a decisive step in priming. This requires simultaneous Cl(-) uptake through granular ClC-3 Cl(-) channels. Accordingly, granule acidification and priming are inhibited by agents that prevent transgranular Cl(-) fluxes, such as 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS) and an antibody against the ClC-3 channels, but accelerated by increases in the intracellular ATP:ADP ratio or addition of hypoglycemic sulfonylureas. We suggest that this might represent an important mechanism for metabolic regulation of Ca(2+)-dependent exocytosis that is also likely to be operational in other secretory cell types.

Gromada J, Høy M, Olsen HL, Gotfredsen CF, Buschard K, Rorsman P, Bokvist K. 2001. Gi2 proteins couple somatostatin receptors to low-conductance K+ channels in rat pancreatic alpha-cells. Pflugers Arch, 442 (1), pp. 19-26. | Show Abstract | Read more

Somatostatin hyperpolarized rat pancreatic alpha-cells and inhibited spontaneous electrical activity by activating a low-conductance K+ channel (0.9 pS with physiological ionic gradients). This channel was insensitive to tolbutamide (a blocker of ATP-sensitive K+ channels) and apamin (an inhibitor of small-conductance Ca(2+)-activated K+ channels). Channel activation was prevented by pre-treating the cells with pertussis toxin, indicating the involvement of G-proteins. A direct interaction between an inhibitory G-protein and the somatostatin-activated K+ channel is suggested by the finding that intracellular application of guanosine 5'-O-(3-thiotriphosphate) (GTP gamma-S) and the G beta gamma subunit of G-proteins resulted in a transient stimulation of the current. Activation of the K+ current by somatostatin was inhibited by intracellular dialysis with specific antibodies to Gi1/2 and was not seen in cells treated with antisense oligonucleotides against G-proteins of the subtype Gi2. We conclude that somatostatin suppresses alpha-cell electrical activity by a Gi2-protein-dependent mechanism, which culminates in the activation of a sulphonylurea- and apamin-insensitive low-conductance K+ channel.

Larsson-Nyrén G, Sehlin J, Rorsman P, Renström E. 2001. Perchlorate stimulates insulin secretion by shifting the gating of L-type Ca2+ currents in mouse pancreatic B-cells towards negative potentials. Pflugers Arch, 441 (5), pp. 587-595. | Show Abstract | Read more

The effects of the chaotrophic anion perchlorate (ClO4-) on glucose-induced electrical activity, exocytosis and ion channel activity in mouse pancreatic B-cells were investigated by patch-clamp recordings and capacitance measurements. ClO4- stimulated glucose-induced electrical activity and increased the action potential frequency by 70% whilst not affecting the membrane potential when applied in the presence of a subthreshold concentration of the sugar. ClO4- did not influence ATP-dependent K (KATP) channel activity and voltage-gated delayed K+ current. Similarly, ClO4- had no effect on Ca2+-dependent exocytosis. The stimulation of electrical activity and insulin secretion was instead attributable to an enhancement of the whole-cell Ca2+ current. This effect was particularly pronounced at voltages around the threshold for action potential initiation and a doubling of the current amplitude was observed at -30 mV. This was due to a 7-mV shift in the gating of the Ca2+ current towards negative voltages. The action of ClO4- was more pronounced when added in the presence of 0.1 mM BAY K8644, whereas no stimulation was observed when applied at a maximal concentration of the agonist (1 mM). Single-channel recordings revealed that the effect of ClO4- on whole-cell currents was principally due to a 60% increase in the mean duration of the long openings and the number of active channels. We propose that ClO4- stimulates insulin secretion and electrical activity by exerting a BAY K8644-like action on Ca2+ channel gating.

Göpel SO, Kanno T, Barg S, Weng XG, Gromada J, Rorsman P. 2000. Regulation of glucagon release in mouse -cells by KATP channels and inactivation of TTX-sensitive Na+ channels. J Physiol, 528 (Pt 3), pp. 509-520. | Show Abstract | Read more

The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial glucagon-secreting alpha-cells in intact mouse pancreatic islets. alpha-cells were distinguished from the beta- and delta-cells by the presence of a large TTX-blockable Na+ current, a TEA-resistant transient K+ current sensitive to 4-AP (A-current) and the presence of two kinetically separable Ca2+ current components corresponding to low- (T-type) and high-threshold (L-type) Ca2+ channels. The T-type Ca2+, Na+ and A-currents were subject to steady-state voltage-dependent inactivation, which was half-maximal at -45, -47 and -68 mV, respectively. Pancreatic alpha-cells were equipped with tolbutamide-sensitive, ATP-regulated K+ (KATP) channels. Addition of tolbutamide (0.1 mM) evoked a brief period of electrical activity followed by a depolarisation to a plateau of -30 mV with no regenerative electrical activity. Glucagon secretion in the absence of glucose was strongly inhibited by TTX, nifedipine and tolbutamide. When diazoxide was added in the presence of 10 mM glucose, concentrations up to 2 microM stimulated glucagon secretion to the same extent as removal of glucose. We conclude that electrical activity and secretion in the alpha-cells is dependent on the generation of Na+-dependent action potentials. Glucagon secretion depends on low activity of KATP channels to keep the membrane potential sufficiently negative to prevent voltage-dependent inactivation of voltage-gated membrane currents. Glucose may inhibit glucagon release by depolarising the alpha-cell with resultant inactivation of the ion channels participating in action potential generation.

Göpel SO, Kanno T, Barg S, Rorsman P. 2000. Patch-clamp characterisation of somatostatin-secreting -cells in intact mouse pancreatic islets. J Physiol, 528 (Pt 3), pp. 497-507. | Show Abstract | Read more

The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact mouse pancreatic islets. Three types of electrical activity were observed corresponding to alpha-, beta- and delta-cells. The delta-cells were electrically active in the presence of glucose but lacked the oscillatory pattern seen in the beta-cells. By contrast, the alpha-cells were electrically silent at high glucose concentrations but action potentials could be elicited by removal of the sugar. Both alpha- and beta-cells contained transient voltage-activated K+ currents. In the delta-cells, the K+ currents activated above -20 mV and were completely blocked by TEA (20 mM). The alpha-cells differed from the delta-cells in possessing a TEA-resistant K+ current activating already at -40 mV. Immunocytochemistry revealed the presence of Kv3.4 channels in delta-cells and TEA-resistant Kv4.3 channels in alpha-cells. Thus the presence of a transient TEA-resistant current can be used to functionally separate the delta- and alpha-cells. A TTX-sensitive Na+ current developed in delta-cells during depolarisations beyond -30 mV and reached a peak amplitude of 350 pA. Steady-state inactivation of this current was half-maximal at -28 mV. The delta-cells were also equipped with a sustained Ca2+ current that activated above -30 mV and reached a peak of 60 pA when measured at 2.6 mM extracellular Ca2+. A tolbutamide-sensitive KATP channel conductance was observed in delta-cells exposed to glucose-free medium. Addition of tolbutamide (0.1 mM) depolarised the delta-cell and evoked electrical activity. We propose that the KATP channels in delta-cells serve the same function as in the beta-cell and couple an elevation of the blood glucose concentration to stimulation of hormone release.

Barg S, Galvanovskis J, Göpel SO, Rorsman P, Eliasson L. 2000. Tight coupling between electrical activity and exocytosis in mouse glucagon-secreting alpha-cells. Diabetes, 49 (9), pp. 1500-1510. | Show Abstract | Read more

alpha-Cells were identified in preparations of dispersed mouse islets by immunofluorescence microscopy. A high fraction of alpha-cells correlated with a small cell size measured as the average cell diameter (10 microm) and whole-cell capacitance (<4 pF). The alpha-cells generated action potentials at a low frequency (1 Hz) in the absence of glucose. These action potentials were reversibly inhibited by elevation of the glucose concentration to 20 mmol/l. The action potentials originated from a membrane potential more negative than -50 mV, had a maximal upstroke velocity of 5 V/s, and peaked at +1 mV. Voltage-clamp experiments revealed the ionic conductances underlying the generation of action potentials. alpha-Cells are equipped with a delayed tetraethyl-ammonium-blockable outward current (activating at voltages above -20 mV), a large tetrodotoxin-sensitive Na+ current (above -30 mV; peak current 200 pA at +10 mV), and a small Ca2+ current (above -50 mV; peak current 30 pA at +10 mV). The latter flowed through omega-conotoxin GVIA (25%)- and nifedipine-sensitive (50%) Ca(2+)-channels. Mouse alpha-cells contained, on average, 7,300 granules, which undergo Ca(2+)-induced exocytosis when the alpha-cell is depolarized. Three functional subsets of granules were identified, and the size of the immediately releasable pool was estimated as 80 granules, or 1% of the total granule number. The maximal rate of exocytosis (1.5 pF/s) was observed 21 ms after the onset of the voltage-clamp depolarization, which is precisely the duration of Ca(2+)-influx during an action potential. Our results suggest that the secretory machinery of the alpha-cell is optimized for maximal efficiency in the use of Ca2+ for exocytosis.

Høy M, Olsen HL, Bokvist K, Buschard K, Barg S, Rorsman P, Gromada J. 2000. Tolbutamide stimulates exocytosis of glucagon by inhibition of a mitochondrial-like ATP-sensitive K+ (KATP) conductance in rat pancreatic A-cells. J Physiol, 527 Pt 1 (1), pp. 109-120. | Show Abstract | Read more

1. Capacitance measurements were used to examine the effects of the sulphonylurea tolbutamide on Ca2+-dependent exocytosis in isolated glucagon-secreting rat pancreatic A-cells. 2. When applied extracellularly, tolbutamide stimulated depolarization-evoked exocytosis 4.2-fold without affecting the whole-cell Ca2+ current. The concentration dependence of the stimulatory action was determined by intracellular application through the recording pipette. Tolbutamide produced a concentration-dependent increase in cell capacitance. Half-maximal stimulation was observed at 33 microM and the maximum stimulation corresponded to a 3.4-fold enhancement of exocytosis. 3. The stimulatory action of tolbutamide was dependent on protein kinase C activity. The action of tolbutamide was mimicked by the general K+ channel blockers TEA (10 mM) and quinine (10 microM). A similar stimulation was elicited by 5-hydroxydecanoate (5-HD; 10 microM), an inhibitor of mitochondrial ATP-sensitive K+ (KATP) channels. 4. Tolbutamide-stimulated, but not TEA-induced, exocytosis was antagonized by the K+ channel openers diazoxide, pinacidil and cromakalim. 5. Dissipating the transgranular K+ gradient with nigericin and valinomycin inhibited tolbutamide- and Ca2+-evoked exocytosis. Furthermore, tolbutamide- and Ca2+-induced exocytosis were abolished by the H+ ionophore FCCP or by arresting the vacuolar (V-type) H+-ATPase with bafilomycin A1 or DCCD. Finally, ammonium chloride stimulated exocytosis to a similar extent to that obtained with tolbutamide. 6. We propose that during granular maturation, a granular V-type H+-ATPase pumps H+ into the secretory granule leading to the generation of a pH gradient across the granular membrane and the development of a positive voltage inside the granules. The pumping of H+ is facilitated by the concomitant exit of K+ through granular K+ channels with pharmacological properties similar to those of mitochondrial KATP channels. Release of granules that have been primed is then facilitated by the addition of K+ channel blockers. The resulting increase in membrane potential promotes exocytosis by unknown mechanisms, possibly involving granular alkalinization.

Rorsman P, Eliasson L, Renström E, Gromada J, Barg S, Göpel S. 2000. The Cell Physiology of Biphasic Insulin Secretion. News Physiol Sci, 15 (2), pp. 72-77. | Show Abstract

Glucose-stimulated insulin secretion consists of a transient first phase followed by a sustained second phase. Diabetes (type II) is associated with abnormalities in this release pattern. Here we review the evidence that biphasic insulin secretion reflects exocytosis of two functional subsets of secretory granules and the implications for diabetes.

Bokvist K, Holmqvist M, Gromada J, Rorsman P. 2000. Compound exocytosis in voltage-clamped mouse pancreatic beta-cells revealed by carbon fibre amperometry. Pflugers Arch, 439 (5), pp. 634-645. | Show Abstract | Read more

Capacitance measurements of exocytosis were combined with carbon fibre amperometry for time-resolved measurements of the properties of secretion in single, insulin-secreting, mouse pancreatic beta-cells pre-loaded with the amine serotonin (5-HT). Glucose-induced electrical activity was associated with the appearance of brief and transient amperometric currents reflecting the serotonin co-released with insulin. The integrated amperometric responses resulting from voltage-clamp depolarisations were proportional to the corresponding increase in cell capacitance. Both parameters exhibited U-shaped relationships to the membrane potential with maximums around 0 mV. There was a variable latency (40-730 ms, average 230 ms) between the onset of the depolarisation and the amperometric current. During high-frequency repetitive stimulation, a progressive decrease in the exocytotic capacity ("depression") was observed. This was paralleled by a corresponding reduction of the amperometric responses. Using the carbon fibre to map the beta-cell for release sites indicated that exocytosis was confined to the part of the cell containing the highest density of secretory granules. Two types of amperometric responses were observed. In about 50% of the cells, a smooth increase was observed with no discernible discrete events. In the remaining cells, the amperometric records contained large spikes. These were ten or more times larger than that expected for the fusion of individual secretory granules. We propose that these large spikes reflect the exocytosis of multigranular complexes formed inside the beta-cell prior to exocytosis.

Galvanovskis J, Gopel S, Rorsman P. 2000. Modelling of electrical activity in pancreatic islets: The role of a novel K-Ca-channel. BIOPHYSICAL JOURNAL, 78 (1), pp. 210A-210A.

Göpel S, Kanno T, Barg S, Galvanovskis J, Rorsman P. 1999. Voltage-gated and resting membrane currents recorded from B-cells in intact mouse pancreatic islets. J Physiol, 521 Pt 3 (3), pp. 717-728. | Show Abstract | Read more

1. The perforated patch whole-cell configuration of the patch-clamp technique was applied to superficial cells in intact pancreatic islets. Immunostaining in combination with confocal microscopy revealed that the superficial cells consisted of 35 % insulin-secreting B-cells and 65 % non-B-cells (A- and D-cells). 2. Two types of cell, with distinct electrophysiological properties, could be functionally identified. One of these generated oscillatory electrical activity when the islet was exposed to 10 mM glucose and had the electrophysiological characteristics of isolated B-cells maintained in tissue culture. 3. The Ca2+ current recorded from B-cells in situ was 80 % larger than that of isolated B-cells. It exhibited significant (70 %) inactivation during 100 ms depolarisations. The inactivation was voltage dependent and particularly prominent during depolarisations evoking the largest Ca2+ currents. 4. Voltage-dependent K+ currents were observed during depolarisations to membrane potentials above -20 mV. These currents inactivated little during a 200 ms depolarisation and were unaffected by varying the holding potential between -90 and -30 mV. 5. The maximum resting conductance in the absence of glucose, which reflects the conductance of ATP-regulated K+ (KATP) channels, amounted to approximately 4 nS. Glucose produced a concentration-dependent reduction of KATP channel conductance with half-maximal inhibition observed with 5 mM glucose. 6. Combining voltage- and current-clamp recording allowed the estimation of the gap junction conductance between different B-cells. These experiments indicated that the input conductance of the B-cell at stimulatory glucose concentrations ( approximately 1 nS) is almost entirely accounted for by coupling to neighbouring B-cells.

Rorsman P, Renström E. 1999. Cell biology. Glutamate primes the pump. Nature, 402 (6762), pp. 595-596. | Read more

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Gopel SO, Kanno T, Barg S, Eliasson L, Galvanovskis J, Renstrom E, Rorsman P. 1999. Activation of Ca2+-dependent K+ channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells JOURNAL OF GENERAL PHYSIOLOGY, 114 (6), pp. 759-769. | Show Abstract | Read more

We have applied the perforated patch whole-cell technique to 13 cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (< 0.8 nS) K+ conductance. The current was dependent on Ca2+ influx but unaffected by apamin and charybdotoxin, two blockers of Ca2+-activated K+ channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K+ channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca2+-activated K+ current plays an important role in the generation of oscillatory electrical activity in the β cell.

Göpel SO, Kanno T, Barg S, Eliasson L, Galvanovskis J, Renström E, Rorsman P. 1999. Activation of Ca(2+)-dependent K(+) channels contributes to rhythmic firing of action potentials in mouse pancreatic beta cells. J Gen Physiol, 114 (6), pp. 759-770. | Show Abstract

We have applied the perforated patch whole-cell technique to beta cells within intact pancreatic islets to identify the current underlying the glucose-induced rhythmic firing of action potentials. Trains of depolarizations (to simulate glucose-induced electrical activity) resulted in the gradual (time constant: 2.3 s) development of a small (<0.8 nS) K(+) conductance. The current was dependent on Ca(2+) influx but unaffected by apamin and charybdotoxin, two blockers of Ca(2+)-activated K(+) channels, and was insensitive to tolbutamide (a blocker of ATP-regulated K(+) channels) but partially (>60%) blocked by high (10-20 mM) concentrations of tetraethylammonium. Upon cessation of electrical stimulation, the current deactivated exponentially with a time constant of 6.5 s. This is similar to the interval between two successive bursts of action potentials. We propose that this Ca(2+)-activated K(+) current plays an important role in the generation of oscillatory electrical activity in the beta cell.

Bokvist K, Olsen HL, Høy M, Gotfredsen CF, Holmes WF, Buschard K, Rorsman P, Gromada J. 1999. Characterisation of sulphonylurea and ATP-regulated K+ channels in rat pancreatic A-cells. Pflugers Arch, 438 (4), pp. 428-436. | Show Abstract | Read more

We have monitored whole-cell and single channel ATP-sensitive K+ (KATP) currents in isolated rat glucagon-secreting pancreatic A-cells. Tolbutamide produced a concentration-dependent decrease in the whole-cell KATP conductance (Ki = 6 microM) and initiated action potential firing. The K+ channel opener diazoxide, but not cromakalim or pinacidil, inhibited electrical activity and increased the whole-cell K+ conductance fourfold. ATP applied to the intracellular face of the membrane inhibited KATP channel activity with a Ki of 17 microM, an effect that could be counteracted by Mg-ADP and Mg-GDP. GTP and UTP did not affect KATP channel activity. Phosphatidylinositol 4,5-bisphosphate activated KATP channels inhibited by ATP after a delay of 90 s. In situ hybridisation demonstrated the expression of the mRNA encoding KATP channel subunits Kir6.2 and SUR1 but not Kir6.1 and SUR2. We conclude that rat pancreatic A-cells express KATP channels with the nucleotide-, sulphonylurea- and K+ channel-opener sensitivities expected for a channel formed by Kir6.2 and SUR1 subunits.

Gromada J, Høy M, Renström E, Bokvist K, Eliasson L, Göpel S, Rorsman P. 1999. CaM kinase II-dependent mobilization of secretory granules underlies acetylcholine-induced stimulation of exocytosis in mouse pancreatic B-cells. J Physiol, 518 ( Pt 3) (3), pp. 745-759. | Show Abstract | Read more

1. Measurements of cell capacitance were used to investigate the mechanisms by which acetylcholine (ACh) stimulates Ca2+-induced exocytosis in single insulin-secreting mouse pancreatic B-cells. 2. ACh (250 microM) increased exocytotic responses elicited by voltage-clamp depolarizations 2.3-fold. This effect was mediated by activation of muscarinic receptors and dependent on elevation of the cytoplasmic Ca2+ concentration ([Ca2+]i) attributable to mobilization of Ca2+ from intracellular stores. The latter action involved interference with the buffering of [Ca2+]i and the time constant (tau) for the recovery of [Ca2+]i following a voltage-clamp depolarization increased 5-fold. As a result, Ca2+ was present at concentrations sufficient to promote the replenishment of the readily releasable pool of granules (RRP; > 0.2 microM) for much longer periods in the presence than in the absence of the agonist. 3. The effect of Ca2+ on exocytosis was mediated by activation of CaM kinase II, but not protein kinase C, and involved both an increased size of the RRP from 40 to 140 granules and a decrease in tau for the refilling of the RRP from 31 to 19 s. 4. Collectively, the effects of ACh on the RRP and tau result in a > 10-fold stimulation of the rate at which granules are supplied for release.

Barg S, Renström E, Berggren PO, Bertorello A, Bokvist K, Braun M, Eliasson L, Holmes WE, Köhler M, Rorsman P, Thévenod F. 1999. The stimulatory action of tolbutamide on Ca2+-dependent exocytosis in pancreatic beta cells is mediated by a 65-kDa mdr-like P-glycoprotein. Proc Natl Acad Sci U S A, 96 (10), pp. 5539-5544. | Show Abstract | Read more

Intracellular application of the sulfonylurea tolbutamide during whole-cell patch-clamp recordings stimulated exocytosis >5-fold when applied at a cytoplasmic Ca2+ concentration of 0.17 microM. This effect was not detectable in the complete absence of cytoplasmic Ca2+ and when exocytosis was elicited by guanosine 5'-O-(3-thiotriphosphate) (GTPgammaS). The stimulatory action could be antagonized by the sulfonamide diazoxide, by the Cl--channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), by intracellular application of the antibody JSB1 [originally raised against a 170-kDa multidrug resistance (mdr) protein], and by tamoxifen (an inhibitor of the mdr- and volume-regulated Cl- channels). Immunocytochemistry and Western blot analyses revealed that JSB1 recognizes a 65-kDa protein in the secretory granules. This protein exhibited no detectable binding of sulfonylureas and is distinct from the 140-kDa sulfonylurea high-affinity sulfonylurea receptors also present in the granules. We conclude that (i) tolbutamide stimulates Ca2+-dependent exocytosis secondary to its binding to a 140-kDa high-affinity sulfonylurea receptor in the secretory granules; and (ii) a granular 65-kDa mdr-like protein mediates the action. The processes thus initiated culminate in the activation of a granular Cl- conductance. We speculate that the activation of granular Cl- fluxes promotes exocytosis (possibly by providing the energy required for membrane fusion) by inducing water uptake and an increased intragranular hydrostatic pressure.

Gall D, Gromada J, Susa I, Rorsman P, Herchuelz A, Bokvist K. 1999. Significance of Na/Ca exchange for Ca2+ buffering and electrical activity in mouse pancreatic beta-cells. Biophys J, 76 (4), pp. 2018-2028. | Show Abstract | Read more

We have combined the patch-clamp technique with microfluorimetry of the cytoplasmic Ca2+ concentration ([Ca2+]i) to characterize Na/Ca exchange in mouse beta-cells and to determine its importance for [Ca2+]i buffering and shaping of glucose-induced electrical activity. The exchanger contributes to Ca2+ removal at [Ca2+]i above 1 microM, where it accounts for >35% of the total removal rate. At lower [Ca2+]i, thapsigargin-sensitive Ca2+-ATPases constitute a major (70% at 0.8 microM [Ca2+]i) mechanism for Ca2+ removal. The beta-cell Na/Ca exchanger is electrogenic and has a stoichiometry of three Na+ for one Ca2+. The current arising from its operation reverses at approximately -20 mV (current inward at more negative voltages), has a conductance of 53 pS/pF (14 microM [Ca2+]i), and is abolished by removal of external Na+ or by intracellularly applied XIP (exchange inhibitory peptide). Inhibition of the exchanger results in shortening (50%) of the bursts of action potentials of glucose-stimulated beta-cells in intact islets and a slight (5 mV) hyperpolarization. Mathematical simulations suggest that the stimulatory action of glucose on beta-cell electrical activity may be accounted for in part by glucose-induced reduction of the cytoplasmic Na+ concentration with resultant activation of the exchanger.

Wiser O, Trus M, Hernández A, Renström E, Barg S, Rorsman P, Atlas D. 1999. The voltage sensitive Lc-type Ca2+ channel is functionally coupled to the exocytotic machinery. Proc Natl Acad Sci U S A, 96 (1), pp. 248-253. | Show Abstract | Read more

Although N- and P-type Ca2+ channels predominant in fast-secreting systems, Lc-type Ca2+ channels (C-class) can play a similar role in certain secretory cells and synapses. For example, in retinal bipolar cells, Ca2+ entry through the Lc channels triggers ultrafast exocytosis, and in pancreatic beta-cells, evoked secretion is highly sensitive to Ca2+. These findings suggest that a rapidly release pool of vesicles colocalizes with the Ca2+ channels to allow high Ca2+ concentration and a tight coupling of the Lc channels at the release site. In binding studies, we show that the Lc channel is physically associated with synaptotagmin (p65) and the soluble N-ethylmaleimide-sensitive attachment proteins receptors: syntaxin and synaptosomal-associated protein of 25 kDa. Soluble N-ethylmaleimide-sensitive attachent proteins receptors coexpressed in Xenopus oocytes along with the Lc channel modify the kinetic properties of the channel. The modulatory action of syntaxin can be overcome by coexpressing p65, where at a certain ratio of p65/syntaxin, the channel regains its unaltered kinetic parameters. The cytosolic region of the channel, Lc753-893, separating repeats II-III of its alpha1C subunit, interacts with p65 and "pulls" down native p65 from rat brain membranes. Lc753-893 injected into single insulin-secreting beta-cell, inhibits secretion in response to channel opening, but not in response to photolysis of caged Ca2+, nor does it affect Ca2+ current. These results suggest that Lc753-893 competes with the endogenous channel for the synaptic proteins and disrupts the spatial coupling with the secretory apparatus. The molecular organization of the Lc channel and the secretory machinery into a multiprotein complex (named excitosome) appears to be essential for an effective depolarization evoked exocytosis.

Mulder H, Holst LS, Svensson H, Degerman E, Sundler F, Ahrén B, Rorsman P, Holm C. 1999. Hormone-sensitive lipase, the rate-limiting enzyme in triglyceride hydrolysis, is expressed and active in beta-cells. Diabetes, 48 (1), pp. 228-232. | Show Abstract | Read more

Triglycerides in the beta-cell may be important for stimulus-secretion coupling, through provision of a lipid-derived signal, and for pathogenetic events in NIDDM, where lipids may adversely affect beta-cell function. In adipose tissues, hormone-sensitive lipase (HSL) is rate-limiting in triglyceride hydrolysis. Here, we investigated whether this enzyme is also expressed and active in beta-cells. Northern blot analysis and reverse transcription-polymerase chain reaction demonstrated that HSL is expressed in rat islets and in the clonal beta-cell lines INS-1, RINm5F, and HIT-T15. Western blot analysis identified HSL in mouse and rat islets and the clonal beta-cells. In mouse and rat, immunocytochemistry showed a predominant occurrence of HSL in beta-cells, with a presumed cytoplasmic localization. Lipase activity in homogenates of the rodent islets and clonal beta-cells constituted 2.1 +/- 0.6% of that in adipocytes; this activity was immunoinhibited by use of antibodies to HSL. The established HSL expression and activity in beta-cells offer a mechanism whereby lipids are mobilized from intracellular stores. Because HSL in adipocytes is activated by cAMP-dependent protein kinase (PKA), PKA-regulated triglyceride hydrolysis in beta-cells may participate in the regulation of insulin secretion, possibly by providing a lipid-derived signal, e.g., long-chain acyl-CoA and diacylglycerol.

Rorsman P, Renström E. 1999. Glutamate primes the pump Nature, 402 (6762), pp. 595-596. | Read more

Gromada J, Holst JJ, Rorsman P. 1998. Cellular regulation of islet hormone secretion by the incretin hormone glucagon-like peptide 1. Pflugers Arch, 435 (5), pp. 583-594. | Show Abstract | Read more

Glucagon-like peptide 1 is a gastrointestinally derived hormone with profound effects on nutrient-induced pancreatic hormone release. GLP-1 modulates insulin, glucagon and somatostatin secretion by binding to guanine nucleotide binding protein-coupled receptors resulting in the activation of adenylate cyclase and generation of cyclic adenosine monophosphate (cAMP). In the B-cell, cAMP, via activation of protein kinase A, interacts with a plethora of signal transduction processes including ion channel activity, intracellular Ca2+ handling and exocytosis of the insulin-containing granules. The stimulatory action of GLP-1 on insulin secretion, contrary to that of the currently used hypoglycaemic sulphonylureas, is glucose dependent and requires the presence of normal or elevated concentrations of the sugar. For this reason, GLP-1 attracts much interest as a possible novel principle for the treatment of human type-2 diabetes. Here we review the actions of GLP-1 on islet cell function and attempt to integrate current knowledge into a working model for the control of pancreatic hormone secretion.

Fuhlendorff J, Rorsman P, Kofod H, Brand CL, Rolin B, MacKay P, Shymko R, Carr RD. 1998. Stimulation of insulin release by repaglinide and glibenclamide involves both common and distinct processes. Diabetes, 47 (3), pp. 345-351. | Show Abstract | Read more

The action of repaglinide, a novel insulin secretagogue, was compared with the sulfonylurea glibenclamide with regard to the hypoglycemic action in vivo, binding to betaTC-3 cells, insulin secretion from perifused mouse islets, and capacity to stimulate exocytosis by direct interaction with the secretory machinery in single voltage-clamped mouse beta-cells. Two binding sites were identified: a high-affinity repaglinide (KD = 3.6 nmol/l) site having lower affinity for glibenclamide (14.4 nmol/l) and one high-affinity glibenclamide (25 nmol/l) site having lower affinity for repaglinide (550 nmol/l). In contrast to glibenclamide, repaglinide (in concentrations as high as 5 micromol/l) lacked the ability to enhance exocytosis in voltage-clamped beta-cells. Repaglinide was more potent than glibenclamide in stimulating insulin release from perifused mouse islets (EC50 29 vs. 80 nmol/l). The greater potency of repaglinide in vitro was paralleled by similar actions in vivo. The ED50 values for the hypoglycemic action were determined to be 10.4 and 15.6 microg/kg after intravenous and oral administration, respectively. The corresponding values for glibenclamide were 70.3 microg/kg (intravenous) and 203.2 microg/kg (oral). Further, repaglinide (1 mg/kg p.o.) was effective (P < 0.001) as an insulin-releasing agent in a rat model (low-dose streptozotocin) of type 2 diabetes. These observations suggest that the insulinotropic actions of repaglinide and glibenclamide in vitro and in vivo are secondary to their binding to the high-affinity repaglinide site and that the insulinotropic action of repaglinide involves both distinct and common cellular mechanisms.

Gromada J, Bokvist K, Ding WG, Holst JJ, Nielsen JH, Rorsman P. 1998. Glucagon-like peptide 1 (7-36) amide stimulates exocytosis in human pancreatic beta-cells by both proximal and distal regulatory steps in stimulus-secretion coupling. Diabetes, 47 (1), pp. 57-65. | Show Abstract | Read more

The effect of glucagon-like peptide 1(7-36) amide [GLP-1(7-36) amide] on membrane potential, whole-cell ATP-sensitive potassium channel (K[ATP]) and Ca2+ currents, cytoplasmic Ca2+ concentration, and exocytosis was explored in single human beta-cells. GLP-1(7-36) amide induced membrane depolarization that was associated with inhibition of whole-cell K(ATP) current. In addition, GLP-1(7-36) amide (and forskolin) produced greater than fourfold potentiation of Ca2+-dependent exocytosis. The latter effect resulted in part (40%) from acceleration of Ca2+ influx through voltage-dependent (L-type) Ca2+ channels. More importantly, GLP-1(7-36) amide (via generation of cyclic AMP and activation of protein kinase A) potentiated exocytosis at a site distal to a rise in the cytoplasmic Ca2+ concentration. Photorelease of caged cAMP produced a two- to threefold potentiation of exocytosis when the cytoplasmic Ca2+ concentrations were clamped at > or =170 nmol/l. The effect of GLP-1(7-36) amide was antagonized by the islet hormone somatostatin. Similar effects on membrane potential, ion conductances, and exocytosis were observed with glucose-dependent insulinotropic polypeptide (GIP), the second major incretin. The present data suggest that the strong insulinotropic action of GLP-1(7-36) amide and GIP in humans results from its interaction with several proximal as well as distal important regulatory steps in the stimulus-secretion coupling.

Gromada J, Bokvist K, Ding WG, Barg S, Buschard K, Renström E, Rorsman P. 1997. Adrenaline stimulates glucagon secretion in pancreatic A-cells by increasing the Ca2+ current and the number of granules close to the L-type Ca2+ channels. J Gen Physiol, 110 (3), pp. 217-228. | Show Abstract | Read more

We have monitored electrical activity, voltage-gated Ca2+ currents, and exocytosis in single rat glucagon-secreting pancreatic A-cells. The A-cells were electrically excitable and generated spontaneous Na+- and Ca2+-dependent action potentials. Under basal conditions, exocytosis was tightly linked to Ca2+ influx through omega-conotoxin-GVIA-sensitive (N-type) Ca2+ channels. Stimulation of the A-cells with adrenaline (via beta-adrenergic receptors) or forskolin produced a greater than fourfold PKA-dependent potentiation of depolarization-evoked exocytosis. This enhancement of exocytosis was due to a 50% enhancement of Ca2+ influx through L-type Ca2+ channels, an effect that accounted for <30% of the total stimulatory action. The remaining 70% of the stimulation was attributable to an acceleration of granule mobilization resulting in a fivefold increase in the number of readily releasable granules near the L-type Ca2+ channels.

Gromada J, Ding WG, Barg S, Renström E, Rorsman P. 1997. Multisite regulation of insulin secretion by cAMP-increasing agonists: evidence that glucagon-like peptide 1 and glucagon act via distinct receptors. Pflugers Arch, 434 (5), pp. 515-524. | Show Abstract | Read more

The mechanisms by which glucagon-like peptide 1(7-36)amide (GLP-1[7-36]amide) potentiates insulin secretion were investigated by measurements of whole-cell K+ and Ca2+ currents, membrane potential, the cytoplasmic Ca2+ concentration ([Ca2+]i) and exocytosis in mouse pancreatic B-cells. GLP-1(7-36)amide (10 nM) stimulated glucose-induced (10 mM) electrical activity in intact pancreatic islets. The effect was manifested as a 34% increase in the duration of the bursts of action potentials and a corresponding 28% shortening of the silent intervals. GLP-1(7-36)amide had no effect on the electrical activity at subthreshold glucose concentrations (< or = 6.5 mM). In cultured B-cells, GLP-1(7-36)amide produced a decrease of the whole-cell ATP-sensitive K+ (KATP) conductance remaining at 5 mM glucose by approximately 30%. This effect was associated with membrane depolarization and the initiation of electrical activity. GLP-1(7-36)amide produced a protein-kinase-A-(PKA-) and glucose-dependent fourfold potentiation of Ca(2+)-induced exocytosis whilst only increasing the Ca2+ current marginally. The stimulatory action of GLP-1(7-36)amide on exocytosis was mimicked by the pancreatic hormone glucagon and exendin-4, a GLP-1 receptor agonist. Whereas the stimulatory action of GLP-1(7-36)amide could be antagonized by exendin-(9-39), this peptide did not interfere with the ability of glucagon to stimulate exocytosis. We suggest that GLP-1(7-36)amide and glucagon stimulate insulin secretion by binding to distinct receptors. The GLP-1(7-36)amide-induced stimulation of electrical activity and Ca2+ influx can account for (maximally) a doubling of insulin secretion. The remainder of its stimulatory action results from a cAMP/PKA-dependent potentiation of Ca(2+)-dependent exocytosis exerted at a stage distal to the elevation of [Ca2+]i.

Eliasson L, Renström E, Ding WG, Proks P, Rorsman P. 1997. Rapid ATP-dependent priming of secretory granules precedes Ca(2+)-induced exocytosis in mouse pancreatic B-cells. J Physiol, 503 ( Pt 2) (2), pp. 399-412. | Show Abstract | Read more

1. The glucose and ATP dependence of exocytosis were investigated in single mouse pancreatic B-cells by monitoring changes in cell capacitance evoked by voltage-clamp depolarizations, infusion of high [Ca2+]i buffers or photorelease of caged Ca2+ or ATP. 2. In intact B-cells, using the perforated patch whole-cell technique, glucose (5 mM) increased exocytotic responses evoked by membrane depolarization 5-fold over that observed in the absence of the sugar. Increasing the glucose concentration to 20 mM produced a further doubling of exocytosis. The stimulatory action of glucose was attributable to glucose metabolism and abolished by mannoheptulose, an inhibitor of glucose phosphorylation. 3. Exocytosis triggered by infusion of high [Ca2+]i and ATP was reduced by 80% when ATP was replaced by its non-hydrolysable analogue adenosine 5'-[beta, gamma-methylene]triphosphate (AMP-PCP) in standard whole-cell experiments. Exocytosis elicited by GTP gamma S was similarly affected by replacement of ATP with the stable analogue. 4. Photoreleasing ATP in the presence of 170 nM [Ca2+]i, following the complete wash-out of endogenous ATP produced a prompt (latency, < 400 ms) and biphasic stimulation of exocytosis. 5. Elevation of [Ca2+]i to exocytotic levels by photorelease from Ca(2+)-nitrophenyl EGTA preloaded into the cell evoked a biphasic stimulation in the presence of Mg-ATP. The response consisted of an initial rapid (completed in < 200 ms) phase followed by a slower (lasting > or = 10 s) sustained component. Replacement of ATP with AMP-PCP abolished the late component but did not affect the initial phase. The latency between elevation of [Ca2+]i and exocytosis was determined as < 45 ms. Inclusion of N-ethylmaleimide (NEM; 0.5 mM for 3 min) in the intracellular solution exerted effects similar to those obtained by substituting AMP-PCP for ATP. 6. We conclude that the B-cell contains a small pool (40 granules) of primed granules which are immediately available for release and which are capable of undergoing exocytosis in an ATP-independent fashion. We propose that this pool of granules is preferentially released during first phase glucose-stimulated insulin secretion. The short latency between the application of ATP and the onset of exocytosis finally suggests that priming takes place with sufficient speed to participate in the rapid adjustment of the secretory capacity of the B-cell.

Renström E, Eliasson L, Rorsman P. 1997. Protein kinase A-dependent and -independent stimulation of exocytosis by cAMP in mouse pancreatic B-cells. J Physiol, 502 ( Pt 1) (1), pp. 105-118. | Show Abstract | Read more

1. The mechanisms by which cAMP stimulates Ca(2+)-dependent insulin secretion were investigated by combining measurements of whole-cell Ca2+ currents, the cytoplasmic free Ca2+ concentration ([Ca2+]i) and membrane capacitance in single mouse B-cells maintained in tissue culture. 2. Cyclic AMP stimulated exocytosis > 4-fold in whole-cell experiments in which secretion was evoked by intracellular dialysis with a Ca(2+)-EGTA buffer with a [Ca2+]i of 1.5 microM. This effect was antagonized by inhibitors of protein kinase A (PKA). 3. Photorelease of cAMP from a caged precursor potentiated exocytosis at Ca2+ concentrations which were themselves stimulatory (> or = 60 nM), but was without effect in the complete absence of Ca2+. 4. Elevation of intracellular cAMP (by exposure to forskolin) evoked a 6-fold PKA-dependent enhancement of the maximal exocytotic response (determined as the maximum increase in cell capacitance that could be elicited by a train of depolarizations) in perforated-patch whole-cell recordings. 5. Exocytosis triggered by single depolarizations in standard whole-cell recordings was strongly potentiated by cAMP, but in this case the effect was unaffected by PKA inhibition. 6. When exocytosis was triggered by Ca2+ released from Ca(2+)-NP-EGTA ('caged Ca2+'), cAMP exerted a dual stimulatory effect on secretion: a rapid (initiated within 80 ms) PKA-independent phase and a late PKA-dependent component. 7. We conclude that cAMP stimulates insulin secretion both by increasing the release probability of secretory granules already in the readily releasable pool and by accelerating the refilling of this pool.

Ding WG, Renström E, Rorsman P, Buschard K, Gromada J. 1997. Glucagon-like peptide I and glucose-dependent insulinotropic polypeptide stimulate Ca2+-induced secretion in rat alpha-cells by a protein kinase A-mediated mechanism. Diabetes, 46 (5), pp. 792-800. | Show Abstract | Read more

High-resolution capacitance measurements were used to explore the effects of the gut hormones GLP-I(7-36) amide [glucagon-like peptide I(7-36) amide] and GIP (glucose-dependent insulinotropic polypeptide) on Ca2+-dependent exocytosis in glucagon-secreting rat pancreatic alpha-cells. Both peptides produced a greater than threefold potentiation of secretion evoked by voltage-clamp depolarizations, an effect that was associated with an approximately 35% increase of the Ca2+ current. The stimulatory actions of GLP-I(7-36) amide and GIP were mimicked by forskolin and antagonized by the protein kinase A (PKA)-inhibitor Rp-8-Br-cAMPS. The islet hormone somatostatin inhibited the stimulatory action of GLP-I(7-36) amide and GIP via a cyclic AMP-independent mechanism, whereas insulin had no effect on exocytosis. These data suggest that the alpha-cells are equipped with receptors for GLP-I and GIP and that these peptides, in addition to their well-established insulinotropic capacity, also stimulate glucagon secretion. We propose that the reported inhibitory action of GLP-I on glucagon secretion is accounted for by a paracrine mechanism (e.g., mediated by stimulated release of somatostatin that in turn suppresses exocytosis in the alpha-cell).

Rorsman P. 1997. The pancreatic beta-cell as a fuel sensor: an electrophysiologist's viewpoint. Diabetologia, 40 (5), pp. 487-495. | Show Abstract | Read more

The pancreatic beta cell serves as the fuel sensor of the entire body and controls, via secretion of the hypoglycaemic hormone insulin, the blood glucose concentrations within narrow limits by regulation of glucose uptake and release. During the last 30 years, a combination of biochemical and ultrastructural approaches has resulted in dramatic progress in the understanding of the processes by which glucose and other nutrients modulate the release of insulin. The beta cells have also been investigated using electrophysiological techniques and were thus found to be electrically excitable and to undergo complex changes in their membrane potential when exposed to glucose and other stimulators of secretion. The application of the patch-clamp technique to the pancreatic islet preparations has revolutionized the understanding of how bioelectrical processes participate in the fuel-sensing of the beta cell. An important achievement was the identification of an ATP-sensitive K(+)-channel as the resting and glucose-sensitive membrane conductance of the beta cell. This channel also constitutes the target of the hypoglycaemic sulphonylureas: a group of compounds which have been used successfully in the treatment of insulin-dependent diabetes mellitus for several decades.

Gilon P, Yakel J, Gromada J, Zhu Y, Henquin JC, Rorsman P. 1997. G protein-dependent inhibition of L-type Ca2+ currents by acetylcholine in mouse pancreatic B-cells. J Physiol, 499 ( Pt 1) (1), pp. 65-76. | Show Abstract | Read more

1. The effect of acetylcholine (ACh) on voltage-dependent Ca2+ currents in mouse pancreatic B-cells was studied using the whole-cell configuration of the patch-clamp technique. 2. ACh (0.25-250 microM) reversibly and dose-dependently inhibited the Ca2+ current elicited by depolarizations from -80 mV to +10 mV. Maximal inhibition was observed at concentrations > 25 microM where it amounted to approximately 35%. The effect was voltage independent and prevented by atropine (10 microM) suggesting that it was mediated by muscarinic receptors. 3. The inhibitory action of ACh on the Ca2+ current was abolished when the cytoplasmic solution contained GDP beta S (2 mM) and became irreversible when the non-hydrolysable GTP analogue GTP gamma S (10 microM) was included in the pipette. This indicates the participation of G proteins in the inhibitory effect of ACh but pretreatment of the cells with either pertussis or cholera toxin failed to prevent the effect of ACh on the Ca2+ current. 4. ACh remained equally effective as an inhibitor of the whole-cell Ca2+ current in the presence of the L-type Ca2+ channel agonist (-)-Bay K 8644 and after partial inhibition of the current by nifedipine. Addition of omega-agatoxin IVA, omega-conotoxin GVIA or omega-conotoxin MVIIC neither affected the peak Ca2+ current amplitude nor the extent of inhibition produced by ACh. These pharmacological properties indicate that ACh acts by inhibiting L-type Ca2+ channels. 5. The inhibitory action of ACh on the B-cell Ca2+ current was not secondary to elevation of [Ca2+]i and ACh remained equally effective as an inhibitor when Ba2+ was used as the charge carrier, when [Ca2+]i was buffered to low concentrations using EGTA and under experimental conditions preventing the mobilization of Ca2+ from intracellular stores. 6. These results suggest that ACh reduces the whole-cell Ca2+ current in the B-cell through a G protein-regulated, voltage- and Ca(2+)-independent inhibition of L-type Ca2+ channels.

Proks P, Eliasson L, Ammälä C, Rorsman P, Ashcroft FM. 1996. Ca(2+)- and GTP-dependent exocytosis in mouse pancreatic beta-cells involves both common and distinct steps. J Physiol, 496 ( Pt 1) (1), pp. 255-264. | Show Abstract | Read more

1. The effects of GTP and Ca2+ on secretion from single pancreatic beta-cells were studied using capacitance measurements as an indicator of exocytosis. 2. GTP or GTP gamma S produced a concentration-dependent increase in cell capacitance in the absence of intracellular calcium. There was no effect of cyclic AMP or BAPTA an GTP-induced secretion. 3. In the absence of GTP, the relationship between intracellular calcium concentration and the maximum rate of secretion was fitted by the Hill equation with a slope factor of 2.5 and half-maximal activation at 1.6 microM intracellular Ca2+. Similar values were obtained in the presence of GTP gamma S, suggesting GTP does not alter the sensitivity of the secretory machinery to Ca2+. 4. GDP beta S alone had no effect on cell capacitance but caused a dose-dependent inhibition of exocytosis induced by infusion of either GTP gamma S or Ca2+, suggesting both stimuli involve G-protein activation. GDP beta S was without effect on exocytosis evoked by depolarization-mediated Ca2+ entry. 5. The time course of exocytosis following rapid elevation of GTP gamma S by photolysis of a caged precursor was dependent on the intracellular Ca2+ and cyclic AMP concentrations. 6. Our results are interpreted in terms of a model in which the secretory pathways stimulated by Ca2+ and GTP contain both common and separate parts.

Renström E, Ding WG, Bokvist K, Rorsman P. 1996. Neurotransmitter-induced inhibition of exocytosis in insulin-secreting beta cells by activation of calcineurin. Neuron, 17 (3), pp. 513-522. | Show Abstract | Read more

Neurotransmitters and hormones such as somatostatin, galanin, and adrenalin reduce insulin secretion. Their inhibitory action involves direct interference with the exocytotic machinery. We have examined the molecular processes underlying this effect using high resolution measurements of cell capacitance. Suppression of exocytosis was maximal at concentrations that did not cause complete inhibition of glucose-stimulated electrical activity. This action was dependent on activation of G proteins but was not associated with inhibition of the voltage-dependent Ca2+ currents or adenylate cyclase activity. The molecular processes initiated by the agonists culminate in the activation of the Ca(2+)-dependent protein phosphatase calcineurin, and suppression of the activity of this enzyme abolishes their action on exocytosis. We propose that mechanisms similar to those we report here may contribute to adrenergic and peptidergic inhibition of secretion in other neuroendocrine cells and in nerve terminals.

Renström E, Eliasson L, Bokvist K, Rorsman P. 1996. Cooling inhibits exocytosis in single mouse pancreatic B-cells by suppression of granule mobilization. J Physiol, 494 ( Pt 1) (1), pp. 41-52. | Show Abstract | Read more

1. The mechanisms by which cooling inhibits insulin secretion were investigated by capacitance measurements of exocytosis in single mouse pancreatic B-cells maintained in short-term tissue culture. 2. A reduction of the bath temperature from 34 to 24 degrees C produced a gradual inhibition of exocytosis. Inhibition of exocytosis was use dependent rather than time dependent. The steady-state inhibition amounted to 90%, which was paralleled by a 30% reduction of the peak Ca2+ current. 3. The Q10 values (between 27 and 37 degrees C) for inhibition of exocytosis and the peak Ca2+ current amplitude were determined as > 5 and 1.6, respectively. From the temperature dependence of exocytosis, an energy of activation was estimated as 145 kJ mol-1. 4. Suppression of exocytosis was not the result of a reduction of Ca2+ influx. When the Ca2+ currents were blocked by 30% (comparable to that produced by cooling) by using a low concentration of Co2+, exocytosis was reduced by < 25%. 5. Elevation of cytoplasmic free Ca2+, by photorelease of 'caged' Ca2+ from Ca(2+)-nitrophenyl-EGTA preloaded into the B-cell, was equally effective at eliciting exocytosis at 24 and 34 degrees C. 6. Cooling produced 70% inhibition of exocytosis evoked by infusion of Ca2+ through the recording electrode. Omission of either MgATP or cAMP from the electrode solution resulted in a comparable reduction of exocytosis. Cooling had no additional inhibitory effect when exocytosis was already suppressed by removal of cytoplasmic MgATP. 7. Our data indicate that exocytosis of granules already docked beneath the membrane is little affected by cooling in the B-cell. Instead, the high overall temperature sensitivity of insulin secretion arises because the replenishment of the readily releasable pool is temperature dependent.

Eliasson L, Proks P, Ammälä C, Ashcroft FM, Bokvist K, Renström E, Rorsman P, Smith PA. 1996. Endocytosis of secretory granules in mouse pancreatic beta-cells evoked by transient elevation of cytosolic calcium. J Physiol, 493 ( Pt 3) (3), pp. 755-767. | Show Abstract | Read more

1. To investigate the mechanisms regulating the reuptake of secretory granule membranes following regulated exocytosis, we have monitored changes in cell capacitance in single pancreatic beta-cells. 2. Membrane retrieval (endocytosis) occurred both in a continuous manner and in abrupt steps, corresponding to the simultaneous retrieval of 50-100 granules. The large endocytotic steps were associated with a conductance change of about 1 nS which we attribute to the formation of a fission pore with a pore radius of approximately 1 nm. 3. In some cells, we observed large amplitude capacitance fluctuations, suggesting that aggregates of granules are connected to the plasma membrane by a single pore and are subsequently retrieved as a single unit. 4. Endocytosis was evoked by elevation of cytosolic [Ca2+]i, but once initiated, a sustained increase in [Ca2+]i was not required for endocytosis to continue. 5. The [Ca2+]i dependence of exo- and endocytosis was studied by photorelease of Ca2+ from the 'caged' precursor Ca(2+)-nitrophenyl-EGTA (Ca(2+)-NP-EGTA). Both exo- and endocytosis were initiated at between 0.5 and 2 microM Cai(2+). The rate of endocytosis saturated above 2 microM Cai(2+), whereas exocytosis continued to increase up to 4 microM Cai(2+). The maximum rate of endocytosis was < 25% of that of exocytosis. 6. Unlike exocytosis, endocytosis proceeded equally well in the presence or absence of Mg-ATP. 7. Our data indicate that in the pancreatic beta-cell, exocytosis and endocytosis are regulated by different mechanisms.

Gromada J, Dissing S, Rorsman P. 1996. Desensitization of glucagon-like peptide 1 receptors in insulin-secreting beta TC3 cells: role of PKA-independent mechanisms. Br J Pharmacol, 118 (3), pp. 769-775. | Show Abstract | Read more

1. The cellular processes involved in the desensitization of the glucagon-like peptide 1 receptors were investigated by measurements of the glucagon-like peptide 1(7-36)amide (GLP-1(7-36)amide)-induced increases in intracellular free Ca2+ concentration ([Ca2+]i) in insulin-secreting beta TC3 cells. 2. In the presence of 11.2 mM glucose, stimulation with GLP-1(7-36)amide led to a small membrane depolarization (< 10 mV), induction of electrical activity and a rapid increase in [Ca2+]i. The increase in [Ca2+]i was not observed in the presence of the L-type Ca(2+)-channel antagonist nifedipine. However, nifedipine was ineffective when applied after addition of GLP-1(7-36)amide. 3. The increase in [Ca2+]i evoked by GLP-1-(7-36)amide was transient and even in the continued presence of the agonist, [Ca2+]i returned to the basal value within 4-5 min. The latter process was slowed, but not prevented, by inhibition of protein kinase C (PKC) by staurosporine and Ro31-8220. 4. Short pretreatment of the cells with the phorbol ester, 4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA), an activator of PKC, reduced the GLP-1(7-36)amide-evoked increase in [Ca2+]i by 75%. This effect of PMA was fully reversed by staurosporine and Ro31-8220. 5. The ability of GLP-1(7-36)amide to increase [Ca2+]i disappeared upon pre-exposure of the cells to the hormone (desensitization). This process was maximal within 5 min of exposure to the agonist. Following removal of the agonist from the medium, the ability to respond to subsequent stimulation by GLP-1(7-36)amide recovered gradually with time; half and complete recovery requiring > 20 min and 60 min, respectively. The desensitizing action of GLP-1(7-36)amide persisted in the presence of either staurosporine or forskolin and did not require an elevation of [Ca2+]i. 6. Our data suggest that the GLP-1(7-36)amide-evoked increase in [Ca2+]i is initiated by Ca(2+)-influx though voltage-dependent and nifedipine-sensitive L-type Ca2+ channels but depends principally on Ca2+ mobilization from internal stores for its maintenance. The desensitization of the GLP-1 receptors that occurs in the continued presence of the agonist does not result from the activation of protein kinase A or Ca(2+)-dependent kinases/phosphatases. Our data indicate that activation of PKC may contribute to the desensitization of the GLP-1 receptors but that other (PKC-independent) mechanisms also participate in this process.

Gromada J, Rorsman P, Dissing S, Wulff BS. 1996. Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK 293 cells induces cAMP-dependent activation of calcium-induced calcium release (vol 373, pg 182, 1995) FEBS LETTERS, 381 (3), pp. 272-272.

Eliasson L, Renström E, Ammälä C, Berggren PO, Bertorello AM, Bokvist K, Chibalin A, Deeney JT et al. 1996. PKC-dependent stimulation of exocytosis by sulfonylureas in pancreatic beta cells. Science, 271 (5250), pp. 813-815. | Show Abstract | Read more

Hypoglycemic sulfonylureas represent a group of clinically useful antidiabetic compounds that stimulate insulin secretion from pancreatic beta cells. The molecular mechanisms involved are not fully understood but are believed to involve inhibition of potassium channels sensitive to adenosine triphosphate (KATP channels) in the beta cell membrane, causing membrane depolarization, calcium influx, and activation of the secretory machinery. In addition to these effects, sulfonylureas also promoted exocytosis by direct interaction with the secretory machinery not involving closure of the plasma membrane KATP channels. This effect was dependent on protein kinase C (PKC) and was observed at therapeutic concentrations of sulfonylureas, which suggests that it contributes to their hypoglycemic action in diabetics.

Lebrun P, Renström E, Antoine MH, Bokvist K, Holmquist M, Rorsman P, Malaisse WJ. 1996. Dynamics of the cationic, bioelectrical and secretory responses to formycin A in pancreatic islet cells. Pflugers Arch, 431 (3), pp. 353-362. | Show Abstract | Read more

The dynamics of the cationic, bioelectrical and secretory responses to formycin A were monitored in pancreatic islet cells in order to assess whether this adenosine analogue, which is known to be converted to formycin A 5'-triphosphate in isolated islets, triggers the same sequence of ionic events as that otherwise involved in the process of nutrient-stimulated insulin release and currently attributed to an increase in adenosine 5'-triphosphate (ATP) generation rate. Unexpectedly, formycin A first increased 86Rb outflow, decreased 45Ca outflow and inhibited insulin release from prelabelled islets perifused at physiological or higher concentrations of D-glucose. This early inhibitory effect of formycin A upon insulin release coincided, in perforated patch whole-cell recordings, with an initial transient increase of ATP-sensitive K+ channel activity. A positive secretory response to formycin A, still not associated with any decrease in K+ conductance, was only observed either immediately after formycin A administration to islets already exposed to glibenclamide or during prolonged exposure to the adenosine analogue. This coincided with an increase of cytosolic Ca2+ concentration in intact B-cells and a greater increase of membrane capacitance in response to depolarization in B-cells examined in the perforated patch whole-cell configuration. The latter stimulation of exocytotic activity could not be attributed, however, to any increase in peak or integrated Ca2+ current. Thus, the mode of action of formycin A, or its 5'-triphosphate ester, in islet cells obviously differs from that currently ascribed to endogenous ATP in the process of nutrient-stimulated insulin release.

Gromada J, Rorsman P, Dissing S, Wulff BS. 1995. Stimulation of cloned human glucagon-like peptide 1 receptor expressed in HEK 293 cells induces cAMP-dependent activation of calcium-induced calcium release. FEBS Lett, 373 (2), pp. 182-186. | Show Abstract | Read more

The actions of glucagon-like peptide-1(7-36)amide (GLP-1(7-36)amide) on cellular signalling were studied in human embryonal kidney 293 (HEK 293) cells stably transfected with the cloned human GLP-1 receptor. The cloned GLP-1 receptor showed a single high-affinity binding site (Kd = 0.76 nM). Binding of GLP-1(7-36)amide stimulated cAMP production in a dose-dependent manner (EC50 = 0.015 nM) and caused an increase in the intracellular free Ca2+ concentration ([Ca2+]i). The latter effect reflected Ca(2+)-induced Ca2+ release and was suppressed by ryanodine. We propose that the ability of GLP-1(7-36)amide to increase [Ca2+]i results from sensitization of the ryanodine receptors by a protein kinase A dependent mechanism.

Gromada J, Dissing S, Bokvist K, Renström E, Frøkjaer-Jensen J, Wulff BS, Rorsman P. 1995. Glucagon-like peptide I increases cytoplasmic calcium in insulin-secreting beta TC3-cells by enhancement of intracellular calcium mobilization. Diabetes, 44 (7), pp. 767-774. | Show Abstract | Read more

In the insulin-secreting beta-cell line beta TC3, stimulation with 11.2 mmol/l glucose caused a rise in the intracellular free Ca2+ concentration ([Ca2+]i) in only 18% of the tested cells. The number of glucose-responsive cells increased after pretreatment of the cells with glucagon-like peptide I (GLP-I)(7-36)amide and at 10(-11) mol/l; 84% of the cells responded to glucose with a rise in [Ca2+]i. GLP-I(7-36)amide induces a rapid increase in [Ca2+]i only in cells exposed to elevated glucose concentrations (> or = 5.6 mmol/l). The action of GLP-I(7-36)amide and forskolin involved a 10-fold increase in cytoplasmic cAMP concentration and was mediated by activation of protein kinase A. It was not associated with an effect on the membrane potential but required some (small) initial entry of Ca2+ through voltage-dependent L-type Ca2+ channels, which then produced a further increase in [Ca2+]i by mobilization from intracellular stores. The latter effect reflected Ca(2+)-induced Ca2+ release and was blocked by ryanodine. Similar increases in [Ca2+]i were also observed in voltage-clamped cells, although there was neither activation of a background (Ca(2+)-permeable) inward current nor enhancement of the voltage-dependent L-type Ca2+ current. These observations are consistent with GLP-I(7-36) amide inducing glucose sensitivity by promoting mobilization of Ca2+ from intracellular stores. We propose that this novel action of GLP-I(7-36)amide represents an important factor contributing to its insulinotropic action.

Bokvist K, Eliasson L, Ammälä C, Renström E, Rorsman P. 1995. Co-localization of L-type Ca2+ channels and insulin-containing secretory granules and its significance for the initiation of exocytosis in mouse pancreatic B-cells. EMBO J, 14 (1), pp. 50-57. | Show Abstract

We have monitored L-type Ca2+ channel activity, local cytoplasmic Ca2+ transients, the distribution of insulin-containing secretory granules and exocytosis in individual mouse pancreatic B-cells. Subsequent to the opening of the Ca2+ channels, exocytosis is initiated with a latency < 100 ms. The entry of Ca2+ that precedes exocytosis is unevenly distributed over the cell and is concentrated to the region with the highest density of secretory granules. In this region, the cytoplasmic Ca2+ concentration is 5- to 10-fold higher than in the remainder of the cell reaching concentrations of several micromolar. Single-channel recordings confirm that the L-type Ca2+ channels are clustered in the part of the cell containing the secretory granules. This arrangement, which is obviously reminiscent of the 'active zones' in nerve terminals, can be envisaged as being favourable to the B-cell as it ensures that the Ca2+ transient is maximal and restricted to the part of the cell where it is required to rapidly initiate exocytosis whilst at the same time minimizing the expenditure of metabolic energy to subsequently restore the resting Ca2+ concentration.

Best L, Ammala C, Rorsman P, Tomlinson S. 1994. Intracellular pH, cytosolic calcium concentration and electrical activity in RINm5F insulinoma cells. Biochim Biophys Acta, 1192 (1), pp. 107-111. | Show Abstract | Read more

The addition of L-lactate or acetate to RINm5F cells caused a transient intracellular acidification, an increase in [Ca2+]i and induced electrical activity. The subsequent withdrawal of lactate or acetate resulted in an intracellular alkalinization with no apparent changes in [Ca2+]i nor electrical activity. Intracellular alkalinization and acidification by application by application and withdrawal of NH4Cl were both accompanied by transient increases in [Ca2+]i in the absence of electrical activity. The induction of electrical activity by lactate was associated with the appearance of inward whole cell currents. Changes in intracellular pH may affect [Ca2+]i though not necessarily by altering plasma membrane potential. The inward currents associated with lactate application may represent an organic anion conductance contributing towards the stimulation of electrical activity by organic acids.

Ammälä C, Eliasson L, Bokvist K, Berggren PO, Honkanen RE, Sjöholm A, Rorsman P. 1994. Activation of protein kinases and inhibition of protein phosphatases play a central role in the regulation of exocytosis in mouse pancreatic beta cells. Proc Natl Acad Sci U S A, 91 (10), pp. 4343-4347. | Show Abstract | Read more

The mechanisms that regulate insulin secretion were investigated using capacitance measurements of exocytosis in single beta cells maintained in tissue culture. Exocytosis was stimulated by voltage-clamp depolarizations to activate the voltage-dependent Ca2+ channels that mediate Ca2+ influx into the beta cell. Under basal conditions, the exocytotic responses were small despite large Ca2+ currents. The exocytotic responses were dramatically increased (10- to 20-fold) by conditions that promote protein phosphorylation, such as activation of protein kinases A and C or inhibition of protein phosphatases. The stimulation of secretion was not due to an enhancement of Ca2+ influx and both peak and integrated Ca2+ currents were largely unaffected. Our data indicate that exocytosis in the insulin-secreting pancreatic beta cell is determined by a balance between protein phosphorylation and dephosphorylation. They further suggest that although Ca2+ is required for the initiation of exocytosis, modulation of exocytosis by protein kinases and phosphatases, at a step distal to the elevation of Ca2+, is of much greater quantitative importance. Thus an elevation of Ca2+ may represent a permissive rather than a decisive factor in the regulation of the insulin secretory process.

Malaisse WJ, Conget I, Sener A, Rorsman P. 1994. Insulinotropic action of AICA riboside. II. Secretory, metabolic and cationic aspects. Diabetes Res, 25 (1), pp. 25-37. | Show Abstract

Preincubation of rat pancreatic islets with AICA riboside (0.1 to 1.0mM) caused a concentration-related stimulation of both 45Ca net uptake and insulin release evoked by 8.3 mM D-glucose, but failed to affect the conversion of D-[5-3H]glucose to 3HOH, the generation of 14CO2 and 14C-labelled amino acids or acidic metabolites from D-[U-14C]glucose, and the islet content in ATP, ADP or AMP. The secretory response to AICA riboside was not suppressed in islets preincubated with methotrexate. AICA riboside caused a progressive decrease in 86Rb outflow from prelabelled islets perifused at 2.8 or 6.0mM D-glucose. This effect faded out at a higher concentration of D-glucose (16.7 mM), in which case AICA riboside nevertheless provoked a delayed, progressive and not rapidly reversible enhancement of insulin output. At concentrations up to 0.4 mM, ZTP only exerted a modest effect on the activity of KATP-channels in inside-out patches of dispersed mouse islet cells. These findings raise the question whether the insulinotropic action of AICA riboside may be attributable to the sequential generation of ZMP, ZDP and ZTP from the nucleoside.

Ashcroft FM, Proks P, Smith PA, Ammälä C, Bokvist K, Rorsman P. 1994. Stimulus-secretion coupling in pancreatic beta cells. J Cell Biochem, 55 Suppl (S1994A), pp. 54-65. | Show Abstract | Read more

Insulin secretion is triggered by a rise in the intracellular Ca2+ concentration that results from the activation of voltage-gated Ca2+ channels in the beta-cell plasma membrane. Multiple types of beta-cell Ca2+ channel have been identified in both electrophysiological and molecular biological studies, but it appears that the L-type Ca2+ channel plays a dominant role in regulating Ca2+ influx. Activity of this channel is potentiated by protein kinases A and C and is inhibited by GTP-binding proteins, which may mediate the effects of potentiators and inhibitors of insulin secretion on Ca2+ influx, respectively. The mechanisms by which elevation of intracellular Ca2+ leads to the release of insulin granules is not fully understood but appears to involve activation of Ca2+/calmodulin-dependent protein kinase. Phosphorylation by either protein kinase A or C, probably at different substrates, potentiates insulin secretion by acting at some late stage in the secretory process. There is also evidence that small GTP-binding proteins are involved in regulating exocytosis in beta cells. The identification and characterisation of the proteins involved in exocytosis in beta cells and clarification of the mechanism(s) of action of Ca2+ is clearly an important goal for the future.

Ammälä C, Eliasson L, Bokvist K, Larsson O, Ashcroft FM, Rorsman P. 1993. Exocytosis elicited by action potentials and voltage-clamp calcium currents in individual mouse pancreatic B-cells. J Physiol, 472 (1), pp. 665-688. | Show Abstract | Read more

1. Measurements of membrane capacitance, as an indicator of exocytosis, and intracellular Ca2+ concentration ([Ca2+]i) were used to determine the Ca2+ dependence of secretion in single pancreatic B-cells. 2. Exocytosis was dependent on a rise in [Ca2+]i and could be evoked by activation of voltage-dependent Ca2+ currents. The threshold for depolarization-induced release was 0.5 microM [Ca2+]i. Once the [Ca2+]i threshold was exceeded, exocytosis was rapidly (< 50 ms) initiated. When individual pulses were applied, exocytosis stopped immediately upon repolarization and the Ca2+ channels closed, although [Ca2+]i remained elevated for several seconds. 3. During repetitive stimulation (1 Hz), when [Ca2+]i attained micromolar levels, exocytosis also took place during the interpulse intervals albeit at a slower rate than during the depolarizations. 4. Exocytosis could be initiated by simulated action potentials. Whereas a single action potential only produced a small capacitance increase, and in some cells even failed to stimulate release, larger and more consistent responses were obtained with > or = four action potentials. 5. Comparison of the rates of exocytosis measured in response to depolarization, mobilization of Ca2+ from intracellular stores or infusion of Ca2+ through the patch pipette suggests that [Ca2+]i at the secretory sites attains a concentration of several micromolar. This is much higher than the average [Ca2+]i detected by microfluorimetry suggesting the existence of steep spatial gradients of [Ca2+]i within the B-cell. 6. Inclusion of inhibitors of Ca2+/calmodulin-dependent protein kinase II in the intracellular solution reduced the depolarization-induced exocytotic responses suggesting this enzyme may be involved in the coupling between elevation of [Ca2+]i to stimulation of the secretory machinery. 7. The size of the unitary exocytotic event was 2 fF, corresponding to a secretory granule diameter of 250 nm. 8. Over short periods, exocytosis may be extremely fast (1 pF/s or 500 granules/s), which is much higher than the rate of endocytosis (18 fF/s or 9 granules/s). Since the latter is in better agreement with the maximum rate of insulin secretion from islets (approximately 2 granules/s), we suggest that membrane retrieval may set an upper limit on the rate of exocytosis during extended periods of secretion.

Sjöholm A, Arkhammar P, Welsh N, Bokvist K, Rorsman P, Hallberg A, Nilsson T, Welsh M, Berggren PO. 1993. Enhanced stimulus-secretion coupling in polyamine-depleted rat insulinoma cells. An effect involving increased cytoplasmic Ca2+, inositol phosphate generation, and phorbol ester sensitivity. J Clin Invest, 92 (4), pp. 1910-1917. | Show Abstract | Read more

To extend previous observations on the role of polyamines in insulin production, metabolism, and replication of insulin-secreting pancreatic beta cells, we have studied the role of polyamines in the regulation of the stimulus-secretion coupling of clonal rat insulinoma cells (RINm5F). For this purpose, RINm5F cells were partially depleted in their polyamine contents by use of the specific ornithine decarboxylase inhibitor difluoromethylornithine (DFMO), which led to an increase in cellular insulin and ATP contents. Analysis of different parts of the signal transduction pathway revealed that insulin secretion and the increase in cytoplasmic free Ca2+ concentration ([Ca2+]i) after K(+)-induced depolarization were markedly enhanced in DFMO-treated cells. These effects were paralleled by increased voltage-activated Ca2+ currents, as judged by whole-cell patch-clamp analysis, probably reflecting increased channel activity rather than elevated number of channels per cell. DFMO treatment also rendered phospholipase C in these cells more sensitive to the muscarinic receptor agonist carbamylcholine, as evidenced by enhanced generation of inositol phosphates, increase in [Ca2+]i and insulin secretion, despite an unaltered ligand binding to muscarinic receptors and lack of effect on protein kinase C activity. In addition, the tumor promoter 12-O-tetradecanoylphorbol 13-acetate, at concentrations suggested to be specific for protein kinase C activation, evoked an increased insulin output in polyamine-deprived cells compared to control cells. The stimulatory effects of glucose or the cyclic AMP raising agent theophylline on insulin release were not increased by DFMO treatment. In spite of increased binding of sulfonylurea in DFMO-treated cells, there was no secretory response or altered increase in [Ca2+]i in response to the drug in these cells. It is concluded that partial polyamine depletion sensitizes the stimulus-secretion coupling at multiple levels in the insulinoma cells, including increased voltage-dependent Ca2+ influx and enhanced responsiveness to activators of phospholipase C and protein kinase C. In their entirety, our present results indicate that the behavior of the stimulus-secretion coupling of polyamine-depleted RINm5F insulinoma cells changes towards that of native beta cells, thus improving the usefulness of this cell line for studies of beta cell insulin secretion.

Juntti-Berggren L, Larsson O, Rorsman P, Ammälä C, Bokvist K, Wåhlander K, Nicotera P, Dypbukt J, Orrenius S, Hallberg A. 1993. Increased activity of L-type Ca2+ channels exposed to serum from patients with type I diabetes. Science, 261 (5117), pp. 86-90. | Show Abstract | Read more

Type I diabetes [insulin-dependent diabetes mellitus (IDDM)] is an autoimmune disease associated with the destruction of pancreatic beta cells. Serum from patients with IDDM increased L-type calcium channel activity of insulin-producing cells and of GH3 cells derived from a pituitary tumor. The subsequent increase in the concentration of free cytoplasmic Ca2+ ([Ca2+]i) was associated with DNA fragmentation typical of programmed cell death or apoptosis. These effects of the serum were prevented by adding a blocker of voltage-activated L-type Ca2+ channels. When the serum was depleted of immunoglobulin M (IgM), it no longer affected [Ca2+]i. An IgM-mediated increase in Ca2+ influx may thus be part of the autoimmune reaction associated with IDDM and contribute to the destruction of beta cells in vivo.

Ammälä C, Ashcroft FM, Rorsman P. 1993. Calcium-independent potentiation of insulin release by cyclic AMP in single beta-cells. Nature, 363 (6427), pp. 356-358. | Show Abstract | Read more

How does cyclic AMP potentiate insulin secretion from pancreatic islet beta-cells? This question is fundamental to understanding how hormones such as glucagon, which elevates cAMP, stimulate insulin secretion and so contribute to the normal secretory response of the islet. It is well established that a rise in the cytoplasmic Ca2+ concentration ([Ca2+]i) is essential for insulin secretion and therefore cAMP has been proposed to act by elevating [Ca2+]i. But studies on permeabilized beta-cells indicate that cAMP increases insulin release even when [Ca2+]i is held constant. We have used microfluorimetry and the patch-clamp technique to measure changes simultaneously in Ca2+ currents, [Ca2+]i and exocytosis in a single beta-cell in response to cAMP. We show here that cAMP, through activation of protein kinase A, increases Ca(2+)-influx through voltage-dependent L-type Ca2+ channels, thereby elevating [Ca2+]i and accelerating exocytosis. More importantly, cAMP also promotes insulin release by a direct interaction with the secretory machinery, which accounts for as much as 80% of its effect.

Larsson O, Ammälä C, Bokvist K, Fredholm B, Rorsman P. 1993. Stimulation of the KATP channel by ADP and diazoxide requires nucleotide hydrolysis in mouse pancreatic beta-cells. J Physiol, 463 (1), pp. 349-365. | Show Abstract | Read more

1. The mechanisms by which ADP and the hyperglycaemic compound diazoxide stimulate the activity of the ATP-regulated K+ channel (KATP channel) were studied using inside-out patches isolated from mouse pancreatic beta-cells maintained in tissue culture. 2. The ability of diazoxide and ADP to increase KATP channel activity declined with time following patch excision and no stimulation was observed after 15-40 min. 3. Activation of KATP channels by ADP required the presence of intracellular Mg2+. The stimulatory effect of ADP was mimicked by AMP but only in the presence of ATP. Replacement of ATP with the non-hydrolysable analogue beta, gamma-methylene ATP did not interfere with the ability of ADP to stimulate KATP channel activity. By contrast, enhancement of KATP channel activity was critically dependent on hydrolysable ADP and no stimulation was observed after substitution of alpha,beta-methylene ADP for standard ADP. 4. The ability of diazoxide to enhance KATP channel activity was dependent on the presence of both internal Mg2+ and ATP. Diazoxide stimulation of KATP channel activity was not observed after substitution of beta,gamma-methylene ATP for ATP. However, in the presence of ADP, at a concentration which in itself had no stimulatory action (10 microM), diazoxide was stimulatory also in the presence of the stable ATP analogue. 5. The stimulatory action of diazoxide on KATP channel activity in the presence of ATP was markedly enhanced by intracellular ADP. This potentiating effect of ADP was not reproduced by the stable analogue alpha,beta-methylene ADP and was conditional on the presence of intracellular Mg2+. A similar enhancement of channel activity was also observed with AMP (0.1 mM). In the absence of ATP, diazoxide was still capable of stimulating channel activity provided ADP was present. This effect was not reproduced by AMP. 6. In both nucleotide-free solution and in the presence of 0.1 mM ATP, the distribution of the KATP channel open times were described by a single exponential with a time constant of approximately 20 ms. Addition of ADP or diazoxide resulted in the appearance of a second component with a time constant of > 100 ms which comprised 40-70% of the total number of events. Under the latter experimental conditions, the open probability of the channel increased more than fivefold relative to that observed in the presence of ATP alone.(ABSTRACT TRUNCATED AT 400 WORDS)

Ammälä C, Bokvist K, Larsson O, Berggren PO, Rorsman P. 1993. Demonstration of a novel apamin-insensitive calcium-activated K+ channel in mouse pancreatic B cells. Pflugers Arch, 422 (5), pp. 443-448. | Show Abstract | Read more

The whole-cell configuration of the patch-clamp technique was used to characterize the biophysical and pharmacological properties of an oscillating K(+)-current that can be induced by intracellular application of GTP[gamma S] in mouse pancreatic B cells (Ammälä et al. 1991). These K+ conductance changes are evoked by periodic increases in the cytoplasmic Ca2+ concentration ([Ca2+]i) and transiently repolarize the B cell, thus inhibiting action-potential firing and giving rise to a bursting pattern. GTP[gamma S]-evoked oscillations in K+ conductance were reversibly suppressed by a high (300 microM) concentration of carbamylcholine. By contrast, alpha 2-adrenoreceptor stimulation by 20 microM clonidine did not interfere with the oscillatory behaviour but evoked a small sustained outward current. At 0 mV membrane potential, the oscillating K(+)-current elicited by GTP[gamma S] was highly sensitive to extracellular tetraethylammonium (TEA; 70% block by 1 mM). The TEA-resistant component, which carried approximately 80% of the current at -40 mV, was affected neither by apamin (1 microM) nor by tolbutamide (500 microM). The current evoked by internal GTP[gamma S] was highly selective for K+, as demonstrated by a 51-mV change in the reversal potential for a sevenfold change in [K+]o. Stationary fluctuation analysis indicated a unitary conductance of 0.5 pS when measured with symmetric (approximately 140 mM) KCl solutions. The estimated single-channel conductance with physiological ionic gradients is 0.1 pS. The results indicate the existence of a novel Ca(2+)-gated K+ conductance in pancreatic B cells. Activation of this K+ current may contribute to the generation of the oscillatory electrical activity characterizing the B cell at intermediate glucose concentrations.

BEST L, AMMALA C, RORSMAN P. 1993. EVIDENCE FOR AN ELECTROGENIC LACTATE CONDUCTANCE IN MOUSE BETA-CELLS AND RINM5F INSULINOMA CELLS JOURNAL OF PHYSIOLOGY-LONDON, 467 pp. P253-P253.

Juntti-Berggren L, Rorsman P, Siffert W, Berggren PO. 1992. Intracellular pH and the stimulus-secretion coupling in insulin-producing RINm5F cells. Biochem J, 287 ( Pt 1) (1), pp. 59-66. | Show Abstract | Read more

The regulation of intracellular pH (pHi) and its role in the insulin-secretory process were evaluated, by using the clonal insulin-secreting cell line RINm5F. Glyceraldehyde, lactate and dihydroxyacetone decreased pHi, but only the first two released insulin. In the presence of extracellular Na+ the cells counteracted the acidification. Blocking the Na+/H+ exchange in acidic cells resulted in a drastic further lowering of pHi, an effect not obtained under basal conditions. Whereas glyceraldehyde depolarized the cells, lactate was without effect. Dihydroxyacetone hyperpolarized the cells in the presence of extracellular Na+, but this effect disappeared when Na+ was excluded from the medium. Stimulation with glyceraldehyde resulted in increased free cytoplasmic Ca2+ concentration ([Ca2+]i). Dihydroxyacetone and lactate had no effect on [Ca2+]i in the presence of Na+, but lactate induced a decrease in [Ca2+]i in Na(+)-deficient medium. In RINm5F cells the activity of the Na+/H+ antiport could not be augmented by activation of protein kinase C (PKC). Hence, in insulin-secreting cells a PKC-insensitive Na+/H+ antiport is the major mechanism restoring a decrease in pHi. Acidification itself does not affect membrane potential, but may directly interact with the mechanisms regulating exocytosis.

Rorsman P, Ammälä C, Berggren PO, Bokvist K, Larsson O. 1992. Cytoplasmic calcium transients due to single action potentials and voltage-clamp depolarizations in mouse pancreatic B-cells. EMBO J, 11 (8), pp. 2877-2884. | Show Abstract

Changes in the cytoplasmic free calcium concentration ([Ca2+]i) in pancreatic B-cells play an important role in the regulation of insulin secretion. We have recorded [Ca2+]i transients evoked by single action potentials and voltage-clamp Ca2+ currents in isolated B-cells by the combination of dual wavelength emission spectrofluorimetry and the patch-clamp technique. A 500-1000 ms depolarization of the B-cell from -70 to -10 mV evoked a transient rise in [Ca2+]i from a resting value of approximately 100 nM to a peak concentration of 550 nM. Similar [Ca2+]i changes were associated with individual action potentials. The depolarization-induced [Ca2+]i transients were abolished by application of nifedipine, a blocker of L-type Ca2+ channels, indicating their dependence on influx of extracellular Ca2+. Following the voltage-clamp step, [Ca2+]i decayed with a time constant of approximately 2.5 s and summation of [Ca2+]i occurred whenever depolarizations were applied with an interval of less than 2 s. The importance of the Na(+)-Ca2+ exchange for B-cell [Ca2+]i maintenance was evidenced by the demonstration that basal [Ca2+]i rose to 200 nM and the magnitude of the depolarization-evoked [Ca2+]i transients was markedly increased after omission of extracellular Na+. However, the rate by which [Ca2+]i returned to basal was not affected, suggesting the existence of additional [Ca2+]i buffering processes.

Kindmark H, Köhler M, Efendić S, Rorsman P, Larsson O, Berggren PO. 1992. Protein kinase C activity affects glucose-induced oscillations in cytoplasmic free Ca2+ in the pancreatic B-cell. FEBS Lett, 303 (1), pp. 85-90. | Show Abstract | Read more

Acute stimulation of protein kinase C (PKC) inhibited glucose-induced slow oscillations in cytoplasmic free Ca(2+)-concentration, [Ca2+]i, in mouse pancreatic B-cells. In PKC-depleted cells glucose induced rapid transients in [Ca2+]i, lasting for approximately 10 s, superimposed on the slow oscillations in [Ca2+]i. It was demonstrated that the transients did not occur in the absence of extracellular Ca2+. Each transient typically was preceded by a slow increase in [Ca2+]i, representing the rising phase of an ordinary glucose-induced slow oscillation, and the [Ca2+]i, immediately after a transient was lower than just before the spike. These data further emphasize the interplay between voltage-dependent Ca(2+)-channels and the phospholipase C system in the regulation of B-cell [Ca2+]i-oscillations.

RORSMAN P, BERGGREN P. 1992. ELECTRICAL BURSTING IN ISLET BETA-CELLS - REPLY NATURE, 357 (6373), pp. 28-28. | Read more

Islam MS, Rorsman P, Berggren PO. 1992. Ca(2+)-induced Ca2+ release in insulin-secreting cells. FEBS Lett, 296 (3), pp. 287-291. | Show Abstract | Read more

The sulphydryl reagent thimerosal (50 microM) released Ca2+ from a non-mitochondrial intracellular Ca2+ pool in a dose-dependent manner in permeabilized insulin-secreting RINm5F cells. This release was reversed after addition of the reducing agent dithiothreitol. Ca2+ was released from an Ins(1,4,5)P3-insensitive pool, since release was observed even after depletion of the Ins(1,4,5)P3-sensitive pool by a supramaximal dose of Ins(2,4,5)P3 or thapsigargin. The Ins(1,4,5)P3-sensitive pool remained essentially unaltered by thimerosal. Thimerosal-induced Ca2+ release was potentiated by caffeine. These findings suggest the existence of Ca(2+)-induced Ca2+ release also in insulin-secreting cells.

Ammälä C, Berggren PO, Bokvist K, Rorsman P. 1992. Inhibition of L-type calcium channels by internal GTP [gamma S] in mouse pancreatic beta cells. Pflugers Arch, 420 (1), pp. 72-77. | Show Abstract | Read more

Pretreatment of pancreatic beta cells with pertussis toxin resulted in a 30% increase in peak whole-cell Ca2+ currents recorded in the absence of exogenous intracellular guanine nucleotides. Intracellular application of 90 microM GTP[gamma S], by liberation from a caged precursor, resulted in 40% reduction of the peak Ca2+ current irrespective of whether the current was carried by Ca2+ or Ba2+. Effects on the delayed outward K+ current were small and restricted to a transient Ca(2+)-dependent K+ current component. Inhibition by GTP[gamma S] of the Ca2+ current was not mimicked by standard GTP and could not be prevented either by pretreatment with pertussis toxin or by inclusion of GDP[beta S] or cyclic AMP in the intracellular medium. The inhibitory effect of GTP[gamma S] could be counteracted by a prepulse to a large depolarizing voltage. A similar effect of a depolarizing prepulse was observed in control cells with no exogenous guanine nucleotides. These observations indicate that inhibition of beta cell Ca2+ current by G protein activation results from direct interaction with the channel and does not involve second-messenger systems. Our findings also suggest that the beta cell Ca2+ current is subject to resting inhibition by G proteins.

Juntti-Berggren L, Arkhammar P, Nilsson T, Rorsman P, Berggren PO. 1991. Glucose-induced increase in cytoplasmic pH in pancreatic beta-cells is mediated by Na+/H+ exchange, an effect not dependent on protein kinase C. J Biol Chem, 266 (35), pp. 23537-23541. | Show Abstract

Glucose-induced changes in cytoplasmic pH (pHi) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Glucose, at concentrations above 3-5 mM, depolarized the beta-cell and increased pHi, cytoplasmic free Ca2+ ([Ca2+]i), and insulin release. This increase in pHi was dependent on the presence of extracellular Na+ and was inhibited by 5-(N-ethyl-N-isopropyl) amiloride, a blocker of Na+/H+ exchange. Stimulation of protein kinase C with phorbol ester also induced an alkalinization. However, when protein kinase C activity was down-regulated, glucose stimulation still induced alkalinization. At 20 mM glucose, 10 mM NH4Cl induced a marked rise in pHi, paralleled by repolarization, inhibition of electrical activity, and decreases in both [Ca2+]i and insulin release. Reduction in [Ca2+]i was prevented by 200 microM tolbutamide, but not by 10 mM tetraethylammonium. At 4 mM glucose, NH4Cl induced a transient increase in insulin release, without changing [Ca2+]i. Exposure of beta-cells to 10 mM sodium acetate caused a persistent decrease in pHi, an effect paralleled by a small transient increase in [Ca2+]i. Acidification per se did not change the beta-cell sensitivity to glucose, not excluding that the activity of the ATP-regulated K+ channels may be modulated by changes in pHi.

Ammälä C, Larsson O, Berggren PO, Bokvist K, Juntti-Berggren L, Kindmark H, Rorsman P. 1991. Inositol trisphosphate-dependent periodic activation of a Ca(2+)-activated K+ conductance in glucose-stimulated pancreatic beta-cells. Nature, 353 (6347), pp. 849-852. | Show Abstract | Read more

Glucose-stimulated insulin secretion is associated with the appearance of electrical activity in the pancreatic beta-cell. At intermediate glucose concentrations, beta-cell electrical activity follows a characteristic pattern of slow oscillations in membrane potential on which bursts of action potentials are superimposed. The electrophysiological background of the bursting pattern remains unestablished. Activation of Ca(2+)-activated large-conductance K+ channels (KCa channel) has been implicated in this process but seems unlikely in view of recent evidence demonstrating that the beta-cell electrical activity is unaffected by the specific KCa channel blocker charybdotoxin. Another hypothesis postulates that the bursting arises as a consequence of two components of Ca(2+)-current inactivation. Here we show that activation of a novel Ca(2+)-dependent K+ current in glucose-stimulated beta-cells produces a transient membrane repolarization. This interrupts action potential firing so that action potentials appear in bursts. Spontaneous activity of this current was seen only rarely but could be induced by addition of compounds functionally related to hormones and neurotransmitters present in the intact pancreatic islet. K+ currents of the same type could be evoked by intracellular application of GTP, the effect of which was mediated by mobilization of Ca2+ from inositol 1,4,5-trisphosphate (InsP3)-sensitive intracellular Ca2+ stores. These observations suggest that oscillatory glucose-stimulated electrical activity, which is correlated with pulsatile release of insulin, results from the interaction between the beta-cell and intraislet hormones and neurotransmitters. Our data also provide evidence for a close interplay between ion channels in the plasma membrane and InsP3-induced mobilization of intracellular Ca2+ in an excitable cell.

Kindmark H, Köhler M, Nilsson T, Arkhammar P, Wiechel KL, Rorsman P, Efendić S, Berggren PO. 1991. Measurements of cytoplasmic free Ca2+ concentration in human pancreatic islets and insulinoma cells. FEBS Lett, 291 (2), pp. 310-314. | Show Abstract | Read more

In human pancreatic islets an increase in the glucose concentration from 3 to 20 mM raised the free cytoplasmic Ca2+ concentration [( Ca2+]i), an effect being reversible upon withdrawal of the sugar. Depolarization with a high concentration of K+ or the sulphonylurea tolbutamide also raised [Ca2+]i. Addition of extracellular ATP produced a transient rapid rise in [Ca2+]i. Oscillations in [Ca2+]i were observed in the presence of 10 mM glucose. Insulinoma cells responded to glucose and tolbutamide with increases in [Ca2+]i, whereas the sulphonamide diazoxide caused a decrease in [Ca2+]i. These findings confirm previous results obtained in rodent beta-cells.

Islam MS, Nilsson T, Rorsman P, Berggren PO. 1991. Interaction with the inositol 1,4,5-trisphosphate receptor promotes Ca2+ sequestration in permeabilised insulin-secreting cells. FEBS Lett, 288 (1-2), pp. 27-29. | Show Abstract | Read more

Electropermeabilised insulin-secreting RINm5F cells sequestered Ca2+, resulting in a steady-state level of the ambient free Ca2+ concentration corresponding to 723 +/- 127 nM (mean +/- SEM, n = 10), as monitored by a Ca(2+)-selective minielectrode. Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) promoted a rapid and pronounced release of Ca2+. This Ca2+ was resequestered and a new steady-state Ca2+ level was attained, which was always lower (460 +/- 102 nM, n = 10, P less than 0.001) than the steady-state Ca2+ level maintained before the addition of Ins(1,4,5)P3. Whereas the initial reuptake of Ca2+ subsequent to Ins(1,4,5)P3 stimulation was relatively slow, the later part of reuptake was fast as compared to the reuptake phases of a pulse addition of extraneous Ca2+. In the latter case the uptake of Ca2+ resulted in a steady-state level similar to that found in the absence of Ins(1,4,5)P3. Addition of Ins(1,4,5)P3 under this condition resulted in a further Ca2+ uptake and thus a lower steady-state Ca2+ level. Heparin, which binds to the Ins(1,4,5)P3 receptor, also lowered the steady-state free Ca2+ concentration. In contrast to Ins(1,4,5)P3, inositol 1,3,4,5-tetrakisphosphate was without effect on Ca2+ sequestration. These findings are consistent with the presence of a high-affinity Ins(1,4,5)P3 receptor promoting continuous release of Ca2+ under basal conditions and/or the Ins(1,4,5)P3 receptor being actively involved in Ca2+ sequestration.

Rorsman P, Ashcroft FM, Berggren PO. 1991. Regulation of glucagon release from pancreatic A-cells. Biochem Pharmacol, 41 (12), pp. 1783-1790. | Read more

Bokvist K, Ammälä C, Berggren PO, Rorsman P, Wåhlander K. 1991. Alpha 2-adrenoreceptor stimulation does not inhibit L-type calcium channels in mouse pancreatic beta-cells. Biosci Rep, 11 (3), pp. 147-157. | Show Abstract | Read more

The effects of alpha 2-adrenergic stimulation on the Ca(2+)-current in mouse pancreatic beta-cells were investigated using the patch-clamp technique. When using the conventional whole-cell recording configuration (dialysis of cell interior with pipette solution), addition of adrenaline (1 microM) or the alpha 2-adrenergic agonist clonidine (5 microM) failed to reduce the Ca(2+)-current, irrespective of whether intracellular GTP (or GTP gamma S) was present or not and at both physiological (1.3 mM) and elevated (10.2 mM) Ca(2+)-concentrations. In fact, in the absence of added guanine nucleotides, the agonists tended to increase the Ca(2+)-current amplitude in the presence of the higher Ca(2+)-concentration. Ca(2+)-channel activation measured at 1.3 mM Ca2+ was not affected by clonidine. Half-maximal activation was observed at approximately -20 mV. In addition, when Ca(2+)-currents were recorded from intact beta-cells, using the perforated patch whole-cell configuration, clonidine (1 microM) also failed to detectably affect the Ca(2+)-current. It is therefore suggested that the inhibition of beta-cell electrical activity and insulin-secretion produced by alpha 2-adrenoreceptor stimulation does not result from suppression of the L-type Ca(2+)-current.

Ammälä C, Bokvist K, Galt S, Rorsman P. 1991. Inhibition of ATP-regulated K(+)-channels by a photoactivatable ATP-analogue in mouse pancreatic beta-cells. Biochim Biophys Acta, 1092 (3), pp. 347-349. | Show Abstract | Read more

The effects of a photoactivable (DMNPE-caged) ATP-analogue on ATP-regulated K(+)-channels (KATP-channel) in mouse pancreatic beta-cells were investigated using the inside-out patch configuration of the patch-clamp technique. The caged precursor caused a concentration-dependent reduction of channel activity with a Ki of 17 microM; similar to the 11 microM obtained for standard Mg-ATP. The small difference in the blocking capacity between the precursor and ATP is probably the reason why no change in channel activity was observed upon photolysis of the caged molecule and liberation of ATP. It is suggested that the part of the ATP molecule involved in the blocking reaction of the KATP-channel is not sufficiently protected in DMNPE-caged ATP making this compound unsuitable for studying the rapid kinetics of ATP-induced KATP-channel inhibition.

Bokvist K, Ammälä C, Ashcroft FM, Berggren PO, Larsson O, Rorsman P. 1991. Separate processes mediate nucleotide-induced inhibition and stimulation of the ATP-regulated K(+)-channels in mouse pancreatic beta-cells. Proc Biol Sci, 243 (1307), pp. 139-144. | Show Abstract | Read more

The mechanisms by which nucleotides stimulate the activity of the ATP-regulated K(+)-channel (KATP-channel) were investigated using inside-out patches from mouse pancreatic beta-cells. ATP produces a concentration-dependent inhibition of channel activity with a Ki of 18 microns. The inhibitory action of ATP was counteracted by ADP (0.1 mM) and GDP (0.2 mM) but not GTP (1 mM). Stimulation of channel activity was also observed when ADP, GDP and GTP were applied in the absence of ATP. The ability of ADP and GDP to reactivate KATP-channels blocked by ATP declined with time following patch excision and after 30-60 min these nucleotides were without effect. During the same time period the ability of ADP and GTP to stimulate the channel in the absence of ATP was lost. In fact, ADP now blocked channel activity with 50% inhibition being observed at approximately 0.1 mM. By contrast, GDP remained a stimulator in the absence of ATP even when its ability to evoke channel activity in the presence of ATP was lost. These observations show that nucleotide-induced activation of the KATP-channel does not involve competition with ATP for a common inhibitory site but involves other processes. The data are consistent with the idea that nucleotides modulate KATP-channel activity by a number of different mechanisms that may include both regulation of cytosolic constituents and direct interaction with the channel and associated control proteins.

Rorsman P, Bokvist K, Ammälä C, Arkhammar P, Berggren PO, Larsson O, Wåhlander K. 1991. Activation by adrenaline of a low-conductance G protein-dependent K+ channel in mouse pancreatic B cells. Nature, 349 (6304), pp. 77-79. | Show Abstract | Read more

Insulin is produced and secreted by the B cells in the endocrine pancreas. In vivo, insulin secretion is under the control of a number of metabolic, neural and hormonal substances. It is now clear that stimulation of insulin release by fuel secretagogues, such as glucose, involves the closure of K+ channels that are sensitive to the intracellular ATP concentration (KATP channels). This leads to membrane depolarization and the generation of Ca2(+)-dependent action potentials. The mechanisms whereby hormones and neurotransmitters such as adrenaline, galanin and somatostatin, which are released by intraislet nerve endings and the pancreatic D cells, produce inhibition of insulin secretion are not clear. Here we show that adrenaline suppresses B-cell electrical activity (and thus insulin secretion) by a G protein-dependent mechanism, which culminates in the activation of a sulphonylurea-insensitive low-conductance K+ channel distinct from the KATP channel.

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RORSMAN P, BERGGREN P, BOKVIST K, EFENDIC S. 1990. ATP-REGULATED K+ CHANNELS AND DIABETES-MELLITUS NEWS IN PHYSIOLOGICAL SCIENCES, 5 pp. 143-147.

Smith PA, Bokvist K, Arkhammar P, Berggren PO, Rorsman P. 1990. Delayed rectifying and calcium-activated K+ channels and their significance for action potential repolarization in mouse pancreatic beta-cells. J Gen Physiol, 95 (6), pp. 1041-1059. | Show Abstract | Read more

The contribution of Ca2(+)-activated and delayed rectifying K+ channels to the voltage-dependent outward current involved in spike repolarization in mouse pancreatic beta-cells (Rorsman, P., and G. Trube. 1986. J. Physiol. 374:531-550) was assessed using patch-clamp techniques. A Ca2(+)-dependent component could be identified by its rapid inactivation and sensitivity to the Ca2+ channel blocker Cd2+. This current showed the same voltage dependence as the voltage-activated (Cd2(+)-sensitive) Ca2+ current and contributed 10-20% to the total beta-cell delayed outward current. The single-channel events underlying the Ca2(+)-activated component were investigated in cell-attached patches. Increase of [Ca2+]i invariably induced a dramatic increase in the open state probability of a Ca2(+)-activated K+ channel. This channel had a single-channel conductance of 70 pS [( K+]o = 5.6 mM). The Ca2(+)-independent outward current (constituting greater than 80% of the total) reflected the activation of an 8 pS [( K+]o = 5.6 mM; [K+]i = 155 mM) K+ channel. This channel was the only type observed to be associated with action potentials in cell-attached patches. It is suggested that in mouse beta-cells spike repolarization results mainly from the opening of the 8-pS delayed rectifying K+ channel.

ASHCROFT F, FEWTRELL C, OOSAWA Y, RORSMAN P, SMITH P. 1990. ATP-REGULATED K CHANNELS MODULATE ELECTRICAL-ACTIVITY IN MOUSE ISOLATED PANCREATIC BETA-CELLS JOURNAL OF PHYSIOLOGY-LONDON, 424 pp. P27-P27.

Rorsman P, Berggren PO, Smith PA. 1990. Glucose in glucagon release. Nature, 344 (6268), pp. 716. | Read more

Betsholtz C, Baumann A, Kenna S, Ashcroft FM, Ashcroft SJ, Berggren PO, Grupe A, Pongs O, Rorsman P, Sandblom J. 1990. Expression of voltage-gated K+ channels in insulin-producing cells. Analysis by polymerase chain reaction. FEBS Lett, 263 (1), pp. 121-126. | Show Abstract | Read more

We have used the polymerase chain reaction (PCR) with primers against the S5 and S6 regions of voltage-gated K+ channels to identify 8 different specific amplification products using poly(A)+ RNA isolated from islets of Langerhans from obese hyperglycemic (ob/ob) mice and from the two insulin-producing cell lines HIT T15 and RINm5F. Sequence analysis suggests that they derive from mRNAs coding for a family of voltage-gated K+ channels; 5 of these have been recently identified in mammalian brain and 3 are novel. These hybridize in classes to different mRNAs which distribute differently to a number of tissues and cell lines including insulin-producing cells.

Bokvist K, Rorsman P, Smith PA. 1990. Block of ATP-regulated and Ca2(+)-activated K+ channels in mouse pancreatic beta-cells by external tetraethylammonium and quinine. J Physiol, 423 (1), pp. 327-342. | Show Abstract | Read more

1. The whole-cell and outside-out patch configurations of the patch-clamp technique were used to investigate the effects of extracellular tetraethylammonium ions (TEA+) and quinine on both Ca2(+)-activated and ATP-regulated K+ channels in mouse pancreatic beta-cells. 2. The Ca2(+)-activated K+ channel has a single-channel K+ permeability of 4.7 x 10(-13) cm3 s-1 when recorded with physiological ionic gradients. This value decreased to 2.9 x 10(-13) cm3 s-1 after addition of 0.3 mM-TEA+. 3. Two exponentials with time constants of 0.2 and 4.7 ms were required to describe the distribution of the channel openings suggesting that the Ca2(+)-activated K+ channel has at least two open states. The fast and slow components comprised 16 and 84% of the total number of openings respectively. 4. TEA+ caused a concentration-dependent decrease in the single-channel amplitude and open probability of the Ca2(+)-activated K+ channel. A Kd for the reduction in the mean current of 0.14 mM was observed. The stoichiometry was approximately 1:1. 5. Quinine blocked the Ca2(+)-activated K+ channel in a concentration-dependent manner. Half-maximal block was observed at 0.10 mM and binding was 1:1. Inhibition by 20 microM-quinine was not associated with a decrease in channel amplitude but markedly reduced the lifetime of the channel openings. Two exponentials, with time constants of 0.5 and 1.3 ms, were required to describe the channel openings. The rapid component contained 55% of the events. 6. TEA+ reduced the single-channel amplitude of the ATP-regulated K+ channel in a concentration-dependent manner. Kd for the block was 22 mM and the binding approximately 1:1. The block was not associated with changes in the open probability or channel kinetics. Two exponentials were required to describe the distribution of the open times. The time constants for the fast and slow components were approximately 2 and approximately 20 ms respectively. The rapid component accounted for approximately 35% of the events. 7. Quinine (10-20 microM) almost abolished activity of the ATP-regulated K+ channels. Inhibition was characterized by slow onset and reversibility but not associated with a change in the appearance of the single-channel events. Quinine-induced block could not be reversed by diazoxide. 8. We conclude that TEA+ produces rapid block of both Ca2(+)-activated and ATP-regulated K+ channels.(ABSTRACT TRUNCATED AT 400 WORDS)

Bokvist K, Rorsman P, Smith PA. 1990. Effects of external tetraethylammonium ions and quinine on delayed rectifying K+ channels in mouse pancreatic beta-cells. J Physiol, 423 (1), pp. 311-325. | Show Abstract | Read more

1. The whole-cell and outside-out patch configurations of the patch-clamp technique were used to study the mechanisms of block produced by external tetraethylammonium ions (TEA+) and quinine on delayed rectifying K+ channels in mouse pancreatic beta-cells. 2. In whole-cell recordings, TEA+ blocks the delayed outward current (which reflects the activity of delayed rectifying K+ channels by greater than 85%) in a concentration-dependent manner. The block appeared to be 1:1 with a Kd of approximately 1.4 mM at a membrane potential of 0 mV. The value of Kd varied with the membrane potential and there was an e-fold increase for a 70 mV depolarization. 3. Single-channel recordings revealed that delayed rectifying K+ channels have a unitary conductance of 8.5 pS ([K+]1 = 155 mM; [K+]o = 5.6 mM) and a single-channel K+ permeability of 2.8 X 10(-14) cm3 s-1. 4. First latency histograms of channel openings during voltage pulses from -70 to 0 mV peaked after 4 ms. A reaction scheme involving two closed states adequately but not perfectly described the distribution of the first latencies. The openings of the channels were grouped in bursts and the distribution of the closed times required two exponentials with time constants of 2.0 and 13 ms, respectively. The distribution of the open times could be described by a single exponential with a time constant of 25 ms. 5. Channel block produced by TEA+ (1 mM) was associated with a 40% decrease of the single-channel current amplitudes and a reduction in single-channel K+ permeability to 1.9 X 10(-14) cm3 s-1 but did not measurably affect the single-channel kinetics suggesting that the blocking reaction is very rapid. 6. Quinine blocked the whole-cell delayed outward current in a concentration-dependent manner. Half-maximal inhibition was attained at approximately 4 microM and the binding appeared to be 2:1. 7. Single-channel recordings indicated that the inhibition produced by quinine (10 microM) resulted from a decrease in the duration of the openings to a mean value of 6.7 ms. The time constants for the distribution of the closures were increased by approximately 30%. Quinine did not affect the amplitude of the openings. The rate constant of the blocking reaction (kB) was 15 mM-1 ms-1 at 0 mV.

Smith PA, Ashcroft FM, Rorsman P. 1990. Simultaneous recordings of glucose dependent electrical activity and ATP-regulated K(+)-currents in isolated mouse pancreatic beta-cells. FEBS Lett, 261 (1), pp. 187-190. | Show Abstract | Read more

Membrane potential and membrane currents were recorded from single mouse pancreatic beta-cells using the perforated patch whole-cell recording technique at 30 degrees C. Single beta-cells maintained in primary tissue culture exhibited glucose-dependent electrical activity similar to that reported for freshly isolated intact islets. The resting input conductance (5.1 +/- 0.9 nS) was determined by ATP-regulated K+ (KATP) channels as it was blocked by 1 mM tolbutamide. 8 mM glucose decreased the input conductance by 80%. The input conductance at -70 mV was of a similar value during the plateau phase and during the silent phase of electrical activity in 8 mM glucose. This suggests that oscillations of KATP channel activity do not underlie the slow waves.

Arkhammar P, Hallberg A, Kindmark H, Nilsson T, Rorsman P, Berggren PO. 1990. Extracellular ATP increases cytoplasmic free Ca2+ concentration in clonal insulin-producing RINm5F cells. A mechanism involving direct interaction with both release and refilling of the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool. Biochem J, 265 (1), pp. 203-211. | Show Abstract | Read more

Effects of extracellularly applied ATP (added as disodium salt) on stimulus-secretion coupling were investigated in clonal insulin-producing RINm5F cells. Cytoplasmic free Ca2+ concentration [( Ca2+]i), electrical activity, membrane potential, formation of InsP3 and insulin release were measured. Addition of ATP in a Ca2(+)-containing medium promoted a rapid rise in [Ca2+]i, which was followed by a slow decline towards the basal level. In a Ca2(+)-free medium, the ATP-induced increase in [Ca2+]i was smaller, but still enough to elicit insulin secretion. Upon normalization of the extracellular Ca2+ concentration, the response to ATP recovered instantaneously. The presence of glucose in the incubation medium was a prerequisite to obtain a pronounced effect of ATP in the absence of extracellular Ca2+. However, glucose did not enhance the response to ATP in a Ca2(+)-containing medium. The effect of ATP was dose-dependent, with a clearly detectable increase in [Ca2+]i at 1 microM and a maximal response being obtained at 200 microM-ATP. The response to ATP was unaffected by activating adenylate cyclase by forskolin, but was abolished by 10 nM of the phorbol ester phorbol 12-myristate 13-acetate. The effects of ATP on [Ca2+]i could not be accounted for by a generalized increase in plasma-membrane permeability, as evident from the failure of the nucleotide to increase the fluorescence of the nuclear stain ethidium bromide. After stimulation with ATP there was an increase in membrane potential, in both the absence and the presence of extracellular Ca2+. Blockage of the voltage-activated Ca2+ channals with D-600, in a Ca2(+)-containing medium, decreased the effect of ATP on [Ca2+]i slightly. Patch-clamp measurements using the cell-attached patch configuration revealed that the RINm5F cells produce spontaneous action potentials, the frequency of which increased markedly on addition of ATP. Whole-cell recordings demonstrated that the increase in spike frequency was not associated with the development of an inward current, but was rather accountable for by a decrease in the activity of the ATP-regulated K+ channels. Addition of 200 microM-ATP stimulated phospholipase C activity, as evident from the formation of InsP3, both in the absence and in the presence of extracellular Ca2+. Thus in the absence of extracellular Ca2+ the stimulatory effect of ATP on insulin release can be explained by InsP3-induced mobilization of intracellularly bound Ca2+. Hence, in the RINm5F cells extracellular ATP acts in a manner similar to other Ca2(+)-mobilizing agents.(ABSTRACT TRUNCATED AT 400 WORDS)

Smith PA, Rorsman P, Ashcroft FM. 1989. Modulation of dihydropyridine-sensitive Ca2+ channels by glucose metabolism in mouse pancreatic beta-cells. Nature, 342 (6249), pp. 550-553. | Show Abstract | Read more

Glucose stimulates insulin secretion from the pancreatic beta-cell by increasing the cytosolic calcium concentration. It is believed that this increment results mainly from Ca2+ influx through dihydropyridine-sensitive calcium channels because insulin secretion is abolished by dihydropyridine antagonists and is potentiated by dihydropyridine agonists. Glucose may influence Ca2+ influx through these channels in two ways: either by regulating the beta-cell membrane potential or by biochemical modulation of the channel itself. The former mechanism is well established. Glucose metabolism, by closing ATP-sensitive K+ channels, depolarizes the beta-cell membrane and initiates Ca2+-dependent electrical activity, with higher glucose concentrations further increasing Ca2+ influx by raising the frequency of action potentials. We show here that glucose metabolism also increases calcium influx directly, by modulating the activity of dihydropyridine-sensitive Ca2+ channels.

Rorsman P, Berggren PO, Bokvist K, Ericson H, Möhler H, Ostenson CG, Smith PA. 1989. Glucose-inhibition of glucagon secretion involves activation of GABAA-receptor chloride channels. Nature, 341 (6239), pp. 233-236. | Show Abstract | Read more

The endocrine part of the pancreas plays a central role in blood-glucose regulation. It is well established that an elevation of glucose concentration reduces secretion of the hyperglycaemia-associated hormone glucagon from pancreatic alpha 2 cells. The mechanisms involved, however, remain unknown. Electrophysiological studies have demonstrated that alpha 2 cells generate Ca2+-dependent action potentials. The frequency of these action potentials, which increases under conditions that stimulate glucagon release, is not affected by glucose or insulin. The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) is present in the endocrine part of the pancreas at concentrations comparable to those encountered in the central nervous system, and co-localizes with insulin in pancreatic beta cells. We now describe a mechanism whereby GABA, co-secreted with insulin from beta cells, may mediate part of the inhibitory action of glucose on glucagon secretion by activating GABAA-receptor Cl- channels in alpha 2 cells. These observations provide a model for feedback regulation of glucagon release, which may be of significance for the understanding of the hypersecretion of glucagon frequently associated with diabetes.

FINDLAY I, ASHCROFT F, KELLY R, RORSMAN P, PETERSEN O, TRUBE G. 1989. CALCIUM CURRENTS IN INSULIN-SECRETING BETA-CELLS ANNALS OF THE NEW YORK ACADEMY OF SCIENCES, 560 (1 Calcium Chann), pp. 403-409. | Read more

Rorsman P, Arkhammar P, Bokvist K, Hellerström C, Nilsson T, Welsh M, Welsh N, Berggren PO. 1989. Failure of glucose to elicit a normal secretory response in fetal pancreatic beta cells results from glucose insensitivity of the ATP-regulated K+ channels. Proc Natl Acad Sci U S A, 86 (12), pp. 4505-4509. | Show Abstract | Read more

Fetal pancreatic beta cells demonstrate a deficient insulin release in response to glucose, but the underlying mechanism at the cellular level is unknown. By using beta cells from 21-day fetal rats we made an attempt to clarify the mechanism(s) behind this reduced glucose response. In addition to measuring insulin release, glucose metabolism, and cellular ATP content, ATP-regulated K+ channels (G channels) and voltage-activated Ca2+ currents were investigated with the patch-clamp technique. It was thus demonstrated that the ATP-regulated K+ channels in fetal beta cells were not sensitive to glucose but otherwise had similar characteristics as those of adult beta cells. Also, the characteristics of the voltage-activated Ca2+ currents were similar in adult and fetal beta cells. However, as judged from measurements of both glucose oxidation and glucose utilization, glucose metabolism was impaired in fetal beta cells. In addition, there was no increase in the ATP content, even when the cells were stimulated for 30 min. It is therefore concluded that the attenuated glucose-induced insulin release in fetal pancreatic beta cells is due to an immature glucose metabolism resulting in impaired regulation of the ATP-sensitive K+ channels. These findings may be relevant to the understanding of the deficient stimulus-secretion coupling associated with non-insulin-dependent diabetes.

Ahrén B, Berggren PO, Bokvist K, Rorsman P. 1989. Does galanin inhibit insulin secretion by opening of the ATP-regulated K+ channel in the beta-cell? Peptides, 10 (2), pp. 453-457. | Show Abstract | Read more

The intrapancreatic neuropeptide galanin potently inhibits glucose-induced insulin secretion. This effect is in part due to a repolarization of the beta-cells and ensuring reduction in the cytoplasmic free Ca2+ concentration, [Ca2+]i. We propos that galanin inhibition of beta-cell action potentials is associated with the appearance of ATP-regulated K+ channels. Galanin opens K+ channels in a patch membrane when applied to the external solution in the cell-attached patch configuration. However, galanin does not detectably increase K+ permeability during whole-cell experiments, even when GTP was included in the internal solution. Our findings are not consistent with a direct effect of galanin on the K+ channels, but rather indicate that the effect of the neuropeptide is mediated by some intracellular coupling factor(s).

Smith PA, Bokvist K, Rorsman P. 1989. Demonstration of A-currents in pancreatic islet cells. Pflugers Arch, 413 (4), pp. 441-443. | Show Abstract | Read more

Voltage-activated K+ currents resistant to TEA but blockable by 4-AP were recorded from mouse pancreatic islet cells. These currents first become observable during depolarizations to voltages more positive than -40 mV, reaching a peak amplitude of 120 +/- 34 pA at +6 mV (n = 4), display rapid turn on (tau = 3.3 +/- 1.1 ms at +6 mV) and inactivate completely within 250 ms (tau = 65 +/- 5 at +6 mV). The current is subject to steady-state inactivation. The midpoint (Vh) of the inactivation curve (h infinity) was observed at -72 +/- 2 mV. The properties of this current resemble those reported for the A-current in neurons.

Nilsson T, Arkhammar P, Rorsman P, Berggren PO. 1989. Suppression of insulin release by galanin and somatostatin is mediated by a G-protein. An effect involving repolarization and reduction in cytoplasmic free Ca2+ concentration. J Biol Chem, 264 (2), pp. 973-980. | Show Abstract

The effects of galanin and somatostatin on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration [( Ca2+]i) were investigated using beta-cells isolated from obese hyperglycemic mice. Whereas insulin release was measured in a column perifusion system, membrane potential and [Ca2+]i were measured with the fluorescent indicators bisoxonol (bis-(1,3-diethylthiobarbiturate)trimethineoxonol) and quin 2, in cell suspensions in a cuvette. Galanin (16 nM) and somatostatin (400 nM) suppressed glucose-stimulated insulin release in parallel to promoting repolarization and a reduction in [Ca2+]i. The reduction in [Ca2+]i comprised an initial nadir followed by a slow rise and the establishment of a new steady state level. The slow rise in [Ca2+]i was abolished by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. Both peptides suppressed insulin release even when [Ca2+]i was raised by 25 mM K+. Under these conditions the inhibition of insulin release was partly reversed by an increase in the glucose concentration. Addition of 5 mM Ca2+ to a cell suspension, incubated in the presence of 20 mM glucose and either galanin, somatostatin, or the alpha 2-adrenergic agonist clonidine (10 nM), induced oscillations in [Ca2+]i, this effect disappearing subsequent to the addition of D-600. The effects of galanin, somatostatin, and clonidine on [Ca2+]i were abolished in beta-cells treated with pertussis toxin. In accordance with measurements of [Ca2+]i, treatment with pertussis toxin reversed the inhibitory effect of galanin on insulin release. The inhibitory action of galanin and somatostatin on insulin release is probably accounted for by not only a repolarization-induced reduction in [Ca2+]i and a decreased sensitivity of the secretory machinery to Ca2+, but also by a direct interaction with the exocytotic process. It is proposed that these effects are mediated by a pertussis toxin-sensitive GTP-binding protein.

Ashcroft FM, Rorsman P. 1989. Electrophysiology of the pancreatic beta-cell. Prog Biophys Mol Biol, 54 (2), pp. 87-143. | Read more

Ashcroft FM, Ashcroft SJ, Berggren PO, Betzholz C, Rorsman P, Trube G, Welsh M. 1988. Expression of K channels in Xenopus laevis oocytes injected with poly(A+) mRNA from the insulin-secreting beta-cell line, HIT T15. FEBS Lett, 239 (2), pp. 185-189. | Show Abstract | Read more

Two types of exogenous K channel were identified in Xenopus laevis oocytes injected with poly(A+) mRNA from the insulin-secreting cell line HIT T15. One of these was the ATP-regulated K channel (G channel) as evidenced by its conductance and inhibition by tolbutamide. The other resembled the Ca-activated K channel from beta-cells.

Rorsman P, Ashcroft FM, Trube G. 1988. Single Ca channel currents in mouse pancreatic B-cells. Pflugers Arch, 412 (6), pp. 597-603. | Show Abstract | Read more

Barium currents flowing through single Ca2+ channels were recorded from outside-out patches isolated from mouse pancreatic B-cells. Only one type of Ca2+ channels was observed. In 110 mM Ba2+, the single channel conductance was 24pS (at negative membrane potentials) and the current amplitude at 0 mV was -0.7 pA. Channel openings were activated by depolarisations more positive than -30 mV and showed little inactivation during 200 ms pulses. Open times were increased by BAY K 8644 an decreased by micromolar Cd2+. Channel activity was subject to rundown in excised patches and little activity remained after 10 min. These properties resemble those of L-type Ca2+ channels in other tissues. It is suggested that this Ca2+ channel participates in the generation of the B-cell action potential and mediates the increase in Ca2+ influx required for insulin secretion.

Flatt PR, Abrahamsson H, Arkhammar P, Berggren PO, Rorsman P, Swanston-Flatt SK. 1988. Measurements of membrane potential, transmembrane 45Ca fluxes, cytoplasmic free Ca2+ concentration and insulin release by transplantable rat insulinoma cells maintained in tissue culture. Br J Cancer, 58 (1), pp. 22-29. | Show Abstract | Read more

Regulation of insulin release, membrane potential, transmembrane 45Ca fluxes and cytoplasmic free Ca2+ concentration, [Ca2+]i, was examined using suspensions of transplantable NEDH rat insulinoma cells previously cultured for 2-3 days to eliminate necrotic tumour cells and counter prior hypoglycaemia. Insulinoma cells displayed a resting [Ca2+]i of 94 +/- 8 nM (n = 17) and released 104 +/- 15 ng insulin 10(-6) cells (n = 7) during 60 min incubations with uptake of 2.7 +/- 0.2 nmol 45Ca 10(-6) cells (n = 7). High concentrations of glucose did not affect membrane potential, transmembrane 45Ca fluxes, [Ca2+]i or insulin release by insulinoma cells. K+ at 25 mM depolarised the plasma membrane, induced a small increase in 45Ca efflux and increased [Ca2+]i by 65%. This modest action was not associated with demonstrable effects on 45Ca uptake and insulin release. The effect of 25 mMK+ on [Ca2+]i was counteracted by D-600, but this blocker of voltage-activated Ca2+ channels and verapamil lacked effects on transmembrane 45Ca fluxes and insulin release. The Ca2+-calmodulin antagonist, trifluoroperazine, was also without effect on 45Ca fluxes and insulin release. Ca2+ ionophore ionomycin increased [Ca2+]i, whereas A23187 and X537A did not affect transmembrane 45Ca fluxes. Moreover, insulin release was independent of extracellular Ca2+ over the range 0-20.4 mM despite marked affects on transmembrane 45Ca fluxes and a greater than 4-fold change of [Ca2+]i. Dibutyryl cyclic AMP increased insulin release by 55% without affecting transmembrane 45Ca fluxes or [Ca2+]i. The phosphodiesterase inhibitor, theophylline, also enhanced insulin release by 10-36% with no change of 45Ca uptake. The effectiveness of theophylline was independent of extracellular Ca2+ over the range 0-10.2 mM. These results indicate that inappropriate Ca2+ regulation is a key pathogenic feature underlying the inappropriate insulin secretion of rat insulinoma cells.

Ahrén B, Rorsman P, Berggren PO. 1988. Galanin and the endocrine pancreas. FEBS Lett, 229 (2), pp. 233-237. | Show Abstract | Read more

Galanin is a 29 amino acid peptide, initially isolated from the porcine small intestine. The peptide has been shown to occur in intrapancreatic nerves in close association to the islets. Its effects on islet hormone secretion and its possible mechanisms behind these effects are reviewed. Galanin has been shown to inhibit basal and stimulated insulin secretion both in vivo and in vitro under a variety of experimental conditions. The peptide has also been shown to inhibit somatostatin secretion and the secretion of pancreatic polypeptide (PP). With regard to glucagon secretion, however, results in the literature are not consistent since both stimulatory and inhibitory effects have been reported. A direct interaction with the pancreatic beta-cells has been proposed behind its inhibitory action on insulin secretion, since galanin inhibits insulin secretion from isolated beta-cells from obese, hyperglycaemic, mice. Galanin has thereby also been shown to induce repolarization and to reduce the free Ca2+ concentration, [Ca2+]i. The reduction in [Ca2+]i is probably not due to a direct interference with the voltage-activated Ca2+ channels, since there is no effect of galanin when these channels are opened by depolarization induced by high concentrations of K+. Instead, preliminary studies indicate that galanin activates the K+ channels that are regulated by ATP, in turn inducing a repolarization-induced reduction in [Ca2+]i resulting in reduced insulin secretion. However, the possibility that galanin inhibits the insulin secretory mechanism at a step distal to the regulation of cytoplasmic free Ca2+ concentration should not be overlooked.

Nilsson T, Arkhammar P, Rorsman P, Berggren PO. 1988. Inhibition of glucose-stimulated insulin release by alpha 2-adrenoceptor activation is parallelled by both a repolarization and a reduction in cytoplasmic free Ca2+ concentration. J Biol Chem, 263 (4), pp. 1855-1860. | Show Abstract

Effects of the alpha 2-adrenergic agonist clonidine on insulin release, membrane potential, and cytoplasmic free Ca2+ concentration ([Ca2+]i) were investigated using pancreatic beta-cells isolated from obese hyperglycemic mice. Addition of 2 microM clonidine promptly inhibited glucose-stimulated insulin release, an effect accompanied by a lowering in both membrane potential and [Ca2+]i. Within minutes, the effect on Ca2+ was partly reversed, [Ca2+]i attaining a new level, although still significantly lower than in the absence of agonist. This late increase in [Ca2+]i was inhibited by 50 microM D-600, a blocker of voltage-activated Ca2+ channels. The inhibitory effects of clonidine on membrane potential, [Ca2+]i, and insulin release were abolished by 5 microM of the alpha 2-adrenergic antagonist yohimbine. Depolarization with high K+ increased [Ca2+]i also in the presence of clonidine, conditions accompanied by only a minute release of insulin. Secretion was, however, partly restored by subsequent addition of 20 mM glucose. Addition of 5 mM Ca2+ transiently reversed the effects of clonidine on both membrane potential and [Ca2+]i. Although the clonidine-induced repolarization should be enough for closing the voltage-activated Ca2+ channels with a resulting decrease in [Ca2+]i, a direct interaction of the agonist with these channels cannot be excluded. The fact that it was possible to increase [Ca2+]i with only a minor effect on insulin release suggests that the inhibitory effect of clonidine not only is due to a reduction in [Ca2+]i, but also involves interference with some more distal step in the insulin secretory machinery.

Rorsman P. 1988. Two types of Ca2+ currents with different sensitivities to organic Ca2+ channel antagonists in guinea pig pancreatic alpha 2 cells. J Gen Physiol, 91 (2), pp. 243-254. | Show Abstract | Read more

The possibility that guinea pig pancreatic alpha 2 cells are equipped with more than one type of Ca2+ channel was explored using the patch-electrode voltage-clamp technique. At a holding potential of -100 mV, a slowly developing (tau m approximately 5 ms at -40 mV assuming m2 kinetics) Ca2+ current appeared. This conductance first became detectable at potentials of about -60 mV and reached a maximum amplitude of 50-100 pA between -30 and -20 mV. During long depolarizations, it inactivated completely (tau h approximately 100 ms at -40 mV). Half-maximal steady state inactivation was observed at about -60 mV. A second, more rapidly developing (tau m approximately 2 ms at 0 mV) Ca2+ current was observed during pulses to -40 mV and above. It had a peak amplitude of 150-200 pA between 0 and 10 mV, was less dependent on the holding potential, and inactivated very little, even during long pulses. Both conductances were blocked by Co2+ but were unaffected by tetrodotoxin. The rapidly developing current differed from the slowly developing one in being sensitive to the antagonists D-600 and nifedipine, conducting Ba2+ better than Ca2+, increasing upon exposure to forskolin, and showing time-dependent decay (rundown). These findings indicate that the alpha 2 cells are equipped with two kinds of Ca2+ channels.

Rorsman P, Hellman B. 1988. Voltage-activated currents in guinea pig pancreatic alpha 2 cells. Evidence for Ca2+-dependent action potentials. J Gen Physiol, 91 (2), pp. 223-242. | Show Abstract | Read more

Glucagon-secreting alpha 2 cells were isolated from guinea pig pancreatic islets and used for electrophysiological studies of voltage-activated ionic conductances using the patch-clamp technique. The alpha 2 cells differed from beta cells in producing action potentials in the absence of glucose. The frequency of these potentials increased after addition of 10 mM arginine but remained unaffected in the presence of 5-20 mM glucose. When studying the conductances underlying the action potentials, we identified a delayed rectifying K+ current, an Na+ current, and a Ca2+ current. The K+ current activated above -20 mV and then increased with the applied voltage. The Na+ current developed at potentials above -50 mV and reached a maximal peak amplitude of 550 pA during depolarizing pulses to -15 mV. The Na+ current inactivated rapidly (tau h approximately 0.7 ms at 0 mV). Half-maximal steady state inactivation was attained at -58 mV, and currents could no longer be elicited after conditioning pulses to potentials above -40 mV. The Ca2+ current first became detectable at -50 mV and reached a maximal amplitude of 90 pA (in extracellular [Ca2+] = 2.6 mM) at about -10 mV. Unlike the Na+ current, it inactivated little or not at all. Membrane potential measurements demonstrated that both the Ca2+ and Na+ currents contribute to the generation of the action potential. Whereas there was an absolute requirement of extracellular Ca2+ for action potentials to be elicited at all, suppression of the much larger Na+ current only reduced the upstroke velocity of the spikes. It is suggested that this behavior reflects the participation of a low-threshold Ca2+ conductance in the pacemaking of alpha 2 cells.

Arkhammar P, Nilsson T, Rorsman P, Berggren PO. 1987. Inhibition of ATP-regulated K+ channels precedes depolarization-induced increase in cytoplasmic free Ca2+ concentration in pancreatic beta-cells. J Biol Chem, 262 (12), pp. 5448-5454. | Show Abstract

The effects of glucose, diazoxide, K+, and tolbutamide on the activity of K+ channels, membrane potential, and cytoplasmic free Ca2+ concentration were investigated in beta-cells from the Uppsala colony of obese hyperglycemic mice. With [K+]e = [K+]i = 146 mM, it was demonstrated that the dominating channel at the resting potential is a K+ channel with a single-channel conductance of about 65 picosiemens and a reversal potential of about +70 mV (pipette potential). This channel is characterized by complex kinetics with openings grouped in bursts. The channel was completely inhibited by 20 mM glucose in intact cells or by intracellularly applied Mg-ATP (1 mM). The number of active channels was markedly reduced already by 5 mM glucose. However, the single channel current of the channels remaining active was unaffected, indicating no major depolarization. To evoke a substantial depolarization of the membrane and thereby action potentials, a total block in channel activity was necessary. This could be achieved either by increasing the concentration of glucose to 20 mM or by combining 5 mM glucose with 100 microM tolbutamide. In both cases, the effect was counteracted by the hyperglycemic sulfonamide diazoxide. The effects on single channel activity were paralleled by changes in membrane potential and cytoplasmic free Ca2+ concentration, also when the latter measurements were performed at room temperature. The transient increase in the number of active channels and the resulting hyperpolarization observed after raising the glucose concentration to 20 mM probably reflected a drop in cytoplasmic ATP concentration. It is suggested that ATP works as a key regulator of the beta-cell membrane potential and thereby the opening of voltage-activated Ca2+ channels.

Rorsman P, Arkhammar P, Berggren PO. 1986. Voltage-activated Na+ currents and their suppression by phorbol ester in clonal insulin-producing RINm5F cells. Am J Physiol, 251 (6 Pt 1), pp. C912-C919. | Show Abstract

The whole-cell configuration of the patch-clamp technique was applied on the clonal insulin-producing cell line RINm5). Thus attempts were made to characterize voltage-activated inward and outward membrane currents and to examine to what extent these were affected by both long-term and acute exposure to the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). Current responses to voltage-clamp steps up to -40 mV were small. A pulse to -28 mV evoked an inward current, and slowly activating outward currents developed at potentials above -20 mV. The inward current had a V-shaped current-voltage relationship, reaching a peak between -10 and 0 mV, whereas the outward current increased linearly at potentials beyond -20 mV. It was demonstrated that the inward currents are carried primarily by Ca2+ and Na+ and the outward current by K+. After long-term exposure to TPA, there was a suppression of Na+ currents in one-third of the cells, whereas the Ca2+ and K+ currents were unaffected. Acute exposure to the phorbol ester increased the Ca2+ currents with little effect on the Na+ currents. The extent to which the differences in effects on membrane currents initiated by respective acute and long-term exposure to TPA may reflect two separate mechanisms of protein kinase C activation, the latter related to regulation of differentiation of the RINm5F cells, merits further investigation.

Trube G, Rorsman P, Ohno-Shosaku T. 1986. Opposite effects of tolbutamide and diazoxide on the ATP-dependent K+ channel in mouse pancreatic beta-cells. Pflugers Arch, 407 (5), pp. 493-499. | Show Abstract | Read more

The influence of the antidiabetic sulphonylurea tolbutamide on K+ channels of mouse pancreatic beta-cells was investigated using different configurations of the patch clamp technique. The dominant channel in resting cells is a K+ channel with a single-channel conductance of 60 pS that is inhibited by intracellular ATP or, in intact cells, by stimulation with glucose. In isolated patches of beta-cells membrane, this channel was blocked by tolbutamide (0.1 mM) when applied to either the intracellular or extracellular side of the membrane. The dose-dependence of the tolbutamide-induced block was obtained from whole-cell experiments and revealed that 50% inhibition was attained at approximately 7 microM. In cell-attached patches low concentrations of glucose augmented the action of tolbutamide. Thus, the simultaneous presence of 5 mM glucose and 0.1 mM tolbutamide abolished channel activity and induced action potentials. These were not produced when either of these substances was added alone at these concentrations. The inhibitory action of tolbutamide or glucose on the K+ channel was counteracted by the hyperglycaemic sulphonamide diazoxide (0.4 mM). Tolbutamide (1 mM) did not affect Ca2+-dependent K+ channels. It is concluded that the hypo- and hyperglycaemic properties of tolbutamide and diazoxide reflect their ability to induce the closure or opening, respectively, of ATP-regulated K+ channels.

Rorsman P, Trube G. 1986. Calcium and delayed potassium currents in mouse pancreatic beta-cells under voltage-clamp conditions. J Physiol, 374 (1), pp. 531-550. | Show Abstract | Read more

Pancreatic islets of NMRI mice were dissociated into single cells which were kept in tissue culture for 1-3 days. The whole-cell configuration of the patch-clamp technique was used to study inward and delayed outward currents of beta-cells under voltage-clamp conditions at 20-22 degrees C. Outward currents were suppressed by substituting the impermeant cation N-methyl-D-glucamine for intracellular K+. The remaining inward current had a V-shaped current-voltage relation reaching a peak value of 39 +/- 4 pA (mean +/- S.E. of mean) around -15 mV. It was identified as a Ca2+ current, because the peak amplitude was increased 1.6 times by increasing external [Ca2+] ([Ca2+]o) from 2.6 mM to 10 mM and it was blocked by Co2+ (5 mM) or nifedipine (5 microM) but not by TTX (20 microM). The activation time constant of the inward current at -10 mV was 1.28 +/- 0.08 ms. The relation between the degree of activation (estimated from the size of the tail currents) and membrane potential V followed the sigmoidal function f = 1/(1 + exp [(Vh-V)/k]) with half-maximal activation potential, Vh = 4 +/- 1 mV and slope factor, k = 14 +/- 1 mV (for [Ca2+]o 10 mM). The inward current inactivated only weakly during depolarizing pulses of 0.1-1 s duration. The delayed outward current (in experiments with 155 mM-internal [K+] ([K+]i)) had a linear voltage dependence at potentials above -20 mV; its amplitude at -10 mV was 210 +/- 30 pA. Tail currents related to the activation of the outward current had K+-dependent reversal potentials. The current was blocked by extracellularly applied tetraethylammonium (20 mM) and 4-aminopyridine (2 mM). It was not affected by glibenclamide (3 microM), tolbutamide (0.2 mM) and alterations of intracellular [Ca2+] (1 nM-1 microM). The activation time constant of the outward current at -10 mV was 21 +/- 3 ms. The voltage dependence of activation could be described by the sigmoidal function (see above) with Vh = 19 +/- 1 mV and k = 5.6 +/- 0.4 mV. The outward current inactivated during long (15 s) depolarizing pre-pulses (time constant at -10 mV: 2.6 +/- 0.6 s). 50% inactivation occurred at Vh = -36 +/- 2 mV, k was -4.1 +/- 0.2 mV. Inward and outward currents during depolarizing voltage pulses in beta-cells are similar to Ca2+ and delayed K+ currents in other cell types. These currents seem sufficient to generate the action potentials of the beta-cell.

Arkhammar P, Berggren PO, Rorsman P. 1986. Direct evidence for opposite effects of D-glucose and D-glyceraldehyde on cytoplasmic pH of mouse pancreatic beta-cells. Biosci Rep, 6 (4), pp. 355-361. | Show Abstract | Read more

The effects of D-glucose, D-glyceraldehyde, glibenclamide, D-600, NH4+ and high concentrations of K+ on cytoplasmic pH (pHi) were investigated in dispersed and cultured pancreatic beta-cells from ob/ob mice. The cytoplasmic pH was measured with the fluorescent H+-indicator quene 1. The average pHi value in resting beta-cells was 6.71. Addition of 20 mM of the physiological stimulus D-glucose increased pHi with 0.05 units. Both glibenclamide and high concentrations of K+ decreased pHi. The latter effects were completely reversed by D-600, supporting the notion that free cytoplasmic Ca2+ can be involved in the regulation of pHi. In contrast to D-glucose, 10 mM of D-glyceraldehyde decreased pHi by 0.09 units, an effect persisting even in the presence of D-600. From the present study it is evident that D-glyceraldehyde and D-glucose have opposite effects on pHi in pancreatic beta-cells.

Trube G, Rorsman P. 1986. Calcium and potassium currents recorded from pancreatic beta-cells under voltage clamp control. Adv Exp Med Biol, 211 pp. 167-175.

Rorsman P, Trube G. 1985. Glucose dependent K+-channels in pancreatic beta-cells are regulated by intracellular ATP. Pflugers Arch, 405 (4), pp. 305-309. | Show Abstract | Read more

The resting conductance of cultured beta-cells from murine pancreases was investigated using the whole-cell, cell-attached and isolated patch modes of the patch-clamp technique. Whole-cell experiments revealed a high input resistance of the cells (greater than 20 G omega per cell or greater than 100 k omega X cm2), if the medium dialysing the cell interior contained 3 mM ATP. The absence of ATP evoked a large additional K+ conductance. In cell-attached patches single K+-channels were observed in the absence of glucose. Addition of glucose (20 mM) to the bath suppressed the channel activity and initiated action potentials. Similar single-channel currents were recorded from isolated patches. In this case the channels were reversibly blocked by adding ATP (3 mM) to the solution at the intracellular side of the membrane. The conductances (51 pS and 56 pS for [K+]0 = 145 mM, T = 21 degrees C) and kinetics (at -70 mV: tau open = 2.2 ms and 1.8 ms, tau closed = 0.38 ms and 0.33 ms) of the glucose- and ATP-dependent channels were found to be very similar. It is concluded that both channels are identical. The result suggests that glucose could depolarize the beta-cell by increasing the cytoplasmic concentration of ATP.

Rorsman P, Abrahamsson H. 1985. Cyclic AMP potentiates glucose-induced insulin release from mouse pancreatic islets without increasing cytosolic free Ca2+. Acta Physiol Scand, 125 (4), pp. 639-647. | Show Abstract | Read more

The effects of various stimulants of insulin release on cytosolic free Ca2+, [Ca2+]i, in dispersed and cultured pancreatic beta-cells from ob/ob-mice were studied using the indicator quin-2, which in itself has only slight effects on the glucose-induced insulin release and the metabolism of the sugar. The resting [Ca2+]i was 158 +/- 7 nM. After increasing glucose to 20 mM there was a lag-period of 1-2 min before [Ca2+]i gradually rose, reaching a new plateau 60% higher after 5-6 min. Increasing intracellular cyclic AMP by adding forskolin did not further increase [Ca2+]i; on the contrary there was a slight temporary reduction despite a doubling of insulin secretion. The maintenance of the beta-cell function was evident from a marked increase of cytosolic [Ca2+]i after depolarization evoked by high extracellular K+. Also dibutyryl cyclic AMP and theophylline lacked the ability to raise [Ca2+]i beyond that obtained by glucose. The results suggest that cyclic AMP potentiates glucose-induced insulin release by sensitizing the secretory machinery to changes of [Ca2+]i rather than by increasing the cytosolic concentration of the ion.

Abrahamsson H, Berggren PO, Rorsman P. 1985. Direct measurements of increased free cytoplasmic Ca2+ in mouse pancreatic beta-cells following stimulation by hypoglycemic sulfonylureas. FEBS Lett, 190 (1), pp. 21-24. | Show Abstract | Read more

The effects of the hypoglycemic sulfonylureas tolbutamide and glibenclamide on free cytoplasmic Ca2+, [Ca2+]i, were compared with that of a depolarizing concentration of K+ in dispersed and cultured pancreatic beta-cells from ob/ob mice. [Ca2+]i was measured with the fluorescent Ca2+-indicator quin2. The basal level corresponded to 150 nM and increased to 600 nM after exposure to 30.9 mM K+. The corresponding levels after stimulation with 1 microM glibenclamide and 100 microM tolbutamide were 390 and 270 nM respectively. K+ depolarization increased [Ca2+]i more rapidly than either of the sulfonylureas. It is suggested that the increased [Ca2+]i obtained after stimulation by sulfonylureas is due to depolarization of the beta-cells with subsequent entry of Ca2+ through voltage-dependent channels.

Larsson R, Wallfelt C, Abrahamsson H, Gylfe E, Ljunghall S, Rastad J, Rorsman P, Wide L, Akerström G. 1984. Defective regulation of the cytosolic Ca2+ activity in parathyroid cells from patients with hyperparathyroidism. Biosci Rep, 4 (11), pp. 909-915. | Show Abstract | Read more

The parathyroid hormone (PTH) release and cytosolic Ca2+ activity were determined in normal bovine parathyroid cells and parathyroid cells obtained from patients with hyperparathyroidism (HPT). There was a sigmoid relation between the cytosolic Ca2+ activity and the extracellular calcium concentration between 0.5 and 6.0 mmol/l. The PTH release was inhibited in parallel with the rise in the cytosolic Ca2+ activity. Both the hormone release and the cytosolic Ca2+ activity were lower in cells from human adenomas and hyperplastic glands, and in comparison with the bovine preparations these cells had higher set points for the cytosolic Ca2+ activity and PTH release. There was a close correlation between the individual set points for the cytosolic Ca2+ activity and PTH release in a material containing both normal and pathological cells. The results indicate that the abnormal PTH release characteristic of HPT is due to a defective regulation of the cytosolic Ca2+ activity.

Rorsman P, Abrahamsson H, Gylfe E, Hellman B. 1984. Dual effects of glucose on the cytosolic Ca2+ activity of mouse pancreatic beta-cells. FEBS Lett, 170 (1), pp. 196-200. | Show Abstract | Read more

The cytosolic Ca2+ activity of mouse pancreatic beta-cells was studied with the intracellular fluorescent indicator quin2 . When the extracellular Ca2+ concentration was 1.20 mM, the basal cytosolic Ca2+ activity was 162 +/- 9 nM. Stimulation with 20 mM glucose increased this Ca2+ activity by 40%. In the presence of only 0.20 mM Ca2+ or after the addition of the voltage-dependent Ca2+ -channel blocker D-600, glucose had an opposite and more prompt effect in reducing cytosolic Ca2+ by about 15%. It is concluded that an early result of glucose exposure is a lowering of the cytosolic Ca2+ activity and that this effect tends to be masked by a subsequent increase of the Ca2+ activity due to influx of Ca2+ through the voltage-dependent Ca2+ channels.

Rorsman P, Hellman B. 1983. The interaction between manganese and calcium fluxes in pancreatic beta-cells. Biochem J, 210 (2), pp. 307-314. | Show Abstract | Read more

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets isolated from ob/ob mice. The efflux from preloaded islets was estimated from the amounts remaining after 30 min of subsequent test incubations in the absence of Mn2+. An increase in the extracellular Mg2+ concentration promoted the Mn2+ efflux and removal of Na+ from a Ca2+-deficient medium had the opposite effect. Addition of 25 mM-K+ failed to affect Mn2+ outflow as did 3-isobutyl-1-methylxanthine and dibutyryl cyclic AMP. Whereas tolbutamide caused retention of manganese, the ionophore Br-X537A promoted an efflux. D-Glucose was equally potent in retaining the islet manganese when the external Ca2+ concentration ranged from 15 microM to 6.30 mM. Subcellular-fractionation experiments indicated a glucose-stimulated incorporation of manganese into all fractions except the microsomes. The effect was most pronounced in the mitochondrial fraction, being as high as 164%. The glucose-induced uptake of intracellular 45Ca was abolished in the presence of 0.25 mM-Mn2+. When added to medium containing 2.5 mM-Mn2+, glucose even tended to decrease 45Ca2+ uptake. The inhibitory effect of Mn2+ was apparent also from a diminished uptake of 45Ca into all subcellular fractions. The efflux of 45Ca2+ was markedly influenced by Mn2+ as manifested in a prominent stimulation followed by inhibition. In addition to demonstrating marked interactions between fluxes of Mn2+ and Ca2+, the present studies support the view that the glucose inhibition of the efflux of bivalent cations from pancreatic beta-cells is accounted for by their accumulation in the mitochondria.

Rorsman P, Berggren PO, Gylfe E, Hellman B. 1983. Reduction of the cytosolic calcium activity in clonal insulin-releasing cells exposed to glucose. Biosci Rep, 3 (10), pp. 939-946. | Show Abstract | Read more

The cytosolic Ca2+ activity of insulin-releasing clonal cells (RINm5F) was studied with the intracellular fluorescent indicator quin-2. When the extracellular Ca2+ concentration was 1 mM, the basal cytosolic Ca2+ activity was 101 +/- 5 nM. Depolarization with 25 mM K+ increased this Ca2+ activity to at least 318 nM, an effect completely reversed by the voltage-dependent channel blocker D-600. In the presence of K+ alone these channels appeared to have a half-life of 6.7 +/- 0.8 min. In contrast to the action of K+, exposure of the RINm5F cells to 4 mM glucose resulted in a reduction of the cytosolic Ca2+ activity. This effect was observed during K+ depolarization but was more pronounced under basal conditions when it amounted to 20%. The data provide the first direct evidence that glucose can decrease the cytosolic Ca2+ activity in beta-cells. Unlike the case in normal beta-cells the glucose effect on the voltage-dependent Ca2+ channels in the RINm5F cells is apparently not sufficient to overcome the intracellular buffering of Ca2+. A defective depolarization is therefore a probable cause of the failing insulin secretion of RINm5F cells exposed to glucose.

Gylfe E, Andersson T, Rorsman P, Abrahamsson H, Arkhammar P, Hellman P, Hellman B, Oie HK, Gazdar AF. 1983. Depolarization-independent net uptake of calcium into clonal insulin-releasing cells exposed to glucose. Biosci Rep, 3 (10), pp. 927-937. | Show Abstract | Read more

Insulin release, net fluxes of Ca2+, and glucose metabolism were studied in a clonal cell line (RINm5F) established from a transplantable rat islet tumor. The insulin content amounted to only 0.03% of that of the total protein and decreased even further with subsequent passages. The insulin secretion was as high as 10 to 20% of the total hormone content per hour. Insulin release was stimulated by K+ depolarization but not by exposure to glucose. In contrast to this secretory pattern, glucose but not K+ stimulated the net uptake of Ca2+ at micromolar concentrations of the ion. The glucose effect was not mimicked by 20 mM 3-O-methylglucose. It was as pronounced at 1 mM as at 20 mM of the sugar and corresponded to an uptake of 119 fmol cm-2 s-1. Glucose metabolism was typical for tumor cells with a high glycolytic flux and an oxidation-to-utilization ratio as low as 0.05-0.15. Maximal oxidative degradation was attained already at 1 mM. This concentration was also equivalent to the Km for glucose utilization, indicating a substantial left-hand shift of the normal dose-response curve. It is suggested that glucose induces a depolarization-independent net uptake of Ca2+ by favouring intracellular buffering of the cation.

Rorsman P, Berggren PO, Hellman B. 1982. Manganese accumulation in pancreatic beta-cells and its stimulation by glucose. Biochem J, 202 (2), pp. 435-444. | Show Abstract | Read more

Electrothermal atomic-absorption spectroscopy was employed for measuring manganese in beta-cell-rich pancreatic islets microdissected from ob/ob mice. The islet content of endogenous manganese was 80 mumol/kg dry wt., which is about half as much as found in the exocrine pancreas. The initial uptake was characterized by two components, with approximate Km values of 35 microM and 3.7 microM respectively. After 60 min of incubation with 0.25 mM-Mn2+, the intracellular concentration of manganese corresponded to an almost 25-fold accumulation compared with that of the extracellular medium. When exposed to 20 mM-D-glucose, the islets retained more manganese, owing to suppression of its mobilization. The glucose inhibition of efflux was prompt and reversible, as indicated from direct recordings of manganese in a perifusion medium. D-Glucose was an equally potent inhibitor of efflux in the presence of 15 microM- and 1.28 mM-Ca2+. The inhibitory action disappeared when metabolism was suppressed by adding 0.1 mM-N-ethylmaleimide or by lowering the temperature from 37 degrees C to 2 degrees C. At a concentration of 0.25 mM, Mn2+ abolished the insulin-releasing action of D-glucose, exerting only moderate suppression of its metabolism. The addition of Mn2+ resulted in inhibition of basal insulin release in the presence of 1.28 mM-Ca2+, but not in a Ca2+-deficient medium. The studies indicate that the previously observed phenomenon of glucose inhibition of 45Ca efflux has a counterpart in the suppression of manganese mobilization from the pancreatic islets. With the demonstration of a pronounced glucose inhibition of manganese efflux, it is evident that Mn2+ may represent a useful tool for exploring the mechanism of glucose-induced retention of calcium in the pancreatic beta-cells.

RORSMAN P, BERGGREN P. 1982. DIRECT DETERMINATION OF MANGANESE IN MICROGRAM AMOUNTS OF PANCREATIC TISSUE BY ELECTROTHERMAL ATOMIC-ABSORPTION SPECTROMETRY ANALYTICA CHIMICA ACTA, 140 (1), pp. 325-329. | Show Abstract | Read more

Manganese is determined by the direct insertion of freeze-dried biological samples (1-15 μg) or by injection of 2-μl samples of perifusion medium into the graphite furnace. At the most sensitive wavelength (279.5 nm), down to 0.2 pmol of manganese can be measured in the perifusion medium as well as the endogenous manganese in the endocrine and exocrine parts of the pancreas. The latter values were 0.08 ± 0.01 and 0.16 ± 0.01 mmol Mn kg-1 (dry wt.), respectively. A less sensitive wavelength (403.1 nm) is employed for measuring the larger amounts obtained after incubating the specimens in the presence of manganese(II). © 1982.

Rorsman P. 1982. Glucose promotes intracellular sequestration of manganese in pancreatic beta-cells. Acta Biol Med Ger, 41 (12), pp. 1221-1224. | Show Abstract

D-glucose reduces the outflow of manganese from pancreatic beta-cells in analogy to what has previously been reported for 45Ca. The decrease can be accounted for by an enhanced intracellular sequestration. The effect of D-glucose on manganese fluxes seems to be restricted to inhibition of the efflux; no stimulation of the influx being noted. The glucose-dependence of manganese uptake is hyperbolic with half maximal stimulation between 5 and 6 mM D-glucose.

Hellman B, Andersson T, Berggren PO, Rorsman P. 1980. Calcium and pancreatic beta-cell function. XI. Modification of 45Ca fluxes by Na+ removal. Biochem Med, 24 (2), pp. 143-152. | Show Abstract | Read more

Pancreatic islets were isolated from noninbred ob/ob mice and used for evaluating how removal of extracellular Na+ affects 45Ca fluxes in the insulin-producing β cells. Isoosmotic replacement of Na+ with Li+ was less efficient in stimulating the incorporation of intracellular (La3+ nondisplaceable) 45Ca than substitution with choline+ or sucrose. Ample support for the significance of Na+-Ca2+ exchange for the net efflux of Ca2+ was obtained with the observation that Na+ deprivation markedly inhibited the 45Ca washout in a Ca2+-deficient medium. The inhibitory action of Na+ deficiency exceeded that obtained with 20 mM glucose. The 45Ca incorporated after removal of Na+ differed from that taken in response to glucose in not being exchanged with the Ca2+ entering the β cells during glucose stimulation and in appearing also in the postmicrosomal supernatant obtained by differential centrifugation.

Ramracheya RD, McCulloch LJ, Clark A, Wiggins D, Johannessen H, Olsen MK, Cai X, Zhao CM, Chen D, Rorsman P. 2016. PYY-Dependent Restoration of Impaired Insulin and Glucagon Secretion in Type 2 Diabetes following Roux-En-Y Gastric Bypass Surgery. Cell Rep, 15 (5), pp. 944-950. | Show Abstract | Read more

Roux-en-Y gastric bypass (RYGB) is a weight-reduction procedure resulting in rapid resolution of type 2 diabetes (T2D). The role of pancreatic islet function in this restoration of normoglycemia has not been fully elucidated. Using the diabetic Goto-Kakizaki (GK) rat model, we demonstrate that RYGB restores normal glucose regulation of glucagon and insulin secretion and normalizes islet morphology. Culture of isolated islets with serum from RYGB animals mimicked these effects, implicating a humoral factor. These latter effects were reversed following neutralization of the gut hormone peptide tyrosine tyrosine (PYY) but persisted in the presence of a glucagon-like peptide-1 (GLP-1) receptor antagonist. The effects of RYGB on secretion were replicated by chronic exposure of diabetic rat islets to PYY in vitro. These findings indicate that the mechanism underlying T2D remission may be mediated by PYY and suggest that drugs promoting PYY release or action may restore pancreatic islet function in T2D.

Shigeto M, Ramracheya R, Tarasov AI, Cha CY, Chibalina MV, Hastoy B, Philippaert K, Reinbothe T et al. 2015. GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation. J Clin Invest, 125 (12), pp. 4714-4728. | Show Abstract | Read more

Strategies aimed at mimicking or enhancing the action of the incretin hormone glucagon-like peptide 1 (GLP-1) therapeutically improve glucose-stimulated insulin secretion (GSIS); however, it is not clear whether GLP-1 directly drives insulin secretion in pancreatic islets. Here, we examined the mechanisms by which GLP-1 stimulates insulin secretion in mouse and human islets. We found that GLP-1 enhances GSIS at a half-maximal effective concentration of 0.4 pM. Moreover, we determined that GLP-1 activates PLC, which increases submembrane diacylglycerol and thereby activates PKC, resulting in membrane depolarization and increased action potential firing and subsequent stimulation of insulin secretion. The depolarizing effect of GLP-1 on electrical activity was mimicked by the PKC activator PMA, occurred without activation of PKA, and persisted in the presence of PKA inhibitors, the KATP channel blocker tolbutamide, and the L-type Ca(2+) channel blocker isradipine; however, depolarization was abolished by lowering extracellular Na(+). The PKC-dependent effect of GLP-1 on membrane potential and electrical activity was mediated by activation of Na(+)-permeable TRPM4 and TRPM5 channels by mobilization of intracellular Ca(2+) from thapsigargin-sensitive Ca(2+) stores. Concordantly, GLP-1 effects were negligible in Trpm4 or Trpm5 KO islets. These data provide important insight into the therapeutic action of GLP-1 and suggest that circulating levels of this hormone directly stimulate insulin secretion by β cells.

Collins SC, Do HW, Hastoy B, Hugill A, Adam J, Chibalina MV, Galvanovskis J, Godazgar M et al. 2016. Increased Expression of the Diabetes Gene SOX4 Reduces Insulin Secretion by Impaired Fusion Pore Expansion. Diabetes, 65 (7), pp. 1952-1961. | Show Abstract | Read more

The transcription factor Sox4 has been proposed to underlie the increased type 2 diabetes risk linked to an intronic single nucleotide polymorphism in CDKAL1 In a mouse model expressing a mutant form of Sox4, glucose-induced insulin secretion is reduced by 40% despite normal intracellular Ca(2+) signaling and depolarization-evoked exocytosis. This paradox is explained by a fourfold increase in kiss-and-run exocytosis (as determined by single-granule exocytosis measurements) in which the fusion pore connecting the granule lumen to the exterior expands to a diameter of only 2 nm, which does not allow the exit of insulin. Microarray analysis indicated that this correlated with an increased expression of the exocytosis-regulating protein Stxbp6. In a large collection of human islet preparations (n = 63), STXBP6 expression and glucose-induced insulin secretion correlated positively and negatively with SOX4 expression, respectively. Overexpression of SOX4 in the human insulin-secreting cell EndoC-βH2 interfered with granule emptying and inhibited hormone release, the latter effect reversed by silencing STXBP6 These data suggest that increased SOX4 expression inhibits insulin secretion and increased diabetes risk by the upregulation of STXBP6 and an increase in kiss-and-run exocytosis at the expense of full fusion. We propose that pharmacological interventions promoting fusion pore expansion may be effective in diabetes therapy.

Zhang Q, Ramracheya R, Lahmann C, Tarasov A, Bengtsson M, Braha O, Braun M, Brereton M et al. 2013. Role of KATP channels in glucose-regulated glucagon secretion and impaired counterregulation in type 2 diabetes. Cell Metab, 18 (6), pp. 871-882. | Show Abstract | Read more

Glucagon, secreted by pancreatic islet α cells, is the principal hyperglycemic hormone. In diabetes, glucagon secretion is not suppressed at high glucose, exacerbating the consequences of insufficient insulin secretion, and is inadequate at low glucose, potentially leading to fatal hypoglycemia. The causal mechanisms remain unknown. Here we show that α cell KATP-channel activity is very low under hypoglycemic conditions and that hyperglycemia, via elevated intracellular ATP/ADP, leads to complete inhibition. This produces membrane depolarization and voltage-dependent inactivation of the Na(+) channels involved in action potential firing that, via reduced action potential height and Ca(2+) entry, suppresses glucagon secretion. Maneuvers that increase KATP channel activity, such as metabolic inhibition, mimic the glucagon secretory defects associated with diabetes. Low concentrations of the KATP channel blocker tolbutamide partially restore glucose-regulated glucagon secretion in islets from type 2 diabetic organ donors. These data suggest that impaired metabolic control of the KATP channels underlies the defective glucose regulation of glucagon secretion in type 2 diabetes.

Cited:

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Ashcroft FM, Rorsman P. 2013. KATP channels and islet hormone secretion: New insights and controversies Nature Reviews Endocrinology, 9 (11), pp. 660-669. | Show Abstract | Read more

ATP-sensitive potassium channels (K ATP channels) link cell metabolism to electrical activity by controlling the cell membrane potential. They participate in many physiological processes but have a particularly important role in systemic glucose homeostasis by regulating hormone secretion from pancreatic islet cells. Glucose-induced closure of K ATP channels is crucial for insulin secretion. Emerging data suggest that K ATP channels also play a key part in glucagon secretion, although precisely how they do so remains controversial. This Review highlights the role of K ATP channels in insulin and glucagon secretion. We discuss how K ATP channels might contribute not only to the initiation of insulin release but also to the graded stimulation of insulin secretion that occurs with increasing glucose concentrations. The various hypotheses concerning the role of K ATP channels in glucagon release are also reviewed. Furthermore, we illustrate how mutations in K ATP channel genes can cause hyposecretion or hypersecretion of insulin, as in neonatal diabetes mellitus and congenital hyperinsulinism, and how defective metabolic regulation of the channel may underlie the hypoinsulinaemia and the hyperglucagonaemia that characterize type 2 diabetes mellitus. Finally, we outline how sulphonylureas, which inhibit K ATP channels, stimulate insulin secretion in patients with neonatal diabetes mellitus or type 2 diabetes mellitus, and suggest their potential use to target the glucagon secretory defects found in diabetes mellitus. © 2013 Macmillan Publishers Limited. All rights reserved.

Rorsman P, Braun M. 2013. Regulation of insulin secretion in human pancreatic islets. Annu Rev Physiol, 75 (1), pp. 155-179. | Show Abstract | Read more

Pancreatic β cells secrete insulin, the body's only hormone capable of lowering plasma glucose levels. Impaired or insufficient insulin secretion results in diabetes mellitus. The β cell is electrically excitable; in response to an elevation of glucose, it depolarizes and starts generating action potentials. The electrophysiology of mouse β cells and the cell's role in insulin secretion have been extensively investigated. More recently, similar studies have been performed on human β cells. These studies have revealed numerous and important differences between human and rodent β cells. Here we discuss the properties of human pancreatic β cells: their glucose sensing, the ion channel complement underlying glucose-induced electrical activity that culminates in exocytotic release of insulin, the cellular control of exocytosis, and the modulation of insulin secretion by circulating hormones and locally released neurotransmitters. Finally, we consider the pathophysiology of insulin secretion and the interactions between genetics and environmental factors that may explain the current diabetes epidemic.

Rosengren AH, Braun M, Mahdi T, Andersson SA, Travers ME, Shigeto M, Zhang E, Almgren P et al. 2012. Reduced insulin exocytosis in human pancreatic β-cells with gene variants linked to type 2 diabetes. Diabetes, 61 (7), pp. 1726-1733. | Show Abstract | Read more

The majority of genetic risk variants for type 2 diabetes (T2D) affect insulin secretion, but the mechanisms through which they influence pancreatic islet function remain largely unknown. We functionally characterized human islets to determine secretory, biophysical, and ultrastructural features in relation to genetic risk profiles in diabetic and nondiabetic donors. Islets from donors with T2D exhibited impaired insulin secretion, which was more pronounced in lean than obese diabetic donors. We assessed the impact of 14 disease susceptibility variants on measures of glucose sensing, exocytosis, and structure. Variants near TCF7L2 and ADRA2A were associated with reduced glucose-induced insulin secretion, whereas susceptibility variants near ADRA2A, KCNJ11, KCNQ1, and TCF7L2 were associated with reduced depolarization-evoked insulin exocytosis. KCNQ1, ADRA2A, KCNJ11, HHEX/IDE, and SLC2A2 variants affected granule docking. We combined our results to create a novel genetic risk score for β-cell dysfunction that includes aberrant granule docking, decreased Ca(2+) sensitivity of exocytosis, and reduced insulin release. Individuals with a high risk score displayed an impaired response to intravenous glucose and deteriorating insulin secretion over time. Our results underscore the importance of defects in β-cell exocytosis in T2D and demonstrate the potential of cellular phenotypic characterization in the elucidation of complex genetic disorders.

Ramracheya R, Ward C, Shigeto M, Walker JN, Amisten S, Zhang Q, Johnson PR, Rorsman P, Braun M. 2010. Membrane potential-dependent inactivation of voltage-gated ion channels in alpha-cells inhibits glucagon secretion from human islets. Diabetes, 59 (9), pp. 2198-2208. | Show Abstract | Read more

OBJECTIVE: To document the properties of the voltage-gated ion channels in human pancreatic alpha-cells and their role in glucagon release. RESEARCH DESIGN AND METHODS: Glucagon release was measured from intact islets. [Ca(2+)](i) was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patch-clamping in isolated alpha-cells identified by immunocytochemistry. RESULT: Glucose inhibited glucagon secretion from human islets; maximal inhibition was observed at 6 mmol/l glucose. Glucagon secretion at 1 mmol/l glucose was inhibited by insulin but not by ZnCl(2). Glucose remained inhibitory in the presence of ZnCl(2) and after blockade of type-2 somatostatin receptors. Human alpha-cells are electrically active at 1 mmol/l glucose. Inhibition of K(ATP)-channels with tolbutamide depolarized alpha-cells by 10 mV and reduced the action potential amplitude. Human alpha-cells contain heteropodatoxin-sensitive A-type K(+)-channels, stromatoxin-sensitive delayed rectifying K(+)-channels, tetrodotoxin-sensitive Na(+)-currents, and low-threshold T-type, isradipine-sensitive L-type, and omega-agatoxin-sensitive P/Q-type Ca(2+)-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, omega-agatoxin, and isradipine. The [Ca(2+)](i) oscillations depend principally on Ca(2+)-influx via L-type Ca(2+)-channels. Capacitance measurements revealed a rapid (<50 ms) component of exocytosis. Exocytosis was negligible at voltages below -20 mV and peaked at 0 mV. Blocking P/Q-type Ca(2+)-currents abolished depolarization-evoked exocytosis. CONCLUSIONS: Human alpha-cells are electrically excitable, and blockade of any ion channel involved in action potential depolarization or repolarization results in inhibition of glucagon secretion. We propose that voltage-dependent inactivation of these channels underlies the inhibition of glucagon secretion by tolbutamide and glucose.

De Marinis YZ, Salehi A, Ward CE, Zhang Q, Abdulkader F, Bengtsson M, Braha O, Braun M et al. 2010. GLP-1 inhibits and adrenaline stimulates glucagon release by differential modulation of N- and L-type Ca2+ channel-dependent exocytosis. Cell Metab, 11 (6), pp. 543-553. | Show Abstract | Read more

Glucagon secretion is inhibited by glucagon-like peptide-1 (GLP-1) and stimulated by adrenaline. These opposing effects on glucagon secretion are mimicked by low (1-10 nM) and high (10 muM) concentrations of forskolin, respectively. The expression of GLP-1 receptors in alpha cells is <0.2% of that in beta cells. The GLP-1-induced suppression of glucagon secretion is PKA dependent, is glucose independent, and does not involve paracrine effects mediated by insulin or somatostatin. GLP-1 is without much effect on alpha cell electrical activity but selectively inhibits N-type Ca(2+) channels and exocytosis. Adrenaline stimulates alpha cell electrical activity, increases [Ca(2+)](i), enhances L-type Ca(2+) channel activity, and accelerates exocytosis. The stimulatory effect is partially PKA independent and reduced in Epac2-deficient islets. We propose that GLP-1 inhibits glucagon secretion by PKA-dependent inhibition of the N-type Ca(2+) channels via a small increase in intracellular cAMP ([cAMP](i)). Adrenaline stimulates L-type Ca(2+) channel-dependent exocytosis by activation of the low-affinity cAMP sensor Epac2 via a large increase in [cAMP](i).

Hoppa MB, Collins S, Ramracheya R, Hodson L, Amisten S, Zhang Q, Johnson P, Ashcroft FM, Rorsman P. 2009. Chronic palmitate exposure inhibits insulin secretion by dissociation of Ca(2+) channels from secretory granules. Cell Metab, 10 (6), pp. 455-465. | Show Abstract | Read more

Long-term (72 hr) exposure of pancreatic islets to palmitate inhibited glucose-induced insulin secretion by >50% with first- and second-phase secretion being equally suppressed. This inhibition correlated with the selective impairment of exocytosis evoked by brief (action potential-like) depolarizations, whereas that evoked by long ( approximately 250 ms) stimuli was unaffected. Under normal conditions, Ca(2+) influx elicited by brief membrane depolarizations increases [Ca(2+)](i) to high levels within discrete microdomains and triggers the exocytosis of closely associated insulin granules. We found that these domains of localized Ca(2+) entry become dispersed by long-term (72 hr), but not by acute (2 hr), exposure to palmitate. Importantly, the release competence of the granules was not affected by palmitate. Thus, the location rather than the magnitude of the Ca(2+) increase determines its capacity to evoke exocytosis. In both mouse and human islets, the palmitate-induced secretion defect was reversed when the beta cell action potential was pharmacologically prolonged.

Braun M, Ramracheya R, Bengtsson M, Zhang Q, Karanauskaite J, Partridge C, Johnson PR, Rorsman P. 2008. Voltage-gated ion channels in human pancreatic beta-cells: electrophysiological characterization and role in insulin secretion. Diabetes, 57 (6), pp. 1618-1628. | Show Abstract | Read more

OBJECTIVE: To characterize the voltage-gated ion channels in human beta-cells from nondiabetic donors and their role in glucose-stimulated insulin release. RESEARCH DESIGN AND METHODS: Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated beta-cells. The ion channel complement was determined by quantitative PCR. RESULTS: Human beta-cells express two types of voltage-gated K(+) currents that flow through delayed rectifying (K(V)2.1/2.2) and large-conductance Ca(2+)-activated K(+) (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K(V)2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na(+) currents (Na(V)1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na(+) currents with TTX reduced glucose-stimulated (6-20 mmol/l) insulin secretion by 55-70%. Human beta-cells are equipped with L- (Ca(V)1.3), P/Q- (Ca(V)2.1), and T- (Ca(V)3.2), but not N- or R-type Ca(2+) channels. Blockade of L-type channels abolished glucose-stimulated insulin release, while inhibition of T- and P/Q-type Ca(2+) channels reduced glucose-induced (6 mmol/l) secretion by 60-70%. Membrane potential recordings suggest that L- and T-type Ca(2+) channels participate in action potential generation. Blockade of P/Q-type Ca(2+) channels suppressed exocytosis (measured as an increase in cell capacitance) by >80%, whereas inhibition of L-type Ca(2+) channels only had a minor effect. CONCLUSIONS: Voltage-gated T-type and L-type Ca(2+) channels as well as Na(+) channels participate in glucose-stimulated electrical activity and insulin secretion. Ca(2+)-activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca(2+) influx through P/Q-type Ca(2+) channels.

MacDonald PE, De Marinis YZ, Ramracheya R, Salehi A, Ma X, Johnson PR, Cox R, Eliasson L, Rorsman P. 2007. A K ATP channel-dependent pathway within alpha cells regulates glucagon release from both rodent and human islets of Langerhans. PLoS Biol, 5 (6), pp. e143. | Show Abstract | Read more

Glucagon, secreted from pancreatic islet alpha cells, stimulates gluconeogenesis and liver glycogen breakdown. The mechanism regulating glucagon release is debated, and variously attributed to neuronal control, paracrine control by neighbouring beta cells, or to an intrinsic glucose sensing by the alpha cells themselves. We examined hormone secretion and Ca(2+) responses of alpha and beta cells within intact rodent and human islets. Glucose-dependent suppression of glucagon release persisted when paracrine GABA or Zn(2+) signalling was blocked, but was reversed by low concentrations (1-20 muM) of the ATP-sensitive K(+) (KATP) channel opener diazoxide, which had no effect on insulin release or beta cell responses. This effect was prevented by the KATP channel blocker tolbutamide (100 muM). Higher diazoxide concentrations (>/=30 muM) decreased glucagon and insulin secretion, and alpha- and beta-cell Ca(2+) responses, in parallel. In the absence of glucose, tolbutamide at low concentrations (<1 muM) stimulated glucagon secretion, whereas high concentrations (>10 muM) were inhibitory. In the presence of a maximally inhibitory concentration of tolbutamide (0.5 mM), glucose had no additional suppressive effect. Downstream of the KATP channel, inhibition of voltage-gated Na(+) (TTX) and N-type Ca(2+) channels (omega-conotoxin), but not L-type Ca(2+) channels (nifedipine), prevented glucagon secretion. Both the N-type Ca(2+) channels and alpha-cell exocytosis were inactivated at depolarised membrane potentials. Rodent and human glucagon secretion is regulated by an alpha-cell KATP channel-dependent mechanism. We propose that elevated glucose reduces electrical activity and exocytosis via depolarisation-induced inactivation of ion channels involved in action potential firing and secretion.

Zhang Q, Bengtsson M, Partridge C, Salehi A, Braun M, Cox R, Eliasson L, Johnson PR et al. 2007. R-type Ca(2+)-channel-evoked CICR regulates glucose-induced somatostatin secretion. Nat Cell Biol, 9 (4), pp. 453-460. | Show Abstract | Read more

Pancreatic islets have a central role in blood glucose homeostasis. In addition to insulin-producing beta-cells and glucagon-secreting alpha-cells, the islets contain somatostatin-releasing delta-cells. Somatostatin is a powerful inhibitor of insulin and glucagon secretion. It is normally secreted in response to glucose and there is evidence suggesting its release becomes perturbed in diabetes. Little is known about the control of somatostatin release. Closure of ATP-regulated K(+)-channels (K(ATP)-channels) and a depolarization-evoked increase in cytoplasmic free Ca(2+) concentration ([Ca(2+)](i)) have been proposed to be essential. Here, we report that somatostatin release evoked by high glucose (>or=10 mM) is unaffected by the K(ATP)-channel activator diazoxide and proceeds normally in K(ATP)-channel-deficient islets. Glucose-induced somatostatin secretion is instead primarily dependent on Ca(2+)-induced Ca(2+)-release (CICR). This constitutes a novel mechanism for K(ATP)-channel-independent metabolic control of pancreatic hormone secretion.

MacDonald PE, Braun M, Galvanovskis J, Rorsman P. 2006. Release of small transmitters through kiss-and-run fusion pores in rat pancreatic beta cells. Cell Metab, 4 (4), pp. 283-290. | Show Abstract | Read more

Exocytosis of secretory vesicles begins with a fusion pore connecting the vesicle lumen to the extracellular space. This pore may then expand or it may close to recapture the vesicle intact. The contribution of the latter, termed kiss-and-run, to exocytosis of pancreatic beta cell large dense-core vesicles (LDCVs) is controversial. Examination of single vesicle fusion pores demonstrated that rat beta cell LDCVs can undergo exocytosis by rapid pore expansion, by the formation of stable pores, or via small transient kiss-and-run fusion pores. Elevation of cAMP shifted LDCV fusion pore openings to the transient mode. Under this condition, the small fusion pores were sufficient for release of ATP, stored within LDCVs together with insulin. Individual ATP release events occurred coincident with amperometric "stand alone feet" representing kiss-and-run. Therefore, the LDCV kiss-and-run fusion pores allow small transmitter release but likely retain the larger insulin peptide. This may represent a mechanism for selective intraislet signaling.

Effect of a local islet DPP-IV system on human pancreatic hormone secretion: elucidating its role and mechanism of action.

Diabetes mellitus is the non-communicable disease epidemic of the 21st century and it has been predicted that in the UK alone there will be over 4 million people living with the condition by 2025. Currently >10% of the healthcare budget is spent on treating diabetes, amounting to an alarming £1 million per hour. The incretin effect, which in normal health is responsible for ~80% of insulin secretion at mealtimes, is impaired in type 2 diabetes1. GLP-1 is the most important incretin hormone ...

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Unravelling the mechanisms by which GLP-1 modulates glucagon secretion

The secretion of glucagon by pancreatic alpha-cells plays a critical role in the regulation of glycaemia1. This endocrine hormone is essential to prevent hypoglycaemia. Generally, it opposes the actions of insulin by promoting hepatic glucose production, thereby increasing blood glucose concentrations. Despite the importance of glucagon, very little is known about the precise regulatory machinery that controls its secretion from alpha-cells.It has been established that, in addition to glucose, ...

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