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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A. Confocal image of mouse islet stained against insulin (red), glucagon (green) and somatostatin (blue). B. Electrin micrograph of mouse beta-cell showing the secretory granules lined up along the plasma membrane. C. Electrical activity evoked by glucose in a mouse beta-cell.

A. Confocal image of mouse islet stained against insulin (red), glucagon (green) and somatostatin ...

Stimulus secretion coupling of the human beta-cell. The red rectangles highlight important differences between rodent and human beta-cells. In particular, the low resting KATP-channel activity might contribute (together with the presence of low-threshold T-type calcium channels) to the fact that human beta-cells respond to lower glucose concentrations than their rodent counterparts.

Stimulus secretion coupling of the human beta-cell. The red rectangles highlight important ...

Effects of elevating glucose from 1 to 6 to 20 mM on glucagon and insulin secretion measured using the in situ perfused mouse pancreas preparation in which islets are are perfused at a the physiological rate (0.3ml/min) and in the right direction. Note that glucagon secretion (blue) is suppressed by 6 mM before there is any detectable stimulation of insulin secretion, suggesting that glucose regulation of glucagon secretion in alpha-cells is not mediated by a paracrine mechanism via insulin secreted from the beta-cells

Effects of elevating glucose from 1 to 6 to 20 mM on glucagon and insulin secretion measured using ...

A revised model for regulation of glucagon secretion by exogenous insulin involving insulin-induced stimulation of somatostatin secretion that inhibits glucagon secretion by a paracrine mechanism. The same mechanism may also become activated in diabetic patients, characterised by high basal insulin secretion. This mechanism may contribute to the regulation of glucagon secretion by glucose but does not exclude that glucose also modulates glucagon secretion by a direct effect on the alpha-cell.

The micrograph to the left shows a section through a human pancreatic islet. Note that architecture of human islet is more 'chaotic' than that of mouse islets and that beta- and non-beta-cells are juxtaposed to each other in a way that can be expected to facilitate paracrine interactions.

A revised model for regulation of glucagon secretion by exogenous insulin involving insulin-induced ...

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
Erik Renstrom Lund University Sweden
Dr Patrick J Pollard (NDM) Centre for Cellular and Molecular Physiology University of Oxford United Kingdom

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. Read 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. Hide abstract

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

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

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. Read 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. Hide abstract

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. Read 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. Hide abstract

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. Read 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). Hide abstract

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. Read 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. Hide abstract

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. Read 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. Hide abstract

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. Read 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. Hide abstract

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. Read 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. Hide abstract

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. Read 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. Hide abstract

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. Read 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. Hide abstract

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. Read 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. Hide abstract

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. Read 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. Hide abstract