Prof Patrik Rorsman
|Research Area:||Cell and Molecular Biology|
|Technology Exchange:||Immunohistochemistry, Microscopy (Confocal), Microscopy (EM) and Transgenesis|
|Keywords:||diabetes, calcium, exocytosis, insulin, glucagon and somatostatin|
A. Confocal image of mouse islet stained against insulin (red), glucagon (green) and somatostatin ...
Exocytosis in human beta-celss (measured as a capacitance increase representing an increase in cell ...
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.
|Francis Ashcroft||Department of Physiology||The University of Oxford||UK|
|Per-Olof Berggren||Karolinska Institute||Sweden|
|Erik Renstrom||Lund University||Sweden|
|Thomas Jentsch||Max-Delbruck-Zentrum, Berlin||Germany|
|Franz Hofmann||Technishce Universitat Munich||Germany|
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
To characterize the voltage-gated ion channels in human beta-cells from nondiabetic donors and their role in glucose-stimulated insulin release. Hide abstract
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
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
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
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
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
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
Functional analysis of how diabetes genes affect β-cell function
Type-2 diabetes results from reduced insulin secretion. The recent genome wide scan association studies (GWAS) have identified ~50 gene variants that increase diabetes risk. Most of these genes are believed to affect β-cell function but (with few exceptions) the precise mechanisms remain obscure. The elucidation of how these gene variants impact on β-cell function and insulin secretion represents one of the greatest challenges in experimental diabetes research. Insulin is secreted from the ...
Regulation of glucagon secretion: physiology and pathophysiology
Diabetes is a bihormonal disorder resulting from insufficient secretion of insulin from the pancreatic beta-cells and oversecretion of glucagon from the alpha-cells. The decisive role of glucagon in diabetes is illustrated by the recent observation that even complete destruction of the insulin-producing beta-cells does not result in increased in plasma glucose provided glucagon signalling is simultaneously prevented (by genetic ablation of the glucagon receptors). Whereas we have a good ...
Effects of glucagon-like peptide–1 (GLP-1) amides on insulin and glucagon secretion in human islets: is a ‘novel’ GLP-1 receptor involved?
Diabetes mellitus is the non-communicable disease epidemic of the 21st Century with 380 million cases expected worldwide by 2025 and many national economies spending >10% of their healthcare budget on diabetes. Type-2 diabetes (T2DM) accounts for >80% of all cases. Elevated blood glucose levels in T2DM results from impaired insulin secretion from beta-cells and hypersecretion of glucagon from alpha-cells of the pancreatic islets 1 . The incretin effect, which in normal health is responsible ...