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The somatostatin-secreting δ-cells comprise ~5% of the cells of the pancreatic islets. The δ-cells have complex morphology and might interact with many more islet cells than suggested by their low numbers. δ-Cells contain ATP-sensitive potassium channels, which open at low levels of glucose but close when glucose is elevated. This closure initiates membrane depolarization and electrical activity and increased somatostatin secretion. Factors released by neighbouring α-cells or β-cells amplify the glucose-induced effects on somatostatin secretion from δ-cells, which act locally within the islets as paracrine or autocrine inhibitors of insulin, glucagon and somatostatin secretion. The effects of somatostatin are mediated by activation of somatostatin receptors coupled to the inhibitory G protein, which culminates in suppression of the electrical activity and exocytosis in α-cells and β-cells. Somatostatin secretion is perturbed in animal models of diabetes mellitus, which might explain the loss of appropriate hypoglycaemia-induced glucagon secretion, a defect that could be mitigated by somatostatin receptor 2 antagonists. Somatostatin antagonists or agents that suppress somatostatin secretion have been proposed as an adjunct to insulin therapy. In this Review, we summarize the cell physiology of somatostatin secretion, what might go wrong in diabetes mellitus and the therapeutic potential of agents targeting somatostatin secretion or action. [ABSTRACT FROM AUTHOR]

The pancreatic β-cell plays a key role in glucose homeostasis by secreting insulin, the only hormone capable of lowering the blood glucose concentration. Impaired insulin secretion results in the chronic hyperglycemia that characterizes type 2 diabetes (T2DM), which currently afflicts >450 million people worldwide. The healthy β-cell acts as a glucose sensor matching its output to the circulating glucose concentration. It does so via metabolically induced changes in electrical activity, which culminate in an increase in the cytoplasmic Ca2+ concentration and initiation of Ca2+-dependent exocytosis of insulin-containing secretory granules. Here, we review recent advances in our understanding of the β-cell transcriptome, electrical activity, and insulin exocytosis. We highlight salient differences between mouse and human β-cells, provide models of how the different ion channels contribute to their electrical activity and insulin secretion, and conclude by discussing how these processes become perturbed in T2DM. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

ATP-sensitive potassium channels (KATP 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 KATP channels is crucial for insulin secretion. Emerging data suggest that KATP channels also play a key part in glucagon secretion, although precisely how they do so remains controversial. This Review highlights the role of KATP channels in insulin and glucagon secretion. We discuss how KATP 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 KATP channels in glucagon release are also reviewed. Furthermore, we illustrate how mutations in KATP 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 KATP 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. [ABSTRACT FROM AUTHOR]

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. INSET: SUMMARY POINTS. [ABSTRACT FROM AUTHOR]

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. [Copyright &y& Elsevier]

Abstract: 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 (KATP-channels), small-conductance Ca2+-activated K+-channels and voltage-gated Ca2+-channels in the generation of the bursts. Our data indicate that KATP-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. [Copyright &y& Elsevier]

Glucagon, secreted by the α-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 β-cells (paracrine control). However, the observation that glucose can regulate glucagon secretion under conditions when insulin secretion does not occur argues that the α-cell is also equipped with its own intrinsic (exerted within the α-cell itself) glucose sensing. Here we consider the possible mechanisms involved with a focus on ATP-regulated K+-channels and changes in α-cell membrane potential. [Copyright &y& Elsevier]

The article focuses on the insulin secretion in pancreatic beta cells. The insulin is secreted from the pancreas when blood glucose levels are high, and it acts to maintain glucose homeostasis by promoting glucose uptake and storage in muscle, fat, and liver. Glucose-dependent insulin secretion from beta cells is referred to as stimulus-secretion coupling. It is further noted that the secretion of insulin is dependent on electrical activity and calcium entry.

Focuses on the characteristic biphasic time course followed by insulin secretion. Influence of glucose-induced electrical activity on the secretion process; Description of the Ca2+ channel density in β-cell plasma membranes; Capability of the βcell of remarkably high rates of exocytosis.

Throughout the 20th Century, regulatory peptide discovery advanced from the identification of gut hormones to the extraction and characterization of hypothalamic hypophysiotropic factors, and to the isolation and cloning of multiple brain neuropeptides. These discoveries were followed by the discovery of G‐protein‐coupled and other membrane receptors for these peptides. Subsequently, the systems physiology associated with some of these multiple regulatory peptides and receptors has been comprehensively elucidated and has led to improved therapeutics and diagnostics and their approval by the US Food and Drug Administration. In light of this wealth of information and further potential, it is truly a time of renaissance for regulatory peptides. In this perspective, we review what we have learned from the pioneers in exemplified fields of gut peptides, such as cholecystokinin, enterochromaffin‐like‐cell peptides, and glucagon, from the trailblazing studies on the key stress hormone, corticotropin‐releasing factor, as well as from more recently characterized relaxin‐family peptides and receptors. The historical viewpoints are based on our understanding of these topics in light of the earliest phases of research and on subsequent studies and the evolution of knowledge, aiming to sharpen our vision of the current state‐of‐the‐art and those studies that should be prioritized in the future. [ABSTRACT FROM AUTHOR]

By secreting insulin and glucagon, the β‐ and α‐cells of the pancreatic islets play a central role in the regulation of systemic metabolism. Both cells are equipped with ATP‐regulated potassium (KATP) channels that are regulated by the intracellular ATP/ADP ratio. In β‐cells, KATP channels are active at low (non‐insulin‐releasing) glucose concentrations. An increase in glucose leads to KATP channel closure, membrane depolarization and electrical activity that culminates in elevation of [Ca2+]i and initiation of exocytosis of the insulin‐containing secretory granules. The α‐cells are also equipped with KATP channels but they are under strong tonic inhibition at low glucose, explaining why α‐cells are electrically active under hypoglycaemic conditions and generate large Na+‐ and Ca2+‐dependent action potentials. Closure of residual KATP channel activity leads to membrane depolarization and an increase in action potential firing but this stimulation of electrical activity is associated with inhibition rather than acceleration of glucagon secretion. This paradox arises because membrane depolarization reduces the amplitude of the action potentials by voltage‐dependent inactivation of the Na+ channels involved in action potential generation. Exocytosis in α‐cells is tightly linked to the opening of voltage‐gated P/Q‐type Ca2+ channels, the activation of which is steeply voltage‐dependent. Accordingly, the inhibitory effect of the reduced action potential amplitude exceeds the stimulatory effect resulting from the increased action potential frequency. These observations highlight a previously unrecognised role of the action potential amplitude as a key regulator of pancreatic islet hormone secretion. [ABSTRACT FROM AUTHOR]

The coordinated electrical activity of β-cells within the pancreatic islet drives oscillatory insulin secretion. A recent hypothesis postulates that specially equipped "hub" or "leader" cells within the β-cell network drive islet oscillations and that electrically silencing or optically ablating these cells suppresses coordinated electrical activity (and thus insulin secretion) in the rest of the islet. In this Perspective, we discuss this hypothesis in relation to established principles of electrophysiological theory. We conclude that whereas electrical coupling between β-cells is sufficient for the propagation of excitation across the islet, there is no obvious electrophysiological mechanism that explains how hyperpolarizing a hub cell results in widespread inhibition of islet electrical activity and disruption of their coordination. Thus, intraislet diffusible factors should perhaps be considered as an alternate mechanism. [ABSTRACT FROM AUTHOR]

Whole-body glucose homeostasis is coordinated through secretion of glucagon and insulin from pancreatic islets. When glucose is low, glucagon is released from α-cells to stimulate hepatic glucose production. However, the mechanisms that regulate glucagon secretion from pancreatic α-cells remain unclear. Here we show that in α-cells, the interaction between fatty acid oxidation and glucose metabolism controls glucagon secretion. The glucose-dependent inhibition of glucagon secretion relies on pyruvate dehydrogenase and carnitine palmitoyl transferase 1a activity and lowering of mitochondrial fatty acid oxidation by increases in glucose. This results in reduced intracellular ATP and leads to membrane repolarization and inhibition of glucagon secretion. These findings provide a new framework for the metabolic regulation of the α-cell, where regulation of fatty acid oxidation by glucose accounts for the stimulation and inhibition of glucagon secretion. Article Highlights: It has become clear that dysregulation of glucagon secretion and α-cell function plays an important role in the development of diabetes, but we do not know how glucagon secretion is regulated. Here we asked whether glucose inhibits fatty acid oxidation in α-cells to regulate glucagon secretion. We found that fatty acid oxidation is required for the inhibitory effects of glucose on glucagon secretion through reductions in ATP. These findings provide a new framework for the regulation of glucagon secretion by glucose. [ABSTRACT FROM AUTHOR]

Examines the role of glutamate in preparing the release of insulin-secreting pancreatic beta cells. Stimulation of the pancreatic beta-cells to secrete insulin; Glutamate as the metabolic signal on the phases of insulin secretion; Reduction of granular membrane potential for larger ph gradient development across granular membrane.

Glucagon-like peptide-1 (GLP-1) is an endogenous glucose-lowering hormone and GLP-1 receptor agonists are currently being used as antidiabetic drugs clinically. The canonical signalling pathway (including cAMP, Epac2, protein kinase A (PKA) and K channels) is almost universally accepted as the main mechanism of GLP-1-stimulated insulin secretion. This belief is based on in vitro studies that used nanomolar (1-100 nM) concentrations of GLP-1. Recently, it was found that the physiological concentrations (1-10 pM) of GLP-1 also stimulate insulin secretion from isolated islets, induce membrane depolarization and increase of intracellular [Ca] in isolated β cells/pancreatic islets. These responses were unaffected by PKA inhibitors and occurred without detectable increases in intracellular cAMP and PKA activity. These PKA-independent actions of GLP-1 depend on protein kinase C (PKC), involve activation of the standard GLP-1 receptor (GLP1R) and culminate in activation of phospholipase C (PLC), leading to an elevation of diacylglycerol (DAG), increased L-type Ca and TRPM4/TRPM5 channel activities. Here, we review these recent data and contrast them against the effects of nanomolar concentrations of GLP-1. The differential intracellular signalling activated by low and high concentrations of GLP-1 could provide a clue to explain how GLP-1 exerts different function in the central nervous system and peripheral organs. [ABSTRACT FROM AUTHOR]

Participants self-administered the oral glibenclamide suspension (0.3-6 mg/day, split dose twice daily), and fasting pre-dose blood samples were taken before each dose increase every 3-4 days (see Figure S1 for trial timeline). CONCLUSION In conclusion, low-dose glibenclamide safely reduced glucagon levels in a subpopulation of patients with T2D who had fasting hyperglucagonaemia, suggesting that inappropriately increased alpha-cell K SB ATP sb activity may be a key component in the metabolic dysregulation of diabetes. Reducing hyperglucagonaemia in type 2 diabetes using low-dose glibenclamide: Results of the LEGEND-A pilot study Keywords: antidiabetic drug; clinical trial; drug mechanism; experimental pharmacology; glucagon; type 2 diabetes EN antidiabetic drug clinical trial drug mechanism experimental pharmacology glucagon type 2 diabetes 1671 1675 5 07/11/22 20220801 NES 220801 INTRODUCTION Diabetes is a multi-hormonal disorder characterized by insufficient insulin secretion and aberrant glucagon secretion, with fasting hyperglucagonaemia leading to increased hepatic glucose production,1,2 further exacerbating hyperglycaemia. [Extracted from the article]

Dysregulated glucagon secretion from pancreatic alpha-cells is a key feature of type-1 and type-2 diabetes (T1D and T2D), yet our mechanistic understanding of alpha-cell function is underdeveloped relative to insulin-secreting beta-cells. Here we show that the enzyme acetyl-CoA-carboxylase 1 (ACC1), which couples glucose metabolism to lipogenesis, plays a key role in the regulation of glucagon secretion. Pharmacological inhibition of ACC1 in mouse islets or αTC9 cells impaired glucagon secretion at low glucose (1 mmol/l). Likewise, deletion of ACC1 in alpha-cells in mice reduced glucagon secretion at low glucose in isolated islets, and in response to fasting or insulin-induced hypoglycaemia in vivo. Electrophysiological recordings identified impaired KATP channel activity and P/Q- and L-type calcium currents in alpha-cells lacking ACC1, explaining the loss of glucose-sensing. ACC-dependent alterations in S-acylation of the KATP channel subunit, Kir6.2, were identified by acyl-biotin exchange assays. Histological analysis identified that loss of ACC1 caused a reduction in alpha-cell area of the pancreas, glucagon content and individual alpha-cell size, further impairing secretory capacity. Loss of ACC1 also reduced the release of glucagon-like peptide 1 (GLP-1) in primary gastrointestinal crypts. Together, these data reveal a role for the ACC1-coupled pathway in proglucagon-expressing nutrient-responsive endocrine cell function and systemic glucose homeostasis. Veprik et al. show that Acetyl-CoA-carboxylase 1 (ACC1), an enzyme that couples glucose metabolism to lipogenesis, is involved in glucagon secretion and regulates S-acylation of critical glucose-sensing proteins. Loss of ACC1 in pancreatic alpha-cells negatively affects both size and number, as well as glucagon content, while in gut enteroendocrine cells leads to reduced release of glucagon-like peptide 1. [ABSTRACT FROM AUTHOR]

Highlights: [•] We studied GPRC5B expression in mouse and human pancreatic islets. [•] Confocal microscopy showed that GPRC5B is co-localized with insulin cells. [•] A much higher expression of GPRC5B was found in islets from diabetic donors. [•] Down-regulation of Gprc5b in mouse islets positively modulated insulin secretion. [•] Down-regulation of Gprc5b protected MIN6 cells against cytokine-induced apoptosis. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

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 β-cells. First, the role of a K+-current dependent on Ca2+-influx for the termination of burst of action potentials in β-cells could be documented. Neither the current, nor the bursting pattern of electrical activity is preserved in isolated β-cells. Second, the conductance of gap junctions (∼1 nS) between β-cells was determined. Third, electrical properties of glucagon-producing α- and somatostatin-secreting δ-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. [Copyright &y& Elsevier]

The vascular network supporting the islets of Langerhans represents a highly specialised system of arterioles, capillaries and venules. Several features of the islet vasculature (density and fenestration of the capillaries) ensure rapid exchange of nutrients and hormones, which is central to the islets' capacity to control of systemic metabolism via reciprocal changes of insulin and glucagon secretion. Here we discuss how changes in islet blood flow may underlie pulsatile insulin secretion, which becomes impaired in type-2 diabetes. Improved understanding of the architecture and regulation of pancreas/islet blood flow may therefore illuminate the causes underlying this common metabolic disorder. The pioneering work of August Krogh on blood flow, oxygen diffusion and capillary anatomy (that was awarded with the Nobel Prize in 1920) is a cornerstone in these efforts and remains relevant to today's research. Unlabelled Image • Pancreatic islets are highly vascularized • Insulin secretion depends on oxidative metabolism • Adaptive increases in islet blood when insulin secretion is stimulated • Blood flow shapes oscillatory insulin secretion? [ABSTRACT FROM AUTHOR]

Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all‐or‐none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all‐or‐none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317±1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310±1127) for each stimulated exocytosis event. Thus, beta cells release only one‐third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type‐2 diabetes. [ABSTRACT FROM AUTHOR]

Recent work has shown that chemical release during the fundamental cellular process of exocytosis in model cell lines is not all‐or‐none. We tested this theory for vesicular release from single pancreatic beta cells. The vesicles in these cells release insulin, but also serotonin, which is detectible with amperometric methods. Traditionally, it is assumed that exocytosis in beta cells is all‐or‐none. Here, we use a multidisciplinary approach involving nanoscale amperometric chemical methods to explore the chemical nature of insulin exocytosis. We amperometrically quantified the number of serotonin molecules stored inside of individual nanoscale vesicles (39 317±1611) in the cell cytoplasm before exocytosis and the number of serotonin molecules released from single cells (13 310±1127) for each stimulated exocytosis event. Thus, beta cells release only one‐third of their granule content, clearly supporting partial release in this system. We discuss these observations in the context of type‐2 diabetes. [ABSTRACT FROM AUTHOR]

By restoring glucose-regulated insulin secretion, glucagon-like peptide-1-based (GLP-1-based) therapies are becoming increasingly important in diabetes care. Normally, the incretins GLP-1 and glucose-dependent insulinotropic polypeptide (GIP) jointly maintain normal blood glucose levels by stimulation of insulin secretion in pancreatic β cells. However, the reason why only GLP-1-based drugs are effective in improving insulin secretion after presentation of diabetes has not been resolved. ATP-sensitive K+ (KATP) channels play a crucial role in coupling the systemic metabolic status to β cell electrical activity for insulin secretion. Here, we have shown that persistent membrane depolarization of β cells due to genetic (β cell-specific Kcnj11-/- mice) or pharmacological (long-term exposure to sulfonylureas) inhibition of the KATP channel led to a switch from Gs to Gq in a major amplifying pathway of insulin secretion. The switch determined the relative insulinotropic effectiveness of GLP-1 and GIP, as GLP-1 can activate both Gq and Gs, while GIP only activates Gs. The findings were corroborated in other models of persistent depolarization: a spontaneous diabetic KK-Ay mouse and nondiabetic human and mouse β cells of pancreatic islets chronically treated with high glucose. Thus, a Gs/Gq signaling switch in β cells exposed to chronic hyperglycemia underlies the differential insulinotropic potential of incretins in diabetes. [ABSTRACT FROM AUTHOR]

Type 2 diabetes is associated with impaired insulin secretion. Both 1st- and 2nd-phase insulin secretion are reduced, but the effect is particularly pronounced for the 1st phase. The processes culminating in impaired insulin secretion are not fully understood, but both genetic and environmental factors are thought to play a role. Over the past 2 years, genome-wide association scans have transformed the genetic landscape of type 2 diabetes susceptibility, with the current gene count close to 20 (1). A couple of common themes have emerged from these studies. First, the majority of the genes identified thus far seem to affect diabetes susceptibility through β-cell dysfunction (2). Second, the risk alleles tend to be common in the population, but their effect on diabetes risk is relatively small (3,4). TCF7L2, the susceptibility gene with the largest effect on disease susceptibility discovered to date, was identified pre-genome-wide association by Grant et al. in 2006, with rapid replication of its consequence on diabetes susceptibility in multiple populations (5-9). TCF7L2 was a positional candidate gene that mapped to a region of genetic linkage to type 2 diabetes in the Icelandic population on chromosome 10. However, the identified TCF7L2 risk allele, which was present in ∼28% of control subjects and ∼36% of type 2 diabetic individuals, could not explain this linkage, so the finding was actually serendipitous (5). The precise genetic defect that causes the association of TCF7L2 with type 2 diabetes remains unclear. There is a large number of highly correlated variants, none of which are obvious functional candidates, that show association with diabetes (5). The most likely candidate is the single nucleotide polymorphism rs7903146, which shows the strongest association with diabetes and resides in a noncoding region with no obvious mutational mechanism. It is clear, however, that the effect of the TCF7L2 risk allele is through a defect in insulin secretion (9). There have been few studies investigating the role of TCF7L2 on insulin secretion in isolated islets. Recently, a study by Shu et al. (10) reported that silencing of TCF7L2 by siRNA resulted in strong suppression of insulin secretion in human and mouse islets. Conversely, overexpression of TCF7L2 stimulated insulin secretion. Exactly how TCF7L2 protein levels modulate insulin secretion was not established in the study by Shu et al. [ABSTRACT FROM AUTHOR]

Somatostatin secretion from pancreatic islet δ-cells is stimulated by elevated glucose levels, but the underlying mechanisms have only partially been elucidated. Here we show that glucose-induced somatostatin secretion (GISS) involves both membrane potential-dependent and -independent pathways. Although glucose-induced electrical activity triggers somatostatin release, the sugar also stimulates GISS via a cAMP-dependent stimulation of CICR and exocytosis of somatostatin. The latter effect is more quantitatively important and in mouse islets depolarized by 70 mM extracellular K+, increasing glucose from 1 mM to 20 mM produced an ∼3.5-fold stimulation of somatostatin secretion, an effect that was mimicked by the application of the adenylyl cyclase activator forskolin. Inhibiting cAMP-dependent pathways with PKI or ESI-05, which inhibit PKA and exchange protein directly activated by cAMP 2 (Epac2), respectively, reduced glucose/forskolin-induced somatostatin secretion. Ryanodine produced a similar effect that was not additive to that of the PKA or Epac2 inhibitors. Intracellular application of cAMP produced a concentration-dependent stimulation of somatostatin exocytosis and elevation of cytoplasmic Ca2+ ([Ca2+]i). Both effects were inhibited by ESI-05 and thapsigargin (an inhibitor of SERCA). By contrast, inhibition of PKA suppressed δ-cell exocytosis without affecting [Ca2+]i. Simultaneous recordings of electrical activity and [Ca2+]i in δ-cells expressing the genetically encoded Ca2+ indicator GCaMP3 revealed that the majority of glucose-induced [Ca2+]i spikes did not correlate with δ-cell electrical activity but instead reflected Ca2+ release from the ER. These spontaneous [Ca2+]i spikes are resistant to PKI but sensitive to ESI-05 or thapsigargin. We propose that cAMP links an increase in plasma glucose to stimulation of somatostatin secretion by promoting CICR, thus evoking exocytosis of somatostatin-containing secretory vesicles in the δ-cell. [ABSTRACT FROM AUTHOR]

High-content real-time imaging of hormone secretion in tissues or cell populations is a challenging task, which is unlikely to be resolved directly, despite immense translational value. We approach this problem indirectly, using compensatory endocytosis, a process that closely follows exocytosis in the cell, as a surrogate read-out for secretion. The tissue is immobilized in an open-air perifusion chamber and imaged using a two-photon microscope. A fluorescent polar tracer, perifused through the experimental circuit, gets trapped into the cells via endocytosis, and is quantified using a feature-detection algorithm. The signal of the tracer that accumulates into the endocytotic system reliably reflects stimulated exocytosis, which is demonstrated via co-imaging of the latter using existing reporters. A high signal-to-noise ratio and compatibility with multisensor imaging affords the real-time quantification of the secretion at the tissue/population level, whereas the cumulative nature of the signal allows imprinting of the “secretory history” within each cell. The technology works for several cell types, reflects disease progression and can be used for human tissue. [ABSTRACT FROM AUTHOR]

Abstract: Glucagon is the body's main hyperglycemic hormone, and its secretion is dysregulated in type 2 diabetes mellitus (T2DM). The incretin hormone glucagon‐like peptide‐1 (GLP‐1) is released from the gut and is used in T2DM therapy. Uniquely, it both stimulates insulin and inhibits glucagon secretion and thereby lowers plasma glucose levels. In this study, we have investigated the action of GLP‐1 on glucagon release from human pancreatic islets. Immunocytochemistry revealed that only <0.5% of the α‐cells possess detectable GLP‐1R immunoreactivity. Despite this, GLP‐1 inhibited glucagon secretion by 50–70%. This was due to a direct effect on α‐cells, rather than paracrine signaling, because the inhibition was not reversed by the insulin receptor antagonist S961 or the somatostatin receptor‐2 antagonist CYN154806. The inhibitory effect of GLP‐1 on glucagon secretion was prevented by the PKA‐inhibitor Rp‐cAMPS and mimicked by the adenylate cyclase activator forskolin. Electrophysiological measurements revealed that GLP‐1 decreased action potential height and depolarized interspike membrane potential. Mathematical modeling suggests both effects could result from inhibition of P/Q‐type Ca2+ channels. In agreement with this, GLP‐1 and ω‐agatoxin (a blocker of P/Q‐type channels) inhibited glucagon secretion in islets depolarized by 70 mmol/L [K+]o, and these effects were not additive. Intracellular application of cAMP inhibited depolarization‐evoked exocytosis in individual α‐cells by a PKA‐dependent (Rp‐cAMPS‐sensitive) mechanism. We propose that inhibition of glucagon secretion by GLP‐1 involves activation of the few GLP‐1 receptors present in the α‐cell membrane. The resulting small elevation of cAMP leads to PKA‐dependent inhibition of P/Q‐type Ca2+ channels and suppression of glucagon exocytosis. [ABSTRACT FROM AUTHOR]

Adrenaline is a powerful stimulus of glucagon secretion. It acts by activation of β-adrenergic receptors, but the downstream mechanisms have only been partially elucidated. Here, we have examined the effects of adrenaline in mouse and human α-cells by a combination of electrophysiology, imaging of Ca2+ and PKA activity, and hormone release measurements. We found that stimulation of glucagon secretion correlated with a PKA- and EPAC2-dependent (inhibited by PKI and ESI-05, respectively) elevation of [Ca2+]i in α-cells, which occurred without stimulation of electrical activity and persisted in the absence of extracellular Ca2+ but was sensitive to ryanodine, bafilomycin, and thapsigargin. Adrenaline also increased [Ca2+]i in α-cells in human islets. Genetic or pharmacological inhibition of the Tpc2 channel (that mediates Ca2+ release from acidic intracellular stores) abolished the stimulatory effect of adrenaline on glucagon secretion and reduced the elevation of [Ca2+]i Furthermore, in Tpc2-deficient islets, ryanodine exerted no additive inhibitory effect. These data suggest that β-adrenergic stimulation of glucagon secretion is controlled by a hierarchy of [Ca2+]i signaling in the α-cell that is initiated by cAMP-induced Tpc2-dependent Ca2+ release from the acidic stores and further amplified by Ca2+-induced Ca2+ release from the sarco/endoplasmic reticulum. [ABSTRACT FROM AUTHOR]

Key points: Na+ current inactivation is biphasic in insulin‐secreting cells, proceeding with two voltage dependences that are half‐maximal at ∼−100 mV and −60 mV. Inactivation of voltage‐gated Na+ (NaV) channels occurs at ∼30 mV more negative voltages in insulin‐secreting Ins1 and primary β‐cells than in HEK, CHO or glucagon‐secreting αTC1‐6 cells. The difference in inactivation between Ins1 and non‐β‐cells persists in the inside‐out patch configuration, discounting an involvement of a diffusible factor. In Ins1 cells and primary β‐cells, but not in HEK cells, inactivation of a single NaV subtype is biphasic and follows two voltage dependences separated by 30–40 mV. We propose that NaV channels adopt different inactivation behaviours depending on the local membrane environment. Abstract: Pancreatic β‐cells are equipped with voltage‐gated Na+ channels that undergo biphasic voltage‐dependent steady‐state inactivation. A small Na+ current component (10–15%) inactivates over physiological membrane potentials and contributes to action potential firing. However, the major Na+ channel component is completely inactivated at −90 to −80 mV and is therefore inactive in the β‐cell. It has been proposed that the biphasic inactivation reflects the contribution of different NaV α‐subunits. We tested this possibility by expression of TTX‐resistant variants of the NaV subunits found in β‐cells (NaV1.3, NaV1.6 and NaV1.7) in insulin‐secreting Ins1 cells and in non‐β‐cells (including HEK and CHO cells). We found that all NaV subunits inactivated at 20–30 mV more negative membrane potentials in Ins1 cells than in HEK or CHO cells. The more negative inactivation in Ins1 cells does not involve a diffusible intracellular factor because the difference between Ins1 and CHO persisted after excision of the membrane. NaV1.7 inactivated at 15‐­20 mV more negative membrane potentials than NaV1.3 and NaV1.6 in Ins1 cells but this small difference is insufficient to solely explain the biphasic inactivation in Ins1 cells. In Ins1 cells, but never in the other cell types, widely different components of NaV inactivation (separated by 30 mV) were also observed following expression of a single type of NaV α‐subunit. The more positive component exhibited a voltage dependence of inactivation similar to that found in HEK and CHO cells. We propose that biphasic NaV inactivation in insulin‐secreting cells reflects insertion of channels in membrane domains that differ with regard to lipid and/or membrane protein composition. [ABSTRACT FROM AUTHOR]

Loss of first-phase insulin secretion is an early sign of developing type 2 diabetes (T2D). Ca2+ entry through voltage-gated L-type Ca2+ channels triggers exocytosis of insulin-containing granules in pancreatic β cells and is required for the postprandial spike in insulin secretion. Using high-resolution microscopy, we have identified a subset of docked insulin granules in human β cells and rat-derived clonal insulin 1 (INS1) cells for which localized Ca2+ influx triggers exocytosis with high probability and minimal latency. This immediately releasable pool (IRP) of granules, identified both structurally and functionally, was absent in β cells from human T2D donors and in INS1 cells cultured in fatty acids that mimic the diabetic state. Upon arrival at the plasma membrane, IRP granules slowly associated with 15 to 20 L-type channels. We determined that recruitment depended on a direct interaction with the synaptic protein Munc13, because expression of the II-III loop of the channel, the C2 domain of Munc13-1, or of Munc13-1 with a mutated C2 domain all disrupted L-type channel clustering at granules and ablated fast exocytosis. Thus, rapid insulin secretion requires Munc13-mediated recruitment of L-type Ca2+ channels in close proximity to insulin granules. Loss of this organization underlies disturbed insulin secretion kinetics in T2D. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM PUBLISHER]

Glucose stimulates insulin secretion from β-cells by increasing intracellular Ca2+. Ca2+ then binds to synaptotagmin-7 as a major Ca2+ 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 Ca2+-stimulated insulin secretion and accounts for the improvement of glucose homeostasis by GLP-1. A phospho-mimetic synaptotagmin-7 mutant enhances Ca2+-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 Ca2+-triggered exocytosis by direct phosphorylation of a synaptotagmin. [ABSTRACT FROM AUTHOR]

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 expressNav1.7 andα-cellsNav1.3. In α-cells, genetically ablating Scn3a reduces theNa+ 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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 proteingated 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/Qtype Ca2+ currents, and directly inhibiting exocytosis. These effects are mediated predominantly by SSTR2 in both cell types. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

OBJECTIVE--To document the properties of the voltage-gated ion channels in human pancreatic α-cells and their role in glucagon release. RESEARCH DESIGN AND METHODS--Glucagon release was measured from intact islets. [Ca[sup 2+]][sub i] was recorded in cells showing spontaneous activity at 1 mmol/l glucose. Membrane currents and potential were measured by whole-cell patchclamping in isolated α-cells identified by immunocytochemistry. RESULTS--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[sub 2]. Glucose remained inhibitory in the presence of ZnCl[sub 2] and after blockade of type-2 somatostatin receptors. Human α-cells are electrically active at 1 mmol/l glucose. Inhibition of KATe-channels with tolbutamide depolarized α-cells by 10 mV and reduced the action potential amplitude. Human α-cells contain heteropodatoxin-sensitive A-type K[sup +]-channels, stromatoxin-sensitive delayed rectifying K[sup +]-channels, tetrodotox-insensitive Na[sup +]-currents, and low-threshold T-type, isradipinesensitive L-type, and ω-agatoxin-sensitive P/Q-type Ca2 +-channels. Glucagon secretion at 1 mmol/l glucose was inhibited by 40-70% by tetrodotoxin, heteropodatoxin-2, stromatoxin, ω-agatoxin, and isradipine. The [Ca[sup 2+]][sub i] oscillations depend principally on Ca[sup 2+]-influx via L-type Ca[sup 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[sup 2+]-currents abolished depolarization-evoked exocytosis. CONCLUSIONS--Human α-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. Diabetes 59:2198-2208, 2010 [ABSTRACT FROM AUTHOR]

Gain-of-function mutations in Kir6.2 (KCNJ11), the pore-forming subunit of the adenosine triphosphate (ATP)-sensitive potassium (KATP) channel, cause neonatal diabetes. Many patients also suffer from hypotonia (weak and flaccid muscles) and balance problems. The diabetes arises from suppressed insulin secretion by overactive KATP channels in pancreatic β-cells, but the source of the motor phenotype is unknown. By using mice carrying a human Kir6.2 mutation (Val59 → Met59) targeted to either muscle or nerve, we show that analogous motor impairments originate in the central nervous system rather than in muscle or peripheral nerves. We also identify locomotor hyperactivity as a feature of KATP channel overactivity. These findings suggest that drugs targeted against neuronal, rather than muscle, KATP channels are needed to treat the motor deficits and that such drugs require high blood-brain barrier permeability. [ABSTRACT FROM AUTHOR]

Objective: Paracrine signaling via gamma-aminobutyric acid (GABA) and GABA(A) receptors (GABA(A)Rs) has been documented in rodent islets. Here we have studied the importance of GABAergic signaling in human pancreatic islets.Research Design and Methods: Expression of GABA(A)Rs in islet cells was investigated by quantitative PCR, immunohistochemistry, and patch-clamp experiments. Hormone release was measured from intact islets. GABA release was monitored by whole-cell patch-clamp measurements after adenoviral expression of alpha(1)beta(1) GABA(A)R subunits. The subcellular localization of GABA was explored by electron microscopy. The effects of GABA on electrical activity were determined by perforated patch whole-cell recordings.Results: PCR analysis detected relatively high levels of the mRNAs encoding GABA(A)R alpha(2), beta(3,) gamma(2), and pi subunits in human islets. Patch-clamp experiments revealed expression of GABA(A)R Cl(-) channels in 52% of beta-cells (current density 9 pA/pF), 91% of delta-cells (current density 148 pA/pF), and 6% of alpha-cells (current density 2 pA/pF). Expression of GABA(A)R subunits in islet cells was confirmed by immunohistochemistry. beta-Cells secreted GABA both by glucose-dependent exocytosis of insulin-containing granules and by a glucose-independent mechanism. The GABA(A)R antagonist SR95531 inhibited insulin secretion elicited by 6 mmol/l glucose. Application of GABA depolarized beta-cells and stimulated action potential firing in beta-cells exposed to glucose.Conclusions: Signaling via GABA and GABA(A)R constitutes an autocrine positive feedback loop in human beta-cells. The presence of GABA(A)R in non-beta-cells suggests that GABA may also be involved in the regulation of somatostatin and glucagon secretion. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Paracrine signaling via γ-aminobutyric acid (GABA) and GABAA receptors (GABAARS) has been documented in rodent islets. Here we have studied the importance of GABAergic signaling in human pancreatic islets. RESEARCH DESIGN AND METHODS--Expression of GABAARS in islet cells was investigated by quantitative PCR, immunohistochemistry, and patch-clamp experiments. Hormone release was measured from intact islets. GABA release was monitored by whole-cell patch-clamp measurements after adenoviral expression of α1β1 GABAAR subunits. The subcellular localization of GABA was explored by electron microscopy. The effects of GABA on electrical activity were determined by perforated patch whole-cell recordings. RESULTS--PCR analysis detected relatively high levels of the mRNAs encoding GABAAR α2, γ2 γ2, and πsubunits in human islets. Patch-clamp experiments revealed expression of GABA[sub A]R Cl- channels in 52% of β-cells (current density 9 pA/pF), 91% of δ-cells (current density 148 pA/pF), and 6% of α-cells (current density 2 pA/pF). Expression of GABA[sub A]R subunits in islet cells was confirmed by immunohistochemistry. β-Cells secreted GABA both by glucose-dependent exocytosis of insulin-containing granules and by a glucose-independent mechanism. The GABAAR antagonist SR95531 inhibited insulin secretion elicited by 6 mmol/l glucose. Application of GABA depolarized β-cells and stimulated action potential firing in β-cells exposed to glucose. CONCLUSIONS--Signaling via GABA and GABAAR constitutes an autocrine positive feedback loop in human β-cells. The presence of GABAAR in non-β-cells suggests that GABA may also be involved in the regulation of somatostatin and glucagon secretion. Diabetes 59:1694-1701, 2010 [ABSTRACT FROM AUTHOR]

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[sup 2+]][sub i]) was measured by microfluorimetry. Total internal reflection fluorescence microscope (TIRFM) was used for optical imaging of exocytosis and submembrane depolarization-evoked [Ca[sup 2+]][sub 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 β-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[sup 2+] ][sub 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 ins) less affected. The loss of rapid exocytosis correlated with dispersion of Ca[sup 2+] entry in HFD β-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[sup 2+] entry from exocytosis. These observations suggest a novel explanation to the well-established link between obesity and diabetes. Diabetes 59:1192-1201, 2010 [ABSTRACT FROM AUTHOR]