Modes of exocytosis and electrogenesis underlying canine biphasic insulin secretion.

Biphasic insulin secretion in response to glucose consists of a transient first phase followed by a progressive second phase. It is a well described feature of whole perfused pancreases as well as isolated pancreatic islets of Langerhans. Applying to single cell assays of exocytosis (capacitance monitoring and amperometry) to single canine Beta-cells we have examined the time courses of granule exocytosis in response to voltage-clamp depolarizations that mimic two modes of glucose-induced electrical activity, and then compared these to biphasic insulin secretion. Action potentials evoked in short trains at frequencies similar those recorded during first phase insulin secretion trigger phasic exocytosis from a small pool of insulin granules that are likely docked near voltage-activated Ca²âº channels. In contrast, prolonged voltage-clamp pulses mimicking plateau depolarizations occur during second phase insulin secretion and trigger tonic or continuous exocytosis. Comparing the latter results with ones obtained using photorelease of caged Ca²âº in other insulin-secreting cells, we suggest that tonic exocytosis likely results from granule release from a highly Ca²âº-sensitive pool of insulin granules, likely located further from Ca²âº channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine Beta-cells we propose that two distinct modes of exocytosis, tuned to two types of electrical activity, may underlay biphasic insulin secretion.

The isolated pancreatic islet as a micro-organ and its transplantation to cure diabetes: celebrating the legacy of Paul Lacy.

Over the past three decades the pancreatic islet of Langerhans has taken center stage as an endocrine micro-organ whose glucoregulatory function is highly explicable on the basis of the increasingly well understood activities of three highly interactive secretory cells. Islet dysfunction underlies both type 1 and type 2 diabetes mellitus (DM); its protection from immune attack and gluco-and lipo-toxicity may prevent the development of DM; and its replacement by non-surgical transplantation may be curative of DM. During a career marked by vision, focus and tenacity, Paul Lacy contributed substantially to the development of each of these concepts. In this review we focus on Lacy's contribution to the development of the concept of the islet as a micro-organ, how this foreshadowed our current detailed understanding of single cell function and cell-cell interactions and how this led to a reduced model of islet function encouraging islet transplantation. Next, we examine how clinical allotransplantation, first undertaken by Lacy, has contributed to a more complex view of the interaction of islet endocrine cells with its circulation and neighboring tissues, both "in situ" and after transplantation. Lastly, we consider recent developments in some alternative approaches to treatment of DM that Lacy could glimpse on the horizon but did not have the chance to participate in.

Unifying concepts in stimulus-secretion coupling in endocrine cells and some implications for therapeutics.

Stimulus-secretion coupling (SSC) in endocrine cells remains underappreciated as a subject for the study/teaching of general physiology. In the present article, we review key new electrophysiological, electrochemical, and fluorescence optical techniques for the study of exocytosis in single cells that have made this a fertile area for recent research. Based on findings using these techniques, we developed a model of SSC for adrenal chromaffin cells that blends features of Ca(2+) entry-dependent SSC (characteristic of neurons) with G protein receptor-coupled, Ca(2+) release-dependent, and second messenger-dependent SSC (characteristic of epithelial exocrine cells and nucleated blood cells). This model requires two distinct pools of secretory graunules with differing Ca(2+) sensitivities. We extended this model to account for SSC in a wide variety of peripheral and hypothalamic/pituitary-based endocrine cells. These include osmosensitive magnocellular neurosecretory cells releasing antidiuretic hormone, stretch-sensitive atrial myocytes secreting atrial natriuretic peptide, K(+)-sensitive adrenal glomerulosa cells secreting aldosterone, Ca(2+)-sensitive parathyroid chief cells secreting parathyroid hormone, and glucose-sensitive beta- and alpha-cells of pancreatic islets secreting insulin and glucagon, respectively. We conclude this article with implications of this approach for pathophysiology and therapeutics, including defects in chief cell Ca(2+) sensitivity, resulting in the hyperparathyroidism of renal disease, and defects in biphasic insulin secretion, resulting in diabetes mellitus.

Electrical activity and exocytotic correlates of biphasic insulin secretion from beta-cells of canine islets of Langerhans: contribution of tuning two modes of Ca2+ entry-dependent exocytosis to two modes of glucose-induced electrical activity.

Biphasic insulin secretion in response to glucose, consisting of a transient first phase followed by a progressive second phase, is well described in pancreatic islets. Using single canine beta-cells we have compared the time courses of electrical activity and insulin granule exocytosis to biphasic insulin secretion. Short trains of action potentials, similar those found during first phase insulin secretion, trigger phasic exocytosis from a small pool of insulin granules, likely an immediately releasable pool docked near voltage activated Ca(2+) channels. In contrast, plateau depolarizations to between -35 and -20 mV resembling those during second phase insulin secretion, trigger tonic exocytosis from a larger pool of insulin granules, likely a highly Ca(2+)-sensitive pool farther from Ca(2+) channels. Both phasic and tonic modes of exocytosis are enhanced by glucose, via its metabolism. Hence, in canine beta-cells two distinct components of exocytosis, tuned to two components of electrical activity, may contribute significantly to biphasic insulin secretion.

Phasic and tonic modes of depolarization-exocytosis coupling in beta-cells of porcine islets of Langerhans.

In response to depolarizations that open voltage dependent Ca2+ channels single porcine beta-cells display heterogeneous time courses of exocytosis. Some cells display phasic exocytosis that is triggered by individual or short burst of action potentials typically characteristic of glucose-induced electrical activity or brief voltage clamp depolarization. Other cells, singularly or additionally, display tonic exocytosis that (i) is triggered during prolonged (up to seconds-long) depolarizations to voltages (-30 to -20 mV), and (ii) coincides with rises in global cytosolic [Ca2+] >500 nM. We suggest that tonic exocytosis (i) likely results from a recently described pool of granules that is more Ca2+ sensitive and less co-localized with voltage-sensitive Ca2+ entry channels than that contributing to phasic exocytosis and (ii) helps tune exocytosis to glucose-induced electrical activity when the latter consists of spike activity followed by intervals of plateau depolarization to nearly -20 mV.

Ion channels underlying stimulus-exocytosis coupling and its cell-to-cell heterogeneity in beta-cells of transplantable porcine islets of Langerhans.

Given the growing interest in porcine islets as model tissue for studying the pathogenesis of human diabetes mellitus and its treatment by transplantation, we investigated stimulus-exocytosis coupling in single porcine beta-cells using patch clamp electrophysiology, Ca2+ imaging, capacitance tracking and amperometry. We establish that porcine beta-cells display several features prominently seen in beta-cells from human islets of Langerhans. These include: (i) wide heterogeneity of electrical responsiveness to glucose; (ii) dependence of action potential activity on voltage-dependent Na(+) as well as high voltage activated Ca2+ current; (iii) heterogeneity of time course of depolarization-evoked insulin granule exocytosis; and (iv) the dependence of vigorous single cell electrical activity and insulin granule exocytosis on the presence of agents that enhance cytosolic cAMP concentration. These findings promote the usefulness of porcine beta-cells as a model for studying beta-cell function in large mammals, including humans, as well as an appropriate source of tissue for xenotransplantation.

Electrophysiological and immunocytochemical evidence for P2X purinergic receptors in pancreatic beta cells.

OBJECTIVES: Glucose-induced insulin secretion from pancreatic beta cells is modulated by several hormones and transmitters, namely adenosine triphosphate (ATP) via purinergic receptors. Although P2Y receptors are well documented in beta cells, the presence of P2X receptors remains elusive. We present the first electrophysiological evidence for the presence of P2X receptors in single beta cells of different species. METHODS: Ionic currents were recorded from voltage-clamped beta cells near their resting potential using the perforated (nystatin) whole-cell patch-clamp configuration. Receptors were detected by immunocytochemistry. RESULTS: When bathed in substimulatory (2 mM) glucose, mouse beta cells, isolated from islets displaying immunochemical colocalization of P2X1 or P2X3 receptors and insulin, developed large (approximately 250 pA/pF), rapidly activating, and then biexponentially decaying (tau1, approximately 20 milliseconds/tau2, approximately 1 second) inward currents on exposure to micromolar concentrations of ATP and alpha,beta-methylene ATP. The ATP also evoked inward currents (100-300 pA/pF) from porcine and human beta cells, albeit with a slower and more complex inactivation pattern. CONCLUSIONS: The ATP-gated ion channels are present in pancreatic beta cells from different species. Specifically, mouse beta cells express rapidly desensitizing P2X1 and P2X3 receptors. Paracrine or neural activation of these receptors may contribute to the initial outburst of glucose- or acetylcholine-evoked insulin release, thus enhancing the islet secretory response.

Ca2+ controls slow NAD(P)H oscillations in glucose-stimulated mouse pancreatic islets.

Exposure of pancreatic islets of Langerhans to physiological concentrations of glucose leads to secretion of insulin in an oscillatory pattern. The oscillations in insulin secretion are associated with oscillations in cytosolic Ca(2+) concentration ([Ca(2+)](c)). Evidence suggests that the oscillations in [Ca(2+)](c) and secretion are driven by oscillations in metabolism, but it is unclear whether metabolic oscillations are intrinsic to metabolism or require Ca(2+) feedback. To address this question we explored the interaction of Ca(2+) concentration and islet metabolism using simultaneous recordings of NAD(P)H autofluorescence and [Ca(2+)](c), in parallel with measurements of mitochondrial membrane potential (DeltaPsi(m)). All three parameters responded to 10 mm glucose with multiphasic dynamics culminating in slow oscillations with a period of approximately 5 min. This was observed in approximately 90% of islets examined from various mouse strains. NAD(P)H oscillations preceded those of [Ca(2+)](c), but their upstroke was often accelerated during the increase in [Ca(2+)](c), and Ca(2+) influx was a prerequisite for their generation. Prolonged elevations of [Ca(2+)](c) augmented NAD(P)H autofluorescence of islets in the presence of 3 mm glucose, but often lowered NAD(P)H autofluorescence of islets exposed to 10 mm glucose. Comparable rises in [Ca(2+)](c) depolarized DeltaPsi(m). The NAD(P)H lowering effect of an elevation of [Ca(2+)](c) was reversed during inhibition of mitochondrial electron transport. These findings reveal the existence of slow oscillations in NAD(P)H autofluorescence in intact pancreatic islets, and suggest that they are shaped by Ca(2+) concentration in a dynamic balance between activation of NADH-generating mitochondrial dehydrogenases and a Ca(2+)-induced decrease in NADH. We propose that a component of the latter reflects mitochondrial depolarization by Ca(2+), which reduces respiratory control and consequently accelerates oxidation of NADH.

Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans.

Studies of stimulus-secretion coupling in human beta-cells have been hampered by poor availability of tissue due to variability of the supply of cadaver pancreati and in the adequacy of enzymatic liberation of islets as well as by the shunting of isolates into transplant trials. Here we establish that aliquots of islets, several from high-quality but low-yield islet isolates (50,000-100,000 islets), cryopreserved and then thawed as needed, respond to glucose in a calcium- and metabolic-dependent fashion. Insulin secretion is modulated by blockers of voltage-dependent Na+ and Ca2+ channels, and paracrine hormones (glucagon and somatostatin) in manners indistinguishable from fresh tissue preparations. Using single-cell electrophysiological and electrochemical assays we demonstrate that single beta-cells from cryopreserved islets display (1) stimulus-depolarization coupling based on rapid closure of K+ (ATP) channels; (2) action potential electrogenesis with upstrokes based on voltage-dependent Na and Ca currents; and (3) Ca2+ entry-mediated depolarization-exocytosis coupling sustained over multiple bouts of stimulation and modulated by paracrine hormones. All of these features are indistinguishable from those seen in single cells from freshly harvested islets. These results support the utility of cryopreservation, even of low-yield but functional isolates, as a means of ensuring a steady source of repeatedly accessible tissue for research on normal and diabetic islets.

alpha-Latrotoxin increases spontaneous and depolarization-evoked exocytosis from pancreatic islet beta-cells.

alpha-Latrotoxin (alpha-LT), a potent excitatory neurotoxin, increases spontaneous, as well as action potential-evoked, quantal release at nerve terminals and increases hormone release from excitable endocrine cells. We have investigated the effects of alpha-LT on single human, mouse and canine beta-cells. In isolated and combined measurements, alpha-LT, at nanomolar concentrations, induces: (i) rises in cytosolic Ca(2+), into the micromolar range, that are dependent on extracellular Ca(2+); (ii) large conductance non-selective cation channels; and (iii) Ca(2+)-dependent insulin granule exocytosis, measured as increases in membrane capacitance and quantal release of preloaded serotonin. Furthermore, at picomolar concentrations, alpha-LT potentiates depolarization-induced exocytosis often without evidence of inducing channel activity or increasing cytosolic Ca(2+). These results strongly support the hypothesis that alpha-LT, after binding to specific receptors, has at least two complementary modes of action on excitable cells. (i) alpha-LT inserts into the plasma membrane to form Ca(2+) permeable channels and promote Ca(2+) entry thereby triggering Ca(2+)-dependent exocytosis in unstimulated cells. (ii) At lower concentrations, where its channel forming activity is hardly evident, alpha-LT augments depolarization-evoked exocytosis probably by second messenger-induced enhancement of the efficiency of the vesicle recruitment or vesicle fusion machinery. We suggest that both modes of action enhance exocytosis from a newly described highly Ca(2+)-sensitive pool of insulin granules activated by global cytosolic Ca(2+) concentrations in the range of approximately 1 microm.

RyR2 and calpain-10 delineate a novel apoptosis pathway in pancreatic islets.

Cells are programmed to die when critical signaling and metabolic pathways are disrupted. Inhibiting the type 2 ryanodine receptor (RyR2) in human and mouse pancreatic beta-cells markedly increased apoptosis. This mode of programmed cell death was not associated with robust caspase-3 activation prompting a search for an alternative mechanism. Increased calpain activity and calpain gene expression suggested a role for a calpain-dependent death pathway. Using a combination of pharmacological and genetic approaches, we demonstrated that the calpain-10 isoform mediated ryanodine-induced apoptosis. Apoptosis induced by the fatty acid palmitate and by low glucose also required calpain-10. Ryanodine-induced calpain activation and apoptosis were reversed by glucagon-like peptide or short-term exposure to high glucose. Thus RyR2 activity seems to play an essential role in beta-cell survival in vitro by suppressing a death pathway mediated by calpain-10, a type 2 diabetes susceptibility gene with previously unknown function.

Ryanodine receptors in human pancreatic beta cells: localization and effects on insulin secretion.

It is clear that pancreatic beta-cell dysfunction, including basal hyperinsulinemia and reduced insulin release in response to glucose, is a key determinant of disease progression in type 2 diabetes, but the underlying molecular defects are not known. In diabetes, the expression and function of ryanodine receptor (RyR) Ca2+ release channels are reduced. The present studies were undertaken to define the subcellular location and role of RyR in the control of stimulated and basal insulin release from human pancreatic beta cells. Using confocal microscopy, we observed RyR immunoreactivity in a vesicular pattern. RyRs did not colocalize with insulin secretory granules but partially colocalized with endosomes. Direct activation with nanomolar concentrations of ryanodine evoked increases in cytosolic Ca2+ that were coupled to transient insulin release. Insulin release stimulated by 1 nM ryanodine was sensitive to BAPTA-AM preincubation but independent of thapsigargin-sensitive endoplasmic reticulum (ER) Ca2+ pools. Blocking RyRs with micromolar concentrations of ryanodine led to BAPTA-resistant insulin release that was not associated with an increase in cytosolic Ca2+, which implicated alterations in luminal Ca2+. However, neither Ca2+ signals nor insulin release stimulated by glucose was blocked by 10-50 microM ryanodine, which suggests that the CD38/cyclic ADP-ribose/RyR pathway is not a primary mechanism of glucose action in nontransformed beta cells. We provide the first evidence that RyRs directly control insulin secretion in primary beta cells. Unexpectedly, stimulation of insulin secretion by ryanodine occurs independently of glucose and by two mechanisms, including a novel cytosolic Ca2+-independent mechanism likely involving changes in Ca2+ within the lumens of non-ER organelles, such as endosomes.

Increased islet apoptosis in Pdx1+/- mice.

Mice with 50% Pdx1, a homeobox gene critical for pancreatic development, had worsening glucose tolerance with age and reduced insulin release in response to glucose, KCl, and arginine from the perfused pancreas. Surprisingly, insulin secretion in perifusion or static incubation experiments in response to glucose and other secretagogues was similar in islets isolated from Pdx1(+/-) mice compared with Pdx1(+/+) littermate controls. Glucose sensing and islet Ca(2+) responses were also normal. Depolarization-evoked exocytosis and Ca(2+) currents in single Pdx1(+/-) cells were not different from controls, arguing against a ubiquitous beta cell stimulus-secretion coupling defect. However, isolated Pdx1(+/-) islets and dispersed beta cells were significantly more susceptible to apoptosis at basal glucose concentrations than Pdx1(+/+) islets. Bcl(XL) and Bcl-2 expression were reduced in Pdx1(+/-) islets. In vivo, increased apoptosis was associated with abnormal islet architecture, positive TUNEL, active caspase-3, and lymphocyte infiltration. Although similar in young mice, both beta cell mass and islet number failed to increase with age and were approximately 50% less than controls by one year. These results suggest that an increase in apoptosis, with abnormal regulation of islet number and beta cell mass, represents a key mechanism whereby partial PDX1 deficiency leads to an organ-level defect in insulin secretion and diabetes.

Nicotinic acid-adenine dinucleotide phosphate-sensitive calcium stores initiate insulin signaling in human beta cells.

Recent studies suggest a role for autocrine insulin signaling in beta cells, but the mechanism and function of insulin-stimulated Ca(2+) signals is uncharacterized. We examined Ca(2+)-dependent insulin signaling in human beta cells. Two hundred nanomolar insulin elevated [Ca(2+)](c) to 284 +/- 27 nM above baseline in approximately 30% of Fura-4F-loaded cells. Insulin evoked multiple Ca(2+) signal waveforms, 60% of which included oscillations. Although the amplitude of Ca(2+) signals was dose-dependent between 0.002 and 2,000 nM, the percentage of cells responding was highest at 0.2 nM insulin, suggesting the interaction of stimulatory and inhibitory pathways. Ca(2+)-free solutions did not affect the initiation of insulin-stimulated Ca(2+) signals, but abolished the second phase of plateaus/oscillations. Likewise, inositol 1,4,5-trisphosphate (IP(3)) receptor antagonists xestospongin C and caffeine selectively blocked the second phase, but not the initiation of insulin signaling. Thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ) blocked insulin signaling, implicating sarcoplasmic/endoplasmic Ca(2+)-ATPase (SERCA)-containing Ca(2+) stores. Insulin-stimulated Ca(2+) signals were insensitive to ryanodine. Injection of the CD38-derived Ca(2+) mobilizing metabolite, nicotinic acid-adenine dinucleotide phosphate (NAADP), at nanomolar concentrations, evoked oscillatory Ca(2+) signals that could be initiated in the presence of ryanodine, xestospongin C, and Ca(2+)-free solutions. Desensitizing concentrations of NAADP abolished insulin-stimulated Ca(2+) signals. Insulin-stimulated Ca(2+) signals led to a Ca(2+)-dependent increase in cellular insulin contents, but not secretion. These data reveal the complexity of insulin signal transduction and function in human beta cells and demonstrate functional NAADP-sensitive Ca(2+) stores in a human primary cultured cell type.