Suppression of Sulfonylurea- and Glucose-Induced Insulin Secretion In Vitro and In Vivo in Mice Lacking the Chloride Transport Protein ClC-3

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

In pancreatic β-cells myosin 1b regulates glucose-stimulated insulin secretion by modulating an early step in insulin granule trafficking from the Golgi

Pancreatic β-cells secrete insulin, which controls blood glucose levels, and defects in insulin secretion are responsible for diabetes mellitus. The actin cytoskeleton and some myosins support insulin granule trafficking and release, although a role for the class I myosin Myo1b, an actin- and membrane-associated load-sensitive motor, in insulin biology is unknown. We found by immunohistochemistry that Myo1b is expressed in islet cells of the rat pancreas. In cultured rat insulinoma 832/13 cells, Myo1b localized near actin patches, the trans-Golgi network (TGN) marker TGN38, and insulin granules in the perinuclear region. Myo1b depletion by small interfering RNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS) and led to the accumulation of (pro)insulin secretory granules (SGs) at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin, Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing SGs budding from the TGN. The studies indicate for the first time that in pancreatic β-cells Myo1b controls GSIS at least in part by mediating an early stage in insulin granule trafficking from the TGN.     

Exocyst Sec5 Regulates Exocytosis of Newcomer Insulin Granules Underlying Biphasic Insulin Secretion

The exocyst complex subunit Sec5 is a downstream effector of RalA-GTPase which promotes RalA-exocyst interactions and exocyst assembly, serving to tether secretory granules to docking sites on the plasma membrane. We recently reported that RalA regulates biphasic insulin secretion in pancreatic islet β cells in part by tethering insulin secretory granules to Ca²⁺ channels to assist excitosome assembly. Here, we assessed β cell exocytosis by patch clamp membrane capacitance measurement and total internal reflection fluorescence microscopy to investigate the role of Sec5 in regulating insulin secretion. Sec5 is present in human and rodent islet β cells, localized to insulin granules. Sec5 protein depletion in rat INS-1 cells inhibited depolarization-induced release of primed insulin granules from both readily-releasable pool and mobilization from the reserve pool. This reduction in insulin exocytosis was attributed mainly to reduction in recruitment and exocytosis of newcomer insulin granules that undergo minimal docking time at the plasma membrane, but which encompassed a larger portion of biphasic glucose stimulated insulin secretion. Sec5 protein knockdown had little effect on predocked granules, unless vigorously stimulated by KCl depolarization. Taken together, newcomer insulin granules in β cells are more sensitive than predocked granules to Sec5 regulation.     

Regulation of Insulin Secretion in Islets of Langerhans by Ca<Superscript>2+</Superscript>Channels

Insulin secretion from β-cells of the pancreatic islets of Langerhans is triggered by Ca²⁺ influx through voltage-dependent Ca²⁺ channels. Electrophysiological and molecular studies indicate that β-cells express several subtypes of these channels. This review discusses their roles in regulating insulin secretion, focusing on recent studies using β-cells, exogenous expression systems, and Ca²⁺ channel knockout mice. These investigations reveal that L-type Ca²⁺ channels in the β-cell physically interact with the secretory apparatus by binding to synaptic proteins on the plasma membrane and insulin granule. As a result, Ca²⁺ influx through L-type channels efficiently and rapidly stimulates release of a pool of insulin granules in close contact with the channels. Thus, L-type Ca²⁺ channel activity is preferentially coupled to exocytosis in the β-cell, and plays a critical role in regulating the dynamics of insulin secretion. Non-L-type channels carry a significant portion of the total voltage-dependent Ca²⁺ current in β-cells and cell lines from some species, but nevertheless account for only a small fraction of insulin secretion. These channels may regulate exocytosis indirectly by affecting membrane potential or second messenger signaling pathways. Finally, voltage-independent Ca²⁺ entry pathways and their potential roles in β-cell function are discussed. The emerging picture is that Ca²⁺ channels regulate insulin secretion at multiple sites in the stimulus-secretion coupling pathway, with the specific role of each channel determined by its biophysical and structural properties.This revised version was published online in June 2005 with a corrected cover date.     

The Proinsulin C-peptide—A Multirole Model

The C-peptide links the insulin A and B chains in proinsulin, providing thereby a means to promote their efficient folding and assembly in the endoplasmic reticulum during insulin biosynthesis. It then facilitates the intracellular transport, sorting, and proteolytic processing of proinsulin into biologically active insulin in the maturing secretory granules of the β cells. These manifold functions impose significant constraints on the C-peptide structure that are conserved in evolution. After cleavage of proinsulin, the intact C-peptide is stored with insulin in the soluble phase of the secretory granules and is subsequently released in equimolar amounts with insulin, providing a useful independent indicator of insulin secretion. This brief review highlights many aspects of its roles in biosynthesis, as a prelude to consideration of its possible additional role(s) as a physiologically active peptide after its release with insulin into the circulation in vivo.     

Defective Secretion of Islet Hormones in Chromogranin-B Deficient Mice

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

BIG3 inhibits insulin granule biogenesis and insulin secretion

While molecular regulation of insulin granule exocytosis is relatively well understood, insulin granule biogenesis and maturation and its influence on glucose homeostasis are relatively unclear. Here, we identify a novel protein highly expressed in insulin-secreting cells and name it BIG3 due to its similarity to BIG/GBF of the Arf-GTP exchange factor (GEF) family. BIG3 is predominantly localized to insulin- and clathrin-positive trans-Golgi network (TGN) compartments. BIG3-deficient insulin-secreting cells display increased insulin content and granule number and elevated insulin secretion upon stimulation. Moreover, BIG3 deficiency results in faster processing of proinsulin to insulin and chromogranin A to β-granin in β-cells. BIG3-knockout mice exhibit postprandial hyperinsulinemia, hyperglycemia, impaired glucose tolerance, and insulin resistance. Collectively, these results demonstrate that BIG3 negatively modulates insulin granule biogenesis and insulin secretion and participates in the regulation of systemic glucose homeostasis.     

The GTPase RalA Regulates Different Steps of the Secretory Process in Pancreatic β-Cells

Background

RalA and RalB are multifuntional GTPases involved in a variety of cellular processes including proliferation, oncogenic transformation and membrane trafficking. Here we investigated the mechanisms leading to activation of Ral proteins in pancreatic β-cells and analyzed the impact on different steps of the insulin-secretory process.

Methodology/Principal Findings

We found that RalA is the predominant isoform expressed in pancreatic islets and insulin-secreting cell lines. Silencing of this GTPase in INS-1E cells by RNA interference led to a decrease in secretagogue-induced insulin release. Real-time measurements by fluorescence resonance energy transfer revealed that RalA activation in response to secretagogues occurs within 3–5 min and reaches a plateau after 10–15 min. The activation of the GTPase is triggered by increases in intracellular Ca²⁺ and cAMP and is prevented by the L-type voltage-gated Ca²⁺ channel blocker Nifedipine and by the protein kinase A inhibitor H89. Defective insulin release in cells lacking RalA is associated with a decrease in the secretory granules docked at the plasma membrane detected by Total Internal Reflection Fluorescence microscopy and with a strong impairment in Phospholipase D1 activation in response to secretagogues. RalA was found to be activated by RalGDS and to be severely hampered upon silencing of this GDP/GTP exchange factor. Accordingly, INS-1E cells lacking RalGDS displayed a reduction in hormone secretion induced by secretagogues and in the number of insulin-containing granules docked at the plasma membrane.

Conclusions/Significance

Taken together, our data indicate that RalA activation elicited by the exchange factor RalGDS in response to a rise in intracellular Ca²⁺ and cAMP controls hormone release from pancreatic β-cell by coordinating the execution of different events in the secretory pathway.     

Intracellular Serotonin Modulates Insulin Secretion from Pancreatic β-Cells by Protein Serotonylation

While serotonin (5-HT) co-localization with insulin in granules of pancreatic β-cells was demonstrated more than three decades ago, its physiological role in the etiology of diabetes is still unclear. We combined biochemical and electrophysiological analyses of mice selectively deficient in peripheral tryptophan hydroxylase (Tph1−/−) and 5-HT to show that intracellular 5-HT regulates insulin secretion. We found that these mice are diabetic and have an impaired insulin secretion due to the lack of 5-HT in the pancreas. The pharmacological restoration of peripheral 5-HT levels rescued the impaired insulin secretion in vivo. These findings were further evidenced by patch clamp experiments with isolated Tph1−/− β-cells, which clearly showed that the secretory defect is downstream of Ca²⁺-signaling and can be rescued by direct intracellular application of 5-HT via the clamp pipette. In elucidating the underlying mechanism further, we demonstrate the covalent coupling of 5-HT by transglutaminases during insulin exocytosis to two key players in insulin secretion, the small GTPases Rab3a and Rab27a. This renders them constitutively active in a receptor-independent signaling mechanism we have recently termed serotonylation. Concordantly, an inhibition of such activating serotonylation in β-cells abates insulin secretion. We also observed inactivation of serotonylated Rab3a by enhanced proteasomal degradation, which is in line with the inactivation of other serotonylated GTPases. Our results demonstrate that 5-HT regulates insulin secretion by serotonylation of GTPases within pancreatic β-cells and suggest that intracellular 5-HT functions in various microenvironments via this mechanism in concert with the known receptor-mediated signaling.     

3D FIB-SEM reconstruction of microtubule–organelle interaction in whole primary mouse β cells

Microtubules play a major role in intracellular trafficking of vesicles in endocrine cells. Detailed knowledge of microtubule organization and their relation to other cell constituents is crucial for understanding cell function. However, their role in insulin transport and secretion is under debate. Here, we use FIB-SEM to image islet β cells in their entirety with unprecedented resolution. We reconstruct mitochondria, Golgi apparati, centrioles, insulin secretory granules, and microtubules of seven β cells, and generate a comprehensive spatial map of microtubule–organelle interactions. We find that microtubules form nonradial networks that are predominantly not connected to either centrioles or endomembranes. Microtubule number and length, but not microtubule polymer density, vary with glucose stimulation. Furthermore, insulin secretory granules are enriched near the plasma membrane, where they associate with microtubules. In summary, we provide the first 3D reconstructions of complete microtubule networks in primary mammalian cells together with evidence regarding their importance for insulin secretory granule positioning and thus their supportive role in insulin secretion.     

Impaired Glucose Tolerance in a Mouse Model of Sidt2 Deficiency

Sidt2 was identified as a novel integral lysosomal membrane protein recently. We generated global Sidt2 knockout mice by gene targeting. These mice have a comparatively higher random and fasting glucose concentration. Intraperitoneal and oral glucose tolerance tests in Sidt2 knockout mice indicated glucose intolerance and decreased serum insulin level. Notably, the Sidt2^−/− mice had hypertrophic islets compared with control mice. By Western blot and immunofluorescence, Sidt2^−/− mouse islets were shown to have increased insulin protein, which actually contained more insulin secretory granules than their controls, demonstrated by electromicroscopy. Consistent with the in vivo study, isolated islet culture from the Sidt2^−/− mice produced less insulin when stimulated by a high concentration of glucose or a depolarizing concentration of KCl. Under electromicroscope less empty vesicles and more mature ones in Sidt2^−/− mice islets were observed, supporting impaired insulin secretory granule release. In conclusion, Sidt2 may play a critical role in the regulation of mouse insulin secretory granule secretion.     

Granular detail of β cell structures for insulin secretion

Pancreatic β cells secrete insulin in response to increased glucose concentrations. Müller et al. (2021. J. Cell Biol. https://doi.org/10.1083/jcb.202010039) use 3D FIB-SEM to study the architecture of these cells and to elucidate how glucose stimulation remodels microtubules to control insulin secretory granule exocytosis.

The pancreatic islet β cell is a prototypical model for regulated protein secretion, which has been studied extensively because of its importance for diabetes in humans. Upon stimulation by increased glucose concentrations, β cells mobilize insulin-containing secretory vesicles to the cell surface. These “insulin secretory granules” fuse at the plasma membrane to release insulin into the circulation. Insulin then acts on the liver to inhibit glucose production and on muscle and fat cells to stimulate glucose uptake, thus returning blood glucose concentrations to a narrow physiological range. During the development of diabetes, this negative feedback system fails. In type 1 diabetes, β cells are destroyed by an autoimmune process; in type 2 diabetes, attenuated insulin action (“insulin resistance”) occurs together with impaired β cell function. In both cases, blood glucose concentrations rise above the normal range. Overall, glucose homeostasis requires a delicate balance between glucose-stimulated insulin secretion from β cells and insulin-stimulated effects on glucose metabolism in liver, muscle, fat, and other cell types.Each β cell contains 5,000 to ≥10,000 insulin secretory granules, and acute glucose stimulation causes exocytosis of only 1–2% of this pool (1, 2). Both readily releasable and reserve pools of granules contribute to insulin secretion. Classically, the readily releasable pool has been considered as vesicles that are docked at the plasma membrane; the reserve pool, which is larger and more important, resides deeper within the cell and relies on microtubule-based transport to reach the cell surface. Yet, other data show that newly synthesized insulin is preferentially released, and aged insulin granules are targeted for degradation in lysosomes, implying that microtubules play a more complicated role in granule trafficking (3). In the setting of insulin resistance, the flux of insulin through the secretory pathway is increased. Demands are placed upon the machinery for folding of the insulin precursor, proinsulin, for its proteolytic conversion to produce insulin, and for insulin secretory granule maturation. As well, in type 2 diabetes, lipids and other metabolites act directly on β cells to impair glucose-stimulated insulin secretion. What steps are affected by this critical pathophysiologic insult is not well understood, in part because basic mechanisms by which glucose stimulation remodels microtubules to promote insulin release remain undefined.To better understand these processes, Müller et al. used focused ion beam scanning electron microscopy (FIB-SEM) to image microtubules, insulin secretory granules, and other organelles in whole primary mouse β cells and to study the effects of glucose stimulation on these structures (4). Together with advances in sample preparation, image segmentation, and analysis, FIB-SEM is uniquely suited to this task. The resulting 3D images have a voxel size of 4 × 4 × 4 nm and encompass volumes of 20–30 µm in x, y, and z dimensions. This is sufficient to image microtubules, which have an outer diameter of ~25 nm, in whole β cells, which are 10–20 µm in diameter. Moreover, the imaging was performed on intact islets, rather than on dissociated cells, which may better preserve insulin secretory granule dynamics. The images captured seven β cells in all, three in a low-glucose (unstimulated) condition and four in the glucose-stimulated state. Finally, the images were quantified in a way that controlled for the overall geometry of the cells.The data show that the β cell microtubule network is nonradial, dense, tortuous, and mostly not connected to either centrioles or the Golgi complex, so that microtubules appear to be freely positioned in the cytosol (Fig. 1). This is similar to other differentiated cells (5) but in contrast to previous data suggesting that in β cells most microtubules originate at the Golgi (6). The microtubule cytoskeleton negatively regulates insulin granule exocytosis in unstimulated cells (7). Yet, the FIB-SEM data suggest that the effect of glucose is not simply to disinhibit this effect. Glucose stimulated an approximately threefold increase in the number of microtubules and an approximately threefold decrease in the average length of each microtubule, so that total tubulin polymer density remained unchanged (4). The images also show that microtubule ends and insulin secretory granules are enriched near the plasma membrane and indicate an important role for microtubules in positioning the granules for exocytosis. Intriguingly, glucose stimulation did not cause any marked change in the association of secretory granules with microtubules, suggesting that it may act by other mechanisms to increase microtubule-based transport (3). The data also demonstrate an increase in the number of secretory granules near the Golgi in glucose-stimulated cells, raising the possibility that glucose may promote budding of nascent secretory vesicles at the trans-Golgi network. If so, it is not yet clear whether this is a direct effect or if it is secondary to increased flux through the secretory pathway.Open in a separate windowFigure 1.Regulation of β cell architecture by glucose. In low-glucose conditions (left panel), microtubules form a dense, nonradial network. The tubules are mostly not anchored to the Golgi complex or to centrosomes but are associated with insulin secretory granules near the plasma membrane (PM). After stimulation with high-glucose concentrations (right panel), the microtubules are shorter and more numerous, and an increased density of vesicles near the Golgi was observed.The observation that glucose stimulation results in shorter, more numerous microtubules, without changing tubulin polymer density, suggests a possible role for severing enzymes such as katanin, spastin, or fidgetin (8). Whether and how such enzymes may be stimulated by glucose is not known. Glucose stimulates insulin secretion by increasing the ATP/ADP ratio, in part due to local ATP generation by pyruvate kinase, as well as by oxidative phosphorylation (9). Although these microtubule-severing enzymes are AAA ATPases, it is not clear that ATP availability acts physiologically to regulate their activities. Other data show that in β cells, glucose-responsive kinases phosphorylate tau, causing it to dissociate from microtubule plus ends to destabilize microtubules and to promote remodeling (10). Could such kinases regulate severing enzymes as well?The technological achievements of Müller et al. are impressive, and the work will serve as a model for the analysis of cellular architecture in other cell types. In β cells, the results may be extended by using FIB-SEM to study effects of various genetic manipulations, by using EM-visible tags, and by examining diabetes models. It may be informative to image granules of different ages or to use drugs to manipulate microtubules or membrane lipids, or that act on β cells to enhance insulin secretion (3). In this study, Müller and colleagues used a 1-h high-glucose stimulation, but it may be interesting to test other time points to determine the effects of more acute versus chronic glucose exposure. Although FIB-SEM can image only fixed, static cells, the work will complement other studies using super-resolution imaging and live cell microscopy. Aside from these future directions, it is worthwhile to pause and celebrate the present work, which is the first to reconstruct the entire 3D architecture of the microtubule network in a primary mammalian cell during interphase. The movies in the supplement are gorgeous. The work bodes well for the future of FIB-SEM and will stimulate new directions to understand both diabetes physiology and regulated protein secretion.     

Direct identification of prohormone conversion site in insulin-secreting cells

We have localized proinsulin in B cells of human and rat pancreatic islets, using a proinsulin-specific monoclonal antibody revealed by immunocytochemistry. Proinsulin is abundant in Golgi stacks and clathrin-coated secretory granules. It rapidly disappears from these compartments when protein synthesis is inhibited. Depletion of ATP stores prevents movement of proinsulin from the Golgi stacks to the secretory granules; under these conditions, the prohormone in preformed coated granules is converted to insulin, whereas that bound to the Golgi complex is not. Non-coated granules show a low level of proinsulin reactivity under all incubation protocols. These findings provide direct evidence that coated secretory granules are the major, if not the only, cellular site of proinsulin to insulin conversion. They also suggest that the Golgi stack is not involved in conversion, and that intercisternal transport and coated granule formation are hitherto unrecognized energy-requiring steps that precede conversion.     

Sorting ourselves out: seeking consensus on trafficking in the beta-cell

Biogenesis of the regulated secretory pathway in the pancreatic beta-cell involves packaging of products, notably proinsulin, into immature secretory granules derived from the trans -Golgi network. Proinsulin is converted to insulin and C-peptide as granules mature. Secretory proteins not entering granules are conveyed by transport intermediates directly to the plasma membrane for constitutive secretion. One of the co-authors, Peter Arvan, has proposed that in addition, small vesicles bud from granules to traffic to the endosomal system. From there, some proteins are secreted by a (post-granular) constitutive-like pathway. He argues that retention in granules is facilitated by condensation, rendering soluble products (notably C-peptide and proinsulin) more available for constitutive-like secretion. Thus he argues that prohormone conversion is potentially important in secretory granule biogenesis. The other co-author, Philippe Halban, argues that the post-granular secretory pathway is not of physiological relevance in primary beta-cells, and contests the importance of proinsulin conversion for retention in granules. Both, however, agree that trafficking from granules to endosomes is important, purging granules of unwanted newly synthesized proteins and allowing their traffic to other destinations. In this Traffic Interchange, the two co-authors attempt to reconcile their differences, leading to a common vision of proinsulin trafficking in primary and transformed cells.     

Insulin, not C-peptide (proinsulin), is present in crinophagic bodies of the pancreatic B-cell

We have obtained evidence by autoradiography and immunocytochemistry that mature secretory granules of the pancreatic B-cell gain access to a lysosomal compartment (multigranular or crinophagic bodies) where the secretory granule content is degraded. Whereas the mature secretory granule content shows both insulin and C-peptide (proinsulin) immunoreactivities, in crinophagic bodies only insulin, but not C- peptide, immunoreactivity was detectable. The absence of C-peptide (proinsulin) immunoreactivity in multigranular bodies, i.e., in early morphological stages of lysosomal digestion, was compatible with the ready access and breakdown of C-peptide and/or proinsulin by lysosomal degrading enzymes, while the insulin crystallized in secretory granule cores remained relatively protected. However, in the final stage of lysosomal digestion, i.e., in residual bodies where the secretory granule core material is no longer present, insulin immunoreactivity became undetectable. Lysosomal digestion thus appears to be a normal pathway for insulin degradation in the pancreatic B-cell.     

A Novel GLP1 Receptor Interacting Protein ATP6ap2 Regulates Insulin Secretion in Pancreatic Beta Cells

GLP1 activates its receptor, GLP1R, to enhance insulin secretion. The activation and transduction of GLP1R requires complex interactions with a host of accessory proteins, most of which remain largely unknown. In this study, we used membrane-based split ubiquitin yeast two-hybrid assays to identify novel GLP1R interactors in both mouse and human islets. Among these, ATP6ap2 (ATPase H⁺-transporting lysosomal accessory protein 2) was identified in both mouse and human islet screens. ATP6ap2 was shown to be abundant in islets including both alpha and beta cells. When GLP1R and ATP6ap2 were co-expressed in beta cells, GLP1R was shown to directly interact with ATP6ap2, as assessed by co-immunoprecipitation. In INS-1 cells, overexpression of ATP6ap2 did not affect insulin secretion; however, siRNA knockdown decreased both glucose-stimulated and GLP1-induced insulin secretion. Decreases in GLP1-induced insulin secretion were accompanied by attenuated GLP1 stimulated cAMP accumulation. Because ATP6ap2 is a subunit required for V-ATPase assembly of insulin granules, it has been reported to be involved in granule acidification. In accordance with this, we observed impaired insulin granule acidification upon ATP6ap2 knockdown but paradoxically increased proinsulin secretion. Importantly, as a GLP1R interactor, ATP6ap2 was required for GLP1-induced Ca²⁺ influx, in part explaining decreased insulin secretion in ATP6ap2 knockdown cells. Taken together, our findings identify a group of proteins that interact with the GLP1R. We further show that one interactor, ATP6ap2, plays a novel dual role in beta cells, modulating both GLP1R signaling and insulin processing to affect insulin secretion.     

Coupling of metabolic, second messenger pathways and insulin granule dynamics in pancreatic beta-cells: a computational analysis

Insulin secretory responses to nutrient stimuli and hormonal modulators in pancreatic beta-cells are controlled by a variety of secondary messengers. We have analyzed numerous mechanisms responsible for regulated exocytosis in these cells and present an integrated mathematical model of cytosolic Ca²⁺, cAMP and granule dynamics. The insulin-containing granules in the beta-cell were divided into four classes: a large “reserve” granule pool, a smaller pool of the morphologically docked granules that is chemically ‘primed’ for release or the “readily releasable pool”, and a pool of “restless newcomer granules” that undergoes preferential exocytosis. The model incorporates glucose and other aspects of metabolism, the cAMP amplifying pathway, insulin granule dynamics and the exocyst concept for granule binding. The values of most of the model parameters were inferred from available experimental data. The model can generate both the fast first phase and slow biphasic insulin secretion found experimentally in response to a step increase of membrane potential or of glucose. The numerical simulations have also reproduced a variety of experimental conditions, such as periodic stimulation by high K⁺ and the potentiation induced in islets by pre-incubation with cAMP pathway activators. The explicit incorporation of Ca²⁺ channels, Ca²⁺ and cAMP dynamics allows the model to be further connected to current models for calcium and metabolic dynamics and provides an interpretation of the roles of the triggering and amplifying pathways of glucose-stimulated insulin secretion. The model may be important in the identification of pharmacological targets for improving insulin secretion in type 2 diabetes.     

The effects of d- and l-glyceraldehyde on glucose oxidation, insulin secretion and insulin biosynthesis by pancreatic islets of the rat

d-glyceraldehyde stimulated insulin secretion from isolated rat pancreatic islets in static incubation and perifusion systems. At low concentrations (2–4 mM) d-glyceraldehyde was a more potent secretagogue than glucose. The insulinotropic action of 15 mM d-glyceraldehyde was not affected by d-mannoheptulose, was potentiated by cytochalasin B (5 μg/ml) and theophylline (4 mM), and was inhibited by both adrenalin (2 μM) and somatostatin (10 μg/ml). D-glyceraldehyde at a concentration of 1.5 mM produced a 10-fold increase of l-[4,5-³ H]leucine incorporation into proinsulin and insulin without a significant increase into other islet proteins. Glucose at 1.5 mM did not stimulate proinsulin biosynthesis. d-Glyceraldehyde at concentrations higher than 1.5 mM, in marked contrast to glucose, progressively inhibited incorporation of labelled leucine into proinsulin + insulin and other islet proteins. d-glyceraldehyde also inhibited the oxidation of glucose. l-Glyceraldehyde did not stimulate proinsulin biosynthesis and had less effect than the d-isomer on insulin release and glucose oxidation. The results strongly suggest that metabolites below d-glyceraldehyde-3-P are signals for insulin biosynthesisand release. Interaction of d-glyceraldehyde with a “membrane receptor” cannot, however, be excluded with certainty.