Β-cell Exocytosis Research Articles

A role and mechanism for redox sensing by SENP1 in β-cell responses to high fat feeding

Pancreatic β-cells respond to metabolic stress by upregulating insulin secretion, however the underlying mechanisms remain unclear. Here we show, in β-cells from overweight humans without diabetes and mice fed a high-fat diet for 2 days, insulin exocytosis and secretion are enhanced without increased Ca2+ influx. RNA-seq of sorted β-cells suggests altered metabolic pathways early following high fat diet, where we find increased basal oxygen consumption and proton leak, but a more reduced cytosolic redox state. Increased β-cell exocytosis after 2-day high fat diet is dependent on this reduced intracellular redox state and requires the sentrin-specific SUMO-protease-1. Mice with either pancreas- or β-cell-specific deletion of this fail to up-regulate exocytosis and become rapidly glucose intolerant after 2-day high fat diet. Mechanistically, redox-sensing by the SUMO-protease requires a thiol group at C535 which together with Zn+-binding suppresses basal protease activity and unrestrained β-cell exocytosis, and increases enzyme sensitivity to regulation by redox signals.

Dual functions of dynamin in beta cell endocytosis and autophagy trafficking as a membrane fission molecule.

Pancreatic β-cell exocytosis has been a central research theme for decades, but its endocytosis remains less known. Our work has investigated β-cell endocytosis by focusing on a subfamily of dynamin proteins and demonstrated their crucial role in regulating glucose-stimulated insulin secretion (GSIS) (J Clin Invest. 2015, PNAS 2021). Among three mammalian dynamin genes, DNM2 and DNM3 express abundantly in β cells of mice and humans, where they regulate endocytosis in a gene dose-dependent manner. Unlike neurons, most β-cells after tamoxifen-induced triple-dynamin knockout (TKO) are viable, which contrasts with the notion of the housekeeping role of dynamin and suggests an unappreciated role of dynamin-independent endocytosis. DNM2KO impairs the 2nd GSIS phase selectively, and TKO reduces both phases. Moreover, our recent experiments uncover dynamin's role in autophagy. LC3 (microtubule-associated protein 1A/1B light chain 3) staining reveals disrupted autophagic structures in KO β-cells, and these enlarged LC3+-structures are autolysosomes (ALS) rather than other autophagic intermediates as they are lamp1-positive. Starvation exacerbates the defect. Under TIRFM imaging, dynamin-2-EGFP can be recruited onto ALS to induce its fission, as evidenced by transient dynamin-signal increases at ALS fission sites right before ALS fission. This is done by expressing dynamin-2-EGFP in TKO β-cells, which provides a valuable platform for effectively monitoring dynamin dynamics/function because this method removes unlabeled endogenous dynamin molecules to compete with dynamin-2-EGFP. These new results indicate that dynamin regulates β-cell autophagy, in part by directly binding ALS, where it may act as a membrane remodeling and fission protein to transform large ALS into lysosomes. Together, these studies suggest that dynamin is a master regulator of β-cell membrane trafficking in both endocytosis and autophagy, and both processes involve the same intrinsic feature of dynamin as a membrane fission GTPase.

Abstract 1656: Presynaptic scaffold protein, liprin, regulates glucose stimulated insulin secretion and the spatial organisation of exocytosis in pancreatic β-cells

Abstract 1656: Presynaptic scaffold protein, liprin, regulates glucose stimulated insulin secretion and the spatial organisation of exocytosis in pancreatic β-cells

Loss of tetraspanin-7 expression reduces pancreatic β-cell exocytosis Ca2+ sensitivity but has limited effect on systemic metabolism.

Tetraspanin-7 (Tspan7) is an islet autoantigen involved in autoimmune type 1 diabetes and known to regulate β-cell L-type Ca2+ channel activity. However, the role of Tspan7 in pancreatic β-cell function is not yet fully understood. Histological analyses were conducted using immunostaining. Whole-body metabolism was tested using glucose tolerance test. Islet hormone secretion was quantified using static batch incubation or dynamic perifusion. β-cell transmembrane currents, electrical activity and exocytosis were measured using whole-cell patch-clamping and capacitance measurements. Gene expression was studied using mRNA-sequencing and quantitative PCR. Tspan7 is expressed in insulin-containing granules of pancreatic β-cells and glucagon-producing α-cells. Tspan7 knockout mice (Tspan7y/- mouse) exhibit reduced body weight and ad libitum plasma glucose but normal glucose tolerance. Tspan7y/- islets have normal insulin content and glucose- or tolbutamide-stimulated insulin secretion. Depolarisation-triggered Ca2+ current was enhanced in Tspan7y/- β-cells, but β-cell electrical activity and depolarisation-evoked exocytosis were unchanged suggesting that exocytosis was less sensitive to Ca2+ . TSPAN7 knockdown (KD) in human pseudo-islets led to a significant reduction in insulin secretion stimulated by 20 mM K+ . Transcriptomic analyses show that TSPAN7 KD in human pseudo-islets correlated with changes in genes involved in hormone secretion, apoptosis and ER stress. Consistent with rodent β-cells, exocytotic Ca2+ sensitivity was reduced in a human β-cell line (EndoC-βH1) following Tspan7 KD. Tspan7 is involved in the regulation of Ca2+ -dependent exocytosis in β-cells. Its function is more significant in human β-cells than their rodent counterparts.

Metabolism-secretion coupling in glucose-stimulated insulin secretion.

Pancreatic β-cells in the islets of Langerhans secrete insulin in response to blood glucose levels. Precise control of the amount of insulin secreted is of critical importance for maintaining systemic carbohydrate homeostasis. It is now well established that glucose induced production of ATP from ADP and the KATP channel closure elevate cytosolic Ca2+, triggering insulin exocytosis in β-cells. However, for full activation of insulin secretion by glucose, other mechanisms besides Ca2+ elevation are needed. These mechanisms are the targets of current research and include intracellular metabolic pathways branching from glycolysis. They are metabolic pathways originating from the TCA cycle intermediates, the glycerolipid/free fatty acid cycle and the pentose phosphate pathway. Signaling effects of these pathways including degradation (removal) of protein SUMOylation, modulation of insulin vesicular energetics, and lipid modulation of exocytotic machinery may converge to fulfill insulin secretion, though the precise mechanisms have yet to be elucidated. This mini-review summarize recent advances in research on metabolic coupling mechanisms functioning in insulin secretion.

β-Cell Knockout of SENP1 Reduces Responses to Incretins and Worsens Oral Glucose Tolerance in High-Fat Diet-Fed Mice.

SUMOylation reduces oxidative stress and preserves islet mass at the expense of robust insulin secretion. To investigate a role for the deSUMOylating enzyme sentrin-specific protease 1 (SENP1) following metabolic stress, we put pancreas/gut-specific SENP1 knockout (pSENP1-KO) mice on a high-fat diet (HFD). Male pSENP1-KO mice were more glucose intolerant following HFD than littermate controls but only in response to oral glucose. A similar phenotype was observed in females. Plasma glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) responses were identical in pSENP1-KO and wild-type littermates, including the HFD-induced upregulation of GIP responses. Islet mass was not different, but insulin secretion and β-cell exocytotic responses to the GLP-1 receptor agonist exendin-4 (Ex4) and GIP were impaired in islets lacking SENP1. Glucagon secretion from pSENP1-KO islets was also reduced, so we generated β-cell–specific SENP1 KO mice. These phenocopied the pSENP1-KO mice with selective impairment in oral glucose tolerance following HFD, preserved islet mass expansion, and impaired β-cell exocytosis and insulin secretion to Ex4 and GIP without changes in cAMP or Ca2+ levels. Thus, β-cell SENP1 limits oral glucose intolerance following HFD by ensuring robust insulin secretion at a point downstream of incretin signaling.

KATP channel blockers control glucagon secretion by distinct mechanisms: A direct stimulation of α-cells involving a [Ca2+]c rise and an indirect inhibition mediated by somatostatin

ObjectiveGlucagon is secreted by pancreatic α-cells in response to hypoglycemia and its hyperglycemic effect helps to restore normal blood glucose. Insulin and somatostatin (SST) secretions from β- and δ-cells, respectively, are stimulated by glucose by mechanisms involving an inhibition of their ATP-sensitive K+ (KATP) channels, leading to an increase in [Ca2+]c that triggers exocytosis. Drugs that close KATP channels, such as sulfonylureas, are used to stimulate insulin release in type 2 diabetic patients. α-cells also express KATP channels. However, the mechanisms by which sulfonylureas control glucagon secretion are still largely debated and were addressed in the present study. In particular, we studied the effects of KATP channel blockers on α-cell [Ca2+]c and glucagon secretion in the presence of a low (1 mM) or a high (15 mM) glucose concentration and evaluated the role of SST in these effects. MethodsUsing a transgenic mouse model expressing the Ca2+-sensitive fluorescent protein, GCaMP6f, specifically in α-cells, we measured [Ca2+]c in α-cells either dispersed or within whole islets (by confocal microscopy). By measuring [Ca2+]c in α-cells within islets and glucagon secretion using the same perifusion protocols, we tested whether glucagon secretion correlated with changes in [Ca2+]c in response to sulfonylureas. We studied the role of SST in the effects of sulfonylureas using multiple approaches including genetic ablation of SST, or application of SST-14 and SST receptor antagonists. ResultsApplication of the sulfonylureas, tolbutamide, or gliclazide, to a medium containing 1 mM or 15 mM glucose increased [Ca2+]c in α-cells by a direct effect as in β-cells. At low glucose, sulfonylureas inhibited glucagon secretion of islets despite the rise in α-cell [Ca2+]c that they triggered. This glucagonostatic effect was indirect and attributed to SST because, in the islets of SST-knockout mice, sulfonylureas induced a stimulation of glucagon secretion which correlated with an increase in α-cell [Ca2+]c. Experiments with exogenous SST-14 and SST receptor antagonists indicated that the glucagonostatic effect of sulfonylureas mainly resulted from an inhibition of the efficacy of cytosolic Ca2+ on exocytosis. Although SST-14 was also able to inhibit glucagon secretion by decreasing α-cell [Ca2+]c, no decrease in [Ca2+]c occurred during sulfonylurea application because it was largely counterbalanced by the direct stimulatory effect of these drugs on α-cell [Ca2+]c. At high glucose, i.e., in conditions where glucagon release was already low, sulfonylureas stimulated glucagon secretion because their direct stimulatory effect on α-cells exceeded the indirect effect by SST. Our results also indicated that, unexpectedly, SST-14 poorly decreased the efficacy of Ca2+ on exocytosis in β-cells. ConclusionsSulfonylureas exert two opposite actions on α-cells: a direct stimulation as in β-cells and an indirect inhibition by SST. This suggests that any alteration of SST paracrine influence, as described in diabetes, will modify the effect of sulfonylureas on glucagon release. In addition, we suggest that δ-cells inhibit α-cells more efficiently than β-cells.

MEK/ERK Signaling in β-Cells Bifunctionally Regulates β-Cell Mass and Glucose-Stimulated Insulin Secretion Response to Maintain Glucose Homeostasis.

In diabetic pathology, insufficiency in β-cell mass, unable to meet peripheral insulin demand, and functional defects of individual β-cells in production of insulin are often concurrently observed, collectively causing hyperglycemia. Here we show that the phosphorylation of ERK1/2 is significantly decreased in the islets of db/db mice as well as in those of a cohort of subjects with type 2 diabetes. In mice with abrogation of ERK signaling in pancreatic β-cells through deletion of Mek1 and Mek2, glucose intolerance aggravates under high-fat diet-feeding conditions due to insufficient insulin production with lower β-cell proliferation and reduced β-cell mass, while in individual β-cells dampening of the number of insulin exocytosis events is observed, with the molecules involved in insulin exocytosis being less phosphorylated. These data reveal bifunctional roles for MEK/ERK signaling in β-cells for glucose homeostasis, i.e., in regulating β-cell mass as well as in controlling insulin exocytosis in individual β-cells, thus providing not only a novel perspective for the understanding of diabetes pathophysiology but also a potential clue for new drug development for diabetes treatment.

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection.

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic β-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to β-cells improves whole-body glucose homeostasis, enhances β-cell function, and surprisingly, protection of β-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of β-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.

The synthetic glucagon-like peptide-1 receptor agonist liraglutide affects the kinetics of pro-inflammatory kinases and improves insulin sensitivity of 3T3-L1 adipocytes

Abstract Background Obesity is the main risk factor of latent inflammation, which leads to insulin resistance (IR) which can transform to type 2 diabetes mellitus (T2DM). Today several groups of drugs are used to correct T2DM and its complications. The most perspective treatment is the use of glucagon-like peptide-1 (GLP-1) receptor agonists, such as liraglutide, that affect the insulin exocytosis in pancreatic β-cells. The metabolic pathways in these cells are well studied, but liraglutide's effects on other insulin-sensitive tissues, such as adipose tissue, remain unclear. Purpose We wanted to investigate the effect of the synthetic analogue of GLP-1 liraglutide on insulin sensitivity and on inflammatory and mitogenic signaling pathways in 3T3-L1 adipocytes. Methods We studied the effects of liraglutide on the kinetics of inflammatory and mitogenic pathways and insulin sensitivity in 3T3-L1 adipocytes, which were differentiated according to standard protocol with insulin, IBMX and dexamethasone. IR was induced by treatment with 1mM free fatty acids for 24 hours. Activities of insulin, mitogenic and inflammatory signaling pathways were analyzed by Western blotting with phospho-specific antibodies. Insulin sensitivity was also assessed by insulin-stimulated [3H]-deoxyglucose uptake analysis. Results We have showed that liraglutide promotes the decrease in expression of kinases JNK and ERK. So, under these results, we had a hypothesis that liraglutide changes cAMP-dependent regulation of transcription. In order to check it we have used the inhibitor of adenylyl cyclase (SQ22536) to see whether there will be any effects of liraglutide. As we suspected there was no effect, so our hypothesis of the liraglutide-stimulated activation of cAMP-dependent pathway was confirmed. In the model of IR adipocytes liraglutide promotes the increase of total amount of IRS and Akt and increased phosphorylation of pAkt-T308. When SQ22536 was added there was no effect of liraglutide on insulin sensitivity. Conclusion These results suggest the action of liraglutide in 3T3-L1 adipocytes via transcriptional regulation and insulin sensitivity control. According to our hypothesis, GLP-1 analogue liraglutide can influence systemic insulin sensitivity via regulation of adipose tissue metabolism. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): Russian Science Foundation

Improved glucose tolerance with DPPIV inhibition requires β-cell SENP1 amplification of glucose-stimulated insulin secretion.

Pancreatic islet insulin secretion is amplified by both metabolic and receptor‐mediated signaling pathways. The incretin‐mimetic and DPPIV inhibitor anti‐diabetic drugs increase insulin secretion, but in humans this can be variable both in vitro and in vivo. We examined the correlation of GLP‐1 induced insulin secretion from human islets with key donor characteristics, glucose‐responsiveness, and the ability of glucose to augment exocytosis in β‐cells. No clear correlation was observed between several donor or organ processing parameters and the ability of Exendin 4 to enhance insulin secretion. The ability of glucose to facilitate β‐cell exocytosis was, however, significantly correlated with responses to Exendin 4. We therefore studied the effect of impaired glucose‐dependent amplification of insulin exocytosis on responses to DPPIV inhibition (MK‐0626) in vivo using pancreas and β‐cell specific sentrin‐specific protease‐1 (SENP1) mice which exhibit impaired metabolic amplification of insulin exocytosis. Glucose tolerance was improved, and plasma insulin was increased, following either acute or 4 week treatment of wild‐type (βSENP1+/+) mice with MK‐0626. This DPPIV inhibitor was ineffective in βSENP1+/− or βSENP1− / − mice. Finally, we confirm impaired exocytotic responses of β‐cells and reduced insulin secretion from islets of βSENP1− / − mice and show that the ability of Exendin 4 to enhance exocytosis is lost in these cells. Thus, an impaired ability of glucose to amplify insulin exocytosis results in a deficient effect of DPPIV inhibition to improve in vivo insulin responses and glucose tolerance.

Potential Protection Against Type 2 Diabetes in Obesity Through Lower CD36 Expression and Improved Exocytosis in β-Cells.

Obesity is a risk factor for type 2 diabetes (T2D); however, not all obese individuals develop the disease. In this study, we aimed to investigate the cause of differential insulin secretion capacity of pancreatic islets from donors with T2D and non-T2D (ND), especially obese donors (BMI ≥30 kg/m2). Islets from obese donors with T2D had reduced insulin secretion, decreased β-cell exocytosis, and higher expression of fatty acid translocase CD36. We tested the hypothesis that CD36 is a key molecule in the reduced insulin secretion capacity. Indeed, CD36 overexpression led to decreased insulin secretion, impaired exocytosis, and reduced granule docking. This was accompanied by reduced expression of the exocytotic proteins SNAP25, STXBP1, and VAMP2, likely because CD36 induced downregulation of the insulin receptor substrate (IRS) proteins, suppressed the insulin-signaling phosphatidylinositol 3-kinase/AKT pathway, and increased nuclear localization of the transcription factor FoxO1. CD36 antibody treatment of the human β-cell line EndoC-βH1 increased IRS1 and exocytotic protein levels, improved granule docking, and enhanced insulin secretion. Our results demonstrate that β-cells from obese donors with T2D have dysfunctional exocytosis likely due to an abnormal lipid handling represented by differential CD36 expression. Hence, CD36 could be a key molecule to limit β-cell function in T2D associated with obesity.

Chronic hepatitis C virus infection impairs insulin secretion by regulation of p38δ MAPK-dependent exocytosis in pancreatic β-cells.

Chronic hepatitis C virus (HCV) infection has a close association with type 2 diabetes mellitus. Although the mechanisms of insulin resistance in chronic hepatitis C (CHC) patients have been extensively studied, little attention has been given to the role of β-cell function in HCV-associated diabetes. Here, we analysed β-cell function in CHC patients and HCV-infected mouse model and found in addition to insulin resistance, impaired pancreatic β-cell function occurred in CHC patients and HCV-infected C/OTg mice, not only in diabetic individuals but also in individuals with impaired fasting glucose levels. Both first-phase and second-phase insulin secretion were impaired, at least partially due to the reduction of exocytosis of secretory insulin-containing granules following HCV infection. Up-regulated p38δ in HCV-infected β-cells resulted in inactivation of protein kinase D (PKD), which was responsible for impaired insulin secretory capacity of β-cells. Thus, impaired insulin secretion due to HCV infection in β-cells contributes to HCV-associated type 2 diabetes. These findings provided a new inspiration for the important prognostic and therapeutic implications in the management of CHC patients with impaired fasting glucose.

Selectively Bred Diabetes Models: GK Rats, NSY Mice, and ON Mice.

The polygenic background of selectively bred diabetes models mimics the etiology of type 2 diabetes. So far, three different rodent models (Goto-Kakizaki rats, Nagoya-Shibata-Yasuda mice, and Oikawa-Nagao mice) have been established in the diabetes research field by continuous selective breeding for glucose tolerance from outbred rodent stocks. The origin of hyperglycemia in these rodents is mainly insulin secretion deficiency from the pancreatic β-cells and mild insulin resistance in insulin target organs. In this chapter, we summarize backgrounds and phenotypes of these rodent models to highlight their importance in diabetes research. Then, we introduce experimental methodologies to evaluate β-cell exocytosis as a putative common defect observed in these rodent models.

GDP-Bound Rab27a Dissociates from the Endocytic Machinery in a Phosphorylation-Dependent Manner after Insulin Secretion

Glucose-stimulated insulin secretion is controlled by both exocytosis and endocytosis in pancreatic β-cells. Although endocytosis is a fundamental step to maintain cellular responses to the secretagogue, the molecular mechanism of endocytosis remains poorly defined. We have previously shown that in response to high concentrations of glucose, guanosine 5'-diphosphate (GDP)-bound Rab27a is recruited to the plasma membrane where IQ motif-containing guanosine 5'-triphosphatase (GTPase)-activating protein 1 (IQGAP1) is expressed, and that complex formation promotes endocytosis of secretory membranes after insulin secretion. In the present study, the regulatory mechanisms of dissociation of the complex were investigated. Phosphorylation of IQGAP1 on serine (Ser)-1443, a site recognized by protein kinase Cε (PKCε), inhibited the interaction of GDP-bound Rab27a with IQGAP1 in a Cdc42-independent manner. Glucose stimulation caused a translocation of PKCε from the cytosol to the plasma membrane. In addition, glucose-induced endocytosis was inhibited by the knockdown of IQGAP1 with small interfering RNA (siRNA). However, the expression of the non-phosphorylatable or phosphomimetic form of IQGAP1 could not rescue the inhibition, suggesting that a phosphorylation-dephosphorylation cycle of IQGAP1 is required for endocytosis. These results suggest that IQGAP1 phosphorylated by PKCε promotes the dissociation of the IQGAP1-GDP-bound Rab27a complex in pancreatic β-cells, thereby regulating endocytosis of secretory membranes following insulin secretion.

A cryptic non-GPI-anchored cytosolic isoform of CD59 controls insulin exocytosis in pancreatic β-cells by interaction with SNARE proteins.

CD59 is a glycosylphosphatidylinositol (GPI)-anchored cell surface inhibitor of the complement membrane attack complex (MAC). We showed previously that CD59 is highly expressed in pancreatic islets but is down-regulated in rodent models of diabetes. CD59 knockdown but not enzymatic removal of cell surface CD59 led to a loss of glucose-stimulated insulin secretion (GSIS), suggesting that an intracellular pool of CD59 is required. In this current paper, we now report that non-GPI-anchored CD59 is present in the cytoplasm, colocalizes with exocytotic protein vesicle-associated membrane protein 2, and completely rescues GSIS in cells lacking endogenous CD59 expression. The involvement of cytosolic non-GPI-anchored CD59 in GSIS is supported in phosphatidylinositol glycan class A knockout GPI anchor-deficient β-cells, in which GSIS is still CD59 dependent. Furthermore, site-directed mutagenesis demonstrated different structural requirements of CD59 for its 2 functions, MAC inhibition and GSIS. Our results suggest that CD59 is retrotranslocated from the endoplasmic reticulum to the cytosol, a process mediated by recognition of trimmed N-linked oligosaccharides, supported by the partial glycosylation of non-GPI-anchored cytosolic CD59 as well as the failure of N-linked glycosylation site mutant CD59 to reach the cytosol or rescue GSIS. This study thus proposes the previously undescribed existence of non-GPI-anchored cytosolic CD59, which is required for insulin secretion.-Golec, E., Rosberg, R., Zhang, E., Renström, E., Blom, A. M., King, B. C. A cryptic non-GPI-anchored cytosolic isoform of CD59 controls insulin exocytosis in pancreatic β-cells by interaction with SNARE proteins.

Modulation of large dense core vesicle insulin content mediates rhythmic hormone release from pancreatic beta cells over the 24h cycle.

The rhythmic nature of insulin secretion over the 24h cycle in pancreatic islets has been mostly investigated using transcriptomics studies showing that modulation of insulin secretion over this cycle is achieved via distal stages of insulin secretion. We set out to measure β-cell exocytosis using in depth cell physiology techniques at several time points. In agreement with the activity and feeding pattern of nocturnal rodents, we find that C57/Bl6J islets in culture for 24h exhibit higher insulin secretion during the corresponding dark phase than in the light phase (Zeitgeber Time ZT20 and ZT8, respectively, in vivo). Glucose-induced insulin secretion is increased by 21% despite normal intracellular Ca2+ transients and depolarization-evoked exocytosis, as measured by whole-cell capacitance measurements. This paradox is explained by a 1.37-fold increase in beta cell insulin content. Ultramorphological analyses show that vesicle size and density are unaltered, demonstrating that intravesicular insulin content per granule is modulated over the 24h cycle. Proinsulin levels did not change between ZT8 and ZT20. Islet glucagon content was inversely proportional to insulin content indicating that this unique feature is likely to support a physiological role. Microarray data identified the differential expression of 301 transcripts, of which 26 are miRNAs and 54 are known genes (including C2cd4b, a gene previously involved in insulin processing, and clock genes such as Bmal1 and Rev-erbα). Mouse β-cell secretion over the full course of the 24h cycle may rely on several distinct cellular functions but late night increase in insulin secretion depends solely on granule insulin content.

Inositol hexakisphosphate kinase 1 is a metabolic sensor in pancreatic β-cells

Diphosphoinositol pentakisphosphate (IP7) is critical for the exocytotic capacity of the pancreatic β-cell, but its regulation by the primary instigator of β-cell exocytosis, glucose, is unknown. The high Km for ATP of the IP7-generating enzymes, the inositol hexakisphosphate kinases (IP6K1 and 2) suggests that these enzymes might serve as metabolic sensors in insulin secreting β-cells and act as translators of disrupted metabolism in diabetes. We investigated this hypothesis and now show that glucose stimulation, which increases the ATP/ADP ratio, leads to an early rise in IP7 concentration in β-cells. RNAi mediated knock down of the IP6K1 isoform inhibits both glucose-mediated increase in IP7 and first phase insulin secretion, demonstrating that IP6K1 integrates glucose metabolism and insulin exocytosis. In diabetic mouse islets the deranged ATP/ADP levels under both basal and glucose-stimulated conditions are mirrored in both disrupted IP7 generation and insulin release. Thus the unique metabolic sensing properties of IP6K1 guarantees appropriate concentrations of IP7 and thereby both correct basal insulin secretion and intact first phase insulin release. In addition, our data suggest that a specific cell signaling defect, namely, inappropriate IP7 generation may be an essential convergence point integrating multiple metabolic defects into the commonly observed phenotype in diabetes.

Sox5 regulates beta-cell phenotype and is reduced in type 2 diabetes

Type 2 diabetes (T2D) is characterized by insulin resistance and impaired insulin secretion, but the mechanisms underlying insulin secretion failure are not completely understood. Here, we show that a set of co-expressed genes, which is enriched for genes with islet-selective open chromatin, is associated with T2D. These genes are perturbed in T2D and have a similar expression pattern to that of dedifferentiated islets. We identify Sox5 as a regulator of the module. Sox5 knockdown induces gene expression changes similar to those observed in T2D and diabetic animals and has profound effects on insulin secretion, including reduced depolarization-evoked Ca2+-influx and β-cell exocytosis. SOX5 overexpression reverses the expression perturbations observed in a mouse model of T2D, increases the expression of key β-cell genes and improves glucose-stimulated insulin secretion in human islets from donors with T2D. We suggest that human islets in T2D display changes reminiscent of dedifferentiation and highlight SOX5 as a regulator of β-cell phenotype and function.

Thrombin stimulates insulin secretion via protease-activated receptor-3

The disease mechanisms underlying type 2 diabetes (T2D) remain poorly defined. Here we aimed to explore the pathophysiology of T2D by analyzing gene co-expression networks in human islets. Using partial correlation networks we identified a group of co-expressed genes (‘module’) including F2RL2 that was associated with glycated hemoglobin. F2Rl2 is a G-protein-coupled receptor (GPCR) that encodes protease-activated receptor-3 (PAR3). PAR3 is cleaved by thrombin, which exposes a 6-amino acid sequence that acts as a ‘tethered ligand’ to regulate cellular signaling. We have characterized the effect of PAR3 activation on insulin secretion by static insulin secretion measurements, capacitance measurements, studies of diabetic animal models and patient samples. We demonstrate that thrombin stimulates insulin secretion, an effect that was prevented by an antibody that blocks the thrombin cleavage site of PAR3. Treatment with a peptide corresponding to the PAR3 tethered ligand stimulated islet insulin secretion and single β-cell exocytosis by a mechanism that involves activation of phospholipase C and Ca2+ release from intracellular stores. Moreover, we observed that the expression of tissue factor, which regulates thrombin generation, was increased in human islets from T2D donors and associated with enhanced β-cell exocytosis. Finally, we demonstrate that thrombin generation potential in patients with T2D was associated with increased fasting insulin and insulinogenic index. The findings provide a previously unrecognized link between hypercoagulability and hyperinsulinemia and suggest that reducing thrombin activity or blocking PAR3 cleavage could potentially counteract the exaggerated insulin secretion that drives insulin resistance and β-cell exhaustion in T2D.