Islet autoimmunity in human type 1 diabetes: initiation and progression from the perspective of the beta cell.

Type 1 diabetes results from the poorly understood process of islet autoimmunity, which ultimately leads to the loss of functional pancreatic beta cells. Mounting evidence supports the notion that the activation and evolution of islet autoimmunity in genetically susceptible people is contingent upon early life exposures affecting the islets, especially beta cells. Here, we review some of the recent advances and studies that highlight the roles of these changes as well as antigen presentation and stress response pathways in beta cells in the onset and propagation of the autoimmune process in type 1 diabetes. Future progress in this area holds promise for advancing islet- and beta cell-directed therapies that could be implemented in the early stages of the disease and could be combined with immunotherapies.

Association between residual islet beta-cell function and achieving the target of time in range in inpatients with type 2 diabetes undergoing antidiabetic treatment: An observation study.

AIM: To assess whether the beta-cell function of inpatients undergoing antidiabetic treatment influences achieving time in range (TIR) and time above range (TAR) targets. MATERIALS AND METHODS: This cross-sectional study included 180 inpatients with type 2 diabetes. TIR and TAR were assessed by a continuous glucose monitoring system, with target achievement defined as TIR more than 70% and TAR less than 25%. Beta-cell function was assessed by the insulin secretion-sensitivity index-2 (ISSI2). RESULTS: Following antidiabetic treatment, logistic regression analysis showed that lower ISSI2 was associated with a decreased number of inpatients achieving TIR (OR = 3.10, 95% CI: 1.19-8.06) and TAR (OR = 3.40, 95% CI: 1.35-8.55) targets after adjusting for potential confounders. Similar associations still existed in those participants treated with insulin secretagogues (TIR: OR = 2.91, 95% CI: 0.90-9.36, P = .07; TAR, OR = 3.14, 95% CI: 1.01-9.80) or adequate insulin therapy (TIR: OR = 2.84, 95% CI: 0.91-8.81, P = .07; TAR, OR = 3.24, 95% CI: 1.08-9.67). Furthermore, receiver operating characteristic curves showed that the diagnostic value of the ISSI2 for achieving TIR and TAR targets was 0.73 (95% CI: 0.66-0.80) and 0.71 (95% CI: 0.63-0.79), respectively. CONCLUSIONS: Beta-cell function was associated with achieving TIR and TAR targets. Stimulating insulin secretion or exogenous insulin treatment could not overcome the disadvantage of lower beta-cell function on glycaemic control.

Effect of faecal microbial transplant via colonoscopy in patients with severe obesity and insulin resistance: A randomized double-blind, placebo-controlled Phase 2 trial.

AIM: To assess the effects of faecal microbial transplant (FMT) from lean people to subjects with obesity via colonoscopy. MATERIAL AND METHODS: In a double-blind, randomized controlled trial, subjects with a body mass index ≥ 35 kg/m2 and insulin resistance were randomized, in a 1:1 ratio in blocks of four, to either allogenic (from healthy lean donor; n = 15) or autologous FMT (their own stool; n = 13) delivered in the caecum and were followed for 3 months. The main outcome was homeostatic model assessment of insulin resistance (HOMA-IR) and secondary outcomes were glycated haemoglobin levels, lipid profile, weight, gut hormones, endotoxin, appetite measures, intestinal microbiome (IM), metagenome, serum/faecal metabolites, quality of life, anxiety and depression scores. RESULTS: In the allogenic versus autologous groups, HOMA-IR and clinical variables did not change significantly, but IM and metabolites changed favourably (P < 0.05): at 1 month, Coprococcus, Bifidobacterium, Bacteroides and Roseburia increased, and Streptococcus decreased; at 3 months, Bacteroides and Blautia increased. Several species also changed significantly. For metabolites, at 1 month, serum kynurenine decreased and faecal indole acetic acid and butenylcarnitine increased, while at 3 months, serum isoleucine, leucine, decenoylcarnitine and faecal phenylacetic acid decreased. Metagenomic pathway representations and network analyses assessing relationships with clinical variables, metabolites and IM were significantly enhanced in the allogenic versus autologous groups. LDL and appetite measures improved in the allogenic (P < 0.05) but not in the autologous group. CONCLUSIONS: Overall, in those with obeisty, allogenic FMT via colonoscopy induced favourable changes in IM, metabolites, pathway representations and networks even though other metabolic variables did not change. LDL and appetite variables may also benefit.

Higher systolic blood pressure is specifically associated with better islet beta-cell function in T2DM patients with high glycemic level.

BACKGROUND: Patients with type 2 diabetes mellitus (T2DM) usually have higher blood viscosity attributed to high blood glucose that can decrease blood supply to the pancreas. A mild increase in blood pressure (BP) has been reported as a potential compensatory response that can maintain blood perfusion in the islet. However, how BP influences beta-cell function in T2DM subjects remains inconsistent. This study aimed to examine the relationship between BP and beta-cell function in patients with T2DM under different HbA1c levels. METHODS: This is a cross-sectional study of 615 T2DM patients, whose clinical data were extracted from hospital medical records. Beta-cell function was assessed by insulin secretion-sensitivity index-2 (ISSI2). Multivariable linear regression analysis and restricted cubic splines (RCS) analysis were performed to identify the association between systolic BP (SBP) and ISSI2. Mediation analysis was performed to determine whether higher SBP could reduce blood glucose by enhancing beta-cell function. RESULTS: After adjustment of potential confounders, in participants with HbA1c ≥ 10%, the SBP between 140 to150 mmHg had the highest log ISSI2 (b = 0.227, 95% CI 0.053-0.402), an association specific to participants with < 1 year duration of diabetes. RCS analyses demonstrated an inverted U-shaped association between SBP and ISSI2 with the SBP at 144 mmHg corresponding to the best beta-cell function. This higher SBP was "paradoxically" associated with lower 2 h postprandial blood glucose (PBG) when SBP < 150 mmHg that was almost exclusively mediated by ISSI2 (mediating effect = - 0.043, 95%CI - 0.067 to - 0.018; mediating effect percentage = 94.7%, P < 0.01). SBP was however not associated with improvement in ISSI2 or 2 h PBG in participants with HbA1c < 10%. CONCLUSIONS: In early stage of diabetes, a slightly elevated SBP (140-150 mmHg) was transiently associated with better beta-cell function in T2DM patients with HbA1c ≥ 10% but not in those with HbA1c < 10%.

Susceptibility Factors and Cellular Mechanisms Underlying Alcoholic Pancreatitis.

Alcohol is a major cause of acute and chronic pancreatitis. There have been some recent advances in the understanding of the mechanisms underlying alcoholic pancreatitis, which include perturbation in mitochondrial function and autophagy and ectopic exocytosis, with some of these cellular events involving membrane fusion soluble N-ethylmaleimide-sensitive factor receptor protein receptor proteins. Although new insights have been unraveled recently, the precise mechanisms remain complex, and their finer details have yet to be established. The overall pathophysiology of pancreatitis involves not only the pancreatic acinar cells but also the stellate cells and duct cells. Why only some are more susceptible to pancreatitis and with increased severity, while others are not, would suggest that there may be undefined protective factors or mechanisms that enhance recovery and regeneration after injury. Furthermore, there are confounding influences of lifestyle factors such as smoking and diet, and genetic background. Whereas alcohol and smoking cessation and a generally healthy lifestyle are intuitively the advice given to these patients afflicted with alcoholic pancreatitis in order to reduce disease recurrence and progression, there is as yet no specific treatment. A more complete understanding of the pathogenesis of pancreatitis from which novel therapeutic targets could be identified will have a great impact, particularly with the stubbornly high fatality (>30%) of severe pancreatitis. This review focuses on the susceptibility factors and underlying cellular mechanisms of alcohol injury on the exocrine pancreas.

C2 Domains of Munc13-4 Are Crucial for Ca2+-Dependent Degranulation and Cytotoxicity in NK Cells.

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.

Elevated triglyceride-glucose (TyG) index predicts incidence of Prediabetes: a prospective cohort study in China.

BACKGROUND: Prediabetes has become a pandemic. This study aimed to identify a better predictor for the incidence of prediabetes, which we hypothesize to be the triglyceride-glucose (TyG) index, a simplified insulin resistance index. We compared its predictive value with the other common risk factors of prediabetes. METHODS: The participants of this analysis were derived from the Risk Evaluation of cAncers in Chinese diabeTic Individuals: a lONgitudinal (REACTION) study. A total of 4543 participants without initial prediabetes or diabetes were followed up for 3.25 years. Using multivariate logistic regression model, the associations between baseline obesity, lipid profiles and non-insulin-based insulin resistance indices with the incidence of prediabetes were analyzed. To assess which is better predictor for the incidence of prediabetes, the area under curves (AUCs) calculated from the receiver operating characteristic curve analyses were used to evaluate and compare with the predictive value of the different indices. RESULTS: During the 3.25 years, 1071 out of the 4543 participants developed prediabetes. Using the logistic regression analysis adjusted for some potential confounders, the risk of incidence of prediabetes increased 1.38 (1.28-1.48) fold for each 1-SD increment of TyG index. The predictive ability (assessed by AUCs) of TyG index for predicting prediabetes was 0.60 (0.58-0.62), which was superior to the indices of obesity, lipid profiles and other non-insulin-based insulin resistance indices. Although the predictive ability of the TyG index was overall similar to fasting plasma glucose (FPG) (P = 0.4340), TyG index trended higher than FPG in females (0.62 (0.59-0.64) vs. 0.59 (0.57-0.61), P = 0.0872) and obese subjects (0.59 (0.57-0.62) vs. 0.57 (0.54-0.59), P = 0.1313). TyG index had superior predictive ability for the prediabetic phenotype with isolated impaired glucose tolerance compared with FPG (P <  0.05) and other indices. Furthermore, TyG index significantly improved the C statistic (0.62 (0.60-0.64)), integrated discrimination improvement (1.89% (1.44-2.33%)) and net reclassification index (28.76% (21.84-35.67%)) of conventional model in predicting prediabetes than other indices. CONCLUSIONS: TyG could be a potential predictor to identify the high risk individuals of prediabetes.

Kv2.1 clusters on ß-cell plasma membrane act as reservoirs that replenish pools of newcomer insulin granule through their interaction with syntaxin-3.

The voltage-dependent K+ (Kv) channel Kv2.1 is a major delayed rectifier in many secretory cells, including pancreatic ß cells. In addition, Kv2.1 has a direct role in exocytosis at an undefined step, involving SNARE proteins, that is independent of its ion-conducting pore function. Here, we elucidated the precise step in exocytosis. We previously reported that syntaxin-3 (Syn-3) is the key syntaxin that mediates exocytosis of newcomer secretory granules that spend minimal residence time on the plasma membrane before fusion. Using high-resolution total internal reflection fluorescence microscopy, we now show that Kv2.1 forms reservoir clusters on the ß-cell plasma membrane and binds Syn-3 via its C-terminal C1b domain, which recruits newcomer insulin secretory granules into this large reservoir. Upon glucose stimulation, secretory granules were released from this reservoir to replenish the pool of newcomer secretory granules for subsequent fusion, occurring just adjacent to the plasma membrane Kv2.1 clusters. C1b deletion blocked the aforementioned Kv2.1-Syn-3-mediated events and reduced fusion of newcomer secretory granules. These insights have therapeutic implications, as Kv2.1 overexpression in type-2 diabetes rat islets restored biphasic insulin secretion.

Depletion of the membrane-fusion regulator Munc18c attenuates caerulein hyperstimulation-induced pancreatitis.

Epithelial pancreatic acinar cells perform crucial functions in food digestion, and acinar cell homeostasis required for secretion of digestive enzymes relies on SNARE-mediated exocytosis. The ubiquitously expressed Sec1/Munc18 protein mammalian uncoordinated-18c (Munc18c) regulates membrane fusion by activating syntaxin-4 (STX-4) to bind cognate SNARE proteins to form a SNARE complex that mediates exocytosis in many cell types. However, in the acinar cell, Munc18c's functions in exocytosis and homeostasis remain inconclusive. Here, we found that pancreatic acini from Munc18c-depleted mice (Munc18c+/-) and human pancreas (lenti-Munc18c-shRNA-treated) exhibit normal apical exocytosis of zymogen granules (ZGs) in response to physiologic stimulation with the intestinal hormone cholecystokinin (CCK-8). However, when stimulated with supraphysiologic CCK-8 levels to mimic pancreatitis, Munc18c-depleted (Munc18c+/-) mouse acini exhibited a reduction in pathological basolateral exocytosis of ZGs resulting from a decrease in fusogenic STX-4 SNARE complexes. This reduced basolateral exocytosis in part explained the less severe pancreatitis observed in Munc18c+/- mice after hyperstimulation with the CCK-8 analog caerulein. Likely as a result of this secretory blockade, Munc18c-depleted acini unexpectedly activated a component of the endoplasmic reticulum (ER) stress response that contributed to autophagy induction, resulting in downstream accumulation of autophagic vacuoles and autolysosomes. We conclude that Munc18c's role in mediating ectopic basolateral membrane fusion of ZGs contributes to the initiation of CCK-induced pancreatic injury, and that blockade of this secretory process could increase autophagy induction.

Cell polarity defines three distinct domains in pancreatic ß-cells.

The structural organisation of pancreatic ß-cells in the islets of Langerhans is relatively unknown. Here, using three-dimensional (3D) two-photon, 3D confocal and 3D block-face serial electron microscopy, we demonstrate a consistent in situ polarisation of ß-cells and define three distinct cell surface domains. An apical domain located at the vascular apogee of ß-cells, defined by the location of PAR-3 (also known as PARD3) and ZO-1 (also known as TJP1), delineates an extracellular space into which adjacent ß-cells project their primary cilia. A separate lateral domain, is enriched in scribble and Dlg, and colocalises with E-cadherin and GLUT2 (also known as SLC2A2). Finally, a distinct basal domain, where the ß-cells contact the islet vasculature, is enriched in synaptic scaffold proteins such as liprin. This 3D analysis of ß-cells within intact islets, and the definition of distinct domains, provides new insights into understanding ß-cell structure and function.

Pancreatitis-Induced Depletion of Syntaxin 2 Promotes Autophagy and Increases Basolateral Exocytosis.

BACKGROUND & AIMS: Pancreatic acinar cells are polarized epithelial cells that store enzymes required for digestion as inactive zymogens, tightly packed at the cell apex. Stimulation of acinar cells causes the zymogen granules to fuse with the apical membrane, and the cells undergo exocytosis to release proteases into the intestinal lumen. Autophagy maintains homeostasis of pancreatic acini. Syntaxin 2 (STX2), an abundant soluble N-ethyl maleimide sensitive factor attachment protein receptor in pancreatic acini, has been reported to mediate apical exocytosis. Using human pancreatic tissues and STX2-knockout (KO) mice, we investigated the functions of STX2 in zymogen granule-mediated exocytosis and autophagy. METHODS: We obtained pancreatic tissues from 5 patients undergoing surgery for pancreatic cancer and prepared 80-µm slices; tissues were exposed to supramaximal cholecystokinin octapeptide (CCK-8) or ethanol and a low concentration of CCK-8 and analyzed by immunoblot and immunofluorescence analyses. STX2-KO mice and syntaxin 2+/+ C57BL6 mice (controls) were given intraperitoneal injections of supramaximal caerulein (a CCK-8 analogue) or fed ethanol and then given a low dose of caerulein to induce acute pancreatitis, or saline (controls); pancreata were isolated and analyzed by histology and immunohistochemistry. Acini were isolated from mice, incubated with CCK-8, and analyzed by immunofluorescence microscopy or used in immunoprecipitation experiments. Exocytosis was quantified using live-cell exocytosis and Ca2+ imaging analyses and based on formation of exocytotic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes. Dysregulations in autophagy were identified using markers, electron and immunofluorescence microscopy, and protease activation assays. RESULTS: Human pancreatic tissues and dispersed pancreatic acini from control mice exposed to CCK-8 or ethanol plus CCK-8 were depleted of STX2. STX2-KO developed more severe pancreatitis after administration of supramaximal caerulein or a 6-week ethanol diet compared with control. Acini from STX2-KO mice had increased apical exocytosis after exposure to CCK-8, as well as increased basolateral exocytosis, which led to ectopic release of proteases. These increases in apical and basolateral exocytosis required increased formation of fusogenic soluble N-ethyl maleimide sensitive factor attachment protein receptor complexes, mediated by STX3 and STX4. STX2 bound ATG16L1 and prevented it from binding clathrin. Deletion of STX2 from acini increased binding of AT16L1 to clathrin, increasing formation of pre-autophagosomes and inducing autophagy. Induction of autophagy promoted the CCK-8-induced increase in autolysosome formation and the activation of trypsinogen. CONCLUSIONS: In studies of human pancreatic tissues and pancreata from STX2-KO and control mice, we found STX2 to block STX3- and STX4-mediated fusion of zymogen granules with the plasma membrane and exocytosis and prevent binding of ATG16L1 to clathrin, which contributes to induction of autophagy. Exposure of pancreatic tissues to CCK-8 or ethanol depletes acinar cells of STX2, increasing basolateral exocytosis and promoting autophagy induction, leading to activation of trypsinogen.

Ex vivo human pancreatic slice preparations offer a valuable model for studying pancreatic exocrine biology.

A genuine understanding of human exocrine pancreas biology and pathobiology has been hampered by a lack of suitable preparations and reliance on rodent models employing dispersed acini preparations. We have developed an organotypic slice preparation of the normal portions of human pancreas obtained from cancer resections. The preparation was assessed for physiologic and pathologic responses to the cholinergic agonist carbachol (Cch) and cholecystokinin (CCK-8), including 1) amylase secretion, 2) exocytosis, 3) intracellular Ca2+ responses, 4) cytoplasmic autophagic vacuole formation, and 5) protease activation. Cch and CCK-8 both dose-dependently stimulated secretory responses from human pancreas slices similar to those previously observed in dispersed rodent acini. Confocal microscopy imaging showed that these responses were accounted for by efficient apical exocytosis at physiologic doses of both agonists and by apical blockade and redirection of exocytosis to the basolateral plasma membrane at supramaximal doses. The secretory responses and exocytotic events evoked by CCK-8 were mediated by CCK-A and not CCK-B receptors. Physiologic agonist doses evoked oscillatory Ca2+ increases across the acini. Supraphysiologic doses induced formation of cytoplasmic autophagic vacuoles and activation of proteases (trypsin, chymotrypsin). Maximal atropine pretreatment that completely blocked all the Cch-evoked responses did not affect any of the CCK-8-evoked responses, indicating that rather than acting on the nerves within the pancreas slice, CCK cellular actions directly affected human acinar cells. Human pancreas slices represent excellent preparations to examine pancreatic cell biology and pathobiology and could help screen for potential treatments for human pancreatitis.

New Roles of Syntaxin-1A in Insulin Granule Exocytosis and Replenishment.

In type-2 diabetes (T2D), severely reduced islet syntaxin-1A (Syn-1A) levels contribute to insulin secretory deficiency. We generated ß-cell-specific Syn-1A-KO (Syn-1A-ßKO) mice to mimic ß-cell Syn-1A deficiency in T2D. Glucose tolerance tests showed that Syn-1A-ßKO mice exhibited blood glucose elevation corresponding to reduced blood insulin levels. Perifusion of Syn-1A-ßKO islets showed impaired first- and second-phase glucose-stimulated insulin secretion (GSIS) resulting from reduction in readily releasable pool and granule pool refilling. To unequivocally determine the ß-cell exocytotic defects caused by Syn-1A deletion, EM and total internal reflection fluorescence microscopy showed that Syn-1A-KO ß-cells had a severe reduction in the number of secretory granules (SGs) docked onto the plasma membrane (PM) at rest and reduced SG recruitment to the PM after glucose stimulation, the latter indicating defects in replenishment of releasable pools required to sustain second-phase GSIS. Whereas reduced predocked SG fusion accounted for reduced first-phase GSIS, selective reduction of exocytosis of short-dock (but not no-dock) newcomer SGs accounted for the reduced second-phase GSIS. These Syn-1A actions on newcomer SGs were partly mediated by Syn-1A interactions with newcomer SG VAMP8.

Chaperoning of closed syntaxin-3 through Lys46 and Glu59 in domain 1 of Munc18 proteins is indispensable for mast cell exocytosis.

Understanding how Munc18 proteins govern exocytosis is crucial because mutations of this protein cause severe secretion deficits in neuronal and immune cells. Munc18-2 has indispensable roles in the degranulation of mast cell, partly by binding and chaperoning a subset of syntaxin isoforms. However, the key syntaxin that, crucially, participates in the degranulation ­ whose levels and intracellular localization are regulated by Munc18-2 ­ remains unknown. Here, we demonstrate that double knockdown of Munc18-1 and Munc-2 in mast cells results in greatly reduced degranulation accompanied with strikingly compromised expression levels and localization of syntaxin-3. This phenotype is fully rescued by wild-type Munc18 proteins but not by the K46E, E59K and K46E/E59K mutants of Munc-18 domain 1, each of which exhibits completely abolished binding to 'closed' syntaxin-3. Furthermore, knockdown of syntaxin-3 strongly impairs degranulation. Collectively, our data argue that residues Lys46 and Glu59 of Munc18 proteins are indispensable for mediating the interaction between Munc18 and closed syntaxin-3, which is essential for degranulation by chaperoning syntaxin-3. Our results also indicate that the functional contribution of these residues differs between immune cell degranulation and neuronal secretion.

Palmitic acid increases invasiveness of pancreatic cancer cells AsPC-1 through TLR4/ROS/NF-κB/MMP-9 signaling pathway.

Pancreatic cancer (PC) is an aggressive malady with proclivity for early metastasis. Overexpression of toll-like receptor 4 (TLR4) in pancreatic ductal adenocarcinoma, the most common type of pancreatic malignancy, correlates to tumor size, lymph node involvement, venous invasion and pathological stage. Lipopolysaccharides (LPS) are natural TLR4 ligands that have been shown to increase the invasive ability of PC cells. However, rapid inactivation of circulating LPS and low systemic absorption of inhaled LPS from the bronchoalveolar compartment make other agonists such as saturated fatty acids more suitable to be considered for TLR4-related cell invasiveness. Interestingly, PC risk was strongly associated to intake of saturated fat from animal food sources, in particular to consumption of saturated palmitic acid (PA). In the present study, we investigated the influence of PA on the invasive capacity of human PC cells AsPC-1. Using specific inhibitors, we found that PA stimulation of these tumor cells induced a TLR4-mediated cell invasion. Our results also indicate that the signaling events downstream of TLR4 involved generation of reactive oxygen species, activation of nuclear factor-kappa beta, and secretion and activation of matrix metalloproteinase 9 (MMP-9). Furthermore, PA stimulation decreased the levels of the micro RNA 29c (miR-29c). Of note, while inhibition of miR-29c increased MMP-9 mRNA levels, MMP-9 secretion and activation, and invasiveness, miR-29c mimic abrogated all these PA-stimulated effects. These results strongly suggest that miR-29c could be an attractive potential pharmacological agent for antitumoral therapy in PC.

Recent new insights into the role of SNARE and associated proteins in insulin granule exocytosis.

Initial work on the exocytotic machinery of predocked insulin secretory granules (SGs) in pancreatic ß-cells mimicked the SNARE hypothesis work in neurons, which includes SM/SNARE complex and associated priming proteins, fusion clamps and Ca2+ sensors. However, ß-cell SGs, unlike neuronal synaptic vesicles, exhibit a biphasic secretory response that requires additional distinct features in exocytosis including newcomer SGs that undergo minimal docking time at the plasma membrane (PM) before fusion and multi-SG (compound) fusion. These exocytotic events are mediated by Munc18/SNARE complexes distinct from that which mediates predocked SG fusion. We review some recent insights in SNARE complex assembly and the promiscuity in SM/SNARE complex formation, whereby both contribute to conferring different insulin SG fusion kinetics. Some SNARE and associated proteins play non-fusion roles, including tethering SGs to Ca2+ channels, SG recruitment from cell interior to PM, and inhibitory SNAREs that block the action of profusion SNAREs. We discuss new insights into how sub-PM cytoskeletal mesh gates SG access to the PM and the targeting of SG exocytosis to PM domains in functionally polarized ß-cells within intact islets. These recent developments have major implications on devising clever SNARE replacement therapies that could restore the deficient insulin secretion in diabetic islet ß-cells.

Changes in beta cell function occur in prediabetes and early disease in the Lepr (db) mouse model of diabetes.

AIMS/HYPOTHESIS: Type 2 diabetes is a progressive disease that increases morbidity and the risk of premature death. Glucose dysregulation, such as elevated fasting blood glucose, is observed prior to diabetes onset. A decline in beta cell insulin secretion contributes to the later stages of diabetes, but it is not known what, if any, functional beta cell changes occur in prediabetes and early disease. METHODS: The Lepr (db) mouse (age 13-18 weeks) was used as a model of type 2 diabetes and a two-photon granule fusion assay was used to characterise the secretory response of pancreatic beta cells. RESULTS: We identified a prediabetic state in db/db mice where the animals responded normally to a glucose challenge but have elevated fasting blood glucose. Isolated islets from prediabetic animals secreted more and were bigger. Insulin secretion, normalised to insulin content, was similar to wild type but basal insulin secretion was elevated. There was increased glucose-induced granule fusion with a high prevalence of granule-granule fusion. The glucose-induced calcium response was not changed but there was altered expression of the exocytic machinery. db/db animals at the next stage of disease had overt glucose intolerance. Isolated islets from these animals had reduced insulin secretion, reduced glucose-induced granule fusion events and decreased calcium responses to glucose. CONCLUSIONS/INTERPRETATION: Beta cell function is altered in prediabetes and there are further changes in the progression to early disease.

Progesterone receptor membrane component 1 is a functional part of the glucagon-like peptide-1 (GLP-1) receptor complex in pancreatic ß cells.

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that regulates glucose homeostasis. Because of their direct stimulation of insulin secretion from pancreatic ß cells, GLP-1 receptor (GLP-1R) agonists are now important therapeutic options for the treatment of type 2 diabetes. To better understand the mechanisms that control the insulinotropic actions of GLP-1, affinity purification and mass spectrometry (AP-MS) were employed to uncover potential proteins that functionally interact with the GLP-1R. AP-MS performed on Chinese hamster ovary cells or MIN6 ß cells, both expressing the human GLP-1R, revealed 99 proteins potentially associated with the GLP-1R. Three novel GLP-1R interactors (PGRMC1, Rab5b, and Rab5c) were further validated through co-immunoprecipitation/immunoblotting, fluorescence resonance energy transfer, and immunofluorescence. Functional studies revealed that overexpression of PGRMC1, a novel cell surface receptor that associated with liganded GLP-1R, enhanced GLP-1-induced insulin secretion (GIIS) with the most robust effect. Knockdown of PGRMC1 in ß cells decreased GIIS, indicative of positive interaction with GLP-1R. To gain insight mechanistically, we demonstrated that the cell surface PGRMC1 ligand P4-BSA increased GIIS, whereas its antagonist AG-205 decreased GIIS. It was then found that PGRMC1 increased GLP-1-induced cAMP accumulation. PGRMC1 activation and GIIS induced by P4-BSA could be blocked by inhibition of adenylyl cyclase/EPAC signaling or the EGF receptor-PI3K signal transduction pathway. These data reveal a dual mechanism for PGRMC1-increased GIIS mediated through cAMP and EGF receptor signaling. In conclusion, we identified several novel GLP-1R interacting proteins. PGRMC1 expressed on the cell surface of ß cells was shown to interact with the activated GLP-1R to enhance the insulinotropic actions of GLP-1.

RalA GTPase tethers insulin granules to L- and R-type calcium channels through binding α2 δ-1 subunit.

RalA GTPase has been implicated in the regulated delivery of exocytotic vesicles to the plasma membrane (PM) in mammalian cells. We had reported that RalA regulates biphasic insulin secretion, which we have now determined to be contributed by RalA direct interaction with voltage-gated calcium (Cav ) channels. RalA knockdown (KD) in INS-1 cells and primary rat ß-cells resulted in a reduction in Ca(2+) currents arising specifically from L-(Cav 1.2 and Cav 1.3) and R-type (Cav 2.3) Ca(2+) channels. Restoration of RalA expression in RalA KD cells rescued these defects in Ca(2+) currents. RalA co-immunoprecipitated with the Cav α2 δ-1 auxiliary subunit known to bind the three Cav s. Moreover, the functional molecular interactions between Cav α2 δ-1 and RalA on the PM shown by total internal reflection fluorescent microscopy/FRET analysis could be induced by glucose stimulation. KD of RalA inhibited trafficking of α2 δ-1 to insulin granules without affecting the localization of the other Cav subunits. Furthermore, we confirmed that RalA and α2 δ-1 functionally interact since RalA KD-induced inhibition of Cav currents could not be recovered by RalA when α2 δ-1 was simultaneously knocked down. These data provide a mechanism for RalA function in insulin secretion, whereby RalA binds α2 δ-1 on insulin granules to tether these granules to PM Ca(2+) channels. This acts as a chaperoning step prior to and in preparation for sequential assembly of exocyst and excitosome complexes that mediate biphasic insulin secretion.

Spatial and temporal coordination of insulin granule exocytosis in intact human pancreatic islets.

AIMS/HYPOTHESIS: Insulin secretion is widely studied because it plays a central role in glucose homeostasis and diabetes. Processes from insulin granule fusion in beta cells to in vivo insulin secretion have been elucidated, but data at the cellular level do not fully account for several aspects of the macroscopic secretory pattern. Here we investigated how individual secretory events are coordinated spatially and temporally within intact human islets. METHODS: We used the fluorescent probe neuropeptide Y (NPY)-pHluorin to visualise insulin granule secretion in isolated intact human islets. RESULTS: We found that individual beta cells respond to increases in glucose concentration by releasing insulin granules in very discrete bursts with periods consistent with in vivo pulsatile insulin secretion. In successive secretory bursts during prolonged exposure to high glucose levels, secretory events progressively localised to preferential release sites, coinciding with the transition to second phase insulin secretion. Granule secretion was very synchronised in neighbouring beta cells, forming discrete regional clusters of activity. CONCLUSIONS/INTERPRETATION: These results reveal how individual secretory events are coordinated to produce pulsatile insulin secretion from human islets.