KCNH6 channel promotes insulin exocytosis via interaction with Munc18-1 independent of electrophysiological processes.

Glucose-stimulated insulin secretion (GSIS) in pancreatic islet ß-cells primarily relies on electrophysiological processes. Previous research highlighted the regulatory role of KCNH6, a member of the Kv channel family, in governing GSIS through its influence on ß-cell electrophysiology. In this study, we unveil a novel facet of KCNH6's function concerning insulin granule exocytosis, independent of its conventional electrical role. Young mice with ß-cell-specific KCNH6 knockout (ßKO) exhibited impaired glucose tolerance and reduced insulin secretion, a phenomenon not explained by electrophysiological processes alone. Consistently, islets from KCNH6-ßKO mice exhibited reduced insulin secretion, conversely, the overexpression of KCNH6 in murine pancreatic islets significantly enhanced insulin release. Moreover, insulin granules lacking KCNH6 demonstrated compromised docking capabilities and a reduced fusion response upon glucose stimulation. Crucially, our investigation unveiled a significant interaction between KCNH6 and the SNARE protein regulator, Munc18-1, a key mediator of insulin granule exocytosis. These findings underscore the critical role of KCNH6 in the regulation of insulin secretion through its interaction with Munc18-1, providing a promising and novel avenue for enhancing our understanding of the Kv channel in diabetes mechanisms.

Rapid Quantification of First and Second Phase Insulin Secretion Dynamics using an In vitro Platform for Improving Insulin Therapy.

High-throughput quantification of the first- and second-phase insulin secretion dynamics is intractable with current methods. The fact that independent secretion phases play distinct roles in metabolism necessitates partitioning them separately and performing high-throughput compound screening to target them individually. We developed an insulin-nanoluc luciferase reporter system to dissect the molecular and cellular pathways involved in the separate phases of insulin secretion. We validated this method through genetic studies, including knockdown and overexpression, as well as small-molecule screening and their effects on insulin secretion. Furthermore, we demonstrated that the results of this method are well correlated with those of single-vesicle exocytosis experiments conducted on live cells, providing a quantitative reference for the approach. Thus, we have developed a robust methodology for screening small molecules and cellular pathways that target specific phases of insulin secretion, resulting in a better understanding of insulin secretion, which in turn will result in a more effective insulin therapy through the stimulation of endogenous glucose-stimulated insulin secretion.

The signaling protein GIV/Girdin mediates the Nephrin-dependent insulin secretion of pancreatic islet ß cells in response to high glucose.

Glucose-stimulated insulin secretion of pancreatic ß cells is essential in maintaining glucose homeostasis. Recent evidence suggests that the Nephrin-mediated intercellular junction between ß cells is implicated in the regulation of insulin secretion. However, the underlying mechanisms are only partially characterized. Herein we report that GIV is a signaling mediator coordinating glucose-stimulated Nephrin phosphorylation and endocytosis with insulin secretion. We demonstrate that GIV is expressed in mouse islets and cultured ß cells. The loss of function study suggests that GIV is essential for the second phase of glucose-stimulated insulin secretion. Next, we demonstrate that GIV mediates the high glucose-stimulated tyrosine phosphorylation of GIV and Nephrin by recruiting Src kinase, which leads to the endocytosis of Nephrin. Subsequently, the glucose-induced GIV/Nephrin/Src signaling events trigger downstream Akt phosphorylation, which activates Rac1-mediated cytoskeleton reorganization, allowing insulin secretory granules to access the plasma membrane for the second-phase secretion. Finally, we found that GIV is downregulated in the islets isolated from diabetic mice, and rescue of GIV ameliorates the ß-cell dysfunction to restore the glucose-stimulated insulin secretion. We conclude that the GIV/Nephrin/Akt signaling axis is vital to regulate glucose-stimulated insulin secretion. This mechanism might be further targeted for therapeutic intervention of diabetic mellitus.

Recurrent hypoglycemia dampens functional regulation mediated via Neurexin-1, Neuroligin-2 and Mint-1 docking proteins: Intensified complications during diabetes.

Glycemic regulation is important for maintaining critical physiological functions. Extreme variation in levels of circulating glucose are known to affect insulin secretion. Elevated insulin levels result in lowering of circulating glycemic levels culminating into hypoglycemia. Recurrence of hypoglycemia are often noted owing to fasting conditions, untimely meals, irregular dietary consumption, or as a side-effect of disease pathophysiology. Such events of hypoglycemia threaten to hamper the patterns of insulin secretion in diabetic condition. Insulin vesicle docking is a prerequisite phase which ensures anchoring of the vesicles to the ß-cell membrane in order to expel the insulin cargo. Neurexin and Neuroligin are the marker docking proteins which assists in the tethering of the insulin granules to the secretory membrane. However, these cell adhesion molecules indirectly affect the glycemic levels by regulating insulin secretion. The effect of extreme levels of glycemic fluctuations on these molecules, and how it affects the docking machinery remains obscure. Our current study demonstrates down-regulated expression of Neurexin-1, Neuroligin-2 and Mint-1 molecules during hyperglycemia, hypoglycemia and diabetic hypoglycemia in rodents as well as for an in-vitro system using MIN6 cell-line. Studies with fluorescently labelled insulin revealed presence of lessened functional insulin secretory granules, concomitant with the alterations in morphology and as a result of hypoglycemia in control and diabetic condition which was found to be further deteriorating. Our studies indicate towards a feeble vesicular anchorage, which may partly be responsible for dwindled insulin secretion during diabetes. However, hypoglycemia poses as a potent diabetic complication in further deteriorating the docking machinery. To the best of our knowledge this is the first report which demonstrates the effect of hypoglycemic events in affecting insulin secretion by weakening insulin vesicular anchorage in normal and diabetic individuals.

Sulfated Fucogalactan From Laminaria Japonica Ameliorates ß-Cell Failure by Attenuating Mitochondrial Dysfunction via SIRT1-PGC1-α Signaling Pathway Activation.

As mitochondrial metabolism is a major determinant of ß-cell insulin secretion, mitochondrial dysfunction underlies ß-cell failure and type 2 diabetes mellitus progression. An algal polysaccharide of Laminaria japonica, sulfated fucogalactan (SFG) displays various pharmacological effects in a variety of conditions, including metabolic disease. We investigated the protective effects of SFG against hydrogen peroxide (H2O2)-induced ß-cell failure in MIN6 cells and islets. SFG significantly promoted the H2O2-inhibited proliferation in the cells and ameliorated their senescence, and potentiated ß-cell function by regulating ß-cell identity and the insulin exocytosis-related genes and proteins in H2O2-induced ß-cells. SFG also attenuated mitochondrial dysfunction, including alterations in ATP content, mitochondrial respiratory chain genes and proteins expression, and reactive oxygen species and superoxide dismutase levels. Furthermore, SFG resulted in SIRT1-PGC1-α pathway activation and upregulated the downstream Nrf2 and Tfam. Taken together, the results show that SFG attenuates H2O2-induced ß-cell failure by improving mitochondrial function via SIRT1-PGC1-α signaling pathway activation. Therefore, SFG is implicated as a potential agent for treating pancreatic ß-cell failure.

MAFA and MAFB regulate exocytosis-related genes in human ß-cells.

AIMS: Reduced expression of exocytotic genes is associated with functional defects in insulin exocytosis contributing to impaired insulin secretion and type 2 diabetes (T2D) development. MAFA and MAFB transcription factors regulate ß-cell physiology, and their gene expression is reduced in T2D ß cells. We investigate if loss of MAFA and MAFB in human ß cells contributes to T2D progression by regulating genes required for insulin exocytosis. METHODS: Three approaches were performed: (1) RNAseq analysis with the focus on exocytosis-related genes in MafA-/- mouse islets, (2) correlational analysis between MAFA, MAFB and exocytosis-related genes in human islets and (3) MAFA and MAFB silencing in human islets and EndoC-ßH1 cells followed by functional in vitro studies. RESULTS: The expression of 30 exocytosis-related genes was significantly downregulated in MafA-/- mouse islets. In human islets, the expression of 29 exocytosis-related genes correlated positively with MAFA and MAFB. Eight exocytosis-related genes were downregulated in MafA-/- mouse islets and positively correlated with MAFA and MAFB in human islets. From this analysis, the expression of RAB3A, STXBP1, UNC13A, VAMP2, NAPA, NSF, STX1A and SYT7 was quantified after acute MAFA or MAFB silencing in EndoC-ßH1 cells and human islets. MAFA and MAFB silencing resulted in impaired insulin secretion and reduced STX1A, SYT7 and STXBP1 (EndoC-ßH1) and STX1A (human islets) mRNA expression. STX1A and STXBP1 protein expression was also impaired in islets from T2D donors which lack MAFA expression. CONCLUSION: Our data indicate that STXBP1 and STX1A are important MAFA/B-regulated exocytosis genes which may contribute to insulin exocytosis defects observed in MAFA-deficient human T2D ß cells.

Role of the active zone protein, ELKS, in insulin secretion from pancreatic ß-cells.

BACKGROUND: Insulin is stored within large dense-core granules in pancreatic beta (ß)-cells and is released by Ca2+-triggered exocytosis with increasing blood glucose levels. Polarized and targeted secretion of insulin from ß-cells in pancreatic islets into the vasculature has been proposed; however, the mechanisms related to cellular and molecular localization remain largely unknown. Within nerve terminals, the Ca2+-dependent release of a polarized transmitter is limited to the active zone, a highly specialized area of the presynaptic membrane. Several active zone-specific proteins have been characterized; among them, the CAST/ELKS protein family members have the ability to form large protein complexes with other active zone proteins to control the structure and function of the active zone for tight regulation of neurotransmitter release. Notably, ELKS but not CAST is also expressed in ß-cells, implying that ELKS may be involved in polarized insulin secretion from ß-cells. SCOPE OF REVIEW: This review provides an overview of the current findings regarding the role(s) of ELKS and other active zone proteins in ß-cells and focuses on the molecular mechanism underlying ELKS regulation within polarized insulin secretion from islets. MAJOR CONCLUSIONS: ELKS localizes at the vascular-facing plasma membrane of ß-cells in mouse pancreatic islets. ELKS forms a potent insulin secretion complex with L-type voltage-dependent Ca2+ channels on the vascular-facing plasma membrane of ß-cells, enabling polarized Ca2+ influx and first-phase insulin secretion from islets. This model provides novel insights into the functional polarity observed during insulin secretion from ß-cells within islets at the molecular level. This active zone-like region formed by ELKS at the vascular side of the plasma membrane is essential for coordinating physiological insulin secretion and may be disrupted in diabetes.

ELKS/Voltage-Dependent Ca2+ Channel-ß Subunit Module Regulates Polarized Ca2+ Influx in Pancreatic ß Cells.

Pancreatic ß cells secrete insulin by Ca2+-triggered exocytosis. However, there is no apparent secretory site similar to the neuronal active zones, and the cellular and molecular localization mechanism underlying polarized exocytosis remains elusive. Here, we report that ELKS, a vertebrate active zone protein, is used in ß cells to regulate Ca2+ influx for insulin secretion. ß cell-specific ELKS-knockout (KO) mice showed impaired glucose-stimulated first-phase insulin secretion and reduced L-type voltage-dependent Ca2+ channel (VDCC) current density. In situ Ca2+ imaging of ß cells within islets expressing a membrane-bound G-CaMP8b Ca2+ sensor demonstrated initial local Ca2+ signals at the ELKS-localized vascular side of the ß cell plasma membrane, which were markedly decreased in ELKS-KO ß cells. Mechanistically, ELKS directly interacted with the VDCC-ß subunit via the GK domain. These findings suggest that ELKS and VDCCs form a potent insulin secretion complex at the vascular side of the ß cell plasma membrane for polarized Ca2+ influx and first-phase insulin secretion from pancreatic islets.

Characterization of P4 ATPase Phospholipid Translocases (Flippases) in Human and Rat Pancreatic Beta Cells: THEIR GENE SILENCING INHIBITS INSULIN SECRETION.

The negative charge of phosphatidylserine in lipid bilayers of secretory vesicles and plasma membranes couples the domains of positively charged amino acids of secretory vesicle SNARE proteins with similar domains of plasma membrane SNARE proteins enhancing fusion of the two membranes to promote exocytosis of the vesicle contents of secretory cells. Our recent study of insulin secretory granules (ISG) (MacDonald, M. J., Ade, L., Ntambi, J. M., Ansari, I. H., and Stoker, S. W. (2015) Characterization of phospholipids in insulin secretory granules in pancreatic beta cells and their changes with glucose stimulation. J. Biol. Chem. 290, 11075-11092) suggested that phosphatidylserine and other phospholipids, such as phosphatidylethanolamine, in ISG could play important roles in docking and fusion of ISG to the plasma membrane in the pancreatic beta cell during insulin exocytosis. P4 ATPase flippases translocate primarily phosphatidylserine and, to a lesser extent, phosphatidylethanolamine across the lipid bilayers of intracellular vesicles and plasma membranes to the cytosolic leaflets of these membranes. CDC50A is a protein that forms a heterodimer with P4 ATPases to enhance their translocase catalytic activity. We found that the predominant P4 ATPases in pure pancreatic beta cells and human and rat pancreatic islets were ATP8B1, ATP8B2, and ATP9A. ATP8B1 and CDC50A were highly concentrated in ISG. ATP9A was concentrated in plasma membrane. Gene silencing of individual P4 ATPases and CDC50A inhibited glucose-stimulated insulin release in pure beta cells and in human pancreatic islets. This is the first characterization of P4 ATPases in beta cells. The results support roles for P4 ATPases in translocating phosphatidylserine to the cytosolic leaflets of ISG and the plasma membrane to facilitate the docking and fusion of ISG to the plasma membrane during insulin exocytosis.

Discovery of novel vitamin D-regulated proteins in INS-1 cells: a proteomic approach.

BACKGROUND: Experimental evidence indicates that vitamin D may have a beneficial role in pancreatic ß-cell function. Global gene expression studies have shown that the active metabolite 1,25-dihydroxyvitamin D3 [1,25-(OH)2 D3 ] modulates genes involved in ion transport, lipid metabolism and insulin secretion. METHODS: We employed stable isotope labelling by amino acids in cell culture in combination with liquid chromatography-tandem mass spectrometry to quantitatively assess the impact of two vitamin D metabolites, 1,25-(OH)2 D3 and 25-hydroxyvitamin D3 [25-(OH)D3 ], on global protein expression on a model rat ß-cell line, insulinoma-derived INS-1 cells. RESULTS: Although treatment with 1,25-(OH)2 D3 resulted in 31 differentially expressed proteins, 25-(OH)D3 had no impact on protein expression. Of these 31 proteins, 29 were upregulated, whereas two showed a decrease in abundance. Proteins whose expression levels markedly increased in the presence of 1,25-(OH)2 D3 included Crat, Hmgn2, Protein Tmsbl1 and Gdap1. One of the most important findings in this study is upregulation of proteins implicated in insulin granule motility and insulin exocytosis, suggesting a positive effect on insulin secretion. Moreover, modulation of several membrane transport proteins suggests that 1,25-(OH)2 D3 has an impact on the homeostatic regulation of ions, which is critical for most functions in the ß-cell. CONCLUSIONS: In this study, we discovered a number of novel 1,25-(OH)2 D3 -regulated proteins, which may contribute to a better understanding of the reported beneficial effects of vitamin D on pancreatic ß-cells. All in all, our findings should pave the way for future studies providing insights into molecular mechanisms by which 1,25-(OH)2 D3 regulates protein expression in pancreatic ß-cells.