VITAMIN D-INDUCED INCREASE IN CALCIUM CONTENT IN SECRETORY GRANULES OF Β CELLS PLAYS A ROLE IN RECOVERY OF INSULIN SECRETION IN VITAMIN D-DEFICIENT RATS

Pancreatic islet β-cells secrete the hormones insulin, amylin and pancreastatin. To search for further β-cell hormones, we purified peptides from secretory granules isolated from cultured murine βTC6-F7 β-cells. We identified a 34-amino-acid peptide (3948Da), corresponding to Asp69–Leu102 of the proinsulin-like growth factor II E-peptide, which we have termed ‘preptin’. Preptin, is present in islet β-cells and undergoes glucose-mediated co-secretion with insulin. Synthetic preptin increases insulin secretion from glucose-stimulated βTC6-F7 cells in a concentration-dependent and saturable manner. Preptin infusion into the isolated, perfused rat pancreas increases the second phase of glucose-mediated insulin secretion by 30%, while anti-preptin immunoglobulin infusion decreases the first and second phases of insulin secretion by 29 and 26% respectively. These findings suggest that preptin is a physiological amplifier of glucose-mediated insulin secretion.

Download Full-text

Human stem cell model of HNF1A deficiency shows uncoupled insulin to C-peptide secretion with accumulation of abnormal insulin granules

10.1101/2021.01.26.428260 ◽

2021 ◽

Author(s):

Bryan J. González ◽

Haoquan Zhao ◽

Jacqueline Niu ◽

Damian J. Williams ◽

Jaeyop Lee ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Insulin Secretion ◽

Intracellular Calcium ◽

Cell Fate ◽

Endocrine Cells ◽

Secretory Granules ◽

Β Cells ◽

C Peptide ◽

Pancreatic Endocrine Cells

AbstractMutations in HNF1A cause Maturity Onset Diabetes of the Young type 3 (MODY3), the most prevalent form of monogenic diabetes. We generated stem cell-derived pancreatic endocrine cells from human embryonic stem cells (hESCs) with induced hypomorphic mutations in HNF1A. Using these cells, we show that HNF1A orchestrates a transcriptional program required for distinct aspects of β-cell fate and function. During islet cell differentiation, HNF1A deficiency biases islet endocrine cells towards an α-cell fate associated with PAX4 down-regulation. HNF1A- deficient β-cells display impaired basal and glucose stimulated-insulin secretion in association with a reduction in CACNA1A and intracellular calcium levels, and impaired insulin granule exocytosis in association with SYT13 down-regulation. Knockout of PAX4, CACNA1A and SYT13 reproduce the relevant phenotypes. Reduction of insulin secretion is associated with accumulation of enlarged secretory granules, and altered stoichiometry of secreted insulin to C-peptide. In HNF1A deficient β-cells, glibenclamide, a sulfonylurea drug used in the treatment of MODY3 patients, increases intracellular calcium to levels beyond those achieved by glucose, and restores C-peptide and insulin secretion to a normal stoichiometric ratio. To study HNF1A deficiency in the context of a human disease model, we also generated stem cell-derived pancreatic endocrine cells from two MODY3 patient’s induced pluripotent stem cells (iPSCs). While insulin secretion defects are constitutive in cells with complete HNF1A loss of function, β-cells heterozygous for hypomorphic HNF1A mutations are initially normal, but lose the ability to secrete insulin and acquire abnormal stoichiometric secretion ratios. Importantly, the defects observed in these stem cell models are also seen in circulating proportions of insulin:C-peptide in nine MODY3 patients.One sentence of summaryDeficiency of the transcription factor HNF1A biases islet endocrine cell fate towards α-cells, impairs intracellular calcium homeostasis and insulin exocytosis, alters the stoichiometry of insulin to C-peptide release, and leads to an accumulation of abnormal insulin secretory granules in β-cells.

Download Full-text

Vitamin D induces autophagy of pancreatic β-cells and enhances insulin secretion

Molecular Medicine Reports ◽

10.3892/mmr.2016.5531 ◽

2016 ◽

Vol 14 (3) ◽

pp. 2644-2650 ◽

Cited By ~ 18

Author(s):

Yubin Wang ◽

Dawei He ◽

Chengpei Ni ◽

Huiying Zhou ◽

Shuyan Wu ◽

...

Keyword(s):

Vitamin D ◽

Insulin Secretion ◽

Β Cells ◽

Pancreatic Β Cells

Download Full-text

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

Molecular Biology of the Cell ◽

10.1091/mbc.e21-03-0094 ◽

2021 ◽

pp. mbc.E21-03-0094

Author(s):

Hiroshi Tokuo ◽

Shigeru Komaba ◽

Lynne M. Coluccio

Keyword(s):

Insulin Secretion ◽

Islet Cells ◽

Early Stage ◽

Secretory Granules ◽

Β Cells ◽

Pancreatic Β Cells ◽

Glucose Levels ◽

Insulin Granule ◽

Glucose Stimulated Insulin Secretion ◽

Insulinoma Cells

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 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 siRNA in 832/13 cells reduced intracellular proinsulin and insulin content and glucose-stimulated insulin secretion (GSIS), and led to the accumulation of (pro)insulin SGs at the TGN. Using an in situ fluorescent pulse-chase strategy to track nascent proinsulin (Bearrows et al., 2019), Myo1b depletion in insulinoma cells reduced the number of (pro)insulin-containing secretory granules 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.

Download Full-text

SIDT2 is involved in the NAADP-mediated release of calcium from insulin secretory granules

Journal of Molecular Endocrinology ◽

10.1530/jme-15-0227 ◽

2016 ◽

Vol 56 (3) ◽

pp. 249-259 ◽

Cited By ~ 8

Author(s):

Guoying Chang ◽

Rui Yang ◽

Yanan Cao ◽

Aifang Nie ◽

Xuefan Gu ◽

...

Keyword(s):

Insulin Secretion ◽

Response Pattern ◽

Secretory Granules ◽

Peak Height ◽

Bafilomycin A1 ◽

Β Cells ◽

Cd38 Expression ◽

Impaired Insulin ◽

Acidic Compartment

The Sidt2 global knockout mouse (Sidt2−/−) has impaired insulin secretion. The aim of this study was to assess the role of SIDT2 protein in glucose-induced insulin secretion in primary cultured mouse β-cells. The major metabolic and electrophysiological steps of glucose-induced insulin secretion of primary cultured β-cells from Sidt2−/− mice were investigated. The β-cells from Sidt2−/− mice had normal NAD(P)H responses and KATP and KV currents. However, they exhibited a lower [Ca2+]i peak height when stimulated with 20mM glucose compared with those from WT mice. Furthermore, it took a longer time for the [Ca2+]i of β-cell from Sidt2−/− mice to reach the peak. Pretreatment with ryanodine or 2-aminoethoxydiphenyl borate (2-APB) did not change [Ca2+]i the response pattern to glucose in Sidt2−/− cells. Extraordinarily, pretreatment with bafilomycin A1(Baf-A1) led to a comparable [Ca2+]i increase pattern between these two groups, suggesting that calcium traffic from the intracellular acidic compartment is defective in Sidt2−/− β-cells. Bath-mediated application of 50nM nicotinic acid adenine dinucleotide phosphate (NAADP) normalized the [Ca2+]i response of Sidt2−/− β-cells. Finally, glucose-induced CD38 expression increased to a comparable level between Sidt2−/− and WT islets, suggesting that Sidt2−/− islets generated NAADP normally. We conclude that Sidt2 is involved in NAADP-mediated release of calcium from insulin secretory granules and thus regulates insulin secretion.

Download Full-text

Dopamine-Mediated Autocrine Inhibitory Circuit Regulating Human Insulin Secretion in Vitro

Molecular Endocrinology ◽

10.1210/me.2012-1101 ◽

2012 ◽

Vol 26 (10) ◽

pp. 1757-1772 ◽

Cited By ~ 43

Author(s):

Norman Simpson ◽

Antonella Maffei ◽

Matthew Freeby ◽

Steven Burroughs ◽

Zachary Freyberg ◽

...

Keyword(s):

Insulin Secretion ◽

Secretory Granules ◽

Human Islets ◽

Negative Regulator ◽

Β Cells ◽

Regulatory Circuit ◽

Glucose Stimulated Insulin Secretion ◽

Insulin Secretion In Vitro

Abstract We describe a negative feedback autocrine regulatory circuit for glucose-stimulated insulin secretion in purified human islets in vitro. Using chronoamperometry and in vitro glucose-stimulated insulin secretion measurements, evidence is provided that dopamine (DA), which is loaded into insulin-containing secretory granules by vesicular monoamine transporter type 2 in human β-cells, is released in response to glucose stimulation. DA then acts as a negative regulator of insulin secretion via its action on D2R, which are also expressed on β-cells. We found that antagonism of receptors participating in islet DA signaling generally drive increased glucose-stimulated insulin secretion. These in vitro observations may represent correlates of the in vivo metabolic changes associated with the use of atypical antipsychotics, such as increased adiposity.

Download Full-text

α-Synuclein binds the KATP channel at insulin-secretory granules and inhibits insulin secretion

AJP Endocrinology and Metabolism ◽

10.1152/ajpendo.00262.2010 ◽

2011 ◽

Vol 300 (2) ◽

pp. E276-E286 ◽

Cited By ~ 40

Author(s):

Xuehui Geng ◽

Haiyan Lou ◽

Jian Wang ◽

Lehong Li ◽

Alexandra L. Swanson ◽

...

Keyword(s):

Insulin Secretion ◽

Secretory Granules ◽

Cell Types ◽

Β Cells ◽

Glucose Stimulation ◽

Transmission Electron ◽

Parkinson’S Diseases ◽

Granule Density ◽

Numerous Cell ◽

Glucose Stimulated Insulin Secretion

α-Synuclein has been studied in numerous cell types often associated with secretory processes. In pancreatic β-cells, α-synuclein might therefore play a similar role by interacting with organelles involved in insulin secretion. We tested for α-synuclein localizing to insulin-secretory granules and characterized its role in glucose-stimulated insulin secretion. Immunohistochemistry and fluorescent sulfonylureas were used to test for α-synuclein localization to insulin granules in β-cells, immunoprecipitation with Western blot analysis for interaction between α-synuclein and KATP channels, and ELISA assays for the effect of altering α-synuclein expression up or down on insulin secretion in INS1 cells or mouse islets, respectively. Differences in cellular phenotype between α-synuclein knockout and wild-type β-cells were found by using confocal microscopy to image the fluorescent insulin biosensor Ins-C-emGFP and by using transmission electron microscopy. The results show that anti-α-synuclein antibodies labeled secretory organelles within β-cells. Anti-α-synuclein antibodies colocalized with KATP channel, anti-insulin, and anti-C-peptide antibodies. α-Synuclein coimmunoprecipitated in complexes with KATP channels. Expression of α-synuclein downregulated insulin secretion at 2.8 mM glucose with little effect following 16.7 mM glucose stimulation. α-Synuclein knockout islets upregulated insulin secretion at 2.8 and 8.4 mM but not 16.7 mM glucose, consistent with the depleted insulin granule density at the β-cell surface membranes observed in these islets. These findings demonstrate that α-synuclein interacts with KATP channels and insulin-secretory granules and functionally acts as a brake on secretion that glucose stimulation can override. α-Synuclein might play similar roles in diabetes as it does in other degenerative diseases, including Alzheimer's and Parkinson's diseases.

Download Full-text

Vesicular Nucleotide Transporter-Mediated ATP Release Regulates Insulin Secretion

Endocrinology ◽

10.1210/en.2012-1818 ◽

2012 ◽

Vol 154 (2) ◽

pp. 675-684 ◽

Cited By ~ 38

Author(s):

Jessica C. Geisler ◽

Kathryn L. Corbin ◽

Qin Li ◽

Andrew P. Feranchak ◽

Craig S. Nunemaker ◽

...

Keyword(s):

Insulin Secretion ◽

Basal Insulin ◽

Secretory Granules ◽

Atp Release ◽

Extracellular Atp ◽

Β Cells ◽

Pancreatic Β Cells ◽

Hairpin Rna ◽

Small Hairpin Rna ◽

Mouse Islets

Extracellular ATP plays a critical role in regulating insulin secretion in pancreatic β cells. The ATP released from insulin secretory vesicles has been proposed to be a major source of extracellular ATP. Currently, the mechanism by which ATP accumulates into insulin secretory granules remains elusive. In this study, the authors identified the expression of a vesicular nucleotide transporter (VNUT) in mouse pancreas, isolated mouse islets, and MIN6 cells, a mouse β cell line. Immunohistochemistry and immunofluorescence revealed that VNUT colocalized extensively with insulin secretory granules. Functional studies showed that suppressing endogenous VNUT expression in β cells by small hairpin RNA knockdown greatly reduced basal- and glucose-induced ATP release. Importantly, knocking down VNUT expression by VNUT small hairpin RNA in MIN6 cells and isolated mouse islets dramatically suppressed basal insulin release and glucose-stimulated insulin secretion (GSIS). Moreover, acute pharmacologic blockade of VNUT with Evans blue, a VNUT antagonist, greatly attenuated GSIS in a dose-dependent manner. Exogenous ATP treatment effectively reversed the insulin secretion defect induced by both VNUT knockdown and functional inhibition, indicating that VNUT-mediated ATP release is essential for maintaining normal insulin secretion. In contrast to VNUT knockdown, overexpression of VNUT in β cells resulted in excessive ATP release and enhanced basal insulin secretion and GSIS. Elevated insulin secretion induced by VNUT overexpression was reversed by pharmacologic inhibition of P2X but not P2Y purinergic receptors. This study reveals VNUT is expressed in pancreatic β cells and plays an essential and novel role in regulating insulin secretion through vesicular ATP release and extracellular purinergic signaling.

Download Full-text

A fluorescent timer reporter enables sorting of insulin secretory granules by age

Journal of Biological Chemistry ◽

10.1074/jbc.ra120.012432 ◽

2020 ◽

Vol 295 (27) ◽

pp. 8901-8911 ◽

Cited By ~ 2

Author(s):

Belinda Yau ◽

Lori Hays ◽

Cassandra Liang ◽

D. Ross Laybutt ◽

Helen E. Thomas ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Function ◽

Islet Cells ◽

Ex Vivo ◽

Secretory Granules ◽

Β Cells ◽

Β Cell ◽

Short Period ◽

Insulin Granule

Within the pancreatic β-cells, insulin secretory granules (SGs) exist in functionally distinct pools, displaying variations in motility as well as docking and fusion capability. Current therapies that increase insulin secretion do not consider the existence of these distinct SG pools. Accordingly, these approaches are effective only for a short period, with a worsening of glycemia associated with continued decline in β-cell function. Insulin granule age is underappreciated as a determinant for why an insulin granule is selected for secretion and may explain why newly synthesized insulin is preferentially secreted from β-cells. Here, using a novel fluorescent timer protein, we aimed to investigate the preferential secretion model of insulin secretion and identify how granule aging is affected by variation in the β-cell environment, such as hyperglycemia. We demonstrate the use of a fluorescent timer construct, syncollin-dsRedE5TIMER, which changes its fluorescence from green to red over 18 h, in both microscopy and fluorescence-assisted organelle-sorting techniques. We confirm that the SG-targeting construct localizes to insulin granules in β-cells and does not interfere with normal insulin SG behavior. We visualize insulin SG aging behavior in MIN6 and INS1 β-cell lines and in primary C57BL/6J mouse and nondiabetic human islet cells. Finally, we separated young and old insulin SGs, revealing that preferential secretion of younger granules occurs in glucose-stimulated insulin secretion. We also show that SG population age is modulated by the β-cell environment in vivo in the db/db mouse islets and ex vivo in C57BL/6J islets exposed to different glucose environments.

Download Full-text

Pancreatic beta-cell specific deletion of VPS41 causes diabetes due to defects in insulin secretion

10.1101/2020.04.25.043729 ◽

2020 ◽

Author(s):

Christian H. Burns ◽

Belinda Yau ◽

Anjelica Rodriguez ◽

Jenna Triplett ◽

Drew Maslar ◽

...

Keyword(s):

Insulin Secretion ◽

Glucose Homeostasis ◽

Transmembrane Protein ◽

Secretory Granules ◽

Pancreatic Beta Cell ◽

Protein Composition ◽

Β Cells ◽

Regulated Exocytosis ◽

Specific Proteins ◽

Hops Complex

AbstractInsulin secretory granules (SGs) mediate the regulated secretion of insulin, which is essential for glucose homeostasis. The basic machinery responsible for this regulated exocytosis consists of specific proteins present both at the plasma membrane and on insulin SGs. The protein composition of insulin SGs thus dictates their release properties, yet the mechanisms controlling insulin SG formation, which determines this molecular composition, remain poorly understood. VPS41, a component of the endo-lysosomal tethering HOPS complex, was recently identified as a cytosolic factor involved in the formation of neuroendocrine and neuronal granules. We now find that VPS41 is required for insulin SG biogenesis and regulated insulin secretion. Loss of VPS41 in pancreatic β-cells leads to a reduction in insulin SG number, changes in their transmembrane protein composition, and defects in granule regulated exocytosis. Exploring a human point mutation, identified in patients with neurological but no endocrine defects, we show that the effect on SG formation is independent of HOPS complex formation. Finally, we report that mice with a deletion of VPS41 specifically in β-cells develop diabetes due to severe depletion of insulin SG content and a defect in insulin secretion. In sum, our data demonstrate that VPS41 contributes to glucose homeostasis and metabolism.

Download Full-text