Functional Reconstitution of the Insulin-Secreting Porosome Complex in Live Cells

Abstract Supramolecular cup-shaped lipoprotein structures called porosomes embedded in the cell plasma membrane mediate fractional release of intravesicular contents from cells during secretion. The presence of porosomes, have been documented in many cell types including neurons, acinar cells of the exocrine pancreas, GH-secreting cells of the pituitary, and insulin-secreting pancreatic β-cells. Functional reconstitution of porosomes into artificial lipid membranes, have also been accomplished. Earlier studies on mouse insulin-secreting Min6 cells report 100-nm porosome complexes composed of nearly 30 proteins. In the current study, porosomes have been functionally reconstituted for the first time in live cells. Isolated Min6 porosomes reconstituted into live Min6 cells demonstrate augmented levels of porosome proteins and a consequent increase in the potency and efficacy of glucose-stimulated insulin release. Elevated glucose-stimulated insulin secretion 48 hours after reconstitution, reflects on the remarkable stability and viability of reconstituted porosomes, documenting the functional reconstitution of native porosomes in live cells. These results, establish a new paradigm in porosome-mediated insulin secretion in β-cells.

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Fructose-1,6-Bisphosphatase Regulates Glucose-Stimulated Insulin Secretion of Mouse Pancreatic β-Cells

Endocrinology ◽

10.1210/en.2009-1185 ◽

2010 ◽

Vol 151 (10) ◽

pp. 4688-4695 ◽

Cited By ~ 16

Author(s):

Ye Zhang ◽

Zhifang Xie ◽

Guangdi Zhou ◽

Hai Zhang ◽

Jian Lu ◽

...

Keyword(s):

Insulin Secretion ◽

Specific Inhibitor ◽

Physiological Role ◽

Glucose Sensing ◽

Β Cells ◽

Pancreatic Β Cells ◽

Min6 Cells ◽

Fructose 1,6 Bisphosphatase ◽

Glucose Stimulated Insulin Secretion

Pancreatic β-cells can precisely sense glucose stimulation and accordingly adjust their insulin secretion. Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme, but its physiological significance in β-cells is not established. Here we determined its physiological role in regulating glucose sensing and insulin secretion of β-cells. Considerable FBPase mRNA was detected in normal mouse islets and β-cell lines, although their protein levels appeared to be quite low. Down-regulation of FBP1 in MIN6 cells by small interfering RNA could enhance the glucose-stimulated insulin secretion (GSIS), whereas FBP1-overexpressing MIN6 cells exhibited decreased GSIS. Inhibition of FBPase activity in islet β-cells by its specific inhibitor MB05032 led to significant increase of their glucose utilization and cellular ATP to ADP ratios and consequently enhanced GSIS in vitro. Pretreatment of mice with the MB05032 prodrug MB06322 could potentiate GSIS in vivo and improve their glucose tolerance. Therefore, FBPase plays an important role in regulating glucose sensing and insulin secretion of β-cells and serves a promising target for diabetes treatment.

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Cell-wide analysis of secretory granule dynamics in three dimensions in living pancreatic β-cells: evidence against a role for AMPK-dependent phosphorylation of KLC1 at Ser517/Ser520 in glucose-stimulated insulin granule movement

Biochemical Society Transactions ◽

10.1042/bst0380205 ◽

2010 ◽

Vol 38 (1) ◽

pp. 205-208 ◽

Cited By ~ 4

Author(s):

Angela McDonald ◽

Sarah Fogarty ◽

Isabelle Leclerc ◽

Elaine V. Hill ◽

D. Grahame Hardie ◽

...

Keyword(s):

Insulin Secretion ◽

Glutathione Transferase ◽

Three Dimensions ◽

Β Cells ◽

Pancreatic Β Cells ◽

Glucose Levels ◽

Insulin Granules ◽

Elevated Glucose ◽

Glucose Stimulated Insulin Secretion

Glucose-stimulated insulin secretion from pancreatic β-cells requires the kinesin-1/Kif5B-mediated transport of insulin granules along microtubules. 5′-AMPK (5′-AMP-activated protein kinase) is a heterotrimeric serine/threonine kinase which is activated in β-cells at low glucose concentrations, but inhibited as glucose levels increase. Active AMPK blocks glucose-stimulated insulin secretion and the recruitment of insulin granules to the cell surface, suggesting motor proteins may be targets for this kinase. While both kinesin-1/Kif5B and KLC1 (kinesin light chain-1) contain consensus AMPK phosphorylation sites (Thr693 and Ser520, respectively) only recombinant GST (glutathione transferase)–KLC1 was phosphorylated by purified AMPK in vitro. To test the hypothesis that phosphorylation at this site may modulate kinesin-1-mediated granule movement, we developed an approach to study the dynamics of all the resolvable granules within a cell in three dimensions. This cell-wide approach revealed that the number of longer excursions (>10 μm) increased significantly in response to elevated glucose concentration (30 versus 3 mM) in control MIN6 β-cells. However, similar changes were seen in cells overexpressing wild-type KLC1, phosphomimetic (S517D/S520D) or non-phosphorylatable (S517A/S520A) mutants of KLC1. Thus, changes in the phosphorylation state of KLC1 at Ser517/Ser520 seem unlikely to affect motor function.

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Inhibition of Forkhead Box O1 Protects Pancreatic β-Cells against Dexamethasone-Induced Dysfunction

Endocrinology ◽

10.1210/en.2009-0343 ◽

2009 ◽

Vol 150 (9) ◽

pp. 4065-4073 ◽

Cited By ~ 24

Author(s):

Xiongfei Zhang ◽

Wei Yong ◽

Jinghuan Lv ◽

Yunxia Zhu ◽

Jingjing Zhang ◽

...

Keyword(s):

Insulin Secretion ◽

Cell Types ◽

Pancreatic Β Cell ◽

Rinm5f Cells ◽

Β Cells ◽

Β Cell ◽

Rat Islets ◽

Cell Dysfunction ◽

Forkhead Box ◽

Glucose Stimulated Insulin Secretion

Abstract Forkhead Box O1 (FoxO1) is a key transcription regulator of insulin/IGF-I signaling pathway, and its activity can be increased by dexamethasone (DEX) in several cell types. However, the role of FoxO1 in DEX-induced pancreatic β-cell dysfunction has not been fully understood. Therefore, in this study, we investigated whether FoxO1 could mediate DEX-induced β-cell dysfunction and the possible underlying mechanisms in pancreatic β-cell line RINm5F cells and primary rat islet. We found that DEX markedly increased FoxO1 mRNA and protein expression and decreased FoxO1 phosphorylation through the Akt pathway, which resulted in an increase in active FoxO1 in RINm5F cells and isolated rat islets. Activated FoxO1 subsequently inhibited pancreatic duodenal homeobox-1 expression and induced nuclear exclusion of pancreatic duodenal homeobox-1. Knockdown of FoxO1 by RNA interference restored the expression of pancreatic duodenal homeobox-1 and prevented DEX-induced dysfunction of glucose-stimulated insulin secretion in rat islets. Together, the results of present study demonstrate that FoxO1 is integrally involved in DEX-induced inhibition of pancreatic duodenal homeobox-1 and glucose-stimulated insulin secretion dysfunction in pancreatic islet β-cells. Inhibition of FoxO1 can effectively protect β-cells against DEX-induced dysfunction.

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Redox Signaling is Essential for Insulin Secretion

10.5772/intechopen.94312 ◽

2021 ◽

Author(s):

Petr Ježek ◽

Blanka Holendová ◽

Martin Jabůrek ◽

Jan Tauber ◽

Andrea Dlasková ◽

...

Keyword(s):

Plasma Membrane ◽

Insulin Secretion ◽

Redox Signaling ◽

H2o2 Production ◽

Β Cells ◽

Pancreatic Β Cells ◽

Nadph Oxidase 4 ◽

Elevated Glucose ◽

Branched Chain ◽

Glucose Stimulated Insulin Secretion

In this review, we place redox signaling in pancreatic β-cells to the context with signaling pathways leading to insulin secretion, acting for example upon the action of incretins (GLP-1, GIP) and the metabotropic receptor GPR40. Besides a brief description of ion channel participation in depolarization/repolarization of the plasma membrane, we emphasize a prominent role of the elevated glucose level in pancreatic β-cells during glucose-stimulated insulin secretion (GSIS). We focus on our recent findings, which revealed that for GSIS, not only elevated ATP synthesis is required, but also fundamental redox signaling originating from the NADPH oxidase 4- (NOX4-) mediated H2O2 production. We hypothesized that the closing of the ATP-sensitive K+ channel (KATP) is only possible when both ATP plus H2O2 are elevated in INS-1E cells. KATP alone or with synergic channels provides an element of logical sum, integrating both metabolic plus redox homeostasis. This is also valid for other secretagogues, such as branched chain ketoacids (BCKAs); and partly for fatty acids (FAs). Branched chain aminoacids, leucine, valine and isoleucine, after being converted to BCKAs are metabolized by a series of reactions resembling β-oxidation of FAs. This increases superoxide formation in mitochondria, including its portion elevated due to the function of electron transfer flavoprotein ubiquinone oxidoreductase (ETF:QOR). After superoxide conversion to H2O2 the oxidation of BCKAs provides the mitochondrial redox signaling extending up to the plasma membrane to induce its depolarization together with the elevated ATP. In contrast, experimental FA-stimulated insulin secretion in the presence of non-stimulating glucose concentrations is predominantly mediated by GPR40, for which intramitochondrial redox signaling activates phospholipase iPLA2γ, cleaving free FAs from mitochondrial membranes, which diffuse to the plasma membrane and largely amplify the GPR40 response. These events are concomitant to the insulin release due to the metabolic component. Hypothetically, redox signaling may proceed by simple H2O2 diffusion or via an SH-relay enabled by peroxiredoxins to target proteins. However, these aspects have yet to be elucidated.

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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.

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P-Rex1 Mediates Glucose-Stimulated Rac1 Activation and Insulin Secretion in Pancreatic β-Cells

Cellular Physiology and Biochemistry ◽

10.33594/000000310 ◽

2020 ◽

Vol 54 (6) ◽

pp. 1218-1230

Keyword(s):

Insulin Secretion ◽

Cell Types ◽

Human Islets ◽

Pi3 Kinase ◽

Membrane Extraction ◽

Limited Information ◽

Β Cells ◽

Downstream Signaling ◽

Published Evidence ◽

Glucose Stimulated Insulin Secretion

Background/Aims: Despite the published evidence implicating phosphoinositide 3-kinase (PI3-kinase) in the regulation of islet function, limited information is available on the putative contributory roles of its downstream signaling steps, including the phosphatidylinositol-3,4,5-trisphosphate-dependent Rac exchange factor 1 (P-Rex1) signaling pathway in the islet β-cell. Therefore, we investigated potential roles for P-Rex1 in glucose-stimulated Rac1 activation and insulin secretion in insulin-secreting (INS-1 832/13) β-cells. Methods: Glucose-stimulated Insulin secretion (GSIS) was quantified by ELISA. Expression of endogenous P-Rex1 and RhoG was suppressed by siRNA transfection using the DharmaFect1 reagent. Total membrane and cytosolic fractions were isolated using the Mem-PER Plus Membrane Extraction Kit. The degree of activation of Rac1 was determined by the pull-down assay. Results: P-Rex1 is expressed in INS-1 832/13 cells, normal rat islets and human islets. siRNA-mediated knockdown of P-Rex1 attenuated glucose-induced Rac1 activation, membrane association and insulin secretion. RhoG, which has been implicated in PI3-kinase-mediated Rac1 activation in other cell types, appears not to contribute to GSIS since the siRNA-mediated knockdown of RhoG failed to exert significant effects on GSIS. LY294002, a known inhibitor of PI3-kinase, potentiated GSIS without affecting glucose-induced Rac1 activation. Conclusion: Based on these findings, we conclude that P-Rex1 plays a novel regulatory role in glucose-induced Rac1 activation and insulin secretion.

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vH+-ATPase-induced intracellular acidification is critical to glucose-stimulated insulin secretion

10.1101/732222 ◽

2019 ◽

Author(s):

Akshata R. Naik ◽

Brent J. Formosa ◽

Rishika G. Pulvender ◽

Asiri G. Liyanaarachchi ◽

Bhanu P. Jena

Keyword(s):

Plasma Membrane ◽

Insulin Secretion ◽

Secretory Vesicles ◽

Intracellular Acidification ◽

Cell Secretion ◽

Atpase Inhibitor ◽

Cell Plasma Membrane ◽

Min6 Cells ◽

Cell Plasma ◽

Glucose Stimulated Insulin Secretion

ABSTRACTSwelling of secretory vesicles is critical for the regulated expulsion of intra-vesicular contents from cells during secretion. At the secretory vesicle membrane of the exocrine pancreas and neurons, GTP-binding G proteins, vH+-ATPase, potassium channels and AQP water channels, are among the players implicated in vesicle volume regulation. Here we report in insulin secreting MIN6 cells, the requirement of vH+-ATPase-mediated intracellular acidification, on glucose-stimulated insulin release. MIN6 cells exposed to the vH+-ATPase inhibitor Bafilomycin A show decreased acidification of the cytosolic compartment that include insulin-carrying granules. Additionally, a loss of insulin granule association with the cell plasma membrane is demonstrated and results in a decrease in glucose-stimulated insulin secretion and accumulation of intracellular insulin. These results suggest that vH+-ATPase-mediated intracellular acidification is required both at the level of secretory vesicles and the cell plasma membrane for cell secretion.

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Ontology of the apelinergic system in mouse pancreas during pregnancy and relationship with β-cell mass

Scientific Reports ◽

10.1038/s41598-021-94725-0 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Brenda Strutt ◽

Sandra Szlapinski ◽

Thineesha Gnaneswaran ◽

Sarah Donegan ◽

Jessica Hill ◽

...

Keyword(s):

Cell Proliferation ◽

Insulin Secretion ◽

Glucose Transporter ◽

Cell Mass ◽

Elevated Serum ◽

Pancreatic Β Cell ◽

Β Cells ◽

Β Cell ◽

Glucose Transporter 2 ◽

Glucose Stimulated Insulin Secretion

AbstractThe apelin receptor (Aplnr) and its ligands, Apelin and Apela, contribute to metabolic control. The insulin resistance associated with pregnancy is accommodated by an expansion of pancreatic β-cell mass (BCM) and increased insulin secretion, involving the proliferation of insulin-expressing, glucose transporter 2-low (Ins+Glut2LO) progenitor cells. We examined changes in the apelinergic system during normal mouse pregnancy and in pregnancies complicated by glucose intolerance with reduced BCM. Expression of Aplnr, Apelin and Apela was quantified in Ins+Glut2LO cells isolated from mouse pancreata and found to be significantly higher than in mature β-cells by DNA microarray and qPCR. Apelin was localized to most β-cells by immunohistochemistry although Aplnr was predominantly associated with Ins+Glut2LO cells. Aplnr-staining cells increased three- to four-fold during pregnancy being maximal at gestational days (GD) 9–12 but were significantly reduced in glucose intolerant mice. Apelin-13 increased β-cell proliferation in isolated mouse islets and INS1E cells, but not glucose-stimulated insulin secretion. Glucose intolerant pregnant mice had significantly elevated serum Apelin levels at GD 9 associated with an increased presence of placental IL-6. Placental expression of the apelinergic axis remained unaltered, however. Results show that the apelinergic system is highly expressed in pancreatic β-cell progenitors and may contribute to β-cell proliferation in pregnancy.

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