Physiological development of insulin secretion, calcium channels, and GLUT2 expression of pancreatic rat β-cells

Insulin secretion in mature β-cells increases vigorously when extracellular glucose concentration rises. Glucose-stimulated insulin secretion depends on Ca2+ influx through voltage-gated Ca2+ channels. During fetal development, this structured response is not well established, and it is after birth that β-cells acquire glucose sensitivity and a robust secretion. We compared some elements of glucose-induced insulin secretion coupling in β-cells obtained from neonatal and adult rats and found that neonatal cells are functionally immature compared with adult cells. We observed that neonatal cells secrete less insulin and cannot sense changes in extracellular glucose concentrations. This could be partially explained because in neonates Ca2+ current density and synthesis of mRNA α1 subunit Ca2+ channel are lower than in adult cells. Interestingly, immunostaining for α1B, α1C, and α1D subunits in neonatal cells is similar in cytoplasm and plasma membrane, whereas it occurs predominantly in the plasma membrane in adult cells. We also observed that GLUT2 expression in adult β-cells is mostly located in the membrane, whereas in neonatal cells glucose transporters are predominantly in the cytoplasm. This could explain, in part, the insensitivity to extracellular glucose in neonatal β-cells. Understanding neonatal β-cell physiology and maturation contributes toward a better comprehension of type 2 diabetes physiopathology, where alterations in β-cells include diminished L-type Ca2+ channels and GLUT2 expression that results in an insufficient insulin secretion.

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Uncoupling protein-2 contributes significantly to high mitochondrial proton leak in INS-1E insulinoma cells and attenuates glucose-stimulated insulin secretion

Biochemical Journal ◽

10.1042/bj20070954 ◽

2007 ◽

Vol 409 (1) ◽

pp. 199-204 ◽

Cited By ~ 60

Author(s):

Charles Affourtit ◽

Martin D. Brand

Keyword(s):

Insulin Secretion ◽

Uncoupling Protein ◽

Proton Leak ◽

Cell Physiology ◽

Uncoupling Protein 2 ◽

Proton Conductance ◽

Β Cells ◽

Knock Down ◽

Glucose Stimulated Insulin Secretion ◽

Insulinoma Cells

Proton leak exerts stronger control over ATP/ADP in mitochondria from clonal pancreatic β-cells (INS-1E) than in those from rat skeletal muscle, due to the higher proton conductance of INS-1E mitochondria [Affourtit and Brand (2006) Biochem. J. 393, 151–159]. In the present study, we demonstrate that high proton leak manifests itself at the cellular level too: the leak rate (measured as myxothiazol-sensitive, oligomycin-resistant respiration) was nearly four times higher in INS-1E cells than in myoblasts. This relatively high leak activity was decreased more than 30% upon knock-down of UCP2 (uncoupling protein-2) by RNAi (RNA interference). The high contribution of UCP2 to leak suggests that proton conductance through UCP2 accounts for approx. 20% of INS-1E respiration. UCP2 knock-down enhanced GSIS (glucose-stimulated insulin secretion), consistent with a role for UCP2 in β-cell physiology. We propose that the high mitochondrial proton leak in β-cells is a mechanism which amplifies the effect of physiological UCP2 regulators on cytoplasmic ATP/ADP and hence on insulin secretion.

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Calcium-activated K+ Channels of Mouse β-cells are Controlled by Both Store and Cytoplasmic Ca2+

The Journal of General Physiology ◽

10.1085/jgp.20028581 ◽

2002 ◽

Vol 120 (3) ◽

pp. 307-322 ◽

Cited By ~ 49

Author(s):

P.B. Goforth ◽

R. Bertram ◽

F.A. Khan ◽

M. Zhang ◽

A. Sherman ◽

...

Keyword(s):

Insulin Secretion ◽

Action Potentials ◽

Potassium Current ◽

Cytosolic Calcium ◽

Β Cells ◽

Calcium Dependent ◽

Extracellular Glucose Concentration ◽

Extracellular Glucose ◽

Experimental Findings

A novel calcium-dependent potassium current (Kslow) that slowly activates in response to a simulated islet burst was identified recently in mouse pancreatic β-cells (Göpel, S.O., T. Kanno, S. Barg, L. Eliasson, J. Galvanovskis, E. Renström, and P. Rorsman. 1999. J. Gen. Physiol. 114:759–769). Kslow activation may help terminate the cyclic bursts of Ca2+-dependent action potentials that drive Ca2+ influx and insulin secretion in β-cells. Here, we report that when [Ca2+]i handling was disrupted by blocking Ca2+ uptake into the ER with two separate agents reported to block the sarco/endoplasmic calcium ATPase (SERCA), thapsigargin (1–5 μM) or insulin (200 nM), Kslow was transiently potentiated and then inhibited. Kslow amplitude could also be inhibited by increasing extracellular glucose concentration from 5 to 10 mM. The biphasic modulation of Kslow by SERCA blockers could not be explained by a minimal mathematical model in which [Ca2+]i is divided between two compartments, the cytosol and the ER, and Kslow activation mirrors changes in cytosolic calcium induced by the burst protocol. However, the experimental findings were reproduced by a model in which Kslow activation is mediated by a localized pool of [Ca2+] in a subspace located between the ER and the plasma membrane. In this model, the subspace [Ca2+] follows changes in cytosolic [Ca2+] but with a gradient that reflects Ca2+ efflux from the ER. Slow modulation of this gradient as the ER empties and fills may enhance the role of Kslow and [Ca2+] handling in influencing β-cell electrical activity and insulin secretion.

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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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Facilitative Glucose Transporter 9 Expression Affects Glucose Sensing in Pancreatic β-Cells

Endocrinology ◽

10.1210/en.2009-0747 ◽

2009 ◽

Vol 150 (12) ◽

pp. 5302-5310 ◽

Cited By ~ 30

Author(s):

Sarah A. Evans ◽

Manuel Doblado ◽

Maggie M. Chi ◽

John A. Corbett ◽

Kelle H. Moley

Keyword(s):

Plasma Membrane ◽

Insulin Secretion ◽

Small Interfering Rna ◽

Glucose Sensing ◽

Low Density ◽

Β Cells ◽

Microsome Fraction ◽

Glucose Stimulated Insulin Secretion ◽

Rna Knockdown ◽

Interfering Rna

Abstract Facilitative glucose transporters (GLUTs) including GLUT9, accelerate the facilitative diffusion of glucose across the plasma membrane. Studies in GLUT2-deficient mice suggested the existence of another GLUT in the mammalian β-cell responsible for glucose sensing. The objective of this study was to determine the expression and function of GLUT9 in murine and human β-cells. mRNA and protein expression levels were determined for both isoforms of GLUT9 in murine and human isolated islets as well as insulinoma cell lines (MIN6). Immunohistochemistry and subcellular localization were performed to localize the protein within the cell. Small interfering RNA knockdown of GLUT9 was used to determine the effect of this transporter, in the presence of GLUT2, on cell metabolism and insulin secretion in MIN6 and INS cells. In this report we demonstrate that GLUT9a and GLUT9b are expressed in pancreatic islets and that this expression localizes to insulin-containing β-cells. Subcellular localization studies indicate that mGLUT9b is found associated with the plasma membrane as well as in the high-density microsome fraction and low-density microsome fraction, whereas mGLUT9a appears to be located only in the high-density microsome and low-density microsome under basal conditions. Functionally GLUT9 appears to participate in the regulation of glucose-stimulated insulin secretion in addition to GLUT2. small interfering RNA knockdown of GLUT9 results in reduced cellular ATP levels that correlate with reductions in glucose-stimulated insulin secretion in MIN6 and INS cells. These studies confirm the expression of GLUT9a and GLUT9b in murine and human β-cells and suggest that GLUT9 may participate in glucose-sensing in β-cells.

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Effects of olive leaf polyphenols against H₂O₂ toxicity in insulin secreting β-cells.

Acta Biochimica Polonica ◽

10.18388/abp.2011_2284 ◽

2011 ◽

Vol 58 (1) ◽

Cited By ~ 31

Author(s):

Ahmet Cumaoğlu ◽

Lucia Rackova ◽

Milan Stefek ◽

Murat Kartal ◽

Pierre Maechler ◽

...

Keyword(s):

Insulin Secretion ◽

Redox Homeostasis ◽

Ros Generation ◽

Cell Physiology ◽

Olive Leaf ◽

Β Cells ◽

Olive Leaves ◽

Chronic Hyperglycemia ◽

Metabolic Signal ◽

Glucose Stimulated Insulin Secretion

In pancreatic β-cells, although H₂O₂ is a metabolic signal for glucose stimulated insulin secretion, it may induce injury in the presence of increased oxidative stress (OS) as in the case of diabetic chronic hyperglycemia. Olea europea L. (olive) leaves contain polyphenolic compounds that may protect insulin-secreting cells against OS. The major polyphenolic compound in ethanolic olive leaf extract (OLE) is oleuropein (about 20%), thus we compared the effects of OLE with the effects of standard oleuropein on INS-1 cells. The cells were incubated with increasing concentrations of OLE or oleuropein for 24 h followed by exposure to H₂O₂ (0.035 mM) for 45 min. H₂O₂ alone resulted in a significantly decreased viability (MTT assay), depressed glucose-stimulated insulin secretion, increased apoptotic and necrotic cell death (AO/EB staining), inhibited glutathione peroxidase activity (GPx) and stimulated catalase activity that were associated with increased intracellular generation of reactive oxygen species (ROS) (fluorescence DCF). OLE and oleuropein partly improved the viability, attenuated necrotic and apoptotic death, inhibited the ROS generation and improved insulin secretion in H₂O₂-exposed cells. The effects of oleuropein on insulin secretion were more pronounced than those of OLE, while OLE exerted a stronger anti-cytotoxic effect than oleuropein. Unlike OLE, oleuropein had no significant preserving effect on GPx; however, both compounds stimulated the activity of catalase in H₂O₂-exposed cells. These findings indicate different modulatory roles of polyphenolic constituents of olive leaves on redox homeostasis that may have a role in the maintenance of β-cell physiology against OS.

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Overexpression of short heterodimer partner recovers impaired glucose-stimulated insulin secretion of pancreatic β-cells overexpressing UCP2

Journal of Endocrinology ◽

10.1677/joe.1.05675 ◽

2004 ◽

Vol 183 (1) ◽

pp. 133-144 ◽

Cited By ~ 13

Author(s):

Y-H Suh ◽

S-Y Kim ◽

H-Y Lee ◽

B C Jang ◽

J H Bae ◽

...

Keyword(s):

Insulin Secretion ◽

Islet Cells ◽

Uncoupling Protein ◽

Metabolic Inhibitor ◽

Β Cells ◽

Pancreatic Β Cells ◽

Channel Inhibition ◽

Glucose Sensitivity ◽

Glucose Stimulated Insulin Secretion ◽

Short Heterodimer Partner

The short heterodimer partner (SHP) (NR0B2) is an orphan nuclear receptor whose function in pancreatic β-cells is unclear. Mitochondrial uncoupling protein (UCP2) in β-cells is upregulated in obesity-related diabetes, causing impaired glucose-stimulated insulin secretion (GSIS). We investigated whether SHP plays a role in UCP2-induced GSIS impairment. We overexpressed SHP in normal islet cells and in islet cells overexpressing UCP2 by an adenovirus-mediated infection technique. We found that SHP overexpression enhanced GSIS in normal islets, and restored GSIS in UCP2-overexpressing islets. SHP overexpression increased the glucose sensitivity of ATP-sensitive K+ (KATP) channels and enhanced theATP/ADP ratio. A peroxisome proliferator-activated receptor gamma (PPARγ) antagonist, GW9662, did not block the SHP effect on GSIS. SHP overexpression also corrected the impaired sensitivity of UCP2-overexpressing β-cells to methylpyruvate, another energy fuel that bypasses glycolysis and directly enters the Krebs cycle. KATP channel inhibition mediated by dihydroxyacetone, which gives reducing equivalents directly to complex II of the electron transport system, was similar in Ad-Null-, Ad-UCP2- and Ad-UCP2+Ad-SHP-infected cells. The mitochondrial metabolic inhibitor sodium azide totally blocked the effect of SHP overexpression on GSIS. These results suggest that SHP positively regulates GSIS in β-cells and restores glucose sensitivity in UCP2-overexpressing β-cells by enhancing mitochondrial glucose metabolism, independent of PPARγ activation.

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Pericytes contribute to the islet basement membranes to promote beta-cell gene expression

Scientific Reports ◽

10.1038/s41598-021-81774-8 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Lina Sakhneny ◽

Alona Epshtein ◽

Limor Landsman

Keyword(s):

Gene Expression ◽

Cell Function ◽

Basement Membranes ◽

Cell Physiology ◽

Β Cells ◽

Β Cell ◽

Cell Clustering ◽

Β Cell Function ◽

Cell Gene Expression ◽

Glucose Stimulated Insulin Secretion

Abstractβ-Cells depend on the islet basement membrane (BM). While some islet BM components are produced by endothelial cells (ECs), the source of others remains unknown. Pancreatic pericytes directly support β-cells through mostly unidentified secreted factors. Thus, we hypothesized that pericytes regulate β-cells through the production of BM components. Here, we show that pericytes produce multiple components of the mouse pancreatic and islet interstitial and BM matrices. Several of the pericyte-produced ECM components were previously implicated in β-cell physiology, including collagen IV, laminins, proteoglycans, fibronectin, nidogen, and hyaluronan. Compared to ECs, pancreatic pericytes produce significantly higher levels of α2 and α4 laminin chains, which constitute the peri-islet and vascular BM. We further found that the pericytic laminin isoforms differentially regulate mouse β-cells. Whereas α2 laminins promoted islet cell clustering, they did not affect gene expression. In contrast, culturing on Laminin-421 induced the expression of β-cell genes, including Ins1, MafA, and Glut2, and significantly improved glucose-stimulated insulin secretion. Thus, alongside ECs, pericytes are a significant source of the islet BM, which is essential for proper β-cell function.

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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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