Insulin Secretion In INS-1 Research Articles

Low-grade elevation of palmitate and lipopolysaccharide synergistically induced β-cell damage via inhibition of neutral ceramidase

High concentrations of free fatty acids (FFAs) or lipopolysaccharide (LPS) could lead to β-cell apoptosis and dysfunction, while low-grade elevation of FFAs or LPS, which are more common in people with type 2 diabetes mellitus (T2DM) or obesity, have no obvious toxic effect on β-cells. Palmitate is a component closely related to metabolic disorders in FFAs. Recent studies have found that low-grade elevation of palmitate and LPS synergistically affects the sphingolipid signaling pathway by activating Toll-like receptor 4 (TLR4) and further enhances the expression of inflammatory cytokines in immune cells. Previous studies demonstrated that sphingolipids also played an important role in the occurrence and development of T2DM. This study aimed to investigate the synergistic effects of low-grade elevation of palmitate and LPS on viability, apoptosis and insulin secretion in the rat pancreatic β-cell line INS-1 or islets and the role of sphingolipids in this process. We showed that low-grade elevation of palmitate or LPS alone did not affect the viability, apoptosis, glucose-stimulated insulin secretion (GSIS) or intracellular insulin content of INS-1 cells or islets, while the combination of the two synergistically inhibited cell viability, induced apoptosis and decreased basal insulin secretion in INS-1 cells or islets. Treatment with palmitate and LPS markedly upregulated TLR4 protein expression and downregulated neutral ceramidase (NCDase) activity and protein expression. Additionally, low-grade elevation of palmitate and LPS synergistically induced a significant increase in ceramide and a decrease in sphingosine-1-phosphate. Blocking TLR4 signaling or overexpressing NCDase remarkably attenuated INS-1 cell injury induced by the combination of palmitate and LPS. However, inhibition of ceramide synthase did not ameliorate injury induced by palmitate and LPS. Overall, we show for the first time that low-grade elevation of palmitate and LPS synergistically induced β-cell damage by activating TLR4 signaling, inhibiting NCDase activity, and further modulating sphingolipid metabolism, which was different from a high concentration of palmitate-induced β-cell injury by promoting ceramide synthesis.

Vitamin B12 Inhibits Streptozotocin-Induced Islet β-Cell Oxidative Stress and Apoptosis by Activating Peroxisome Proliferator-Activated Receptor-γ Signaling Pathway

The present study aimed to investigate the effects of vitamin B12 on islet β-cell (INS-1) induced by streptozotocin (STZ) and the potential mechanisms. In this study, CCK8 was used to detect cell viability and TUNEL assay was used to detect apoptosis levels of treated INS-1 cells. The expression level of oxidative stress factors was measured by ELISA. Furthermore, western blot assay was used to detect the expression of PPAR-γ and apoptotic factors in treated INS-1 cells. Vitamin B12 improves STZ-induced insulin secretion in INS-1 cells, oxidative stress injury and apoptosis. Moreover, Vitamin B12 can activate the PPAR-γ signaling pathway and inhibit STZ-induced INS-1 cell damage. Taken together, our study demonstrated that vitamin B12 inhibits streptozotocin-induced islet β-cell oxidative stress and apoptosis by activating PPAR-γ signaling pathway.

Acylation of the Incretin Peptide Exendin-4 Directly Impacts Glucagon-Like Peptide-1 Receptor Signaling and Trafficking.

The glucagon-like peptide-1 receptor (GLP-1R) is a class B G protein–coupled receptor and mainstay therapeutic target for the treatment of type 2 diabetes and obesity. Recent reports have highlighted how biased agonism at the GLP-1R affects sustained glucose-stimulated insulin secretion through avoidance of desensitization and downregulation. A number of GLP-1R agonists (GLP-1RAs) feature a fatty acid moiety to prolong their pharmacokinetics via increased albumin binding, but the potential for these chemical changes to influence GLP-1R function has rarely been investigated beyond potency assessments for cAMP. Here, we directly compare the prototypical GLP-1RA exendin-4 with its C-terminally acylated analog, exendin-4-C16. We examine relative propensities of each ligand to recruit and activate G proteins and β-arrestins, endocytic and postendocytic trafficking profiles, and interactions with model and cellular membranes in HEK293 and HEK293T cells. Both ligands had similar cAMP potency, but exendin-4-C16 showed ∼2.5-fold bias toward G protein recruitment and a ∼60% reduction in β-arrestin-2 recruitment efficacy compared with exendin-4, as well as reduced GLP-1R endocytosis and preferential targeting toward recycling pathways. These effects were associated with reduced movement of the GLP-1R extracellular domain measured using a conformational biosensor approach and a ∼70% increase in insulin secretion in INS-1 832/3 cells. Interactions with plasma membrane lipids were enhanced by the acyl chain. Exendin-4-C16 showed extensive albumin binding and was highly effective for lowering of blood glucose in mice over at least 72 hours. Our study highlights the importance of a broad approach to the evaluation of GLP-1RA pharmacology.SIGNIFICANCE STATEMENTAcylation is a common strategy to enhance the pharmacokinetics of peptide-based drugs. This work shows how acylation can also affect various other pharmacological parameters, including biased agonism, receptor trafficking, and interactions with the plasma membrane, which may be therapeutically important.

Typical phthalic acid esters induce apoptosis by regulating the PI3K/Akt/Bcl-2 signaling pathway in rat insulinoma cells

Di-(2-ethylhexyl) phthalate (DEHP) and dibutyl phthalate (DBP) are representative phthalic acid esters (PAEs), a class of environmental endocrine disruptors used as plasticizers. PAEs exposure is associated with glucose metabolism, insulin resistance, and glucose tolerance; however, the mechanism and various PAE effects on human glucose metabolism remain largely unknown. In this study, we investigated the effects of DEHP, DBP, and their mixture on rat insulinoma (INS-1) cell apoptosis and the mechanism involved in vitro. The INS-1 cells were cultured in RPMI-1640 + 10% fetal bovine serum for 24 h and pretreated with dimethyl sulfoxide (vehicle, <0.1%), DEHP (30 μM), DBP (30 μM), and their mixture (30 μM DEHP + 30 μM DBP). The methyl-thiazolyl tetrazolium bromide test was used to measure cell viability. Hoechst 33342/propidium iodide (PI) staining and Annexin V-FITC/PI staining, 2′,7′-dichlorofluorescein diacetate assay, and glucose-induced insulin secretion assay were used to detect cell apoptosis rates, intracellular reactive oxygen species (ROS), and insulin secretion in INS-1, respectively. The mRNA expression levels of Bcl-2, Bax, Caspase 9, Caspase 8, Caspase 3, phosphoinositide 3-kinase (PI3K), and Akt were detected using real-time quantitative reverse transcription PCR; their protein expression levels were detected using western blotting. To the best of our knowledge, this study was the first to show that the combined effect of the two PAEs promotes a ROS-mediated PI3K/Akt/Bcl-2 pathway-induced pancreatic β cell apoptosis that is significantly higher than the effects of each PAE. Thus, safety standards and studies do not consider this effect as a significant oversight when blending PAEs. We assert that this must be addressed and corrected for establishing more impactful and safer standards.

Exposure of Pancreatic β-Cells to Excess Glucose Results in Bimodal Activation of mTORC1 and mTOR-Dependent Metabolic Acceleration.

SummaryChronic exposure of pancreatic β-cells to excess glucose can lead to metabolic acceleration and loss of stimulus-secretion coupling. Here, we examined how exposure to excess glucose (defined here as concentrations above 5 mM) affects mTORC1 signaling and the metabolism of β-cells. Acute exposure to excess glucose stimulated glycolysis-dependent mTORC1 signaling, without changes in the PI3K or AMPK pathways. Prolonged exposure to excess glucose led to hyperactivation of mTORC1 and metabolic acceleration, characterized by higher basal respiration and maximal respiratory capacity, increased energy demand, and enhanced flux through mitochondrial pyruvate metabolism. Inhibition of pyruvate transport to the mitochondria decelerated the metabolism of β-cells chronically exposed to excess glucose and re-established glucose-dependent mTORC1 signaling, disrupting a positive feedback loop for mTORC1 hyperactivation. mTOR inhibition had positive and negative impacts on various metabolic pathways and insulin secretion, demonstrating a role for mTOR signaling in the long-term metabolic adaptation of β-cells to excess glucose.

RORB and RORC associate with human islet dysfunction and inhibit insulin secretion in INS-1 cells

ABSTRACTLittle is known about the expression and function of Retinoic acid-related orphan receptors (RORA, B, and C) in pancreatic β cells. Here in, we utilized cDNA microarray and RNA sequencing approaches to investigate the expression pattern of ROR receptors in normal and diabetic human pancreatic islets. Possible correlations between RORs expression and HbA1c levels as well as insulin secretory capacity in isolated human islets were evaluated. The impact of RORB and RORC expression on insulin secretion in INS-1 (832/13) cells was validated as well. While RORA was the highest expressed gene among the three RORs in human islet cells, RORC was the highest expressed in INS-1 cells (832/13) and while RORB was the lowest expressed gene in human islet cells, RORA was the highest expressed in INS-1 cells (832/13). The expression of RORB and RORC was significantly lower in diabetic/hyperglycemic donors as compared with non-diabetic counterparts. Furthermore, while the expression of RORB correlated positively with insulin secretion and negatively with HbA1c, that of RORC correlated negatively with HbA1c. The expression pattern of RORA did not correlate with either of the two parameters. siRNA silencing of RORB or RORC in INS-1 (832/13) cells resulted in a significant downregulation of insulin mRNA expression and insulin secretion. These findings suggest that RORB and RORC are part of the molecular cascade that regulates insulin secretion in pancreatic β cells; and insight that provides for further work on the potential therapeutic utility of RORB and RORC genes in β cell dysfunction in type 2 diabetes.

The LIM homeodomain protein ISL1 mediates the function of TCF7L2 in pancreatic beta cells.

Pancreatic β-cell Tcf7l2 deletion or its functional knockdown suggested the essential role of this Wnt pathway effector in controlling insulin secretion, glucose homeostasis and β-cell gene expression. As the LIM homeodomain protein ISL1 is a suggested Wnt pathway downstream target, we hypothesize that it mediates metabolic functions of TCF7L2. We aimed to determine the role of ISL1 in mediating the function of TCF7L2 and the incretin hormone GLP-1 in pancreatic β-cells. The effect of dominant negative TCF7L2 (TCF7L2DN) mediated Wnt pathway functional knockdown on Isl1 expression was determined in βTCFDN mouse islets and in the rat insulinoma cell line INS-1 832/13. Luciferase reporter assay and chromatin immunoprecipitation were utilized to determine whether Isl1 is a direct downstream target of Tcf7l2 TCF7L2DN adenovirus infection and siRNA-mediated Isl1 knockdown on β-cell gene expression were compared. Furthermore, Isl1 knockdown on GLP-1 stimulated β-catenin S675 phosphorylation and insulin secretion was determined. We found that TCF7L2DN repressed ISL1 levels in βTCFDN islets and the INS-1 832/13 cell line. Wnt stimulators enhanced Isl1 promoter activity and binding of TCF7L2 on Isl1 promoter. TCF7L2DN adenovirus infection and Isl1 knockdown generated similar repression on expression of β-cell genes, including the ones that encode GLUT2 and GLP-1 receptor. Either TCF7L2DN adenovirus infection or Isl1 knockdown attenuated GLP-1-stimulated β-catenin S675 phosphorylation in INS-1 832/13 cells or mouse islets and GLP-1 stimulated insulin secretion in INS-1 832/13 or MIN6 cells. Our observations support the existence of TCF7L2-ISL1 transcriptional network, and we suggest that this network also mediates β-cell function of GLP-1.

Identification of the signals for glucose-induced insulin secretion in INS1 (832/13) β-cells using metformin-induced metabolic deceleration as a model

Metabolic deceleration in pancreatic β-cells is associated with inhibition of glucose-induced insulin secretion (GIIS), but only in the presence of intermediate/submaximal glucose concentrations. Here, we used acute metformin treatment as a tool to induce metabolic deceleration in INS1 (832/13) β-cells, with the goal of identifying key pathways and metabolites involved in GIIS. Metabolites and pathways previously implicated as signals for GIIS were measured in the cells at 2-25 mm glucose, with or without 5 mm metformin. We defined three criteria to identify candidate signals: 1) glucose-responsiveness, 2) sensitivity to metformin-induced inhibition of the glucose effect at intermediate glucose concentrations, and 3) alleviation of metformin inhibition by elevated glucose concentrations. Despite the lack of recovery from metformin-induced impairment of mitochondrial energy metabolism (glucose oxidation, O2 consumption, and ATP production), insulin secretion was almost completely restored at elevated glucose concentrations. Meeting the criteria for candidates involved in promoting GIIS were the following metabolic indicators and metabolites: cytosolic NAD+/NADH ratio (inferred from the dihydroxyacetone phosphate:glycerol-3-phosphate ratio), mitochondrial membrane potential, ADP, Ca2+, 1-monoacylglycerol, diacylglycerol, malonyl-CoA, and HMG-CoA. On the contrary, most of the purine and nicotinamide nucleotides, acetoacetyl-CoA, H2O2, reduced glutathione, and 2-monoacylglycerol were not glucose-responsive. Overall these results underscore the significance of mitochondrial energy metabolism-independent signals in GIIS regulation; in particular, the candidate lipid signaling molecules 1-monoacylglycerol, diacylglycerol, and malonyl-CoA; the predominance of KATP/Ca2+ signaling control by low ADP·Mg2+ rather than by high ATP levels; and a role for a more oxidized state (NAD+/NADH) in the cytosol during GIIS that favors high glycolysis rates.

Arsenite and methylarsonite inhibit mitochondrial metabolism and glucose-stimulated insulin secretion in INS-1 832/13 β cells.

Growing evidence suggests that exposure to environmental contaminants contributes to the current diabetes epidemic. Inorganic arsenic (iAs), a drinking water and food contaminant, is one of the most widespread environmental diabetogens according to epidemiological studies. Several schemes have been proposed to explain the diabetogenic effects of iAs exposure; however, the exact mechanism remains unknown. We have shown that in vitro exposure to low concentrations of arsenite (iAsIII) or its trivalent methylated metabolites, methylarsonite (MAsIII) and dimethylarsinite (DMAsIII), inhibits glucose-stimulated insulin secretion (GSIS) from isolated pancreatic islets, with little effect on insulin transcription or total insulin content. The goal of this study was to determine if exposure to trivalent arsenicals impairs mitochondrial metabolism, which plays a key role in the regulation of GSIS in β cells. We used a Seahorse extracellular flux analyzer to measure oxygen consumption rate (OCR), a proxy for mitochondrial metabolism, in cultured INS-1 832/13 β cells exposed to iAsIII, MAsIII, or DMAsIII and stimulated with either glucose or pyruvate, a final product of glycolysis and a substrate for the Krebs cycle. We found that 24-h exposure to 2 μM iAsIII or 0.375-0.5 μM MAsIII inhibited OCR in both glucose- and pyruvate-stimulated β cells in a manner that closely paralleled GSIS inhibition. In contrast, 24-h exposure to DMAsIII (up to 2µM) had no effects on either OCR or GSIS. These results suggest that iAsIII and MAsIII may impair GSIS in β cells by inhibiting mitochondrial metabolism, and that at least one target of these arsenicals is pyruvate decarboxylation or downstream reactions.

BDE-47 and BDE-85 stimulate insulin secretion in INS-1 832/13 pancreatic β-cells through the thyroid receptor and Akt

PBDEs (polybrominated diphenyl ethers) are environmental pollutants that have been linked to the development of type 2 diabetes, however, the precise mechanisms are not clear. Particularly, their direct effect on insulin secretion is unknown. In this study, we show that two PBDE congeners, BDE-47 and BDE-85, potentiate glucose-stimulated insulin secretion (GSIS) in INS-1 832/13 cells. This effect of BDE-47 and BDE-85 on GSIS was dependent on thyroid receptor (TR). Both BDE-47 and BDE-85 (10μM) activated Akt during an acute exposure. The activation of Akt by BDE-47 and BDE-85 plays a role in their potentiation of GSIS, as pharmacological inhibition of PI3K, an upstream activator of Akt, significantly lowers GSIS compared to compounds alone. This study shows that BDE-47 and BDE-85 directly act on pancreatic β-cells to stimulate GSIS, and that this effect is mediated by the thyroid receptor (TR) and Akt activation.

5'-AMP-activated protein kinase plays an essential role in geniposide-regulated glucose-stimulated insulin secretion in rat pancreatic INS-1 β cells.

Our previous work showed that geniposide affected glucose-stimulated insulin secretion (GSIS) via regulating glucose uptake and metabolism in pancreatic β cells; however, the molecular mechanisms remain largely unknown. Substantial evidence suggests that activation of 5'-AMP-activated protein kinase (AMPK) plays a central role in GSIS. Here, we aim to determine the role of AMPK on geniposide-regulated GSIS in rat pancreatic INS-1 cells. The results demonstrated that 6-[4-(2-piperidin-1-yletoxy)-phenyl]-3-pyridin-4-yl-pyrazolo[1,5-α] pyrimidine (Compound C; an AMPK inhibitor) significantly attenuated the effects of geniposide on glucose uptake, energy metabolism, and insulin secretion in INS-1 cells. We also observed that geniposide induced phosphorylation of acetyl-CoA carboxylase (ACC), a marker of AMPK activity, in a time-dependent manner in INS-1 cells; however, in the presence of Compound C, the influence of geniposide on ACC phosphorylation was obviously inhibited. Furthermore, the knockdown of AMPK protein with AMPK siRNA treatment decreased the effects of geniposide on glucose uptake, adenosine triphosphate production, and GSIS. All these data indicate that AMPK plays an essential role in geniposide-regulated GSIS in pancreatic β cells.

Integrator of Stress Responses Calmodulin Binding Transcription Activator 1 (Camta1) Regulates miR-212/miR-132 Expression and Insulin Secretion

Altered microRNA profiles have been demonstrated in experimental models of type 2 diabetes, including in islets of the diabetic Goto-Kakizaki (GK) rat. Our bioinformatic analysis of conserved sequences in promoters of microRNAs, previously observed to be up-regulated in GK rat islets, revealed putative CGCG-core motifs on the promoter of the miR-212/miR-132 cluster, overexpression of which has been shown to increase insulin secretion. These motifs are possible targets of calmodulin binding transcription activators Camta1 and Camta2 that have been recognized as integrators of stress responses. We also identified putative NKE elements, possible targets of NK2 homeobox proteins like the essential islet transcription factor Nkx2-2. As Camtas can function as co-activators with NK2 proteins in other tissues, we explored the role of Camta1, Camta2, and Nkx2-2 in the regulation of the miR-212/miR-132 cluster and insulin secretion. We demonstrate that exposure of control Wistar or GK rat islets to 16.7 mm glucose increases miR-212/miR-132 expression but significantly less so in the GK rat. In addition, Camta1, Camta2, and Nkx2-2 were down-regulated in GK rat islets, and knockdown of Camta1 reduced miR-212/miR-132 promoter activity and miR-212/miR-132 expression, even under cAMP elevation. Knockdown of Camta1 decreased insulin secretion in INS-1 832/13 cells and Wistar rat islets but increased insulin content. Furthermore, knockdown of Camta1 reduced K(+)-induced insulin secretion and voltage-dependent Ca(2+) currents. We also demonstrate Camta1 and Nkx2-2 protein interaction. These results indicate that Camta1 is required not only for expression of the miR-212/miR-132 cluster but at multiple levels for regulating beta cell insulin content and secretion.

Disrupted dynamics of F-actin and insulin granule fusion in INS-1 832/13 beta-cells exposed to glucotoxicity: partial restoration by glucagon-like peptide 1

Actin dynamics in pancreatic β-cells is involved in insulin exocytosis but the molecular mechanisms of this dynamics and its role in biphasic insulin secretion in pancreatic β-cells is largely unknown. Moreover, the impact of a glucotoxic environment on the sub-cortical actin network dynamics is poorly studied. In this study, we investigate the behavior of insulin granules and the subcortical actin network dynamics in INS-1 832/13 β-cells submitted to a normal or glucotoxic environment. Our results show that glucose stimulation leads to a reorganization of the subcortical actin network with a rupture of its interactions with t-SNARE proteins (Syntaxin 1A and SNAP-25), promoting insulin secretion in INS-1 832/13 β-cells. Prolonged exposure of INS-1 832/13 β-cells to high-glucose levels (glucotoxicity) leads to the densification of the cortical actin network, which prevents its reorganization under acute glucose, and diminishes the glucose-stimulated insulin secretion, as shown by the decreased number of fusion events. The most interesting in our results is the partial restoration by GLP-1 of the insulin secretion ability from high-glucose treated INS-1 832/13 cells. This improved insulin exocytosis is associated with partial restored actin dynamics and fusion events during the two phases of the secretion, with a preferential involvement of Epac2 signaling in the first phase and a rather involvement of PKA signaling in the second phase of insulin exocytosis. All these data provide some new insights into the mechanism by which current therapeutics may be improving insulin secretion.

Rosuvastatin Treatment Affects Both Basal and Glucose-Induced Insulin Secretion in INS-1 832/13 Cells.

Rosuvastatin is a member of the statin family. Like the other statins it is prescribed to lower cholesterol levels and thereby reduce the risk of cardiovascular events. Rosuvastatin lowers the cholesterol levels by inhibiting the key enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMG-CoA reductase) in the cholesterol producing mevalonate pathway. It has been recognized that apart from their beneficial lipid lowering effects, statins also exhibit diabetogenic properties. The molecular mechanisms behind these remain unresolved. To investigate the effects of rosuvastatin on insulin secretion, we treated INS-1 832/13 cells with varying doses (20 nM to 20 μM) of rosuvastatin for 48 h. At concentrations of 2 μM and above basal insulin secretion was significantly increased. Using diazoxide we could determine that rosuvastatin did not increase basal insulin secretion by corrupting the KATP channels. Glucose-induced insulin secretion on the other hand seemed to be affected differently at different rosuvastatin concentrations. Rosuvastatin treatment (20 μM) for 24–48 h inhibited voltage-gated Ca2+ channels, which lead to reduced depolarization-induced exocytosis of insulin-containing granules. At lower concentrations of rosuvastatin (≤ 2 μM) the stimulus-secretion coupling pathway was intact downstream of the KATP channels as assessed by the patch clamp technique. However, a reduction in glucose-induced insulin secretion could be observed with rosuvastatin concentrations as low as 200 nM. The inhibitory effects of rosuvastatin on glucose-induced insulin secretion could be reversed with mevalonate, but not squalene, indicating that rosuvastatin affects insulin secretion through its effects on the mevalonate pathway, but not through the reduction of cholesterol biosynthesis. Taken together, these data suggest that rosuvastatin has the potential to increase basal insulin secretion and reduce glucose-induced insulin secretion. The latter is possibly an unavoidable side effect of rosuvastatin treatment as it occurs through the same mechanisms as the lipid-lowering effects of the drug.

Inhibition of Monoacylglycerol Lipase Activity Decreases Glucose-Stimulated Insulin Secretion in INS-1 (832/13) Cells and Rat Islets.

Lipid signals derived from lipolysis and membrane phospholipids play an important role in glucose-stimulated insulin secretion (GSIS), though the exact secondary signals remain unclear. Previous reports have documented a stimulatory role of exogenously added mono-acyl-glycerol (MAG) on insulin secretion from cultured β-cells and islets. In this report we have determined effects of increasing intracellular MAG in the β-cell by inhibiting mono-acyl-glycerol lipase (MGL) activity, which catalyzes the final step in triacylglycerol breakdown, namely the hydrolysis of MAG to glycerol and free fatty acid (FA). To determine the role of MGL in GSIS, we used three different pharmacological agents (JZL184, MJN110 and URB602). All three inhibited GSIS and depolarization-induced insulin secretion in INS-1 (832/13). JZL184 significantly inhibited both GSIS and depolarization-induced insulin secretion in rat islets. JZL184 significantly decreased lipolysis and increased both mono- and diacyglycerol species in INS-1 cells. Analysis of the kinetics of GSIS showed that inhibition was greater during the sustained phase of secretion. A similar pattern was observed in the response of Ca2+ to glucose and depolarization but to a lesser degree suggesting that altered Ca2+ handling alone could not explain the reduction in insulin secretion. In addition, a significant reduction in long chain-CoA (LC-CoA) was observed in INS-1 cells at both basal and stimulatory glucose following inhibition of MGL. Our data implicate an important role for MGL in insulin secretion.

Chronic leucine exposure results in reduced but reversible glucose-stimulated insulin secretion in INS-1 cells

Previous studies have demonstrated that sustained high leucine exposure decreases glucose-stimulated insulin secretion (GSIS). However, whether this effect is recoverable following the removal of leucine is unclear. Pancreatic/duodenal homeobox-1 (PDX-1) and its downstream target, glucose transporter 2 (GLUT2), are reported to be positively associated with insulin secretion. However, it also remains unclear whether the effect of leucine on GSIS is accompanied by alterations in PDX-1 and GLUT2. In the present study, insulin secretion, insulin content, PDX-1 and GLUT2 protein expression in INS-1 (rat insulinoma cell line) cells were assessed following a 24-h incubation in 40 mmol/l leucine. Half of the cells were incubated in leucine-free media for a further 24 h to observe the abovementioned effects. In contrast to the control, 40 mmol/l leucine for 24 or 48 h diminished GSIS at high glucose concentrations by 11% (P=0.026) or 22% (P=0.003), insulin content by 14% (P=0.008) or 20% (P=0.002), as well as decreasing PDX-1 and GLUT2 expression. When leucine was removed from the media for a further 24-h incubation, in comparison with those cells that were maintained in leucine treatment for 24 and 48 h, the high GSIS increased by 13% (P=0.032) and 27% (P=0.002), insulin content was augmented by 10% (P=0.014) and 20% (P=0.003), and the protein expression of PDX-1 and GLUT2 also increased. The present study demonstrates that sustained high concentrations of leucine induce a reversible impairment of GSIS and alter insulin content, which is mediated by PDX-1 and GLUT2, in INS-1 cells.

Divergent Effects of Sulforaphane on Basal and Glucose-Stimulated Insulin Secretion in β-Cells: Role of Reactive Oxygen Species and Induction of Endogenous Antioxidants

Oxidative stress is implicated in pancreatic β-cell dysfunction, yet clinical outcomes of antioxidant therapies on diabetes are inconclusive. Since reactive oxygen species (ROS) can function as signaling intermediates for glucose-stimulated insulin secretion (GSIS), we hypothesize that exogenously boosting cellular antioxidant capacity dampens signaling ROS and GSIS. To test the hypothesis, we formulated a mathematical model of redox homeostatic control circuit comprising known feedback and feedforward loops and validated model predictions with plant-derived antioxidant sulforaphane (SFN). SFN acutely (30-min treatment) stimulated basal insulin secretion in INS-1(832/13) cells and cultured mouse islets, which could be attributed to SFN-elicited ROS as N-acetylcysteine or glutathione ethyl ester suppressed SFN-stimulated insulin secretion. The mathematical model predicted an adapted redox state characteristic of strong induction of endogenous antioxidants but marginally increased ROS under prolonged SFN exposure, a state that attenuates rather than facilitates glucose-stimulated ROS and GSIS. We validated the prediction by demonstrating that although 24-h treatment of INS-1(832/13) cells with low, non-cytotoxic concentrations of SFN (2-10 μM) protected the cells from cytotoxicity by oxidative insult, it markedly suppressed insulin secretion stimulated by 20 mM glucose. Our study indicates that adaptive induction of endogenous antioxidants by exogenous antioxidants, albeit cytoprotective, inhibits GSIS in β-cells.

Time-resolved metabolomics analysis of β-cells implicates the pentose phosphate pathway in the control of insulin release

Insulin secretion is coupled with changes in β-cell metabolism. To define this process, 195 putative metabolites, mitochondrial respiration, NADP+, NADPH and insulin secretion were measured within 15 min of stimulation of clonal INS-1 832/13 β-cells with glucose. Rapid responses in the major metabolic pathways of glucose occurred, involving several previously suggested metabolic coupling factors. The complexity of metabolite changes observed disagreed with the concept of one single metabolite controlling insulin secretion. The complex alterations in metabolite levels suggest that a coupling signal should reflect large parts of the β-cell metabolic response. This was fulfilled by the NADPH/NADP+ ratio, which was elevated (8-fold; P<0.01) at 6 min after glucose stimulation. The NADPH/NADP+ ratio paralleled an increase in ribose 5-phosphate (>2.5-fold; P<0.001). Inhibition of the pentose phosphate pathway by trans-dehydroepiandrosterone (DHEA) suppressed ribose 5-phosphate levels and production of reduced glutathione, as well as insulin secretion in INS-1 832/13 β-cells and rat islets without affecting ATP production. Metabolite profiling of rat islets confirmed the glucose-induced rise in ribose 5-phosphate, which was prevented by DHEA. These findings implicate the pentose phosphate pathway, and support a role for NADPH and glutathione, in β-cell stimulus-secretion coupling.

Solution composition impacts fibronectin immobilization on carboxymethyl-dextran surfaces and INS-1 insulin secretion

It is shown that solution composition during immobilization plays a critical role in the properties of fibronectin (FN) surfaces and their bioactivity towards insulinoma (INS-1) cell function. X-ray photoelectron spectroscopy revealed FN grafting onto low-fouling carboxymethyl-dextran (CMD) surfaces was successful with solutions composed of 10μM CaCl2, 10μM MgCl2, 10μM MnCl2, and 10μM and 1mM NaCl, but unsuccessful with those made of 150mM NaCl or 1× PBS. Circular dichroism and photon correlation spectroscopy revealed that regardless of solution composition, no measurable differences in free FN conformation prevail. AFM imaging of FN-CMD revealed, while there are no quantitative differences in surface roughness, there are some subtle qualitative differences in topography. FN surface immobilization scheme does not influence INS-1 cell growth after 3 and 7 days regardless of the underlying substrate or solution composition. INS-1 cell insulin secretion in response to glucose is affected by the substrate and solution composition during FN immobilization.

The level of menadione redox-cycling in pancreatic β-cells is proportional to the glucose concentration: Role of NADH and consequences for insulin secretion

Pancreatic β-cells release insulin in response to elevation of glucose from basal (4–7 mM) to stimulatory (8–16 mM) levels. Metabolism of glucose by the β-cell results in the production of low levels of reactive oxygen intermediates (ROI), such as hydrogen peroxide (H 2O 2), a newly recognized coupling factor linking glucose metabolism to insulin secretion. However, high and toxic levels of H 2O 2 inhibit insulin secretion. Menadione, which produces H 2O 2 via redox cycling mechanism in a dose-dependent manner, was investigated for its effect on β-cell metabolism and insulin secretion in INS-1 832/13, a rat β-cell insulinoma cell line, and primary rodent islets. Menadione-dependent redox cycling and resulting H 2O 2 production under stimulatory glucose exceeded several-fold those reached at basal glucose. This was paralleled by a differential effect of menadione (0.1–10 μM) on insulin secretion, which was enhanced at basal, but inhibited at stimulatory glucose. Redox cycling of menadione and H 2O 2 formation was dependent on glycolytically-derived NADH, as inhibition of glycolysis and application of non-glycogenic insulin secretagogues did not support redox cycling. In addition, activity of plasma membrane electron transport, a system dependent in part on glycolytically-derived NADH, was also inhibited by menadione. Menadione-dependent redox cycling was sensitive to the NQO1 inhibitor dicoumarol and the flavoprotein inhibitor diphenylene iodonium, suggesting a role for NQO1 and other oxidoreductases in this process. These data may explain the apparent dichotomy between the stimulatory and inhibitory effects of H 2O 2 and menadione on insulin secretion.