Inhibition of fatty acid translocase cluster determinant 36 (CD36), stimulated by hyperglycemia, prevents glucotoxicity in INS-1 cells

The purpose of the present study was to determine whether exposure of pancreatic islets to glucotoxic conditions changes fatty acid translocase cluster determinant 36 (CD36) and examine the role of CD36 on the induction of glucotoxicity. We measured the changes of CD36 and insulin secretion in high glucose (30 mM) exposed INS-1 cells and CD36 suppressed INS-1 cells by transfection of CD36 siRNA. The intracellular peroxide level of INS-1 cells increased in the high glucose media compared to normal glucose (5.6mM) media. The mRNA levels of insulin and PDX-1, as well as glucose stimulated insulin secretion (GSIS) were decreased in INS-1 cells exposed to high glucose media compared to normal glucose media, while CD36 and palmitate uptake were significantly elevated with exposure to high glucose media for 12h. The inhibition of CD36 reversed the decreased GSIS and intracellular peroxide level in INS-1 cells. These results suggest that high glucose may exacerbate glucotoxicity via increasing fatty acid influx by elevation of CD36 expression, and that CD36 may be a possible target molecule for preventing glucotoxicity in pancreatic beta-cells.     

Glucotoxic and diabetic conditions induce caspase 6-mediated degradation of nuclear lamin A in human islets,rodent islets and INS-1 832/13 cells

Nuclear lamins form the lamina on the interior surface of the nuclear envelope, and regulate nuclear metabolic events, including DNA replication and organization of chromatin. The current study is aimed at understanding the role of executioner caspase 6 on lamin A integrity in islet β-cells under duress of glucotoxic (20 mM glucose; 24 h) and diabetic conditions. Under glucotoxic conditions, glucose-stimulated insulin secretion and metabolic cell viability were significantly attenuated in INS-1 832/13 cells. Further, exposure of normal human islets, rat islets and INS-1 832/13 cells to glucotoxic conditions leads to caspase 6 activation and lamin A degradation, which is also observed in islets from the Zucker diabetic fatty rat, a model for type 2 diabetes (T2D), and in islets from a human donor with T2D. Z-Val-Glu-Ile-Asp-fluoromethylketone, a specific inhibitor of caspase 6, markedly attenuated high glucose-induced caspase 6 activation and lamin A degradation, confirming that caspase 6 mediates lamin A degradation under high glucose exposure conditions. Moreover, Z-Asp-Glu-Val-Asp-fluoromethylketone, a known caspase 3 inhibitor, significantly inhibited high glucose-induced caspase 6 activation and lamin A degradation, suggesting that activation of caspase 3 might be upstream to caspase 6 activation in the islet β-cell under glucotoxic conditions. Lastly, we report expression of ZMPSTE24, a zinc metallopeptidase involved in the processing of prelamin A to mature lamin A, in INS-1 832/13 cells and human islets; was unaffected by high glucose. We conclude that caspases 3 and 6 could contribute to alterations in the integrity of nuclear lamins leading to metabolic dysregulation and failure of the islet β-cell.     

Metabolomic analysis of pancreatic beta cells following exposure to high glucose

Background

Chronic exposure to hyperglycaemic conditions has been shown to have detrimental effects on beta cell function. The resulting glucotoxicity is a contributing factor to the development of type 2 diabetes. The objective of this study was to combine a metabolomics approach with functional assays to gain insight into the mechanism by which glucotoxicity exerts its effects.

Methods

The BRIN-BD11 and INS-1E beta cell lines were cultured in 25 mM glucose for 20 h to mimic glucotoxic effects. PDK-2 protein expression, intracellular glutathione levels and the change in mitochondrial membrane potential and intracellular calcium following glucose stimulation were determined. Metabolomic analysis of beta cell metabolite extracts was performed using GC–MS, ¹H NMR and ¹³C NMR.

Results

Conditions to mimic glucotoxicity were established and resulted in no loss of cellular viability in either cell line while causing a decrease in insulin secretion. Metabolomic analysis of beta cells following exposure to high glucose revealed a change in amino acids, an increase in glucose and a decrease in phospho-choline, n−3 and n−6 PUFAs during glucose stimulated insulin secretion relative to cells cultured under control conditions. However, no changes in calcium handling or mitochondrial membrane potential were evident.

Conclusions

Results indicate that a decrease in TCA cycle metabolism in combination with an alteration in fatty acid composition and phosphocholine levels may play a role in glucotoxicity induced impairment of glucose stimulated insulin secretion.

General significance

Alterations in certain metabolic pathways play a role in glucotoxicity in the pancreatic beta cell.     

Stevioside improves pancreatic beta-cell function during glucotoxicity via regulation of acetyl-CoA carboxylase

Chronic hyperglycemia is detrimental to pancreatic beta-cells, causing impaired insulin secretion and beta-cell turnover. The characteristic secretory defects are increased basal insulin secretion (BIS) and a selective loss of glucose-stimulated insulin secretion (GSIS). Several recent studies support the view that the acetyl-CoA carboxylase (ACC) plays a pivotal role for GSIS. We have shown that stevioside (SVS) enhances insulin secretion and ACC gene expression. Whether glucotoxicity influences ACC and whether this action can be counteracted by SVS are not known. To investigate this, we exposed isolated mouse islets as well as clonal INS-1E beta-cells for 48 h to 27 or 16.7 mM glucose, respectively. We found that 48-h exposure to high glucose impairs GSIS from mouse islets and INS-1E cells, an effect that is partly counteracted by SVS. The ACC dephosphorylation inhibitor okadaic acid (OKA, 10(-8) M), and 5-aminoimidazole-4-carboxamide-1-beta-d-ribofuranoside (AICAR, 10(-4) M), an activator of 5'-AMP protein kinase that phosphorylates ACC, eliminated the beneficial effect of SVS. 5-Tetrade-cyloxy-2-furancarboxylic acid (TOFA), the specific ACC inhibitor, blocked the effect of SVS as well. During glucotoxity, ACC gene expression, ACC protein, and phosphorylated ACC protein were increased in INS-1E beta-cells. SVS pretreatment further increased ACC gene expression with strikingly elevated ACC activity and increased glucose uptake accompanied by enhanced GSIS. Our studies show that glucose is a potent stimulator of ACC and that SVS to some extent counteracts glucotoxicity via increased ACC activity. SVS possesses the potential to alleviate negative effects of glucotoxicity in beta-cells via a unique mechanism of action.     

AMP-activated protein kinase and pancreatic/duodenal homeobox-1 involved in insulin secretion under high leucine exposure in rat insulinoma β-cells

The effect of leucine on glucose-stimulated insulin secretion (GSIS) in pancreatic β-cells is quite controversial, and mechanism involved in the effect has not been elucidated yet. Consequently, we aimed to investigate effect of leucine on GSIS and its mechanism focusing on contribution of AMP-activated protein kinase (AMPK) and pancreatic/duodenal homeobox-1 (PDX-1). Rat insulinoma β-cells (INS-1, RIN m5F, DN-PDX-1#28 and PDX-1#6) were cultured with or without leucine, AICAR (AMPK agonist) or compound C (AMPK antagonist) for 48 hrs. In contrast to control, AICAR treatment decreased GSIS at high glucose and insulin content, also impaired protein and mRNA expression of PDX-1 and its downstream targets, glucokinase (GCK) and glucose transporter 2 (GLUT2). Compound C treatment had the opposite effects. We observed that neither AICAR nor compound C could affect expression of GCK and GLUT2 when PDX-1 expression was absent. Chronic leucine exposure inhibited GSIS at high glucose and insulin content in a dose-dependent manner, concomitant with an increase in AMPK and a decrease in PDX-1, GCK and GLUT2. The inhibitory effects of leucine was potentiated by AICAR treatment and rescued by compound C treatment. Finally, the inhibition of PDX-1 could potentiate the impaired effects induced by leucine whereas overexpression of PDX-1 could protect the cell from impairment induced by leucine. The study indicated that chronic leucine might result in an increase in AMPK and then a decrease in PDX-l, in turn to depress GCK and GLUT2 resulting in decreased GSIS at high glucose and insulin content.     

High glucose induces dysfunction in insulin secretory cells by different pathways: a proteomic approach

Chronic hyperglycemia is a hallmark of type 2 diabetes and can contribute to progressive beta cell dysfunction and death. The aim of the present study was to identify pathways mediating high glucose-induced beta cell demise by a proteomic approach. INS-1E cells were exposed to 25 mM glucose for a sustained period of 24 h. Protein profiling of INS-1E cells was done by two-dimensional difference gel electrophoresis, covering the pH ranges 4-7 and 6-9 (n = 4). Differentially expressed proteins (P < 0.05) were identified by MALDI-TOF/TOF and proteomic results were confirmed by functional assays. High glucose levels impaired glucose-stimulated insulin secretion and decreased insulin content. 2D-DIGE analysis revealed 100 differentially expressed proteins that were involved in different pathways. Chaperone proteins were down-regulated, protein biosynthesis and ubiquitin-related proteasomal degradation were attenuated and perturbations in intracellular trafficking and vesicle transport and secretion could be observed. Moreover, several pathways were confirmed by functional assays and a direct role for eEF2 in insulin biosynthesis was demonstrated. The present findings provide new insights in glucotoxicity and identify key target proteins for the prevention and treatment of beta cell dysfunction in type 2 diabetes.     

Glucose-dependent expansion of pancreatic beta-cells by the protein p8 in vitro and in vivo

p8 protein expression is known to be upregulated in the exocrine pancreas during acute pancreatitis. Own previous work revealed glucose-dependent p8 expression also in endocrine pancreatic beta-cells. Here we demonstrate that glucose-induced INS-1 beta-cell expansion is preceded by p8 protein expression. Moreover, isopropylthiogalactoside (IPTG)-induced p8 overexpression in INS-1 beta-cells (p8-INS-1) enhances cell proliferation and expansion in the presence of glucose only. Although beta-cell-related gene expression (PDX-1, proinsulin I, GLUT2, glucokinase, amylin) and function (insulin content and secretion) are slightly reduced during p8 overexpression, removal of IPTG reverses beta-cell function within 24 h to normal levels. In addition, insulin secretion of p8-INS-1 beta-cells in response to 0-25 mM glucose is not altered by preceding p8-induced beta-cell expansion. Adenovirally transduced p8 overexpression in primary human pancreatic islets increases proliferation, expansion, and cumulative insulin secretion in vitro. Transplantation of mock-transduced control islets under the kidney capsule of immunosuppressed streptozotocin-diabetic mice reduces blood glucose and increases human C-peptide serum concentrations to stable levels after 3 days. In contrast, transplantation of equal numbers of p8-transduced islets results in a continuous decrease of blood glucose and increase of human C-peptide beyond 3 days, indicating p8-induced expansion of transplanted human beta-cells in vivo. This is underlined by a doubling of insulin content in kidneys containing p8-transduced islet grafts explanted on day 9. These results establish p8 as a novel molecular mediator of glucose-induced pancreatic beta-cell expansion in vitro and in vivo and support the notion of existing beta-cell replication in the adult organism.     

Hyperoxia reverses glucotoxicity-induced inhibition of insulin secretion in rat INS-1 β cells

Chronic hyperglycemia has deleterious effects on pancreatic β-cell function, a process known as glucotoxicity. This study examined whether chronic high glucose (CHG) induces cellular hypoxia in rat INS-1 β cells, and whether hyperoxia (35% O₂) can reverse glucotoxicity-induced inhibition of insulin secretion. CHG (33.3?mm, 96?h) reduced insulin secretion, and down-regulated insulin and pancreatic duodenal homeobox factor 1 gene expression. CHG also increased intracellular pimonidazole-protein adducts, a marker for hypoxia. CHG also enhanced hypoxia-inducible factor 1α (HIF-1α) protein expression and its DNA-binding activity, which was accompanied by a decrease in mRNA expression of glucose transporter 2 (GLUT2), glucokinase and uncoupling protein-2 and an increase in mRNA expression of GLUT1 and pyruvate dehydrogenase kinase 1. Hyperoxia restored the decrease in insulin secretion and the gene expression except for GLUT2, and suppressed intracellular hypoxia and HIF-1α activation. These results suggest that glucotoxicity may cause β-cell hypoxia. Hyperoxia might prevent glucotoxicity-induced β-cell dysfunction and improve insulin secretion.     

Cyclin-dependent kinase-5 is a key molecule in tumor necrosis factor-α-induced insulin resistance

The mechanism of TNF-α-induced insulin resistance has remained unresolved with evidence for down-regulation of insulin effector targets effects or blockade of proximal as well as distal insulin signaling events depending upon the dose, time, and cell type examined. To address this issue we examined the acute actions of TNF-α in differentiated 3T3L1 adipocytes. Acute (5-15 min) treatment with 20 ng/ml (~0.8 nm) TNF-α had no significant effect on IRS1-associated phosphatidylinositol 3-kinase. In contrast, TNF-α increased insulin-stimulated cyclin-dependent kinase-5 (CDK5) phosphorylation on tyrosine residue 15 through an Erk-dependent pathway and up-regulated the expression of the CDK5 regulator protein p35. In parallel, TNF-α stimulation also resulted in the phosphorylation and GTP loading of the Rho family GTP-binding protein, TC10α. TNF-α enhanced the depolymerization of cortical F-actin and inhibited insulin-stimulated glucose transporter-4 (GLUT4) translocation. Treatment with the MEK inhibitor, PD98059, blocked the TNF-α-induced increase in CDK5 phosphorylation and the depolymerization of cortical F-actin. Conversely, siRNA-mediated knockdown of CDK5 or treatment with the MEK inhibitor restored the impaired insulin-stimulated GLUT4 translocation induced by TNF-α. Furthermore, siRNA-mediated knockdown of p44/42 Erk also rescued the TNF-α inhibition of insulin-stimulated GLUT4 translocation. Together, these data demonstrate that TNF-α-mediated insulin resistance of glucose uptake can occur through a MEK/Erk-dependent activation of CDK5.     

Nesfatin-1 stimulates glucagon and insulin secretion and beta cell NUCB2 is reduced in human type 2 diabetic subjects

Nesfatin-1 is a novel anorexigenic regulatory peptide. The peptide is the N-terminal part of nucleobindin 2 (NUCB2) and is expressed in brain areas regulating feeding. Outside the brain, nesfatin-1 expression has been reported in adipocytes, gastric endocrine cells and islet cells. We studied NUCB2 expression in human and rodent islets using immunocytochemistry, in situ hybridization and western blot. Furthermore, we investigated the potential influence of nesfatin-1 on secretion of insulin and glucagon in vitro and in vivo in mice and in INS-1 (832/13) cells. The impact of type 2 diabetes (T2D) and glucolipotoxicity on NUCB2 gene expression in human islets and its relationship to insulin secretory capacity and islet gene expression was studied using microarray. Nesfatin-1 immunoreactivity (IR) was abundant in human and rodent beta cells but absent in alpha, delta, PP and ghrelin cells. Importantly, in situ hybridization showed that NUCB2 mRNA is expressed in human and rat islets. Western blot analysis showed that nesfatin-1 IR represented full length NUCB2 in rodent islets. Human islet NUCB2 mRNA was reduced in T2D subjects but upregulated after culture in glucolipotoxic conditions. Furthermore, a positive correlation between NUCB2 and glucagon and insulin gene expression, as well as insulin secretory capacity, was evident. Nesfatin-1 enhanced glucagon secretion but had no effect on insulin secretion from mouse islets or INS-1 (832/13) cells. On the other hand, nesfatin-1 caused a small increase in insulin secretion and reduced glucose during IVGTT in mice. We conclude that nesfatin-1 is a novel glucagon-stimulatory peptide expressed in the beta cell and that its expression is decreased in T2D islets.