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.     

Silk fibroin has a protective effect against high glucose induced apoptosis in HIT-T15 cells

High glucose levels induce cell death in many cell types, including pancreatic β-cells. Although protective agents against glucotoxicity have been searched for extensively, so far none have been found. In this report, we tested silk fibroin (SF) as a candidate material for antiglucotoxicity in the pancreatic β-cell (HIT-T15 cell) line. Approximately 50% of cells were killed after treatment with 80 mg/mL glucose. This reduction of cell number was recovered by the addition of SF at 50 mg/mL. SF treatment also decreased cellular reactive oxygen species (ROS) and increased proliferating cellular nuclear antigen (PCNA) immunoreactivity. In addition, TUNEL assays demonstrated that SF protects against glucose-induced apoptosis of HIT-T15 cells, suggesting that SF might protect cells from cell death by lowering cellular ROS levels. SF also induced expression of the insulin-like growth factor-1 (IGF-1) gene, and IGF-1 expression may be the cause of SF-induced protection against glucose toxicity. Taken together, these results suggest that SF could serve as a potential therapeutic agent to treat the hyperglycemia-induced death of pancreatic β-cells.     

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca²⁺]_i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca²⁺]_i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48 h to a variety of stressors: cytokines (low-grade inflammation), 28 mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca²⁺]_i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca²⁺]_i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3–11 mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca²⁺]_i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11 mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3 mM glucose) observed for FFAs and also for 28G. We also clamped [Ca²⁺]_i using 30 mM KCl + 250 μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3–11 mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca²⁺]_i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca²⁺]_i but not conventional insulin secretion and ‘metabolic’ stressors (FFAs, 28G, rotenone) impacted insulin secretion.     

Glucotoxicity and pancreatic proteomics

Chronic hyperglycaemia is one of the main characteristics of a diabetic state. This is also the first cause of diabetic complications. However, it is now generally accepted that glucotoxicity also participates in the worsening of type 2 diabetes, by affecting the secretion of β-cells. So far, different mechanisms have been proposed to explain the adverse effects of chronic hyperglycaemia. One of them suggests that the modulation of expression of several key proteins during a hyperglycaemia state, may explain the toxic effect of glucotoxicity. Therefore, proteomic analysis of biological samples represents an interesting method to study the effect of chronic hyperglycaemia on protein expression. The discovery of new proteins for which the expression could be modulated by chronic hyperglycaemia may probably help to better understand the mechanisms underlying glucotoxicity. In this review, we will first present an introduction of the different mechanisms known to be involved in the control of glucose homeostasis and in the development of glucotoxicity. In a second part, some proteomic data linked with the effect of glucotoxicity in pancreas, pancreatic islets and β-cells will be presented and discussed.     

O-GlcNAc modification of FoxO1 increases its transcriptional activity: A role in the glucotoxicity phenomenon?

O-GlcNAc glycosylations on serines or threonines are reversible post-translational modifications that control the localisation, the activity or the stability of cytosolic and nuclear proteins. These dynamic modifications are tightly dependent on the availability of glucose and on its flux through the hexosamine biosynthetic pathway. We recently showed that treatments that increase protein O-GlcNAc glycosylation (high-glucose concentrations, glucosamine) or inhibit their deglycosylation (PUGNAc), induced O-GlcNAc modification of FoxO1 in HEK293 cells. O-GlcNAc glycosylation of FoxO1 resulted in an increased of its activity towards a glucose 6-phosphatase promoter-luciferase reporter gene (G6Pase-luc). This effect appeared to be independent of FoxO1 sub-cellular re-localisation, since it was also observed with the constitutively nuclear FoxO1-AAA mutant. In liver-derived HepG2 cells, glucosamine and PUGNAc increased the expression of G6Pase mRNA, and synergistic effects were observed when both agents were present together. In addition, the expression of PGC1 alpha gene, which is known to be under the control of FoxO1, was also increased by glucosamine and PUGNAc. In HepG2 cells stably expressing the G6Pase-luc reporter gene, glucosamine and PUGNAc also increased the activity of the G6Pase promoter. The stimulation of the G6Pase reporter gene by these agents was abolished by two different FoxO1 siRNAs, thereby demonstrating the involvement of endogenous FoxO1 in the observed effects. Since G6Pase plays a key role in glucose production by the liver, increased in its expression through FoxO1 O-GlcNAc modification may be of considerable importance in the context of glucotoxicity associated with chronic hyperglycaemia. Moreover, since FoxO1 also plays important roles in several aspects of cell biology, including cell proliferation, survival and apoptosis, the regulation of FoxO1 activity by O-GlcNAc modification may have implications for other crucial biological processes.