Apoptotic, regenerative, and immune-related signaling in human islets from type 2 diabetes individuals.

Islet dysfunction is a primary cause of developing type 2 diabetes mellitus (T2DM). Events leading to islet failure are still poorly defined due to the complexity of the disease and scarcity of human T2DM islets. The aim of the present study was to identify cellular mechanisms involved in the T2DM pathophysiology by protein profiling islets obtained from T2DM individuals and age- and weight-matched controls using liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry and surface enhanced laser desorption/ionization time-of-flight mass spectrometry. In T2DM islets, multiple differentially expressed proteins correlated with insulin secretion. When these T2DM islet proteins were analyzed for differential pathway activation, three of the five most activated pathways were pathways of cell arrest and apoptosis (p53, caspase, stress-activated), one represented immune-response (Fas), and the most activated pathway was connected with proliferation and regeneration (E2F). Among the inactivated pathways, three out of five were pathways of proliferation and regeneration (insulin, PRL, PDGF). The present study is the first to report differential activation of specific pathways during T2DM islet deterioration. The information about alterations in pathway signaling patterns may open new ways to develop strategies aimed at restoring islet cell function and survival.

AMP-activated protein kinase agonist dose dependently improves function and reduces apoptosis in glucotoxic beta-cells without changing triglyceride levels.

Prolonged hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and apoptosis in insulin-producing beta-cells. The detrimental effects have been connected with glucose-induced lipid accumulation in the beta-cell. AMP-activated protein kinase (AMPK) agonist, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), promotes utilization of nutrient stores for energy production. It was tested how impaired GSIS and elevated apoptosis observed in insulinoma (INS)-1E cells after prolonged culture at 27 mM glucose were affected by the inclusion of 0.3 or 1 mM AICAR during culture. Glucose-induced impairment of insulin release was reverted by the inclusion of 0.3 but not 1 mM AICAR, which did not affect insulin content. The glucose-induced rise in triglyceride (TG) content observed in the cells cultured at 27 mM glucose was not altered by the inclusion of either 0.3 or 1 mM AICAR. Inclusion of 1 but not 0.3 mM AICAR during culture induced phosphorylation of AMPK and its downstream target acyl-CoA carboxylase. Phosphorylation was paralleled by reduced number of apoptotic cells and lowered expression of pro-apoptotic C/EBP homologous protein (CHOP). In conclusion, AICAR dose dependently improves beta-cell function and reduces apoptosis in beta-cells exposed to prolonged hyperglycaemia without changing TG levels.

Mitochondrial protein patterns correlating with impaired insulin secretion from INS-1E cells exposed to elevated glucose concentrations.

Extended hyperglycaemia leads to impaired glucose-stimulated insulin secretion (GSIS) and eventually beta-cell apoptosis in individuals with type 2 diabetes mellitus. In an attempt to dissect mechanisms behind the detrimental effects of glucose, we focused on measuring changes in expression patterns of mitochondrial proteins. Impaired GSIS was observed from INS-1E cells cultured for 5 days at 20 or 27 mM glucose compared to cells cultured at 5.5 or 11 mM glucose. After culture, mitochondria were isolated from the INS-1E cells by differential centrifugation. Proteins of the mitochondrial fraction were bound to a strong anionic surface (SAX2) protein array and mass spectra generated by SELDI-TOF-MS. Analysis of the spectra revealed proteins with expression levels that correlated with the glucose concentration of the culture medium. Indeed, such differentially expressed proteins created patterns of protein changes, which correlated with impairment of GSIS. In conclusion, the study reveals the first glucose-induced differentially expressed patterns of beta-cell mitochondrial proteins obtained by SELDI-TOF-MS.