| Diabetes is the most common metabolic disease in the world. Over 90% of diabetic patients are diagnosed with type 2 diabetes. Although the primary cause of diabetes is not known, insulin resistance and pancreatic beta-cell failure are considered important factors in the development of type 2 diabetes. Both characteristics of type 2 diabetes may be triggered by mitochondrial dysfunction. Alcoholism is known as a risk factor for type 2 diabetes. Excessive or chronic alcohol consumption leads to increased oxidative stress and mitochondrial dysfunction in beta-cells. Prohibitin (PHB) is an evolutionarily conserved protein that plays a role as a mitochondrial chaperone. Although it has anti-oxidant effects in several cell types, its role in pancreatic beta-cells is not known.;To address these aims, mitochondrial dysfunction was induced in RINm5F and INS-1E pancreatic beta-cell lines by treatment with ethanol, which has been shown in other cell types to cause oxidative stress and apoptosis. First, endogenous PHB was determined by western blot and real time polymerase chain reaction (PCR) in RINm5F rat insulinoma cells. The cellular distribution of PHB was visualized using immunocytochemistry and further confirmed using western blot of different cellular fractions. Alcohol dehydrogenase (ADH) expression was detected by western blot and real time PCR. As well, the metabolism of ethanol was determined by assaying ethanol-induced ADH activity as well as by measuring ethanol concentration left in cell culture media after 24 h incubation. Mitochondrial function was determined using various methods including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, reactive oxygen species (ROS) production, uncoupling protein 2 (UCP2) expression, adenosine triphosphate (ATP) production, mitochondrial respiratory complex activity, and mitochondrial membrane potential. The interaction of PHB to mitochondrial proteins was examined by immunoprecipitation (IP). Cell death was monitored by flow cytometry analysis with fluorescein isothiocyanate (FITC)-annexin V staining and Hoechst 33342 nuclear staining. The pathway involved in apoptosis was identified by immunoblot for activation of c-Jun N-terminal kinase (JNK), expression of the proapoptotic protein Bax, and the cleaved caspase-3 assay. The effect of prohibitin on insulin secretion in beta-cells was determined using a rat insulin enzyme linked immunoassay (ELISA).;PHB was expressed in beta-cells under normal culture conditions and co-localized with the nuclear probe Hoechst 33342 in the nucleus and with the mitochondrial probe Mitofluor in the perinuclear area. Ethanol treatment increased PHB expression in beta-cells and induced PHB translocation from the nucleus to the mitochondria. Alcohol dehydrogenase 5 (ADH5) was expressed in pancreatic beta-cells and increased with ethanol incubation. In addition, the total ADH activity was increased. In ethanol treated beta-cells, MTT reduction and ATP production decreased, whereas ROS, UCP2, p-JNK, Bax and cleaved caspase-3 levels increased. Ethanol treatment decreased the interaction of PHB to mitochondrial proteins, impaired the activity of mitochondrial respiratory complexes I and IV, and resulted in a reduction of mitochondrial membrane potential. In addition, flow cytometry analysis of RINm5F cells showed increased apoptosis, while Hoechst 33342 nuclear staining showed small and condensed nuclei after ethanol treatment. In INS-1E rat insulinoma cells, ethanol decreased glucose induced insulin secretion. Exogenously applied PHB or PHB overexpression attenuated ADH activity, prevented the deleterious effects of ethanol on mitochondria and protected from the apoptotic effects of ethanol, whereas PHB knockdown using small interfering RNA (siRNA) of PHB enhanced ethanol induced apoptotic effects on both pancreatic beta-cells. In addition, PHB increased the levels of the transcription factors: pancreatic and duodenal homeobox 1(PDX-1) and v-maf musculoaponeurotic fibrosacoma oncogene homolog A (MafA). The aggregate result was a restoration of glucose induced insulin secretion by PHB in ethanol exposed pancreatic beta-cells.;In conclusion, ethanol causes mitochondrial dysfunction in pancreatic beta-cells by impairing mitochondrial complexes I and IV, and induces apoptosis via the JNK pathway. These harmful effects of ethanol result in a reduction of insulin secretion. PHB prevents ethanol-induced mitochondrial dysfunction, apoptosis, and beta-cell failure by stabilizing mitochondrial complexes I and IV and partially inhibiting ADH activity during ethanol metabolism. PHB in itself increases the levels of beta-cell transcription factors. As a result, PHB restores insulin secretion in ethanol exposed pancreatic beta-cells.;In this thesis, we have investigated the role of prohibitin in ethanol (EtOH) treated pancreatic beta-cells. The specific aims were to determine: (a) the effects of ethanol on prohibitin expression in beta-cells, (b) the distribution of prohibitin in beta-cells, (c) the effect of prohibitin on alcohol dehydrogenase (ADH) expression in beta-cells, (d) the effects of prohibitin on mitochondrial function in beta-cells, (e) the role of prohibitin in beta-cell survival, (f) the effect of prohibitin on insulin secretion. |
