Department of Physiology, Diabetes Research Group, University of Manitoba, Winnipeg, Canada.
FEBS J. 2010 Jan;277(2):488-500. doi: 10.1111/j.1742-4658.2009.07505.x. Epub 2009 Dec 16.
Pancreatic beta-cell dysfunction is a prerequisite for the development of type 2 diabetes. Alcoholism is a diabetes risk factor and ethanol increases oxidative stress in beta-cells, whereas the mitochondrial chaperone prohibitin (PHB) has antioxidant effects in several cell types. In the present study we investigated whether PHB is expressed in beta-cells and protects these cells against deleterious effects of ethanol, using INS-1E and RINm5F beta-cell lines. Endogenous PHB was detected by western blot and immunocytochemistry. Reactive oxygen species were determined by 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate fluorescence assay, and mitochondrial activity was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) reduction, uncoupling protein 2 expression and ATP production. Cell death was determined by Hoechst 33342 staining, cleaved caspase-3 levels and flow cytometry. PHB was expressed in beta-cells under normal conditions and colocalized with Hoechst 33342 in the nucleus and with the mitochondrial probe Mitofluor in the perinuclear area. In ethanol-treated cells, MTT reduction and ATP production decreased, whereas reactive oxygen species, uncoupling protein 2 and cleaved caspase-3 levels increased. In addition, flow cytometry analysis showed an increase of apoptotic cells. Ethanol treatment increased PHB expression and induced PHB translocation from the nucleus to the mitochondria. PHB overexpression decreased the apoptotic effects of ethanol, whereas PHB knockdown enhanced these effects. The protective effects of endogenous PHB were recapitulated by incubation of the cells with recombinant human PHB. Thus, PHB is expressed in beta-cells, increases with oxidative stress and protects the cells against deleterious effects of ethanol.
胰岛β细胞功能障碍是 2 型糖尿病发生的前提条件。酒精中毒是糖尿病的一个危险因素，乙醇会增加β细胞的氧化应激，而线粒体伴侣蛋白 prohibitin（PHB）在几种细胞类型中具有抗氧化作用。在本研究中，我们使用 INS-1E 和 RINm5F 胰岛β细胞系，研究 PHB 是否在胰岛β细胞中表达，并保护这些细胞免受乙醇的有害影响。通过 Western blot 和免疫细胞化学检测内源性 PHB。通过 5-（和-6）-氯甲基-2'，7'-二氯二氢荧光素二乙酸酯荧光测定法测定活性氧，通过 3-（4，5-二甲基噻唑-2-基）-2,5-二苯基四唑溴盐（MTT）还原、解偶联蛋白 2 表达和 ATP 产生评估线粒体活性。通过 Hoechst 33342 染色、切割的 caspase-3 水平和流式细胞术测定细胞死亡。在正常条件下，β细胞中表达 PHB，与核中的 Hoechst 33342 和核周区域的线粒体探针 Mitofluor 共定位。在乙醇处理的细胞中，MTT 还原和 ATP 产生减少，而活性氧、解偶联蛋白 2 和切割的 caspase-3 水平增加。此外，流式细胞术分析显示凋亡细胞增加。乙醇处理增加 PHB 表达并诱导 PHB 从核易位到线粒体。PHB 过表达减少了乙醇的凋亡作用，而 PHB 敲低增强了这些作用。用重组人 PHB 孵育细胞可再现内源性 PHB 的保护作用。因此，PHB 在胰岛β细胞中表达，随着氧化应激的增加而增加，并保护细胞免受乙醇的有害影响。
