Prohibitin is expressed in pancreatic β‐cells and protects against oxidative and proapoptotic effects of ethanol

Abstract

Pancreatic β‐cell dysfunction is a prerequisite for the development of type 2 diabetes. Alcoholism is a diabetes risk factor and ethanol increases oxidative stress in β‐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 β‐cells and protects these cells against deleterious effects of ethanol, using INS‐1E and RINm5F β‐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 β‐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 β‐cells, increases with oxidative stress and protects the cells against deleterious effects of ethanol.

Nuciferine stimulates insulin secretion from beta cells—An in vitro comparison with glibenclamide

“…INS1-E cells (a kind gift from Dr. M. Wheeler, University of Toronto, and Prof. C. Wollheim, University of Geneva) were seeded in 6-well plates and grown for 48 h before insulin secretion experiments (Lee et al, 2010;Nguyen et al, 2012). The cells were washed twice with KRB containing 3.3 mM glucose and preincubated in KRB containing 3.3 mM glucose for 40 min.…”

“…Cellular ATP content was measured using ATP bioluminescent assay kit from Sigma-Aldrich according to the manufacturer's instructions, as previously described (Lee et al, 2010).…”

Functional characterization of naturally occurring transglutaminase 2 mutants implicated in early-onset type 2 diabetes

“…Cellular reactive oxygen species (ROS) generation in INS-1E cells was determined using the fluorescent probe 5-(and-6)-chloromethyl-2 0 ,7 0 -dichlorodihydrofluorescein diacetate (CM-H 2 DCFDA) as described before (Lee et al 2010). CM-H 2 DCFDA passes freely through cellular membranes and is deacetylated by intracellular esterases.…”

“…The INS-1E cells were incubated with DMSO for 30 min in the dark at room temperature. The production of formazan was quantified by measuring absorbancy at 540 and 630 nm using a FLUOstar OPTIMA plate reader (BMG Labtechnologies GmbH, Ortenberg, Germany) and was expressed as a percentage of the positive control (Lee et al 2010).…”

“…INS-1E cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated fetal bovine serum, 10 mM HEPES, 1 mM sodium pyruvate, 50 mM b-mercaptoethanol, 100 mg/ml penicillin-streptomycin (Gauthier et al 2009, Lee et al 2010.…”