Abstract
Resveratrol, a polyphenol compound, is known for its effects on energy homeostasis. With properties of energy sensors mediating effects of calorie restriction, sirtuins are targets of resveratrol. The mammalian sirtuin homolog SIRT1 is a protein deacetylase playing a role in glucose metabolism and islet function. Here, we investigated the effects of resveratrol and possible link with SIRT1 in β-cells. Insulinoma INS-1E cells and human islets were cultured with resveratrol before analyzing their physiological responses. Treatment of INS-1E cells for 24 h with 25 μM resveratrol resulted in marked potentiation of glucose-stimulated insulin secretion. This effect was associated with elevated glycolytic flux, resulting in increased glucose oxidation, ATP generation, and mitochondrial oxygen consumption. Such changes correlated with up-regulation of key genes for β-cell function, i.e. Glut2, glucokinase, Pdx-1, Hnf-1α, and Tfam. In human islets, chronic resveratrol treatment similarly increased both the glucose secretory response and expression of the same set of genes, eventually restoring the glucose response in islets obtained from one type 2 diabetic donor. Overexpression of Sirt1 in INS-1E cells potentiated resveratrol effects on insulin secretion. Conversely, inhibition of SIRT1 achieved either by expression of an inactive mutant or by using the EX-527 inhibitor, both abolished resveratrol effects on glucose responses. Treatment of INS-1E cells with EX-527 also prevented resveratrol-induced up-regulation of Glut2, glucokinase, Pdx-1, and Tfam. Resveratrol markedly enhanced the glucose response of INS-1E cells and human islets, even after removal of the compound from the medium. These effects were mediated by and fully dependent on active SIRT1, defining a new role for SIRT1 in the regulation of insulin secretion.
Highlights
Increased mitochondrial activation in resveratrol-treated cells was favored by up-regulation of GLUT2 and glucokinase, thereby accelerating the glycolytic rate at high glucose and funneling more substrates into the Krebs cycle
Increased expression of GLUT2 and glucokinase might be secondary to the observed up-regulation of the transcription factors PDX-1 and HNF-1␣ in resveratroltreated cells
Regarding HNF-1␣, its expression is required for GLUT2 transcription in differentiated insulin-producing cells [50, 51] and, necessary to maintain mitochondrial catabolism of the glycolytic product pyruvate [52]
Summary
Cell Culture and Treatments—The clonal rat -cell line INS-1E was cultured as described previously [30] in a humidified atmosphere containing 5% CO2 in RPMI 1640 medium supplemented with 5% (v/v) heat-inactivated fetal calf serum, 2 mM glutamine, 10 mM HEPES, 100 units/ml penicillin, 100 g/ml streptomycin, 1 mM sodium pyruvate, and 50 M 2-mercaptoethanol. After 3– 4 days of culture, cells were incubated in complete RPMI 1640 medium supplemented with the indicated concentrations of resveratrol (Sigma-Aldrich) prepared in ethanol. Adenoviral Treatments of INS-1E Cells—INS-1E cells were seeded in 24-well plates and cultured 3– 4 days before adenovirus transduction with the respective adenoviruses, a kind gift from Joseph Rodgers and Pere Puigserver. Cells were washed and cultured for another 2-day period before the 24-h resveratrol treatment. Quantitative Real-time PCR—Total RNA from INS-1E cells and human islets was extracted using the NucleoSpin RNA II kit (Macherey-Nagel, Duren, Germany) and TRIzol reagent (Invitrogen), respectively. For control of SIRT1 expression, INS-1E cells were transduced with respective adenoviruses, and 3 days later, cells were harvested in radioimmune precipitation assay lysis buffer. The membrane was blocked with 5% milk dissolved in TBS containing 0.1% Tween 20 and probed overnight at 4 °C with different antibodies: mouse anti-SIRT1 (1:1000, Chemicon-Millipore, Zug, Switzerland), rabbit anti-AMPK polyclonal antibody (Cell Signaling, Danvers, MA), mouse monoclonal
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
