Glucose et stress oxydatif dans les cellules beta pancréatiques : rôle du zinc et des métallothionéines

Glucose stimulation of insulin secretion by pancreatic β cells is essential to maintain glucose homeostasis. In addition to this short-term effect, glucose is also important to maintain β cell survival and differentiated phenotype. Indeed, both chronic hypo- and hyper-glycemia alter β-cell gene expression, survival and function. Similarly, in vitro, rat β-cell gene expression, function and survival are optimally preserved by culture in the presence of 10 mM glucose (G10) and markedly impaired by culture in either lower (2 or 5 mmol/l glucose, G2 or G5) or higher (30 mmol/l glucose, G30) glucose concentrations. It therefore seems that glucose stimulation exerts beneficial effects on β cell function and survival between G2 and G10 and a deleterious effect on these parameters between G10 and G30. The precise molecular mechanisms behind such phenotypical plasticity are poorly understood, but they could involve an increase in oxidative stress. Indeed, mRNA levels of oxidative stress-response genes like Metallothionein 1a/2a (Mt1a/2a), Heme oxygenase 1 (Hmox1) or c-Mycfollow an asymmetric V-shaped profile similar to that of β cell dysfunction and apoptosis. We therefore hypothesized that extreme glucose concentrations increases oxidative stress in β cells. In a first study, we developed a method to measure the β cell redox status in our experimental conditions. Our results showed that mt-HyPer, besides its expected sensitivity to H2O2, was also highly pH-sensitive. As glucose stimulation increases mitochondrial pH in β cells, that probe could not be used in our experimental model. In contrast, the fluorescence ratio of mt-roGFP1, which measures the thiol/disulfide equilibrium, was only slightly affected by pH. Using that probe, we demonstrated that mitochondrial thiol oxidation in rat β cells reversibly increases when glucose is lowered from 10 to 2 mmol/l. In contrast, acutely increasing glucose concentration from 10 to 30 mmol/l did not increase mt-roGFP1 oxidation in β cell. In a second study, I first demonstrated that 18 to 24h culture of rat islet cell clusters in the présence of G5 or G30 vs. G10 increases mitochondrial glutathione oxidation, thereby confirming our initial hypothesis. I also tested the effect of ZnCl2, a potent inducer of Mt1a, on β cell alterations induced by prolonged exposure to low and high glucose concentrations. My results show that addition of 50μM ZnCl2partially reduced mt-roGFP oxidation after 18-24h culture in G5, and tended to do so after culture in G30.Addition of 100μM ZnCl2also significantly decreased late β-cell apoptosis after prolonged culture in G5 or G30. Theses protective effects of ZnCl2 did not correct β cell dysfunction induced by culture in G5 and G30. In a third study, using islets from Mt1/2 knock-out mice, I wanted to determine the role of MT1/2 on the beneficial effects of Zn2+. I also wanted to test whether the deficiency in Mt1/2 increases β cell apoptosis and mitochondrial glutathione oxidation induced by culture in a low glucose concentration. In contrast with what I observed in rat islets, ZnCl2, despite increasing Mt1expression, did not protect wild-type mouse islets from apoptosis induced by culture in a low glucose concentration, thereby preventing me from studying the role of MT1/2 expression in this effect. Moreover, deficiency in Mt1/2 in mouse islets did not increase apoptosis induced by culture in a low glucose concentration. These results help us to understand the molecular mechanisms underlying the plasticity of the β cell phenotype and may help in the development of new therapeutic strategies for T2D.