Glucotoxicité et altérations de l'expression génique dans les cellules bêta pancréatiques : rôle du facteur de transcription NFkB et du stress du reticulum endoplasmique

In contrast with type 1 diabetes that is caused by autoimmune destruction of pancreatic b-cells, type 2 diabetes (T2D) results from the combination of insulin resistance and defective insulin secretion due to reduced b-cell mass and function. Although the initial cause of such s-cell dysfunction is not known, it is well established that even moderate hyperglycaemia further alters b-cell function and survival. This process of glucotoxicity contributes thus to the progressive worsening of glucose intolerance in these patients. Therefore, elucidating the molecular mechanisms of b-cell glucotoxicity may provide important insights into the pathophysiology of T2D. Methods: Islets were isolated from male Wistar rats and pre-cultured for one week in the presence of 10 mM glucose. Cell survival, function and gene expression were then measured after further culture for 1 to 7 days in the presence of low, intermediate and high glucose concentrations (2, 5, 10 or 30 mM) plus various test substances. First study: The alterations of s-cell gene expression by high glucose are similar to those caused by IL-1b, a pro-inflammatory cytokine which triggers s-cell apoptosis in T1D following the activation of the transcription factor NFB. This suggested that the effects of high glucose may result from the activation of NFkB, likely as a result of oxidative stress. To test this hypothesis, I compared the effects of IL-1s, high glucose and hydrogen peroxide with or without the antioxidant N-Acetyl-L-cystein (NAC) on NFkB activation and target gene mRNA levels in cultured islets. As expected, exogenous IL-1b rapidly activated NFkB and increased the expression of its target genes c-Myc, Hmox1, iNOS, Fas and IkBa. In contrast, culture in the presence of 30mM glucose (G30) or 50µM hydrogen peroxide stimulated the expression of c-Myc, Hmox1 and Fas without activating NFkB or increasing the expression of its target genes iNOS and IBa. These effects were nevertheless inhibited by NAC and by inhibition of ERK and p38MAPK, suggesting that activation of these kinases by oxidative stress plays a role in s-cell glucotoxicity. Second study: b-cell protein synthesis (predominantly proinsulin) is markedly stimulated by glucose. When the increase in endoplasmic reticulum (ER) synthetic load exceeds the ER folding capacity, the ER stress sensors IRE1, ATF6 and PERK activate the unfolded protein response (UPR). Although this response usually favours cell survival by reducing protein synthesis and improving the ER folding capacity, its sustained activation may contribute to b-cell apoptosis in T2D. To test this hypothesis, I compared the effects of increasing glucose concentrations and ER Ca2+ emptying with thapsigargin (TG) on rat islet UPR. As expected, TG rapidly induced a full ER stress response with increased Xbp1 pre-mRNA splicing by IRE1, increased mRNA levels of protein chaperones, eIF2a phosphorylation by PERK and subsequent expression of ATF4 and its target genes Gadd34, Atf3 and Chop. In comparison, glucose moderately activated IRE1 as a result of the stimulation of protein synthesis, as shown by the increase in Xbp1 pre-mRNA splicing and expression of chaperones between G5 and G30 and by their inhibition by low cycloheximide concentrations. However, glucose exerted complex effects on events downstream eIF2a phosphorylation. This part of the UPR, which can be activated by other types of stresses and is therefore referred to as the integrated stress response (ISR), was strongly activated in G2-G5, minimal in G10, and slightly increased in G30. This asymmetric V-shaped glucose response curve is similar to that described for b-cell apoptosis under the same culture conditions. These results therefore suggest a causal link between ISR activation and stimulation of s-cell apoptosis. Conclusion: s-cell glucotoxicity in cultured rat islets partly results from oxidative stress and ER stress but is independent from NFB activation or IL-1b expression. [. ]