Mechanisms responsible for cytokine and hyperglycemia induced beta-cell dysfunction

ABSTRACT Aims/hypotheses: The two main forms of diabetes, type 1 and type 2, are characterised by progressive â-cell failure. â-cell failure in both types of diabetes is characterised by functional defects [selective loss of glucose stimulated insulin secretion (GSIS)], and reduced â-cell mass due to increased cell death (apoptosis). Hyperglycaemia and increased cytokines are likely causes of the loss of GSIS and â-cell apoptosis but the mechanisms responsible remain unknown. This Master thesis examined the two separate hypotheses: firstly, that hyperglycaemia leads to endoplasmic reticulum (ER) stress in pancreatic â-cells and this contributes to increased apoptosis; and secondly, that cytokines lead to â-cell dedifferentiation and this contributes to the loss of GSIS. Methods: Studies were performed in MIN6 â-cells and in isolated islets from two different mouse strains, C57BL/6J and DBA/2. Islets were handpicked after pancreas digestion. Islets and MIN6 cells were treated with different concentrations of glucose over a time course ranging from 4 to 72 h. At the end of the treatment period, either apoptosis was measured or RNA was extracted and mRNA levels of candidate ER stress genes assessed by real-time PCR. MIN6 cells were treated with cytokines (either IL-1â alone or co-treatment with IL-1â, IFN-ã and TNF-á) for 24 or 48 h, and either insulin secretory function was evaluated or RNA was extracted and mRNA levels of candidate â-cell differentiation genes assessed by real-time PCR. Results: Surprisingly, only a modest increase in apoptosis was observed in MIN6 cells cultured at high glucose. By far the largest increase in apoptosis was observed in MIN6 cells cultured in low glucose medium. In both isolated islets and MIN6 cells, high glucose treatment induced ER stress, as evidenced by upregulation of several genes specific to the unfolded protein response (BiP, ERP72, EDEM1, P58) and increased processing of XBP-1, a transcription factor which is entirely dependent on activation of UPR transducer protein IRE1 as a consequence of ER stress. Upregulation of these ER chaperones, folding enzymes and degradation proteins (BiP, ERP72, EDEM1) would serve to protect the cells from further endoplasmic reticulum stress and apoptosis. On the other hand, MIN6 cells and islets treated with low glucose levels displayed increased mRNA levels of the apoptosis inducer CHOP, which appeared to be independent of ER stress and likely mediated by the integrated stress response. Chronic treatment of MIN6 cells with cytokines led to a reduction in GSIS. This was associated with reduced mRNA levels of several islet associated transcription factors (Pax6, HNF4á, PDX-1, Nkx6.1, BETA2). Moreover this was also associated with alterations in mRNA levels of many genes implicated in b-cell glucose sensing (GLUT2, mGPDH, Kir6.2, SERCA2b). Conversely, several genes that are normally suppressed in â-cells such as Id-1 and iNOS that would theoretically impair â-cell function were increased. The severities of the changes in â-cell gene expression, apoptosis, and insulin secretion were dependent on the time of exposure to hyperglycaemia and cytokines. Conclusions/interpretation: These studies demonstrate that hyperglycaemia induces ER stress in â-cells with UPR activation providing protection from apoptosis. Conversely, hypoglycaemia induces apoptosis which is associated with increased CHOP. Thus, ER stress plays a critical role in the survival of â-cells exposed to abnormal glucose levels. Cytokines lead to alterations in the pattern of islet gene expression consistent with the hypothesis that a gradual loss of differentiation contributes to the dysfunction of b-cells in diabetes. Noteworthy, it needs further performance to confirm if these results are statistically significant