In contrast, glucose increased Tpi1, Ldha, Adm and Car12 mRNA levels to a significantly lesser extent under hyperoxic conditions, supporting the role of the increase in islet O2 consumption in these glucose effects. Of note, the glucose JTP-74057 stimulation of pimonidazole-adduct formation was also suppressed by culture in the presence of 90% O2. Also in INS-1E cells, culture under hyperoxic conditions markedly reduced the glucose stimulation of pimonidazole- protein adduct formation, HIF1a and HIF2a nuclear accumulation, and Adm and Tpi1 mRNA expression. It did not, however, significantly affect GSIS and the stimulation of Gapdh mRNA expression. These results indicate that, depending on the BYL719 PI3K inhibitor HIF-target gene studied, the glucose stimulation of mRNA expression is independent or partly results from hypoxia, not only in isolated islets, but also in INS-1E cells. It has previously been shown that approximately one third of the glucose stimulation of islet O2 consumption is Ca2+ -dependent. The L-type Ca2+ channel blocker nimodipine, which almost fully inhibited insulin secretion during culture in G30, only slightly reduced the mRNA levels of Gapdh but markedly reduced Aldoa and Adm mRNA levels in G30. Interestingly, the latter inhibition was not prevented by addition of exogenous insulin to the medium, indicating that Ca2+ influx contributes to the stimulation of HIF-target gene expression independently from changes in insulin concentration. In contrast, nimodipine exerted opposite effects on Txnip and Aldob mRNA levels. Similar results were obtained with diazoxide, a KATP channel opener that inhibits glucose-induced Ca2+ influx and insulin secretion. Also in INS-1E cells, nimodipine significantly reduced the glucose stimulation of Adm and Aldoa mRNA expression without affecting that of Gapdh. To test whether in vivo hyperglycaemia also induces hypoxia and activates HIF in pancreatic islets, we first measured HIF1a protein levels in islets from diabetic Leprdb/db and non-diabetic Leprdb/+ mice. Interestingly, a few HIF1a-positive nuclei were detected in some islets from diabetic mice, whereas none were observed in sections from non-diabetic mice. That the lack of HIF1a staining did not result from a problem in tissue fixation/ processing was confirmed by the observation, on the same section, of a large number of HIF1a-positive epithelial nuclei in the villi of the intestinal mucosa. For technical reasons, we could not determine whether the few HIF1a-positive islet cells detected in Leprdb/db mice are beta-cells or not. We next measured pimonidazole-protein adducts in the same model of diabetes.