Glucose increases the expression of glycolytic enzymes and other hypoxia-response genes in pancreatic beta-cells. Here, we tested whether this effect results from the activation of Hypoxia-Inducible-factors (HIF) 1 and 2 in a hypoxia-dependent manner. Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO.sub.2 values or treated with the HIF activator CoCl.sub.2 . HIF-target gene mRNA levels and HIF subunit protein levels were measured by real-time RT-PCR, Western Blot and immunohistochemistry. The formation of pimonidazole-protein adducts was used as an indicator of hypoxia. In INS-1E and islet beta-cells, glucose concentration-dependently stimulated formation of pimonidazole-protein adducts, HIF1 and HIF2 nuclear expression and HIF-target gene mRNA levels to a lesser extent than CoCl.sub.2 or a four-fold reduction in pO.sub.2 . Islets also showed signs of HIF activation in diabetic Lepr.sup.db/db but not non-diabetic Lepr.sup.db/+ mice. In vitro, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO.sub.2 or by inhibitors of Ca.sup.2+ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of Hif1alpha and Hif2alpha, alone or in combination, indicated that the stimulation of glycolytic enzyme mRNA levels depended on both HIF isoforms while the vasodilating peptide adrenomedullin was a HIF2-specific target gene. Glucose-induced O.sub.2 consumption creates an intracellular hypoxia that activates HIF1 and HIF2 in rat beta-cells, and this glucose effect contributes, together with the activation of other transcription factors, to the glucose stimulation of expression of some glycolytic enzymes and other hypoxia response genes.