Glucose-Induced O₂ Consumption Activates Hypoxia Inducible Factors 1 and 2 in Rat Insulin-Secreting Pancreatic Beta-Cells

<div><h3>Background</h3><p>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.</p> <h3>Methodology/Principal Findings</h3><p>Isolated rat islets and insulin-secreting INS-1E cells were stimulated with nutrients at various pO₂ values or treated with the HIF activator CoCl₂. 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 CoCl₂ or a four-fold reduction in pO₂. Islets also showed signs of HIF activation in diabetic <em>Lepr^db/db</em> but not non-diabetic <em>Lepr^db/+</em> mice. <em>In vitro</em>, these glucose effects were reproduced by nutrient secretagogues that bypass glycolysis, and were inhibited by a three-fold increase in pO₂ or by inhibitors of Ca²⁺ influx and insulin secretion. In INS-1E cells, small interfering RNA-mediated knockdown of <em>Hif1α</em> and <em>Hif2α</em>, 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.</p> <h3>Conclusions/Significance</h3><p>Glucose-induced O₂ 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.</p>