Hypoxia induces a group of physiologically important genes such as erythropoietin and vascular endothelial growth factor. These genes are transcriptionally upregulated by hypoxia-inducible factor 1 (HIF-1), a global regulator that belongs to the basic helix-loop-helix PAS family. Although HIF-1 is a heterodimer composed of ␣ and ␤ subunits, its activity is primarily determined by hypoxiainduced stabilization of HIF-1␣, which is otherwise rapidly degraded in oxygenated cells. We report the identification of an oxygen-dependent degradation (ODD) domain within HIF-1␣ that controls its degradation by the ubiquitinproteasome pathway. The ODD domain consists of Ϸ200 amino acid residues, located in the central region of HIF-1␣. Because portions of the domain independently confer degradation of HIF-1␣, deletion of this entire region is required to give rise to a stable HIF-1␣, capable of heterodimerization, DNA-binding, and transactivation in the absence of hypoxic signaling. Conversely, the ODD domain alone confers oxygendependent instability when fused to a stable protein, Gal4. Hence, the ODD domain plays a pivotal role for regulating HIF-1 activity and thereby may provide a means of controlling gene expression by changes in oxygen tension.

Beta-cell dysfunction and impaired insulin production are hallmarks of diabetes1, but despite the growing diabetes epidemic the molecular mechanisms involved have remained unclear. We identified thioredoxin-interacting protein (TXNIP), a cellular redox regulator, as a critical factor involved in beta-cell biology and showed that beta-cell TXNIP was upregulated in diabetes, whereas TXNIP deficiency protected against diabetes by preventing beta-cell apoptosis2–3. Here we show that TXNIP and diabetes induce beta-cell expression of a specific microRNA, miR-204, which in turn blocks insulin production by directly targeting and downregulating MafA, a known insulin transcription factor. After discovering miR-204 to be induced by TXNIP in a microRNA microarray, we confirmed the findings using INS-1 beta-cells, islets of TXNIP-deficient mice, diabetic mouse models and primary human islets. We further discovered that TXNIP induces miR-204 by controlling the activity of STAT3, a transcription factor involved in miR-204 regulation4–5 and identified MafA as a novel target downregulated by miR-204. Taken together, our results demonstrate for the first time that TXNIP controls microRNA expression and insulin production, and that miR-204 is involved in beta-cell function. The identified novel TXNIP/miR-204/MafA/insulin pathway may contribute to diabetes progression and provides new insight into TXNIP function and microRNA biology in health and disease.

Although loss of functional β-cell mass is a hallmark of diabetes, no treatment approaches that halt this process are currently available. We recently identified thioredoxin-interacting protein (TXNIP) as an attractive target in this regard. Glucose and diabetes upregulate β-cell TXNIP expression, and TXNIP overexpression induces β-cell apoptosis. In contrast, genetic ablation of TXNIP promotes endogenous β-cell survival and prevents streptozotocin (STZ)- and obesity-induced diabetes. Finding an oral medication that could inhibit β-cell TXNIP expression would therefore represent a major breakthrough. We were surprised to discover that calcium channel blockers inhibited TXNIP expression in INS-1 cells and human islets and that orally administered verapamil reduced TXNIP expression and β-cell apoptosis, enhanced endogenous insulin levels, and rescued mice from STZ-induced diabetes. Verapamil also promoted β-cell survival and improved glucose homeostasis and insulin sensitivity in BTBR ob/ob mice. Our data further suggest that this verapamil-mediated TXNIP repression is conferred by reduction of intracellular calcium, inhibition of calcineurin signaling, and nuclear exclusion and decreased binding of carbohydrate response element–binding protein to the E-box repeat in the TXNIP promoter. Thus, for the first time, we have identified an oral medication that can inhibit proapoptotic β-cell TXNIP expression, enhance β-cell survival and function, and prevent and even improve overt diabetes.

It has been proposed that cells sense hypoxia by a heme protein, which transmits a signal that activates the heterodimeric transcription factor hypoxia-inducible factor 1 (HIF-1), thereby inducing a number of physiologically relevant genes such as erythropoietin (Epo). We have investigated the mechanism by which two heme-binding ligands, carbon monoxide and nitric oxide, affect oxygen sensing and signaling. Two concentrations of CO (10 and 80%) suppressed the activation of HIF-1 and induction of Epo mRNA by hypoxia in a dosedependent manner. In contrast, CO had no effect on the induction of HIF-1 activity and Epo expression by either cobalt chloride or the iron chelator desferrioxamine. The affinity of CO for the putative sensor was much lower than that of oxygen (Haldane coefficient, ϳ0.5). Parallel experiments were done with 100 M sodium nitroprusside, a nitric oxide donor. Both NO and CO inhibited HIF-1 DNA binding by abrogating hypoxiainduced accumulation of HIF-1␣ protein. Moreover, both NO and CO specifically targeted the internal oxygendependent degradation domain of HIF-1␣, and also repressed the C-terminal transactivation domain of HIF-1␣. Thus, NO and CO act proximally, presumably as heme ligands binding to the oxygen sensor, whereas desferrioxamine and perhaps cobalt appear to act at a site downstream.