Hyperglycemia and hypoxia are suggested to play essential pathophysiological roles in the complications of diabetes, which may result from a defective response of the tissues to low oxygen tension. In this study, we show that in primary dermal fibroblasts and endothelial cells, hyperglycemia interferes with the function of hypoxiainducible factor-1 (HIF-1), a transcription factor that is essential for adaptive responses of the cell to hypoxia. Experiments using proteasomal and prolyl hydroxylases inhibitors indicate that hyperglycemia inhibits hypoxiainduced stabilization of HIF-1␣ protein levels against degradation and suggest that mechanisms in addition to proline hydroxylation may be involved. This effect of hyperglycemia was dose dependent and correlates with a lower transcription activation potency of HIF-1␣, as assessed by transient hypoxia-inducible reporter gene assay. Regulation of HIF-1␣ function by hyperglycemia could be mimicked by mannitol, suggesting hyperosmolarity as one critical parameter. The interference of hyperglycemia with hypoxia-dependent stabilization of HIF-1␣ protein levels was confirmed in vivo, where only very low levels of HIF-1␣ protein could be detected in diabetic wounds, as compared with chronic venous ulcers. In conclusion, our data demonstrate that hyperglycemia impairs hypoxia-dependent protection of HIF-1␣ against proteasomal degradation and suggest a mechanism by which diabetes interferes with cellular responses to hypoxia. Diabetes 53:3226 -3232, 2004 C hronic complications of diabetes are a major health problem, and it has become a priority to characterize further their pathophysiological mechanisms to develop novel, rational therapeutic strategies. Even though prolonged exposure of the tissues to hyperglycemia seems to be the primary causative factor, some other factors may play a role as far as intensive blood glucose control reduces chronic complications but does not prevent them altogether (1,2). It has become increasingly evident that hypoxia plays an important role in all diabetes complications (3). In addition to deficient blood supply as a consequence of micro-and macrovascular disease, it has been postulated that hyperglycemia induces a pseudohypoxia state. This theory is based on the finding that high glucose concentrations induce a high NADH ϩ /NAD ϩ ratio in cells even when the oxygen tension is normal (4).Adaptive responses of cells to hypoxia are mediated by the hypoxia-inducible factor-1 (HIF-1), which is a heterodimeric transcription factor composed of two subunits, HIF-1␣ and aryl hydrocarbon receptor nuclear translocator (ARNT), both constitutively expressed in mammalian cells. Regulation of HIF-1 activity is critically dependent on the degradation of the HIF-1␣ subunit in normoxia. The molecular basis of its degradation is the O 2 -dependent hydroxylation of at least one of the two proline residues (5,6) in the oxygen-dependent degradation domain of HIF-1␣ by specific prolyl 4-hydroxylases (PHDs) (7-9). In this form, HIF-1 ␣ binds to the von Hippel-Lind...

Hypoxia-inducible factor 1␣ (HIF-1␣) functions as a transcription factor that is activated by decreased cellular oxygen concentrations to induce expression of a network of genes involved in angiogenesis, erythropoiesis, and glucose homeostasis. Here we demonstrate that two members of the SRC-1/p160 family of transcriptional coactivators harboring histone acetyltransferase activity, SRC-1 and transcription intermediary factor 2 (TIF2), are able to interact with HIF-1␣ and enhance its transactivation potential in a hypoxiadependent manner. HIF-1␣ contains within its C terminus two transactivation domains. The hypoxia-inducible activity of both these domains was enhanced by either SRC-1 or the CREB-binding protein (

The human hypoxia inducible factor 1 (HIF-1) system is activated under various pathological conditions, yet less is known about its physiological regulation in healthy human tissue. We have studied the effect of exercise on the activation of HIF-1 in human skeletal muscle. Employing a model where oxygen consumption increases and oxygen tension can be manipulated, nine healthy male subjects performed 45 min of one-legged knee-extension exercise. Biopsies were taken before, directly after, and 30, 120, and 360 min after exercise. Exercise led to elevated HIF-1alpha protein levels and a more prevalent nuclear staining of HIF-1alpha. Interestingly, a concurrent decrease in von Hippel-Lindau tumor suppressor protein (VHL) levels was detected in some subjects. Moreover, exercise induced an increase in the DNA binding activity of HIF-1alpha. Characterization of gene expression by real-time PCR demonstrated that the HIF-1 target genes VEGF and EPO were activated. VEGF mRNA was further increased when blood flow to the exercising leg was restricted. In conclusion, these data clearly demonstrate that physical activity induces the HIF-1-mediated signaling pathway in human skeletal muscle, providing the first evidence that human HIF-1alpha can be activated during physiologically relevant conditions.