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Inhibition of vascular endothelial growth factor signaling stimulates erythropoiesis and sensitizes hepatic insulin signaling through activation of hepatic hypoxia-inducible factor-2 alpha. [electronic resource]

1. VEGF inhibition increases hematocrit by hepatic Hif-2[Alpha]-dependent erythrocytosis and Hif-2[Alpha]--independent plasma volume contraction. Vascular Endothelial Growth Factor (VEGF) is an essential regulator of angiogenesis whose pharmacological inhibition represents a promising cancer therapeutic modality. We have previously reported that high-grade inhibition of VEGF by adenoviral expression of soluble VEGF receptor (VEGFR) ectodomains and recombinant VEGF Trap protein induce hepatic Epo-dependent erythrocytosis and polycythemia. Here, we demonstrate that VEGF inhibition induces erythrocytosis in a hepatic Hif-2[Alpha]-dependent manner. Conditional inactivation of hepatic Hif-2[Alpha] in Hif2[Alpha] flox/flox mice by either adenovirus Cre injection or albumin-Cre genetic cross suppressed VEGF inhibition-induced hepatic Epo production and blunted hematocrit elevation. Interestingly, a modest rise in hematocrit (5-10%) persisted in VEGF inhibitor-treated Hif-2[Alpha] liver knockout mice despite the absence of hepatic Epo elevation or reticulocytosis. Analysis of red blood cell mass and plasma volume indicate that, in addition to erythrocytosis, VEGF inhibition significantly reduces blood plasma volume (PV) (10-30% decrease) in a EPO- and Hif-2[Alpha]-independent manner. Thus, VEGF inhibition-induced hematocrit elevation in mice is attributable to both hepatic Hif-2[Alpha]-dependent erythrocytosis and Hif-2[Alpha] --independent plasma volume contraction, with accompanying implications for human patients exhibiting elevated haemoglobin levels in response to pharmacologic VEGF inhibition. 2. VEGF Inhibitors Enhance Hepatic insulin Signaling and Ameliorates Diabetes through Activation of Hepatic HIF-2[Alpha]. In the United States, over 18 million people have diabetes and approximately 90% of these cases are the type 2 form of the disease. The central pathophysiologic defect of T2DM is insulin resistance, where tissues such as muscle, fat and liver exhibit reduced responsiveness to insulin, despite normal to elevated levels of production of the hormone. During our studies on the role of vascular endothelial growth factor (VEGF) in erythropoeisis, we made a surprising observation that VEGF inhibition reduces blood glucose in normal lean mice and significantly corrects hyperglycemia in diabetic and insulin-resistant db/db mice. Metabolic profiling of VEGF inhibitor-treated mice revealed enhanced insulin sensitivity at the liver but not at the periphery. In the liver of these mice, expression of insulin receptor substrate-2 (IRS-2) was elevated, accompanied by enhanced Akt activation and suppression of gluconeogenic genes. Hepatic insulin sensitization by VEGF inhibitors requires hypoxia-inducible factor 2-alpha (Hif-2[Alpha]) as genetic deletion of hepatic Hif-2[Alpha] abrogated insulin sensitization and normalizes blood glucose in VEGF inhbitor-treated mice. Conversely, overexpression of a constitutively active allele of Hif-2[Alpha] in liver hepatocytes reduces blood glucose and activates hepatic insulin signaling in vitro and in vivo. Analysis of the human Irs-2 sequence revealed the presence of several potential Hypoxia-Responsive-Elements (HREs) within its 5' promoter. Using a Irs-2 promoter reporter construct, we found that overexpression of Hif-2[Alpha] or addition of a hypoxia-mimetic drug deferoxamine (DFO) resulted in increased Irs-2 transcription. These data suggest Irs-2 is a hypoxia-inducible gene. In this study, we show that VEGF inhibitors improve hepatic insulin action through activation of hepatic HIF-2[Alpha]. Our results suggest Hif-2[Alpha] positively regulates insulin signaling by promoting Irs-2 expression. These data suggest a previously unsuspected link between hypoxia and insulin signaling in the liver and indicate VEGF inhibitors may be useful for the treatment of diabetes.