Densitometric quantitation of g. and sufficient to mediate HIF-2 Ciproxifan and VEGF inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between HIF-2Cmediated hypoxic signaling and Rabbit Polyclonal to KR2_VZVD hepatic insulin action via IRS2 induction, which can be co-opted by VEGF inhibitors to modulate glucose metabolism. These studies also show unique functions in hepatic metabolism for HIF-1, which promotes glycolysis7C9, versus HIF-2, which suppresses gluconeogenesis, and suggest novel treatment methods for type 2 diabetes mellitus. The liver regulates systemic energy reserves by controlling carbohydrate and lipid metabolism in response to dietary and systemic cues. Hepatic insulin activation recruits insulin receptor substrate (IRS) proteins to the insulin receptor, with activation of AKT, GSK3 and mTOR, coordinately suppressing hepatic gluconeogenesis and inducing glycogen synthesis and lipogenesis1,2. The liver perivenous zone experiences relative hypoxia accompanied by suppression of gluconeogenesis3. During normoxia, prolyl hydroxylase domainCcontaining enzymes (PHD1C3) and factor inhibiting HIF (FIH) hydroxylate users of the HIF transcription factor family (HIF1C3), resulting in von Hippel-Lindau (VHL)-dependent proteosomal degradation; hypoxic inhibition of this hydroxylation stabilizes HIFs and induces HIF transcriptional targets10. The VEGF family contains VEGF-A-D and PlGF, each with unique affinities for VEGF receptors 1C3 (VEGFR1C3) and neuropilins. VEGFR1/Flt1 is usually a high-affinity receptor for VEGF-A, -B and PlGF versus VEGFR2/Flk1, which is a low-affinity receptor for VEGF-A, -C and CD11,12. VEGF inhibitor treatment decreases fasting blood glucose levels and enhances glucose tolerance in mice and humans through unclear mechanisms13,14, and specific VEGF-B inhibition enhances glucose tolerance through enhanced peripheral glucose uptake15. Here, we utilized single intravenous injection of adenoviruses encoding the soluble extracellular ligand-binding domains of VEGFR1/Flt1 (Ad sFlt1) or VEGFR2/Flk1 fused to an antibody Fc fragment (Ad sFlk1) to achieve hepatic secretion of Flt1 or Flk1 ectodomains into the blood circulation; both ectodomains elicit potent and durable VEGF-A neutralization mice (Fig. 1b) compared to control treatment as confirmed by AUC analysis (Supplementary Fig. 1aCd). Similar results were obtained with Ad sFlk1 (Fig. 1b and Supplementary Fig. 1c,d). Recombinant aflibercept/VEGF Trap, encoding a VEGFR1/VEGFR2 ectodomain fusion that binds VEGF-A, -B and PlGF18,19, also improved glucose tolerance versus control treatment in C57Bl/6 or mice (Fig. 1c, d and Supplementary Fig. 1e,f), as did both the anti-VEGF-A Ciproxifan monoclonal antibody (mAb) B20.4.1.120, and the anti-VEGFR2 monoclonal antibody DC10121 (Supplementary Fig. 1g,h), neither of which interfere with VEGF-B signaling. Open in a separate window Physique 1 VEGF inhibition enhances hepatic insulin actionaCd. Glucose tolerance assessments (GTT) and insulin tolerance assessments (ITT) in adult C57Bl/6J (a) or mice (b) treated with a single i.v. injection of Ad sFlt1/sVEGFR1, Ad sFlk1/sVEGFR2 or Ad Fc (n=8 each, 109 pfu) after 15 days. The initial average blood glucose levels for ITT in a. were Fc=129, sFlt1=72.8 and in b. were Fc=162, sFlt1=55.4, sFlk1=109, all mg/dL. c,d. GTT of adult SCID mice (n=5) or db/db mice (n=5) treated with aflibercept or hFc after 15 days. e. Hyperinsulinemic euglycemic clamp analysis of aflibercept- and hFc-treated mice after 2 weeks. f. ELISA determination of fasting plasma insulin concentration from mice as in d. g,h. Insulin signaling pathway determination in fasted liver extracts after 14 days. i,j. Analysis of and mRNA by qRT-PCR from liver from Ad Fc, Ad sFlt1 and Ad sFlk1-injected mice (n=5, ad lib, day 14) (i) or adult SCID mice treated with aflibercept, (n=5, fasted, day 14) (j). Values are expressed as mean s.e.m. * = P 0.05. VEGF inhibitors decreased fasting or fed glucose levels (Supplementary Fig. 2aCe) and aflibercept did not increase plasma insulin or decrease glucagon (Supplementary Fig. 2f,g). In a hyperinsulinemic euglycemic clamp study, two-week aflibercept treated mice exhibited higher insulin sensitivity, enhanced insulin-induced suppression of hepatic glucose production (HGP) (Fig. 1e and Supplementary Fig. 3) and substantially improved hyperinsulinemia (Fig. 1f) compared to control hFc-treated mice. This occurred without altering insulin-stimulated whole-body glucose disposal, peripheral tissue-specific glucose uptake, or hepatic CREB or AMPK signaling (Supplementary Fig. 4a,b). The insulin-potentiating effects of VEGF inhibition on HGP prompted evaluation of insulin receptor (IR) signaling in liver. Both aflibercept and Ad sFlt1 Ciproxifan treatment increased phosphorylation of AKT (p-AKT) and GSK3 (p-GSK3), augmented expression of IRS2, but not IRS1 or IR itself (Fig. 1g, h), and suppressed phosphoenolpyruvate kinase (VEGF antagonism (Fig. 2c) versus Fc-treated animals. Ad sFlt1 and aflibercept also decreased functional perfusion in mouse liver upon intravascular biotin infusion (Fig. 2d). Further, microarray analysis of aflibercept-treated mouse liver revealed upregulation of several hypoxia-inducible genes, including (Supplementary Fig. 6a), while HIF-2, but not HIF-1 protein was stabilized in Ad sFlt1-treated mouse liver (Fig. 2e). Notably, Ad.
