A liver Hif-2α-Irs2 pathway sensitizes hepatic insulin signaling and is modulated by Vegf inhibition

Abstract

Insulin initiates diverse hepatic metabolic responses, including gluconeogenic suppression and induction of glycogen synthesis and lipogenesis. The liver possesses a rich sinusoidal capillary network with a higher degree of hypoxia and lower gluconeogenesis in the perivenous zone as compared to the rest of the organ. Here, we show that diverse vascular endothelial growth factor (VEGF) inhibitors improved glucose tolerance in nondiabetic C57BL/6 and diabetic db/db mice, potentiating hepatic insulin signaling with lower gluconeogenic gene expression, higher glycogen storage and suppressed hepatic glucose production. VEGF inhibition induced hepatic hypoxia through sinusoidal vascular regression and sensitized liver insulin signaling through hypoxia-inducible factor-2α (Hif-2α, encoded by Epas1) stabilization. Notably, liver-specific constitutive activation of HIF-2α, but not HIF-1α, was sufficient to augment hepatic insulin signaling through direct and indirect induction of insulin receptor substrate-2 (Irs2), an essential insulin receptor adaptor protein. Further, liver Irs2 was both necessary and sufficient to mediate Hif-2α and Vegf inhibition effects on glucose tolerance and hepatic insulin signaling. These results demonstrate an unsuspected intersection between Hif-2α-mediated hypoxic signaling and hepatic insulin action through Irs2 induction, which can be co-opted by Vegf inhibitors to modulate glucose metabolism. These studies also indicate distinct roles in hepatic metabolism for Hif-1α, which promotes glycolysis, and Hif-2α, which suppresses gluconeogenesis, and suggest new treatment approaches for type 2 diabetes mellitus.

Figure 1. VEGF inhibition improves hepatic insulin action

a–d. Glucose tolerance tests (GTT) and insulin tolerance tests (ITT) in adult C57Bl/6J (a) or db/db 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 db/db 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 G6pc and Pepck 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.

Figure 2. VEGF inhibition induces hepatic vascular regression, liver hypoxia and HIF-2α stabilization to augment hepatic insulin signaling

a, b. Liver CD31 immunofluorescence and vessel area density percentage following Ad Fc, Ad sFlt1, Ad sFlk1, or aflibercept treatment after 14 days. Scale bar = 100 μm. c. FACS determination of hypoxia in mouse hepatocytes 14 days after Ad sFlt1-, Ad Flk1- (blue curves) or control Ad Fc- (red curves) treatment. Hypoxyprobe injection in mice was followed by anti-hydroxyprobe FITC-conjugated secondary antibody in disaggregated hepatocytes. The gray curve indicates a control liver without hypoxyprobe injection. d. Streptavidin-HRP staining of biotinylated L. esculentum lectin liver perfusion 22 days after Ad Fc, Ad sFlt1 and aflibercept treatment. Quantification of perfusion area density percentage is shown (n=3). e. HIF-2α and HIF-1α liver nuclear protein levels after 5 days Ad Fc and Ad sFlt1 treatment. f. GTT in 8–10 week old WT (Hif2α^flox/flox) or HIF2LKO (Hif2α^flox/flox; albumin-Cre) mice (n=6) 14 days after i.v. injection with Ad sFlt1 or Ad Fc. g. Liver Western blot from mice treated as in f. after 16 h fast. h. Densitometric quantitation of g. and G6pc liver qRT-PCR from f. Values are mean ± s.e.m. * = P<0.05.

Figure 3. Activation of hepatic HIF-2α but not HIF-1α signaling is sufficient to improve liver insulin action

a. GTT of C57Bl/6 mice 3d after single i.v. injection of Ad Fc, Ad Hif1αΔODD or Ad Hif2αPN. b, c. Liver extracts from animals in a. were analyzed by Western blot for IRS proteins (b) or for insulin signaling intermediates (c), 5d, ad lib. d. qPCR analysis of liver gluconeogenic gene expression, 5d, fasted. e. Western blot of hepatocytes 24 hours after Ad Fc, Ad Hif1αΔODD or Ad Hif2αPN infection, with or without 100 nM insulin stimulation. f. Western blot of primary mouse hepatocytes infected with Ad Fc or Ad Hif2αPN +/− Ad shRNA luc or Ad shRNA Irs2 for 24h, with and without 100 nM insulin stimulation. Short and long exposures for p-AKT are shown. g. GTT from db/db mice (n=8/group) 4 days after Ad Hif2αPN or Ad Fc treatment ± either Ad shRNA luciferase (shLuc) or Ad shRNA Irs2 (shIRS2). h. Western blot and qRT-PCR analysis of G6pc mRNA from liver of animals in g., ad lib. i. GTT from 8–10 week db/db mice treated with Ad Fc, Ad sFlt1, Ad Hif2αPN or Ad Irs2 after 5 days. j. GTT and AUC from C57Bl/6 mice at 5 days after injection with Ad Fc or Ad sFlt1 ± Ad shRNA luc or Ad shRNA Irs2 (n=6). Values are mean ± s.e.m. * = P<0.05.

Figure 4. HIF-2α regulates IRS2 through Srebp1c-dependent and –independent mechanisms

a. qRT-PCR analysis of Irs1 and Irs2 in C57lBl/6 mouse liver 5 days after Ad Hif2αPN or Hif1αΔODD treatment. b. qRT-PCR analysis of IRS2 in primary hepatocytes 24h post-infection with the indicated adenoviruses. c. Luciferase reporter assay in mouse H2 hepatocytes with human IRS2 promoter-luciferase constructs containing wild-type sequence (wt) or mutations of distal (−900mut) or proximal (−123mut) HREs with Ad GFP, Ad Hif2αΔODD or Ad Hif1αΔODD. d. Srebp1c qRT-PCR from primary hepatocytes infected with Ad Fc or Ad Hif2αPN ± insulin. e. Mouse liver Srebp1c qRT-PCR 5 days after Ad Fc, Ad Hif1αΔODD or Ad Hif2αPN treatment. f. Western blot of primary hepatocytes infected with either Ad Fc or Ad Hif2αPN ± Ad nSrebp-1c. g. IRS2 and nSREPB1c liver Western blot after Ad Fc or Ad Hif2αPN treatment ± Ad nSrebp1c. h. Model. HIF-2α signaling positively regulates the insulin receptor signaling pathway in hepatocytes through induction of IRS2 expression. HIF-2α, but not HIF-1α regulates IRS2 expression directly by promoter trans-activation; both HIF-2α and HIF-1α suppress Srebp1c which can facilitate HIF2α transactivation of IRS2. Physiologic liver hypoxia and VEGF inhibition via vascular regression represent two stimuli capable of activating the HIF2α→IRS2 pathway, possibly mediated by PHD3 (see accompanying manuscript by Taniguchi et al.). Values are expressed as mean ± s.e.m. * = P<0.05.