Cross-talk between hypoxia and insulin signaling through Phd3 regulates hepatic glucose and lipid metabolism and ameliorates diabetes

Journal name:
Nature Medicine
Volume:
19,
Pages:
1325–1330
Year published:
DOI:
doi:10.1038/nm.3294
Received
Accepted
Published online

Signaling initiated by hypoxia and insulin powerfully alters cellular metabolism. The protein stability of hypoxia-inducible factor-1 alpha (Hif-1α) and Hif-2α is regulated by three prolyl hydroxylase domain–containing protein isoforms (Phd1, Phd2 and Phd3). Insulin receptor substrate-2 (Irs2) is a critical mediator of the anabolic effects of insulin, and its decreased expression contributes to the pathophysiology of insulin resistance and diabetes1. Although Hif regulates many metabolic pathways2, it is unknown whether the Phd proteins regulate glucose and lipid metabolism in the liver. Here, we show that acute deletion of hepatic Phd3, also known as Egln3, improves insulin sensitivity and ameliorates diabetes by specifically stabilizing Hif-2α, which then increases Irs2 transcription and insulin-stimulated Akt activation. Hif-2α and Irs2 are both necessary for the improved insulin sensitivity, as knockdown of either molecule abrogates the beneficial effects of Phd3 knockout on glucose tolerance and insulin-stimulated Akt phosphorylation. Augmenting levels of Hif-2α through various combinations of Phd gene knockouts did not further improve hepatic metabolism and only added toxicity. Thus, isoform-specific inhibition of Phd3 could be exploited to treat type 2 diabetes without the toxicity that could occur with chronic inhibition of multiple Phd isoforms.

At a glance

Figures

  1. Phd3 specifically regulates hepatic Hif-2[alpha] expression and glucose metabolism in vivo.
    Figure 1: Phd3 specifically regulates hepatic Hif-2α expression and glucose metabolism in vivo.

    (a) Western blots from nuclear and cytoplasmic lysates for the indicated proteins and genotypes. Phd1fl/fl, Phd2fl/fl and Phd3fl/fl mice treated with adGFP used as expression control. For the other genotypes, mice were treated with adCre by tail vein injection to achieve a liver-specific knockout. Each lane represents lysates from an individual mouse liver. Molecular weights in kDa are shown at left. Adeno, adenovirus; NS, nonspecific. (b) Fasting blood glucose and insulin levels from the indicated knockout animals. (cj) Glucose tolerance tests (GTTs) (cf) and insulin tolerance tests (gj) of mice of the indicated genotypes and adenoviruses. Data are expressed as mean ± s.e.m. (n = 8 male mice per group).

  2. Worsened hepatotoxicity without improved metabolism in combination Phd knockout animals.
    Figure 2: Worsened hepatotoxicity without improved metabolism in combination Phd knockout animals.

    (a) Quantitative PCR for relative mRNA levels of the indicated gluconeogenic and lipogenic genes in the livers in the indicated mice of the indicated genotypes and treatments (n = 8 male mice per group). (b) Gene expression data for Slc2a1 (top) and Cpt1a (bottom) in the livers of animals with the indicated genotypes. (c) Liver triglyceride measurements in control and knockout animals as indicated. Data are expressed as mean ± s.e.m. (n = 6 male mice per group). *P < 0.05 compared to adGFP controls and **P < 0.001 compared to adGFP controls. (d) H&E-stained and oil red O–stained sections of mouse livers of the indicated genotypes treated with adGFP or adCre. Scale bars, 100 μm. (e) Kaplan-Meier analysis of survival after adenoviral injection. (n = 8 male mice per treatment group). Log-rank analysis with P < 0.0001 for the Phd1Phd2Phd3fl/fl + adCre compared to Phd1Phd2Phd3fl/fl + adGFP controls.

  3. A Hif-2[alpha] -mediated increase in Irs2 expression in mice lacking hepatic Phd3 improves insulin action and reverses diabetes.
    Figure 3: A Hif-2α –mediated increase in Irs2 expression in mice lacking hepatic Phd3 improves insulin action and reverses diabetes.

    (a) Quantitative PCR of Irs1 and Irs2 from livers of the indicated genotype and adenovirus treatments. Bars represent mean ± s.e.m. (n = 8 age-matched male mice, *P < 0.05). (b) Insulin-stimulated liver lysates from Phd3fl/fl mice infected with adGFP or adCre; molecular weights in kDa are shown at left. Each lane represents a lysate from a different mouse. p-Akt, phosphorylated Akt; p-FoxO1, phosphorylated FoxO1; Hist, histone. NS, nonspecific. (c) Luciferase reporter assay in Fao hepatoma cells with human IRS2 promoter–luciferase constructs containing wild-type sequence (−956wt) or mutations of distal (−900mut) or proximal (−123mut) HREs with HIF-1α, HIF-2α or GFP controls. (d) In vivo ChIP assay. Eight-week-old male C57/BL6 mice were treated with HA–HIF-1, HA–HIF-2 or control Fc control adenoviruses; liver lysates subjected to a ChIP assay to detect the enrichment HIF binding on the indicated HREs within the mouse Irs2 promoter (mmIRS2) are shown. (e) Glucose tolerance tests on Phd3fl/fl mice that were fed either a HFD or normal chow (NC) then treated with adGFP or adCre to induce deletion of Phd3. Data are expressed as mean ± s.e.m. (n = 8 mice per group).

  4. Both Hif-2 and Irs2 are required for improved metabolism in mice with a liver-specific knockout of Phd3.
    Figure 4: Both Hif-2 and Irs2 are required for improved metabolism in mice with a liver-specific knockout of Phd3.

    (a) Insulin signaling studies on mice lacking hepatic Phd3 with an additional shRNA knockdown of Hif2a. Total adenovirus amount for each group was the same (5 × 109 PFU bolus) but was divided equally between the two indicated adenoviruses (see Online Methods). Each vertical lane represents a different mouse liver of the indicated treatments. Molecular weights in kDa are shown at left. (b,c) Fasting blood glucose (b) and insulin levels (c) from the indicated adenoviral treatments (n = 6 male mice per treatment group; data are mean ± s.e.m.; *P < 0.05). (d) GTTs performed on Phd3fl/fl mice treated with the indicated adenovirus combinations. Data are expressed as mean ± s.e.m. (n = 8 male mice per treatment group). (e) Insulin signaling studies on mice lacking hepatic Phd3 with an additional shRNA knockdown of Irs2. (f,g) Fasting blood glucose (f) and insulin levels (g) from the indicated adenoviral treatments (n = 6 male mice per treatment group; data are mean ± s.e.m.; *P < 0.05). (h) GTTs performed on Phd3fl/fl mice treated with the indicated adenovirus combinations. (i) Proposed model of metabolic improvements and toxicity in Phd knockout animals.

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Author information

Affiliations

  1. Division of Radiation and Cancer Biology, Department of Radiation Oncology, Center for Clinical Sciences Research, Stanford, California, USA.

    • Cullen M Taniguchi,
    • Elizabeth C Finger,
    • Colleen Wu,
    • Anh N Diep,
    • Edward L LaGory &
    • Amato J Giaccia
  2. Department of Obstetrics and Gynecology, University of Kansas Medical Center, Kansas City, Kansas, USA.

    • Adam J Krieg
  3. Division of Hematology, Stanford University, Stanford, California, USA.

    • Kevin Wei,
    • Lisa M McGinnis,
    • Jenny Yuan &
    • Calvin J Kuo

Contributions

C.M.T., E.C.F., A.J.K., E.L.L., K.W. and L.M.M. designed and performed experiments and analyzed data. C.W. and A.N.D. generated the knockout animals and contributed to design of all animal experiments. J.Y. and C.J.K. generated and purified the adenoviruses and contributed to experimental design of all adenovirus experiments. C.M.T. and A.J.G. wrote the manuscript and oversaw all aspects of this project.

Competing financial interests

The authors declare no competing financial interests.

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