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. 2012 Feb 1;26(3):259–270. doi: 10.1101/gad.180406.111

Figure 4.

Figure 4.

Hif1α inactivation promotes Pgc1α deacetylation via Sirt2. (A) Visceral WAT of Hif1α iC(T) and Hif1α icKO(T) mice subjected to the HFD/HFD protocol were assessed for Pgc1α protein expression. β-Actin served as a loading control. (B) Endogenous Pgc1α was immunoprecipitated from HFD/HFD-maintained Hif1α iC(T) and Hif1α icKO(T) visceral WAT lysates and probed by immunoblotting for acetylated lysine and Pgc1α. IgG antibodies were used as negative controls. (C,D) Gene expression profiling of Hif1α iC(T) and Hif1α icKO(T) mice subjected to the NFD/NFD (D, top panel) or HFD/HFD (E, top panel) protocol for Sirt1 and Sirt2 mRNA. All values were normalized internally to 18S RNA expression and to the Hif1α iC(T) control, respectively. (*) P < 0.01 compared with control, set at 1.0. Data are mean ± SEM of values from four mice per group. Visceral WAT of Hif1α iC(T) and Hif1α icKO(T) mice subjected to the NCD/NCD (D, bottom panel) or HFD/HFD (E, bottom panel) protocol were assessed for Hif1α, Sirt1, and Sirt2 protein expression (C,D) and tubulin acetylation (D). β-Actin served as a loading control. (E) 3T3-L1-derived adipocytes were infected with nsRNA, shHif1α, or Hif1α virus and assessed for Sirt1 and Sirt2 mRNA levels (top panel) and protein expression of Hif1α, Sirt1, Sirt2, and tubulin acetylation (bottom panel), with β-actin as a loading control. All qPCR values were normalized internally to 18S RNA expression and to nsRNA values, respectively. (*) P < 0.01 compared with control, set at 1.0. Data are mean ± SEM of values from each group. (F) Sequence analysis of the human, monkey, cow, dog, rat, and mouse Sirt2 promoters showing conserved putative HREs (in bold and red). The core consensus HRE motif is capitalized. (G) Hif1α iC(T) and Hif1α icKO(T) WAT derived from mice subjected to the HFD/HFD protocol were assessed for interaction of Hif1α and Hif1β at the Sirt2 promoter by ChIP. DNA from the respective samples was immunoprecipitated with a Hif1α (IP:Hif1α)-specific and a Hif1β (IP:Hif1β)-specific antibody or with a control isotype-matched antibody (IP:Ig control). Primer control and PCR control refer to reactions performed in the absence of primers or in the absence of DNA input, respectively. (H,I) Sirt2 promoter activity in response to Hif1α was determined by transient cotransfection of wild-type (WT) (H) and HRE-mutated (ΔHRE) (I) Sirt2 promoter, respectively, fused to luciferase and with either an empty vector control or a Hif1α ΔODD expression construct. All transfections contained equal amounts of a β-galactosidase expression vector for normalization of luciferase activity. (*) P < 0.01 compared with control, set at 1.0. Data are mean ± SEM of values from each group.