Fig. 7.

Hepatic overexpression of Insig-1 rescues hepatic steatosis in hepatocyte-specific AMPKα2 knockout mice fed with HFHS diet. a–d AMPK deficiency accelerates the degradation rates of Insig-1. a AMPK is required for Insig-1 induction in response to metformin, A769662 and AICAR. AMPK+/+ or AMPKα1/α2 double knockout (AMPK−/−) mouse primary hepatocytes were infected with adenoviruses encoding Insig-1 (Ad-Insig-1) or GFP for 24 h, followed by treatment with 2 mM metformin or AICAR for 24 h, or 100 μM A769662 for 8 h. b Compound C decreases Insig-1 protein levels in SRD-13A cells. Cells were transfected with pcDNA or plasmid encoding myc-tagged Insig-1 for twenty-four hours, followed by treatment with 100 μM A769662 or 20 μM compound C. c, d Compound C increases the degradation rate of Insig-1 in HEK293 cells stably expressing Insig-1. Cells were treated with 20 μM compound C for 24 h, followed by treatment with 100 ng/ml cycloheximide to inhibit protein synthesis as indicated. Data are presented as the mean ± SEM, n = 4. e–g Adenovirus-mediated overexpression Insig-1 attenuates hepatic steatosis in AMPKα2 LKO mice. Eight-week-old male WT and AMPKα2 LKO mice were fed with HFHS diet for 14 weeks, followed by treatment with adenoviruses encoding Insig-1 or control GFP by tail-vein injection for 2 weeks. e Representative H&E and oil red O staining of liver sections are shown. Scale bar, 50 μm. Expression levels of AMPKα2 in the liver, muscle or heart were measured by immunoblots. f Liver triglyceride levels were assessed. g Transcription levels of SREBP-1c and lipogenic enzymes are decreased in the liver of mice treated with Insig-1. mRNA levels of Insig-1, SREBP-1c and lipogenic enzymes are measured by real-time PCR. Data are presented as the mean ± SEM, unpaired two-tailed Student’s t-test, n = 6–8, *p < 0.05, vs. WT and Ad-GFP; ^#p < 0.05, vs. AMPKα2 LKO and Ad-Insig-1. h The proposed model for the post-translational regulation of lipogenesis via AMPK-mediated phosphorylation of Insig. AMPK phosphorylates Insig and represses its ubiquitination and degradation via inhibiting the interaction between Insig and the ubiquitin ligase gp78, which prevents the proteolytic processing and activation of SREBP-1c. The beneficial effects of metformin on hepatic steatosis are partially mediated by Insig