Fig. 5: The p-AMPK and CCO are downstream effectors of Lepr.

Purified hepatocytes were cultured in the medium either with or without metformin (10 μM), or AMPK inhibitor Dorsomorphin for 96 h, before they were harvested and subjected to Western blotting analysis. Meanwhile, in a subset of the experiment, hepatocytes were pre-treated with cytochrome C oxidase (CCO) inhibitor Daunorubicin for 24 h, before switching to medium supplemented with metformin (10 μM) and leptin (10 μM; Sigma) for 72 h. After that, hepatocytes were harvested and subjected to Western blotting analysis. A Western blotting images of hepatocytes, with and without metformin/or dorsomorphin. B Quantitative analysis of A. Except for p-AMPK (n = 10), n = 12. Band intensities in A were quantified in Image Lab (Bio-Rad), and the protein expression level was normalized to the β-actin level. Data are presented on a logarithmic scale as the fold change in expression relative to the hepatocytes derived from HFD-fed Lepr WT rats in the absence of metformin and dorsomorphin. *p < 0.05 vs. hepatocytes derived from HFD-fed Lepr WT rats in the absence of metformin or dorsomorphin; ^#p < 0.05 vs. hepatocytes derived from HFD-fed Lepr-KO rats in the absence of dorsomorphin. C Western blotting images of hepatocytes, with and without metformin, leptin or daunorubicin. D Quantitative analysis of C. Except p-AMPK (n = 10), n = 12. Band intensities in C were quantified in Image Lab (Bio-Rad), and the protein expression level was normalized to the β-actin level. Data are presented on a logarithmic scale as the fold change in expression relative to hepatocytes derived from HFD-fed Lepr WT rats in the absence of metformin, leptin or daunorubicin. *p < 0.05 vs. hepatocytes derived from HFD-fed Lepr WT rats in the absence of metformin, leptin or daunorubicin; ^#p < 0.05 vs. hepatocytes derived from HFD-fed Lepr-KO rats in the absence of metformin, leptin or daunorubicin; ^Δp < 0.05 vs. hepatocytes derived from HFD-fed Lepr-KO rats in the absence of leptin or daunorubicin; ^†p < 0.05 vs. hepatocytes derived from HFD-fed Lepr-KO rats in the absence of leptin; ^‡p < 0.05 vs. hepatocytes derived from HFD-fed Lepr-KO rats in the absence of daunorubicin.