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. 2012 Sep 13;61(10):2484–2494. doi: 10.2337/db11-1665

FIG. 2.

FIG. 2.

Metformin inhibits GH-induced STAT5 transactivity and PDK4 expression in primary hepatocytes. Rat primary hepatocytes were cultured under serum-free conditions for 24 h. A: After pretreatment with metformin (Met) for 12 h, the cells were treated with GH for 1 h at the indicated dose (500 ng/mL). B: Rat primary hepatocytes were infected with Ad-SHP at a multiplicity of infection (MOI) of 30 or 60 for 36 h. After infection for 36 h, cells were treated with GH (500 ng/mL) for 1 h. Specific proteins were assayed in whole-cell extracts by Western blot analysis with the indicated antibodies and then normalized to an internal control (β-actin level and/or total forms). C: Rat primary hepatocytes were infected with 60 MOI of Ad-siSHP and Ad-Scram for 36 h. After infection with Ad-siSHP and Ad-Scram, cells were treated with GH (500 ng/mL) for 1 h, with or without metformin for 12 h at the indicated dose. Whole-cell extracts were isolated and analyzed by immunoblotting with the indicated antibodies, and then normalized to an internal control (β-actin level and/or total forms). The results shown are representative of at least three independent experiments. D: HepG2 cell lines were cotransfected with SHP in the indicated reporter genes and treated with GH for 1 h or metformin for 12 h after transfection. Luciferase (Luc) activity was measured after 36 h and normalized to β-galactosidase activity. All data are representative of three independently performed experiments and are shown as fold activations relative to the control (± SEM). E and F: HepG2 cell lines were transfected with the oligonucleotide siSHP and siScram. After transfection for 36 h, cells were transfected with the indicated reporter gene (STAT5-Luc [E], mPDK4-Luc [F]) and then treated with GH (500 ng/mL) for 1 h in the presence of metformin for 12 h. Luciferase (Luc) activity was normalized to β-galactosidase activity. The results shown are representative of at least three independent experiments. All data are indicated as fold activations relative to the control (± SEM).

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