Figure 5. SRA promotes insulin-simulated phosphorylation of Akt, ERK1/2 and FoxO1, and inhibits ATGL transcription in an insulin-independent manner.

(a) Left panel: Hepa1-6 cells were transfected with pSCT (Control) or pSCT-SRA (SRA) expression vectors and then cultured for 60 h; middle panel: Hepa1-6 cells were infected with lentivirus of scrambled shRNA (ShContronl) or shRNA against SRA (ShSRA) and then cultured for 72 h; right panel: Primary hepatocytes were isolated from SRAKO mice (KO) or WT littermates and cultured for 16 h. Cells were serum-starved for 5 h followed by treatment with or without insulin (10 nM) for 5 min. Afterwards, cell extracts were immunoblotted with the indicated antibodies. (b) Hepa1-6 cells were transfected with SRA or Control plasmids and then cultured for 60 h. After 5 h serum-starvation, cells were pretreated by MG132 (50 μM) with or without wortmannin (1 μM) or trametinib (1 μM) for 30 min, and finally treated with or without insulin (10 nM) for 5 min before harvesting. Then, proteins extracted from the nucleus, cytoplasm or cell lysates were immunoblotted with indicated antibodies. (c,d) HepG2 cells were cotransfected by −3000/+ 1 LUC ATGL (200 ng) plus pRL-TK-Renilla (10 ng) with either pSCT-SRA or control expression vectors (200 ng). Cells were treated without (c) or with (d) AS1842856 (1 μM) immediately after transfection. 36 h later, cells were serum starved and treated with wortmannin (1 μM) or trametinib (1 μM) plus insulin (10 nM) for further 24 h-culture. ATGL promoter driven luciferase activity were normalized to Renilla luciferase activity and presented as mean ± SE. Data are expressed as fold-change relative to the level of control. *p < 0.05.