Fig. 6.

BAP31-deletion in hepatocytes impairs insulin signal transduction in vivo and in vitro. Mice (8–10 weeks old) fasted overnight were anesthetized with ketamine (150 mg/kg) and xylazine (5 mg/kg) and then perfused with insulin for 5 min (5 U/kg body weight), the liver tissues (A) and gastrocnemius (D) were excised and frozen immediately. The protein levels of Akt, p-Akt (Ser473), p-Akt (Thr308), p-Gsk3β, and IRS1 were determined by immunoblot analysis. Primary hepatocytes were isolated from 2- to 3-month-old male WT and MT mice and cultured in serum-free William’s E medium containing 1% ITS supplement and 100 nM dexamethasone for 16 h. The next day, cells were switched to medium without insulin for 8 h and then treated with various doses of insulin (10 nM or 20 nM) for 30 min (B) or treated with 10 nM insulin for 5 min or 30 min (C). At the end of treatment, cells were harvested in RIPA buffer and the protein levels of Akt, p-Akt (Ser473), p-Akt (Thr308), p-Gsk3β, and IRS1 were determined by immunoblot analysis. GAPDH was used as a loading control.