Figure 4. Inhibition of PKB-mediated insulin signalling and gene induction in hepatocytes treated with PIA6 ether lipid.

Hepatocytes were cultured under standard conditions and stimulated with 3×10⁻⁸ M insulin after 23 h in culture. Ether lipid PIA6 at the indicated concentrations or DMSO as control was added to cells 1 h before insulin. Hepatocytes for extraction of protein were harvested 1 h after the addition of insulin. Hepatocytes for isolation of RNA were harvested 7 h after the addition of insulin. (A) Immunoblots using antibodies to PKB phosphorylated at Ser-473, total PKB, GSK3-α and -β phosphorylated at Ser-21 and Ser-9 respectively, and total GSK3. Protein load per lane was as in Figure 2. In the upper part of the P-GSK3 immunoblot, the image of a longer film exposure is shown to visualize P-GSK3-α. (B) Northern blot of total RNA (16 μg per lane) hybridized with ³²P-labelled cDNA probes for GCK, SREBF1 and ribosomal protein S26. The experiment was repeated twice with identical results.