Fig. 8. Insulin increases Akt phosphorylation and the downstream Akt-mediated phosphorylation of the key glycolytic enzyme, PFKFB2, which is abolished in acinar cells from PACIRKO mice.
Pancreatic acinar cells from IRlox/lox (a–c), PACIRKO (a–c) or Elast-Cre (d–f) were treated with or without 10 nM insulin and/or the PI3K inhibitor, LY294002 (10 μM) for 10 min followed by cell lysis. Proteins were separated by SDS-PAGE and western blotted using antibodies for the insulin receptor (IR, a, c and e), phospho-Akt (Ser473) (a, d), total Akt (b, e) and phospho-PFKFB2 (Ser483) (c and f), which recognizes the specific Akt consensus phosphorylation site. For each representative experiment shown (a–f) separate gels were run and each membrane cut and incubated with each corresponding antibody either in parallel or in series, including IR, Akt, pAkt(S473), pFKFB2 and the loading control actin. These were all sufficiently separated so that they could be resolved on the same gel. Each experiment shown (a–f) is representative of at least three independent experiments.