Figure 3. (A–D) Treatment of human islets with MSDC-0160 activates AMPK and downregulates mTOR.

Islets were cultured for 24 h in cCMRL, 5 mM glucose±MSDC-0160 as indicated. Cell extracts were prepared and samples processed for Western blotting and quantitated by densitometry. β-actin was used as a protein loading control. (A) Representative Western blot for activated mTOR, AMPK and downstream target of AMPK, ACC. (B–D) Densitometry of Western blots shown in (A) for phosphorylated mTOR, pACC and pAMPK, respectively. Data are means±SEM of n = 2 experiments. Data analyzed using one-way ANOVA followed by Newman-Keuls test. (E–F) MSDC-0160 downregulates mTOR target, pS6 to relieve mTOR-mediated negative feedback. Human islets were cultured for 4 days (E) or 90 min (F) in cCMRL, 5 or 8 mM glucose with or without the concentrations of MSDC-0160, IGF-1, LY294002 as indicated. Cell extracts were prepared, samples processed for Western blotting and quantitated by densitometry. Following detection of the phosphorylated proteins, the Western blots were reprobed with total S6 antibodies, used as endogenous control. In each figure, the upper panel shows a representative Western blot and the lower panel shows mean values after quantitation of the data. Data analyzed using one-way ANOVA followed by Newman-Keuls test. Data are means±SEM of n = 3 (E) or n = 4 (F) experiments.