Fig 2: OSM induces inhibitory phosphorylation of IRS1 at serine 612 (S612). 3T3-L1 adipocytes were exposed to 0.5 nM OSM for 2 h, 8 h, or overnight (~16 h). Cells were harvested after OSM exposure, and lysates were subjected to immunoblotting for phospho-IRS1 (S612) and total IRS1; STAT5A was used as a loading control. OSM stimulated inhibitory IRS1 phosphorylation of S612 at all time points depicted. The immunoblot pictured here represents results from two independent experiments, each with 3 wells per condition (n = 6 total wells per condition).

Fig 3: Loss of AMPK-α2 hampers the CR-induced activation of the IRS-1–Akt signal pathway. A: IRS-1 was enriched by immunoprecipitation and then probed with anti–phospho-Tyr antibody to detect the Tyr phosphorylation in skeletal muscle of mice. B–D: In skeletal muscle of mice upon insulin stimulation, the influences of CR on phosphorylation of Akt (B), TBC1D1 (C), and TBC1D4 (D) were impaired by deletion of AMPK-α2. **P < 0.01 vs. WT-AL, #P < 0.05 vs. WT-CR. n = 4 per group. α2KO, AMPK-α2 knockout mice; IP, immunoprecipitation; IB, immunoblot.

Fig 4: T-HGK cKO mice develop insulin resistance mediated by IL-6 and IL-17.(a,b) Fasting insulin (a) and glucose levels (b) in the sera of WT and T-HGK cKO mice. n, at least 18. (c) GTT tests were performed on 12-week-old WT and T-HGK cKO mice. Glucose levels were plotted versus time post glucose injection. n=6. (d) ITT tests were performed on 12-week-old mice. Blood glucose levels were measured at indicated time periods post insulin injection. n=6. (e,f) Immunoblotting analyses of p-IRS-1 (Y896 or S612), IRS-1, p-AKT, AKT or tubulin/actin in hepatocytes, skeletal muscle cells and adipocytes of 16-week-old (e) or 8-week-old (f) WT and T-HGK cKO mice. Mice were injected with 2 g kg−1 glucose for 1 h after 16-h fasting. WT, littermate controls (HGKf/f mice or CD4-Cre mice); HGK cKO, T-HGK cKO mice. *P-value<0.05; **P-value<0.01 (a–d, two-tailed Student’s t-test).

Fig 5: Loss of AMPK-α2 impairs the CR-induced modulation of Ser phosphorylation of IRS-1 and the mTORC1-S6K1 signal pathway. A and B: Effects of CR on four Ser phosphorylation sites (Ser1101, Ser636/639, Ser612, and Ser307) of IRS-1 in skeletal muscle. The decreases of Ser1101 and Ser636/639 phosphorylation of IRS-1 by CR were impaired in AMPK-α2−/− mice. C and D: Effects of CR on phosphorylation of mTOR-S6K1 signal pathway were blocked by deletion of AMPK-α2. *P < 0.05 vs. WT-AL, #P < 0.05 vs. WT-CR. n = 6 per group. α2KO, AMPK-α2 knockout mice.