Figure 4.

Peripheral insulin signaling in 12-month-old mice on methionine restriction (MR) and control diet. Insulin signaling was assessed by administering either a saline (n = 3) or low dose of insulin (0.8 mU g⁻¹) (n = 5–6) to mice, via i.p. injection, after a 5-h fast. Levels of phosphorylated IR (tyr 1162/1163), protein kinase B/Akt (ser473), S6 (ser235/236), IR-β, total Akt, and total S6 were measured by immunoblotting in (A) epididymal white adipose tissue (WAT), (C) liver, and (E) gastrocnemius muscle in 12-month-old mice fed MR or control diet. Immunoblots were normalized to Ponceau S and total protein in (B) epididymal WAT, (D) liver, and (F) gastrocnemius muscle in 12-month-old mice fed MR or control diet. Data were analyzed as fold change relative to control-fed insulin-injected mice. Significance was calculated by two-tailed Student’s t-test (*P < 0.05). Data are represented as mean ± SEM. Black hatch bars, 12-month-old control-fed mice injected with saline; black bars, 12-month-old control-fed mice injected with insulin; white crossed bars, 12-month-old MR-fed mice injected with saline; white bars, 12-month-old MR-fed mice injected with insulin.