Targeted disruption of inducible nitric oxide synthase protects against aging, S-nitrosation, and insulin resistance in muscle of male mice

Abstract

Accumulating evidence has demonstrated that S-nitrosation of proteins plays a critical role in several human diseases. Here, we explored the role of inducible nitric oxide synthase (iNOS) in the S-nitrosation of proteins involved in the early steps of the insulin-signaling pathway and insulin resistance in the skeletal muscle of aged mice. Aging increased iNOS expression and S-nitrosation of major proteins involved in insulin signaling, thereby reducing insulin sensitivity in skeletal muscle. Conversely, aged iNOS-null mice were protected from S-nitrosation-induced insulin resistance. Moreover, pharmacological treatment with an iNOS inhibitor and acute exercise reduced iNOS-induced S-nitrosation and increased insulin sensitivity in the muscle of aged animals. These findings indicate that the insulin resistance observed in aged mice is mainly mediated through the S-nitrosation of the insulin-signaling pathway.

FIG. 1.

Insulin sensitivity in wild-type (WT) and iNOS-null male mice after aging. Total body weight (BW) (A), epididymal fat weight (B), and fasting insulin (8-h fast) (C). Hyperinsulinemic-euglycemic clamp glucose infusion rate (GIR) (D) and variation in the GIR (E). F: Glucose uptake in the isolated soleus muscle after insulin incubation. G: GIR values are expressed as micromoles per gram of muscle variation. The bars represent the mean ± SEM of 8–12 mice. One-way ANOVA was used in A–D and F. Student t test was used in E and G. *P < 0.05 vs. the respective young group. §P < 0.05, young iNOS-null vs. young wild-type. #P < 0.05, vs. wild-type.

FIG. 2.

Insulin signaling and S-nitrosation of IR, IRS-1, and Akt in the skeletal muscle of wild-type and iNOS-null male mice after aging. A: Western immunoblot (IB) analysis was performed to evaluate iNOS, eNOS, and nNOS expression. B: IKKβ (Ser 180) phosphorylation and TNF-α expression. IRβ (Tyr 1162/1163) (C), IRS-1 (Tyr 971) (D), and Akt (Ser 473) (E) phosphorylation in the gastrocnemius muscle. The biotin switch method was used to determine IRβ (F), IRS-1 (G), and Akt S-nitrosation (H) in the gastrocnemius muscle. I: Glucose uptake in isolated soleus muscle inoculated with GSNO (0, 1, 5 and 10 mmol/L) during 30 min in the presence or absence of insulin (10 mUI/mL). J: IRβ (Tyr 1162/1163), IRS-1 (Tyr 971), and Akt (Ser 473) phosphorylation in the isolated soleus muscle in the presence or absence of GSNO (10 mmol/L). IRβ (K), IRS-1 (L), and Akt S-nitrosation (M) in the isolated soleus muscle incubated with GSNO (10 mmol/L). The results of scanning densitometry are expressed as arbitrary units (AU). Bars represent the mean ± SEM of eight mice. One-way ANOVA was used in C–I. The Student t test was used in K–M. *P < 0.05 vs. the respective young group. §P < 0.05 young iNOS-null vs. young wild-type. &P < 0.05 vs. aged wild-type. ¶P < 0.05 vs. insulin without GSNO. ‖P < 0.05 vs. vehicle.

FIG. 3.

Pharmacological and physiological iNOS inhibition improves insulin sensitivity in aged mice. Total body weight (A), epididymal fat weight (B), and fasting insulin (8-h fast) (C) were determined 1 h after the last L-NIL injection or 8 h after the exercise protocol D: Hyperinsulinemic-euglycemic clamp. E: Glucose uptake in the isolated soleus muscle after insulin incubation is expressed as micromoles per gram of muscle. One-way ANOVA was used. Bars represent the mean ± SEM of 8–12 mice. *P < 0.05 vs. young wild-type (WT). #P < 0.05 vs. old mice without L-NIL or exercise.

FIG. 4.

Pharmacological and physiological iNOS inhibition improves insulin signaling and reduces S-nitrosation of IRβ, IRS-1, and Akt in the skeletal muscle of aged mice. iNOS expression (A), endothelial eNOS and neuronal nNOS expression (B), and IKKβ (Ser 180) phosphorylation and TNFα expression (C) were determined 1 h after the last L-NIL injection or 8 h after the exercise protocol by immunoblotting (IB). IRβ (Tyr 1162/1163) (D), IRS-1 (E), and Akt (Ser 473) (F) phosphorylation in the gastrocnemius muscle. The biotin switch method was used to determine IRβ (G), IRS-1 (H), and Akt (I) S-nitrosation in the muscle. The results of scanning densitometry are expressed as arbitrary units (AU). White bars indicate control group; grey bars indicate old mice plus L-NIL treatment; and black bars indicate old mice plus exercise. Bars represent the mean ± SEM of eight mice. One-way ANOVA was used. *P < 0.05 vs. young wild-type (WT). #P < 0.05 vs. aged iNOS-null.