Voluntary Exercise Can Ameliorate Insulin Resistance by Reducing iNOS-Mediated S-Nitrosylation of Akt in the Liver in Obese Rats

Abstract

Voluntary exercise can ameliorate insulin resistance. The underlying mechanism, however, remains to be elucidated. We previously demonstrated that inducible nitric oxide synthase (iNOS) in the liver plays an important role in hepatic insulin resistance in the setting of obesity. In this study, we tried to verify our hypothesis that voluntary exercise improves insulin resistance by reducing the expression of iNOS and subsequent S-nitrosylation of key molecules of glucose metabolism in the liver. Twenty-one Otsuka Long-Evans Tokushima Fatty (OLETF) rats, a model of type 2 diabetes mellitus, and 18 non-diabetic control Long-Evans Tokushima Otsuka (LETO) rats were randomly assigned to a sedentary group or exercise group subjected to voluntary wheel running for 20 weeks. The voluntary exercise significantly reduced the fasting blood glucose and HOMA-IR in the OLETF rats. In addition, the exercise decreased the amount of iNOS mRNA in the liver in the OLETF rats. Moreover, exercise reduced the levels of S-nitrosylated Akt in the liver, which were increased in the OLETF rats, to those observed in the LETO rats. These findings support our hypothesis that voluntary exercise improves insulin resistance, at least partly, by suppressing the iNOS expression and subsequent S-nitrosylation of Akt, a key molecule of the signal transduction pathways in glucose metabolism in the liver.

Fig 1. Hyperglycemia and hyperinsulinemia in the OLETF rats were reversed by voluntary exercise.

The blood glucose (A) and plasma insulin (B) levels were significantly greater in the OLETF rats under sedentary conditions (SED) than in the LETO rats on SED. There were no significant differences between the LETO and OLETF rats with respect to the effects of voluntary exercise (VE) on the plasma insulin levels. The OLETF-SED rats exhibited insulin resistance, as indicated by elevated HOMA insulin resistance index values (C). All values are presented as the mean ± SEM. n = 9–11 per group, *,p<0.05; **,p<0.01 versus sedentary LETO, †,p<0.05 versus voluntary exercise OLETF, ‡,p<0.05 versus voluntary exercise LETO. N.S.: not significant.

Fig 2. Effects of voluntary exercise on the iNOS mRNA expression and S-nitrosylation of Akt and IRS-1 in the liver in the OLETF rats.

The mRNA expression of iNOS was significantly increased in the liver in the sedentary OLETF rats (A). The S-nitrosylated Akt levels were significantly increased in the liver in the sedentary (SED) OLETF rats (B). Voluntary exercise decreased the S-nitrosylated Akt levels in the liver in the voluntary exercise (VE) OLETF rats compared with those observed in the OLETF-SED rats. Similarly, S-nitrosylation of IRS-1 was also increased in the liver of SED OLETF rats (C). The degree of S-nitrosylation was evaluated using a biotin switch analysis. All values are presented as the mean ± SEM. n = 9–11 per group, **,p<0.01 versus sedentary LETO, †,p<0.05; ††,p<0.01 versus voluntary exercise OLETF. N.S.: not significant.

Fig 3. Voluntary exercise did not affect the GSH/GSSG ratio in the liver.

Liver GSH content(A). The liver GSSG content was significantly greater in the OLETF-SED rats than in the LETO-SED rats (B). The GSH/GSSG ratio in the liver did not change with voluntary regular exercise in either the LETO or OLETF rats compared with their respective sedentary counterparts (C). All values are presented as the mean ± SEM. n = 9–11 per group, **,p<0.01 versus sedentary LETO, ††,p<0.01 versus voluntary exercise OLETF. N.S.: not significant.

Fig 4. Exercise suppressed the lipogenic gene expression and prevented the accumulation of TG and activation of JNK in the liver in the OLETF rats.

The triglyceride content in the liver (A) was significantly lower in the voluntary exercise (VE) OLETF rats than in the sedentary (SED) OLETF rats. Consistent with the decreased triglyceride levels, the mRNA expression of Srebp-1 (B) and Scd-1 (C) was significantly decreased in the OLETF-VE rats. It is known that lipid accumulation in the liver increases the JNK activity. The total JNK amount was significantly increased in the OLETF rats compare with that observed in the LETO rats (D and E). The phosphorylation of JNK was significantly increased in the liver in the OLETF-SED rats compared with that observed in the LETO-SED rats (D and F). After 20 weeks of exercise, the activated JNK content in the liver decreased in the OLETF rats. The protein levels of JNK and p-JNK were normalized to that of actin. All values are presented as the mean ± SEM. n = 7–11 per group, *,p<0.05; **,p<0.01 versus sedentary LETO, †,p<0.05; ††,p<0.01 versus voluntary exercise OLETF. ‡,p<0.05; ‡‡,p<0.01 versus voluntary exercise LETO. N.S.: not significant.

Fig 5. Insulin-stimulated phosphorylation of Akt was significantly improved in liver of OLETF rats after voluntary exercise.

At 15 weeks of age, insulin (0.5 U/kg BW) or saline was injected via the portal vein following overnight fasting. At 5 min after the injection, liver was taken under anesthesia. Insulin-stimulated phosphorylation of Akt at threonine 308 (C) and serine 473 (D) was significantly increased in the liver of OLETF rats after exercise, as compared with sedentary condition. Basal (exogenous insulin-naïve) Akt phosphorylation in the liver was not different between the voluntary exercise and sedentary OLETF rat groups. The protein expression of Akt did not differ between voluntary exercise and sedentary condition in the liver of OLETF rats (B). *,p<0.05; **,p<0.01 versus sedentary OLETF with saline, †,p<0.05 versus sedentary OLETF with insulin. N.S.: not significant.