A chronic increase in physical activity inhibits fed-state mTOR/S6K1 signaling and reduces IRS-1 serine phosphorylation in rat skeletal muscle

Abstract

A chronic increase in physical activity and (or) endurance training can improve insulin sensitivity in insulin-resistant skeletal muscle. Cellular mechanisms responsible for the development of insulin resistance are unclear, though one proposed mechanism is that nutrient overload chronically increases available energy, over-activating the mammalian target of rapamycin (mTOR) and ribosomal S6 kinase 1 (S6K1) signaling pathway leading to increased phosphorylation of serine residues on insulin receptor substrate-1 (IRS-1). The objective of this study was to determine if increased physical activity would inhibit mTOR/S6K1 signaling and reduce IRS-1 serine phosphorylation in rat skeletal muscle. Soleus muscle was collected from fed male Sprague-Dawley sedentary rats (Inactive) and rats with free access to running wheels for 9 weeks (Active). Immunoblotting methods were used to measure phosphorylation status of mTOR, S6K1, IRS-1, and PKB/Akt (protein kinase B/AKT), and total abundance of proteins associated with the mTOR pathway. Muscle citrate synthase activity and plasma insulin and glucose concentrations were measured. Phosphorylation of mTOR (Ser2448), S6K1 (Thr389), and IRS-1 (Ser636-639) was reduced in Active rats (p<0.05). Total protein abundance of mTOR, S6K1, IRS-1, 4E-BP1, eEF2, PKB/Akt and AMPKalpha, and phosphorylation of PKB/Akt were unaffected (p>0.05). Total SKAR protein, a downstream target of S6K1, and citrate synthase activity increased in Active rats (p<0.05), though plasma insulin and glucose levels were unchanged (p>0.05). Reduced mTOR/S6K1 signaling during chronic increases in physical activity may play an important regulatory role in the serine phosphorylation of IRS-1, which should be examined as a potential mechanism for attenuation of insulin resistance associated with increased IRS-1 serine phosphorylation.

Figures

Fig. 1

Soleus muscle mTOR phosphorylation at Ser2448 (A), total mTOR protein content (B), S6K1 phosphorylation at Thr389 (C), and total S6K1 protein content (D). Data are expressed relative to an internal loading control and as mean ± SEM, n = 8 per group. Insert shows representative Western blot for duplicate samples for Inactive (left) and Active (right) rats. AU, arbitrary units. Asterisk (*) indicates significant difference from Inactive, p < 0.05.

Fig. 2

Soleus muscle total eEF2 protein content (A), total SKAR protein content (B), total 4E-BP1 protein content (C), total AMPKα protein content (D). Data are expressed relative to an internal standard and as mean ± SEM, n = 8 per group. Insert shows representative Western blot for duplicate samples for Inactive (left) and Active (right) rats. AU, arbitrary units. Asterisk (*) indicates significant difference from Inactive, p < 0.05.

Fig. 3

Soleus muscle IRS-1 phosphorylation at Ser636-639 (A), total IRS-1 protein content (B), PKB/Akt phosphorylation at Ser473 (C), and total PKB/Akt protein content (D). Data are expressed relative to an internal loading control and as mean ± SEM, n = 8 per group. Insert shows representative Western blot for duplicate samples for Inactive (left) and Active (right) rats. AU, arbitrary units. Asterisk (*) indicates significant difference from Inactive, p < 0.05.