Regulation of mTORC 1/2 Formation in Response to a High- fat Diet and Exercise Training.

The "nutrient sensor" mammalian target of rapamycin (mTOR) performs important functions of cell growth and proliferation. mTOR is known to exist in two distinct complexes, the rapamycin-sensitive mTOR complex 1 (mTORC1), which is bound to Raptor; and the rapamycin-insensitive mTOR complex 2 (mTORC2), which is bound to Rictor. mTORC1 is a known activator of the ribosomal protein S6 kinase (S6K1), while mTORC2 has recently been shown to phosphorylate Akt on Serine (S)473. PURPOSE: To determine the effects of a high-fat diet and exercise training on mTORC 1/2 formation and the activation of its downstream substrates. METHODS: We examined the effects of an 8 wk high fat diet (HF) and 4 wktreadmill running (EX) intervention on the mTOR signalling pathway in skeletal muscle. Co-immunoprecipitation of mTOR and its binding partners, Raptor and Rictor, followed by Western blot analysis were used to determine mTORC 1/2 formation. The activation and total protein content of mTORC 1/2 and its downstream substrates were also determined. RESULTS: The association of mTOR and its binding partners Raptor (p=0.02) and Rictor (p=0.03) were increased in HF by ~70% and ~60%, respectively. The increase in mTORC 1/2 formation was attenuated by EX. Compared to HF, EX (p=0.01) increased S2448 phosphorylation of mTOR by ~30%. Both Threonine (T)389 and S421/T424 phosphorylation of S6K1 were increased by ~20% in HF (P=0.004 and p=0.04, respectively). The activation of S6K1 was associated with similar increases in the phosphorylation of its direct sites (S636/S639) on insulin receptor substrate 1 (IRS1) in HF (p=0.01). EX prevented the activation of S6K1 and the serine phosphorylation of IRS1. Insulin stimulated S473 phosphorylation of Akt1 was increased by ~25% in HF (P<0.001). EX prevented the increase of Akt1 S473 phosphorylation induced by HF (P=0.02). No changes were observed in the S473 phosphorylation of the Akt2 isoform. CONCLUSIONS: Our results suggest that high fat feeding increases mTORC 1/2 complex formation, resulting in the activation of Akt1 and S6K1, and inhibitory serine phosphorylation of IRS1. Exercise training reverses the effects of a high-fat diet on mTOR complex formation and downstream signalling events. Supported by an RMIT SMS Grant (SJL), ARC Grant DP0663862 (JAH) and NIH Grant GM-48680, GM-08395 and DK-57625 (BBY)