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