Fig. 7. mTORC1 signaling mediates the effect of Grb10 on lipid metabolism and thermogenesis in brown adipocytes.

(A) Oil red O staining of differentiated Grb10-shRNA-suppressed or scramble brown adipocytes. Post 48 hours induction of differentiation, cells were treated with or without 5 nM rapamycin for 72 hours. (B) The effect of rapamycin administration on body mass of Grb10^fKO mice (KO) and Loxp littermates. 3 and half month-old mice were injected with rapamycin (4mg/Kg) or vehicle solution 3 times per week for 6 weeks. Data are presented as mean ± S.E.M. *P<0.05 (Grb10^fKO mice treated with vehicle vs Grb10^fKO mice treated with rapamycin) and #P<0.05 (Grb10^fKO mice treated with vehicle compared with the Loxp control mice treated with vehicle). (C) The effect of rapamycin treatment on fat mass of the Grb10^fKO mice (KO) and Loxp littermates described in (B). (D) The effect of rapamycin treatment on the size and morphology of distinct fat pad from Grb10^fKO mice (KO) and Loxp littermates described in (B). (E) The effect of rapamycin treatment on fat cell size of Grb10^fKO mice (KO) and control Loxp littermates described in (B). (F) The effect of rapamycin treatment on lipolytic and thermogenic gene expression in BAT of Grb10^fKO mice and control littermates as described in (B). (G) Statistical analysis of the data presented in (F). Data are presented as mean ± S.E.M. *<0.05. (H) A proposed model on the mechanism by which Grb10 regulates insulin and mTORC1 signaling pathways in brown adipocytes. Insulin stimulates the tyrosine phosphorylation of the insulin receptor (IR), leading to the activation of downstream events such as promoting lipogenesis. The tyrosine phosphorylation of IR also creates a binding site for Grb10, providing a feedback mechanism to prevent insulin signaling from uncontrolled amplification. When mTOR is over-activated, it negatively regulates insulin signaling by S6K-mediated serine phosphorylation of IRS1/2. Activated mTOR also phosphorylates Grb10, which switches the binding affinity of Grb10 from the insulin receptor to raptor and inhibits mTORC1 signaling. Thus, mTOR-mediated phosphorylation of Grb10 provides a feedback mechanism to more efficiently and specifically control the mTORC1 signaling events.