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Ge Y, Wu A, Warnes C, Liu J, Zhang C, Kawasome H, Terada N, Boppart MD, Schoenherr CJ, Chen J. mTOR regulates skeletal muscle regeneration in vivo through kinase-dependent and kinase-independent mechanisms. Am J Physiol Cell Physiol 297: C1434-C1444, 2009. First published September 30, 2009; doi: 10.1152/ajpcell.00248.2009.--Rapamycin- sensitive signaling is required for skeletal muscle differentiation and remodeling. In cultured myoblasts, the mammalian target of rapamycin (roTOR) has been reported to regulate differentiation at different stages through distinct mechanisms, including one that is independent of roTOR kinase activity. However, the kinase-independent function of roTOR remains controversial, and no in vivo studies have examined those roTOR myogenic mechanisms previously identified in vitro. In this study, we find that rapamycin impairs injury-induced muscle regeneration. To validate the role of mTOR with genetic evidence and to probe the mechanism of roTOR function, we have generated and characterized transgenic mice expressing two mutants of roTOR under the control of human skeletal actin (HSA) promoter: rapamycin-resistant (RR) and RR/kinase-inactive (RR/KI). Our results show that muscle regeneration in rapamycin-administered mice is restored by RR-mTOR expression. In the RR/KI-mTOR mice, nascent myofiber formation during the early phase of regeneration proceeds in the presence of rapamycin, but growth of the regenerating myofibers is blocked by rapamycin. Igf2 mRNA levels increase drastically during early regeneration, which is sensitive to rapamycin in wild-type muscles but partially resistant to rapamycin in both RR-and RR/KI-mTOR muscles, consistent with roTOR regulation of Igf2 expression in a kinase-independent manner. Furthermore, systemic ablation of S6K1, a target of roTOR kinase, results in impaired muscle growth but normal nascent myofiber formation during regeneration. Therefore, roTOR regulates muscle regeneration through kinase-independent and kinase-dependent mechanisms at the stages of nascent myofiber formation and myofiber growth, respectively. rapamycin; rapamycin-resistant; s6kl; mice; myogenesis doi: 10.1152/ajpcell.00248.2009

Chronically high insulin levels and increased circulating free fatty acids released from adipose tissue through lipolysis are two features associated with insulin resistance. The relationship between chronic insulin exposure and adipocyte lipolysis has been unclear. In the present study we found that chronic insulin exposure in 3T3-L1 adipocytes, as well as in mouse primary adipocytes, increased basal lipolysis rates. This effect of insulin on lipolysis was only observed when the mammalian target of rapamycin (mTOR) pathway was inhibited by rapamycin in the adipocytes. In addition, amino acid deprivation in adipocytes phenocopied the effect of rapamycin in permitting the stimulation of lipolysis by chronic insulin exposure. The phosphatidylinositol 3-kinase-Akt pathway does not appear to be involved in this insulin effect. Furthermore, we found that triacylglycerol hydrolase (TGH) activity was required for the stimulation of lipolysis by combined exposure to insulin and rapamycin. Therefore, we propose that nutrient sufficiency, mediated by an mTOR pathway, suppresses TGH-dependent lipolysis stimulated by chronic insulin exposure in adipocytes. mammalian target of rapamycin; insulin resistance; 3T3-L1 cells; nutrient sufficiency; triacylglycerol hydrolase

Glycerol-3-phosphate acyltransferase (GPAT) activity is highly induced in obese individuals with insulin resistance, suggesting a correlation between GPAT function, triacylglycerol accumulation, and insulin resistance. We asked whether microsomal GPAT4, an isoform regulated by insulin, might contribute to the development of hepatic insulin resistance. Compared with control mice fed a high fat diet, Gpat4(-/-) mice were more glucose tolerant and were protected from insulin resistance. Overexpression of GPAT4 in mouse hepatocytes impaired insulin-suppressed gluconeogenesis and insulin-stimulated glycogen synthesis. Impaired glucose homeostasis was coupled to inhibited insulin-stimulated phosphorylation of Akt(Ser⁴⁷³) and Akt(Thr³⁰⁸). GPAT4 overexpression inhibited rictor's association with the mammalian target of rapamycin (mTOR), and mTOR complex 2 (mTORC2) activity. Compared with overexpressed GPAT3 in mouse hepatocytes, GPAT4 overexpression increased phosphatidic acid (PA), especially di16:0-PA. Conversely, in Gpat4(-/-) hepatocytes, both mTOR/rictor association and mTORC2 activity increased, and the content of PA in Gpat4(-/-) hepatocytes was lower than in controls, with the greatest decrease in 16:0-PA species. Compared with controls, liver and skeletal muscle from Gpat4(-/-)-deficient mice fed a high-fat diet were more insulin sensitive and had a lower hepatic content of di16:0-PA. Taken together, these data demonstrate that a GPAT4-derived lipid signal, likely di16:0-PA, impairs insulin signaling in mouse liver and contributes to hepatic insulin resistance., (Copyright © 2014 the American Physiological Society.)

Animal studies have shown that autophagy is essential in the process of obesity. Here, we performed daily injection of the autophagy inhibitor chloroquine (CQ) in mice and found that systemic administration of CQ blocks high-fat diet-induced obesity. To investigate the potential underlying molecular mechanism, we employed genetic and pharmacological interventions in cultured preadipocytes to investigate the role of autophagy in the control of the expression of the adipogenic regulator peroxisome proliferatior-activated receptor-γ (PPARγ). We show that adipogenic differentiation of 3T3-L1 preadipocytes is associated with activation of autophagy and increased PPARγ2 protein level. Treatment with CQ, shRNA-mediated knockdown, or genetic engineering-induced deletion of autophagy-related gene 5 (Atg5) promoted proteasome-dependent PPARγ2 degradation and attenuated adipogenic differentiation. Therefore, activated autophagy increases PPARγ2 stability and promotes adipogenic differentiation, and inhibition of autophagy may prevent high-fat diet-induced obesity and the consequential type 2 diabetes.