OBJECTIVEIn metazoans, target of rapamycin complex 1 (TORC1) plays the key role in nutrient- and hormone-dependent control of metabolism. However, the role of TORC1 in regulation of triglyceride storage and metabolism remains largely unknown.RESEARCH DESIGN AND METHODSIn this study, we analyzed the effect of activation and inhibition of the mammalian TORC1 (mTORC1) signaling pathway on the expression of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), lipolysis, lipogenesis, and lipid storage in different mammalian cells.RESULTSActivation of mTORC1 signaling in 3T3-L1 adipocytes by ectopic expression of Rheb inhibits expression of ATGL and HSL at the level of transcription, suppresses lipolysis, increases de novo lipogenesis, and promotes intracellular accumulation of triglycerides. Inhibition of mTORC1 signaling by rapamycin or by knockdown of raptor stimulates lipolysis primarily via activation of ATGL expression. Analogous results have been obtained in C2C12 myoblasts and mouse embryonic fibroblasts with genetic ablation of tuberous sclerosis 2 (TSC2) gene. Overexpression of ATGL in these cells antagonized the lipogenic effect of TSC2 knockout.CONCLUSIONSOur findings demonstrate that mTORC1 promotes fat storage in mammalian cells by suppression of lipolysis and stimulation of de novo lipogenesis.

FoxO1 represents a central regulator of metabolism in several cell types. Although FoxO1 is abundant in adipocytes, its biological functions in these cells remain largely unknown. We show here that the promotor region of the rate-limiting lipolytic enzyme, adipose triglyceride lipase (ATGL), has two FoxO1-binding sites, and co-transfection with wild type and unphosphorylated FoxO1 mutant activates the expression of luciferase driven by the ATGL promotor. In 3T3-L1 adipocytes, insulin controls nucleo-cytoplasmic shuttling of FoxO1 and regulates its interaction with endogenous ATGL promotors. Knockdown of FoxO1 in 3T3-L1 adipocytes decreases the expression of ATGL and attenuates basal and isoproterenol-stimulated lipolysis. Infection of mouse embryonic fibroblasts with FoxO1-encoding lentivirus increases ATGL expression and renders it sensitive to regulation by insulin. Thus, FoxO1 may play an important role in the regulation of lipolysis in adipocytes by controlling the expression of ATGL.One of the key physiological functions of insulin in the mammalian organism is to inhibit lipolysis and to promote accumulation and storage of triglycerides in fat tissue. Control of lipolysis plays an important role in energy partitioning and balance and maintains the size of fat depots in the body. In patients with insulin resistance and type 2 diabetes, insulin cannot suppress high levels of free fatty acids (FFA) 2 in the bloodstream (1) so that the availability of FFA exceeds the energy requirements of the body. As a result, FFA are accumulated in the form of lipids in non-adipose peripheral tissues, such as liver and skeletal muscle, aggravating insulin resistance and causing multiple hazardous metabolic effects (2, 3). In addition, excess of FFA may lead to overproduction of VLDL in the liver and predispose the organism to cardiovascular disease.The complete hydrolysis of triglycerides to glycerol and FFA is performed jointly by tri-, di-, and monoacylglyceride lipases (4, 5). Recently discovered adipose triglyceride lipase ATGL (6 -8) is responsible for the bulk of triacylglycerol hydrolase activity in cells and has low affinity to di-and monoacylglycerides (4, 5). Importantly, ATGL is now considered the rate-limiting lipolytic enzyme in mammals (9), flies (10), and yeast (11). The major diacylglyceride lipase in adipocytes is hormone-sensitive lipase, or HSL (5). ATGL-and HSL-mediated hydrolase activity is controlled by catecholamines primarily via the cAMP-mediated-phosphorylation of perilipin (12, 13), the major lipid droplet-forming protein in adipocytes (14). Monoacylglyceride lipase in adipocytes is believed to be hormone-independent (4).The effect of insulin on lipolysis is attributed to the stimulation of activity and/or the expression of cyclic nucleotide phosphodiesterases 3B and 4 (15-17), which decrease intracellular levels of cAMP and reverse the stimulatory effect of cAMP-dependent protein kinase on lipolysis (5). In parallel, insulin may suppress lipolysis in a cAMP-independent fashion by stimulating pro...

Rap1 is a Ras-like guanine-nucleotide-binding protein (GNBP) that is involved in a variety of signal-transduction processes. It regulates integrin-mediated cell adhesion and might activate extracellular signal-regulated kinase. Like other Ras-like GNBPs, Rap1 is regulated by guanine-nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs). These GAPs increase the slow intrinsic GTPase reaction of Ras-like GNBPs by many orders of magnitude and allow tight regulation of signalling. The activation mechanism involves stabilization of the catalytic glutamine of the GNBP and, in most cases, the insertion of a catalytic arginine of GAP into the active site. Rap1 is a close homologue of Ras but does not possess the catalytic glutamine essential for GTP hydrolysis in all other Ras-like and Galpha proteins. Furthermore, RapGAPs are not related to other GAPs and apparently do not use a catalytic arginine residue. Here we present the crystal structure of the catalytic domain of the Rap1-specific Rap1GAP at 2.9 A. By mutational analysis, fluorescence titration and stopped-flow kinetic assay, we demonstrate that Rap1GAP provides a catalytic asparagine to stimulate GTP hydrolysis. Implications for the disease tuberous sclerosis are discussed.

One of the basic functions of insulin in the body is to inhibit lipolysis in adipocytes. Recently, we have found that insulin inhibits lipolysis and promotes triglyceride storage by decreasing transcription of adipose triglyceride lipase via the mTORC1-mediated pathway (P. Chakrabarti et al., Diabetes 59:775-781, 2010), although the mechanism of this effect remained unknown. Here, we used a genetic screen in Saccharomyces cerevisiae in order to identify a transcription factor that mediates the effect of Tor1 on the expression of the ATGL ortholog in yeast. This factor, Msn4p, has homologues in mammalian cells that form a family of early growth response transcription factors. One member of the family, Egr1, is induced by insulin and nutrients and directly inhibits activity of the ATGL promoter in vitro and expression of ATGL in cultured adipocytes. Feeding animals a high-fat diet increases the activity of mTORC1 and the expression of Egr1 while decreasing ATGL levels in epididymal fat. We suggest that the evolutionarily conserved mTORC1-Egr1-ATGL regulatory pathway represents an important component of the antilipolytic effect of insulin in the mammalian organism. C urrent epidemics of metabolic diseases, such as type 2 diabetes, cardiac dysfunction, hypertension, hepatic steatosis, etc., are largely caused by widespread obesity. Although obesity can affect human health via several different mechanisms, the bestestablished connection between obesity and metabolic disease is elevated and/or dysregulated levels of circulating free fatty acids (FFA). In addition to their direct pathological effects, superfluous FFA accumulate in the form of lipids, and their metabolic products in nonadipose peripheral tissues, such as liver, skeletal muscle, heart, and pancreas and cause detrimental effects on human health via mechanisms that are currently under intense investigation (1-5).The levels of circulating FFA depend primarily on the rates of lipolysis in the adipose tissue. One of the key physiological functions of insulin as the major anabolic hormone in the body is to restrain lipolysis and to promote fat storage in adipose tissue in the postprandial state. The failure of insulin to suppress lipolysis in adipocytes has been long considered as a very serious metabolic defect and one of the most important if not the most important causative factor of insulin resistance and diabetes mellitus (6, 7).Complete hydrolysis of triglycerides to glycerol and fatty acids is performed jointly by tri-, di-, and monoacylglyceride lipases (8-11). The recently discovered enzyme, adipose triglyceride lipase (ATGL; also known as desnutrin, PNPLA2, TTS2.2, and iPLA 2 ) (12-14), is responsible for the bulk of triacylglycerol hydrolase activity in various cells and represents the rate-limiting lipolytic enzyme. In every experimental model tested thus far, elevated ATGL expression increases, while attenuated ATGL expression decreases, both basal and cAMP-stimulated lipolysis (12)(13)(14)(15)(16)(17)(18)(19)(20)(21)(22). At the same time, ATGL h...