Nutritional excess and/or obesity represent well-known predisposition factors for the development of noninsulin-dependent diabetes mellitus (NIDDM). However, molecular links between obesity and NIDDM are only beginning to emerge. Here, we demonstrate that nutrients suppress phosphatidylinositol 3 (PI3)-kinase/Akt signaling via Raptor-dependent mTOR (mammalian target of rapamycin)-mediated phosphorylation of insulin receptor substrate 1 (IRS-1). Raptor directly binds to and serves as a scaffold for mTOR-mediated phosphorylation of IRS-1 on Ser636/639. These serines lie close to the Y 632 MPM motif that is implicated in the binding of p85␣/p110␣ PI3-kinase to IRS-1 upon insulin stimulation. Phosphomimicking mutations of these serines block insulin-stimulated activation of IRS-1-associated PI3-kinase. Knockdown of Raptor as well as activators of the LKB1/AMPK pathway, such as the widely used antidiabetic compound metformin, suppress IRS-1 Ser636/639 phosphorylation and reverse mTOR-mediated inhibition on PI3-kinase/Akt signaling. Thus, diabetes-related hyperglycemia hyperactivates the mTOR pathway and may lead to insulin resistance due to suppression of IRS-1-dependent PI3-kinase/Akt signaling.mTOR (mammalian target of rapamycin) is a Ser/Thr kinase that belongs to the phosphatidylinositol (PI) kinase-related protein kinase family (17). In the budding yeast Saccharomyces cerevisiae, TOR functions as a nutrient-dependent mediator of cell autonomous growth (17). In metazoans, TOR participates in both nutrient-and hormone-dependent signaling pathways (17). mTOR partitions between two scaffold proteins, Raptor (14, 27) and Rictor (25, 33, 52). The rapamycin-sensitive Raptor/mTOR complex (TORC1) regulates growth via S6K1 and 4EBP1/PHAS (14, 27). The rapamycin-insensitive Rictor/ mTOR (TORC2) complex regulates cellular proliferation via Akt (51) and cytoskeleton organization via protein kinase C␣ (52) and small GTPases Rho and Rac (25).AMP-activated protein kinase (AMPK) acts as an intracellular energy sensor that controls glucose and lipid metabolism in peripheral tissues (6). Its activation leads to a homeostatic response by down-regulation of anabolic pathways and upregulation of catabolic pathways (6). AMPK-and PI3-kinase/ Akt-dependent signaling pathways converge at the level of tuberus sclerosis complex 2 (TSC2 or tuberin) which functions as a GTPase-activating protein for the small GTPase Rheb (20)(21)(22). Genetic studies in Drosophila melanogaster place Rheb downstream of insulin/insulin-like growth factor I and nutrient-dependent signaling pathways (53). Rheb is negatively regulated by AMPK/TSC (20, 22) and functions as an upstream activator of mTOR (20,34,53). Normal cell growth and homeostasis rely on a proper balance between nutrient-and hormone-dependent regulation of the AMPK/mTOR axis, while its deregulation could lead to metabolic and growth-related diseases (11,20,22,67).Several in vitro and in vivo studies show a beneficial metabolic effect of AMPK activation on glucose homeostasis and peripheral insul...

Impaired translocation of the glucose transporter isoform 4 (Glut4) to the plasma membrane in fat and skeletal muscle cells may represent a primary defect in the development of type 2 diabetes mellitus. Glut4 is localized in specialized storage vesicles (GSVs), the biological nature and biogenesis of which are not known. Here, we report that GSVs are formed in differentiating 3T3-L1 adipocytes upon induction of sortilin on day 2 of differentiation. Forced expression of Glut4 prior to induction of sortilin leads to rapid degradation of the transporter, whereas overexpression of sortilin increases formation of GSVs and stimulates insulin-regulated glucose uptake. Knockdown of sortilin decreases both formation of GSVs and insulin-regulated glucose uptake. Finally, we have reconstituted functional GSVs in undifferentiated cells by double transfection of Glut4 and sortilin. Thus, sortilin is not only essential, but also sufficient for biogenesis of GSVs and acquisition of insulin responsiveness in adipose cells.

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...

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