Mtorc1 Inhibition Via Rapamycin Promotes Triacylglycerol Lipolysis and Release of Free Fatty Acids in 3T3-L1 Adipocytes

Lipids (2010) 45:1089–1100 DOI 10.1007/s11745-010-3488-y ORIGINAL ARTICLE mTORC1 Inhibition via Rapamycin Promotes Triacylglycerol Lipolysis and Release of Free Fatty Acids in 3T3-L1 Adipocytes Ghada A. Soliman • Hugo A. Acosta-Jaquez • Diane C. Fingar Received: 27 August 2010 / Accepted: 7 October 2010 / Published online: 2 November 2010 Ó AOCS 2010 Abstract Signaling by mTOR complex 1 (mTORC1) phosphorylation of hormone sensitive lipase (HSL) on Ser- promotes anabolic cellular processes in response to growth 563 (a PKA site), but had no effect on the phosphorylation factors, nutrients, and hormonal cues. Numerous clinical of HSL S565 (an AMPK site). Additionally, rapamycin did trials employing the mTORC1 inhibitor rapamycin (aka not affect the isoproterenol-mediated phosphorylation of sirolimus) to immuno-suppress patients following organ perilipin, a protein that coats the lipid droplet to initiate transplantation have documented the development of lipolysis upon phosphorylation by PKA. These data dem- hypertriglyceridemia and elevated serum free fatty acids onstrate that inhibition of mTORC1 signaling synergizes (FFA). We therefore investigated the cellular role of with the b-adrenergic-cAMP/PKA pathway to augment mTORC1 in control of triacylglycerol (TAG) metabolism phosphorylation of HSL to promote hormone-induced using cultured murine 3T3-L1 adipocytes. We found that lipolysis. Moreover, they reveal a novel metabolic function treatment of adipocytes with rapamycin reduced insulin- for mTORC1; mTORC1 signaling suppresses lipolysis, stimulated TAG storage *50%. To determine whether thus augmenting TAG storage. rapamycin reduces TAG storage by upregulating lipolytic rate, we treated adipocytes in the absence and presence of Keywords mTOR Á mTORC1 Á Rapamycin Á rapamycin and isoproterenol, a b2-adrenergic agonist that Lipid metabolism Á Lipolysis Á Adipocytes activates the cAMP/protein kinase A (PKA) pathway to promote lipolysis. We found that rapamycin augmented Abbreviations isoproterenol-induced lipolysis without altering cAMP ATGL Adipocyte triacylglycerol lipase levels. Rapamycin enhanced the isoproterenol-stimulated AMPK AMP activated protein kinase ATP Adenosine triphosphate cAMP Cyclic adenosine monophosphate eIF4E Eukaryotic initiation factor-4E G. A. Soliman Á D. C. Fingar Division of Metabolism, Endocrinology, 4EBP1/PHAS-I eIF-4E-binding protein or protein-1/ and Diabetes, Department of Medicine, heat and acid stable-activated by insulin University of Michigan Medical School, 109 Zina Pitcher Place, HEAT Huntington elongation factor 3, the A Ann Arbor, MI 48109-2200, USA subunit of protein phosphatase 2A, and H. A. Acosta-Jaquez Á D. C. Fingar TOR1 Department of Cell and Developmental Biology, HSL Hormone sensitive lipase University of Michigan Medical School, 109 Zina Pitcher Place, FBS Fetal bovine serum Ann Arbor, MI 48109-2200, USA FRAP FKBP-12 rapamycin associated protein G. A. Soliman (&) FRB FKBP12-rapamycin binding domain Department of Family and Consumer Sciences, FFA Free fatty acids Western Michigan University, 1903 W. Michigan Avenue, FKBP12 FK506 binding protein 12 Kalamazoo, MI 49008-5322, USA GAP GTPase activating protein e-mail: [email protected] 123 1090 Lipids (2010) 45:1089–1100 GbL G protein b subunit-like protein also mTORC1, functions as an environmental sensor to promote known as mLST8 anabolic cellular processes such as ribosomal gene tran- GLUT 4 Glucose transporter 4 scription, protein synthesis, lipid synthesis, cell growth/ IRS Insulin receptor substrate size, and cell proliferation in response to sufficient levels of LKB Tumor suppressor protein growth factors, nutrients, and hormones [2, 3]. Conversely, LPAAT Lysophosphatidic acid acyl transferase adverse cellular conditions rapidly downregulate mTORC1 MEFs Mouse embryonic fibroblasts signaling to promote catabolic functions such as autophagy MGL Monoacylglycerol lipase [4]. mTORC1 and mTORC2 contain shared and unique mTOR Mammalian target of rapamycin (TOR) partner proteins, possess unique cellular functions, and mTOR P-S2481 mTOR phosphorylated on serine 2481 exhibit differing sensitivities to rapamycin, a clinically mTORC1 Mammalian target of rapamycin employed immunosuppressive drug [3]. Rapamycin (clin- complex 1 ically known as sirolimus) associates with a cellular protein mTORC2 Mammalian target of rapamycin called FKBP12 [5]. Upon entering the cell, the rapamycin/ complex 2 FKBP12 complex directly binds to the mTOR FRB NCS Newborn calf serum (FKBP12-rapamycin binding) domain, which lies imme- NEFA Nonesterified fatty acids diately N-terminal to the kinase domain [5]. Rapamycin/ RAPA Rapamycin FKBP12 binds assembled mTORC1 but not assembled Raptor Regulatory associated protein of mTORC2 [5]. Thus, rapamycin acutely inhibits mTORC1 mammalian target of rapamycin but not mTORC2 signaling. While the mechanisms by Rheb Ras homolog enriched in brain which rapamycin allosterically inhibits mTORC1 signaling Rictor Rapamycin-insensitive companion of remain incompletely defined, the drug reduces the affinity mTOR between mTOR and raptor, a critical mTORC1 partner RII Regulatory subunit II of PKA protein [5, 6]. Additionally, rapamycin reduces mTORC1 TOS TOR signaling motif intrinsic kinase activity, as monitored by mTOR S2481 PA Phosphatidic acid autophosphorylation [7]. The use of rapamycin in clinical PDE Phosphodiesterase medicine underscores the importance of mTORC1 for PLD Phospholipase D organismal physiology. Not only does rapamycin reduce PI3K Phosphatidylinositol 3-OH kinase organ transplant rejection [8], it reduces the intimal Pol I Polymerase I de-differentiation and hyperplasia of vascular smooth muscle PKA Protein kinase A cells that often follows coronary artery stent restenosis [9]. PKB/AKT Protein kinase B Strikingly, rapamycin extends rodent lifespan [ 10]. Rapa- PPAR c Peroxisome proliferator-activated mycin analogs and second-generation mTOR catalytic receptor-c inhibitors also hold therapeutic promise as anti-cancer PTEN Phosphatase and tensin homologue agents [5]. deleted on chromosome 10 Rapamycin administration incurs adverse side effects, S6K1 p70 ribosomal protein S6 kinase 1 however. Various clinical trials have documented the S6K1 P-T389 S6K1 phosphorylated on threonine 389 development of hypertriglyceridemia or hypercholesterol- TAG Triacylglycerol emia in renal, hepatic, cardiac, and islet transplant patients TSC Tuberous sclerosis complex [11, 12]. Additionally, rapamycin has also been shown to VLDL Very low density lipoprotein elevate serum free fatty acid (FFA) levels and induce glucose intolerance [13] in transplant patients as well as in mice lacking the mTORC1 downstream target S6K1 [14]. Clinical trials have also documented the occurrence of hyperlipidemia upon the administration of the rapamycin Introduction analog CCI-779 (also known as temsirolimus; Wyeth) in the treatment of metastatic melanoma and glioblastoma The mammalian target of rapamycin (mTOR), an evolu- multiforme [15]. Consistent with rapamycin increasing tionarily conserved Ser/Thr protein kinase, coordinates a levels of circulating FFAs and inducing hyperlipidemia, signal transduction network that controls a plethora of mice lacking S6K1 globally or lacking raptor in adipose are fundamental cellular functions [1–3]. mTOR forms the lean with reduced adipose mass and exhibit resistance to catalytic core of at least two functionally distinct signaling diet-induced obesity [14]. Thus, mTORC1 inhibition complexes, mTOR complex 1 (mTORC1) and mTOR induced by pharmacological or genetic ablation disrupts complex 2 (mTORC2) [3]. The better-understood complex, lipid homeostasis in vivo. 123 Lipids (2010) 45:1089–1100 1091 In this study we set out to better understand how Experimental Procedures mTORC1 signaling impacts lipid metabolism in cultured adipocytes. Adipose tissue, once considered an inert energy Materials storage depot, is now recognized as an important metabolic and endocrine organ that coordinates energy intake and Reagents were obtained from the following sources: expenditure critical for energy homeostasis [16]. Adipose CHAPS (3-[(3-cholamidopropyl)- dimethylammonio]-1- tissue removes glucose from the circulation (via the action propanesulfonate) was from Pierce (Rockford, IL, USA); of the insulin-responsive glucose transporter GLUT4) and nitrocellulose membrane (0.45 microns) was from Schlei- stores surplus energy in the form of triacylglycerol (TAG) cher and Schuell Bioscience Inc. (Keene, NH, USA); within lipid droplets. The tissue releases energy as needed autoradiography film (HyBlot CL) was from Denville via the action of lipolysis, the process by which enzymes Scientific Inc. (Metuchen, NJ, USA); reagents for enhanced known as lipases break down TAG into FFA and glycerol, chemiluminescence (ECL) (Immobilon Western-Chemilu- which are then released into the circulation. Diverse minescent HRP Substrate) were from Upstate/Millipore extracellular stimuli regulate lipolysis. Catecholamines (Billerica, MA, USA); all standard chemicals were from (e.g., epinephrine) and glucocorticoids (e.g., cortisol) pro- either Fisher Chemicals (Pittsburgh, PA, USA), Calbio- mote lipolysis while insulin and FFA themselves suppress chem/EMD Chemicals (Gibbstown, NJ, USA), or Sigma- lipolysis [17]. Starvation or stress triggers the release of Aldrich (St. Louis, MI, USA). catecholamines, which promote lipolysis by activating G protein-coupled adrenergic receptors that activate adenyl- Antibodies ate cyclase (AC), which lead to increased production

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