Lipids

, Volume 45, Issue 12, pp 1089–1100 | Cite as

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
Original Article

Abstract

Signaling by mTOR complex 1 (mTORC1) promotes anabolic cellular processes in response to growth factors, nutrients, and hormonal cues. Numerous clinical trials employing the mTORC1 inhibitor rapamycin (aka sirolimus) to immuno-suppress patients following organ transplantation have documented the development of hypertriglyceridemia and elevated serum free fatty acids (FFA). We therefore investigated the cellular role of mTORC1 in control of triacylglycerol (TAG) metabolism using cultured murine 3T3-L1 adipocytes. We found that treatment of adipocytes with rapamycin reduced insulin-stimulated TAG storage ~50%. To determine whether rapamycin reduces TAG storage by upregulating lipolytic rate, we treated adipocytes in the absence and presence of rapamycin and isoproterenol, a β2-adrenergic agonist that activates the cAMP/protein kinase A (PKA) pathway to promote lipolysis. We found that rapamycin augmented isoproterenol-induced lipolysis without altering cAMP levels. Rapamycin enhanced the isoproterenol-stimulated phosphorylation of hormone sensitive lipase (HSL) on Ser-563 (a PKA site), but had no effect on the phosphorylation of HSL S565 (an AMPK site). Additionally, rapamycin did not affect the isoproterenol-mediated phosphorylation of perilipin, a protein that coats the lipid droplet to initiate lipolysis upon phosphorylation by PKA. These data demonstrate that inhibition of mTORC1 signaling synergizes with the β-adrenergic-cAMP/PKA pathway to augment phosphorylation of HSL to promote hormone-induced lipolysis. Moreover, they reveal a novel metabolic function for mTORC1; mTORC1 signaling suppresses lipolysis, thus augmenting TAG storage.

Keywords

mTOR mTORC1 Rapamycin Lipid metabolism Lipolysis Adipocytes 

Abbreviations

ATGL

Adipocyte triacylglycerol lipase

AMPK

AMP activated protein kinase

ATP

Adenosine triphosphate

cAMP

Cyclic adenosine monophosphate

eIF4E

Eukaryotic initiation factor-4E

4EBP1/PHAS-I

eIF-4E-binding protein or protein-1/heat and acid stable-activated by insulin

HEAT

Huntington elongation factor 3, the A subunit of protein phosphatase 2A, and TOR1

HSL

Hormone sensitive lipase

FBS

Fetal bovine serum

FRAP

FKBP-12 rapamycin associated protein

FRB

FKBP12-rapamycin binding domain

FFA

Free fatty acids

FKBP12

FK506 binding protein 12

GAP

GTPase activating protein

GβL

G protein β subunit-like protein also known as mLST8

GLUT 4

Glucose transporter 4

IRS

Insulin receptor substrate

LKB

Tumor suppressor protein

LPAAT

Lysophosphatidic acid acyl transferase

MEFs

Mouse embryonic fibroblasts

MGL

Monoacylglycerol lipase

mTOR

Mammalian target of rapamycin (TOR)

mTOR P-S2481

mTOR phosphorylated on serine 2481

mTORC1

Mammalian target of rapamycin complex 1

mTORC2

Mammalian target of rapamycin complex 2

NCS

Newborn calf serum

NEFA

Nonesterified fatty acids

RAPA

Rapamycin

Raptor

Regulatory associated protein of mammalian target of rapamycin

Rheb

Ras homolog enriched in brain

Rictor

Rapamycin-insensitive companion of mTOR

RII

Regulatory subunit II of PKA

TOS

TOR signaling motif

PA

Phosphatidic acid

PDE

Phosphodiesterase

PLD

Phospholipase D

PI3K

Phosphatidylinositol 3-OH kinase

Pol I

Polymerase I

PKA

Protein kinase A

PKB/AKT

Protein kinase B

PPAR γ

Peroxisome proliferator-activated receptor-γ

PTEN

Phosphatase and tensin homologue deleted on chromosome 10

S6K1

p70 ribosomal protein S6 kinase 1

S6K1 P-T389

S6K1 phosphorylated on threonine 389

TAG

Triacylglycerol

TSC

Tuberous sclerosis complex

VLDL

Very low density lipoprotein

Notes

Acknowledgments

The authors would like to express their gratitude to Drs. Nancy Weigel (Baylor College of Medicine) and Victoria Knutson (University of Texas) for sharing reagents, encouragement, support, and advice. Funding: This work was funded by grants from the National Institutes of Health (K01 DK60654) and the American Heart Association (0750060Z) to GS and NIH-R01 (DK-078135) to DCF.

Conflict of interest

Nothing to disclose; there are no commercial or other associations that may pose a conflict of interest.

References

  1. 1.
    Fingar DC, Blenis J (2004) Target of rapamycin (TOR): an integrator of nutrient and growth factor signals and coordinator of cell growth and cell cycle progression. Oncogene 23:3151–3171CrossRefPubMedGoogle Scholar
  2. 2.
    Soliman GA (2005) The mammalian target of rapamycin signaling network and gene regulation. Curr Opin Lipidol 16:317–323CrossRefPubMedGoogle Scholar
  3. 3.
    Foster KG, Fingar DC (2010) Mammalian target of rapamycin (mTOR): conducting the cellular signaling symphony. J Biol Chem 285:14071–14077CrossRefPubMedGoogle Scholar
  4. 4.
    Jung CH, Ro SH, Cao J, Otto NM, Kim DH (2010) mTOR regulation of autophagy. FEBS Lett 584:1287–1295CrossRefPubMedGoogle Scholar
  5. 5.
    Guertin DA, Sabatini DM (2009) The pharmacology of mTOR inhibition. Sci Signal 2:pe24CrossRefPubMedGoogle Scholar
  6. 6.
    Oshiro N, Yoshino K, Hidayat S, Tokunaga C, Hara K, Eguchi S, Avruch J, Yonezawa K (2004) Dissociation of raptor from mTOR is a mechanism of rapamycin-induced inhibition of mTOR function. Genes Cells 9:359–366CrossRefPubMedGoogle Scholar
  7. 7.
    Soliman GA, Acosta-Jaquez HA, Dunlop EA, Ekim B, Maj NE, Tee AR, Fingar DC (2010) mTOR Ser-2481 autophosphorylation monitors mTORC-specific catalytic activity and clarifies rapamycin mechanism of action. J Biol Chem 285:7866–7879CrossRefPubMedGoogle Scholar
  8. 8.
    Kahan BD (2004) Sirolimus: a ten-year perspective. Transplant Proc 36:71–75CrossRefPubMedGoogle Scholar
  9. 9.
    Morice MC, Serruys PW, Sousa JE, Fajadet J, Ban Hayashi E, Perin M, Colombo A, Schuler G, Barragan P, Guagliumi G, Molnar F, Falotico R (2002) A randomized comparison of a sirolimus-eluting stent with a standard stent for coronary revascularization. N Engl J Med 346:1773–1780CrossRefPubMedGoogle Scholar
  10. 10.
    Harrison DE, Strong R, Sharp ZD, Nelson JF, Astle CM, Flurkey K, Nadon NL, Wilkinson JE, Frenkel K, Carter CS, Pahor M, Javors MA, Fernandez E, Miller RA (2009) Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460:392–395PubMedGoogle Scholar
  11. 11.
    Morrisett JD, Abdel-Fattah G, Hoogeveen R, Mitchell E, Ballantyne CM, Pownall HJ, Opekun AR, Jaffe JS, Oppermann S, Kahan BD (2002) Effects of sirolimus on plasma lipids, lipoprotein levels, and fatty acid metabolism in renal transplant patients. J Lipid Res 43:1170–1180PubMedGoogle Scholar
  12. 12.
    Mathe D, Adam R, Malmendier C, Gigou M, Lontie JF, Dubois D, Martin C, Bismuth H, Jacotot B (1992) Prevalence of dyslipidemia in liver transplant recipients. Transplantation 54:167–170CrossRefPubMedGoogle Scholar
  13. 13.
    Teutonico A, Schena PF, Di Paolo S (2005) Glucose metabolism in renal transplant recipients: effect of calcineurin inhibitor withdrawal and conversion to sirolimus. J Am Soc Nephrol 16:3128–3135CrossRefPubMedGoogle Scholar
  14. 14.
    Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, Fumagalli S, Allegrini PR, Kozma SC, Auwerx J, Thomas G (2004) Absence of S6K1 protects against age- and diet-induced obesity while enhancing insulin sensitivity. Nature 431:200–205CrossRefPubMedGoogle Scholar
  15. 15.
    Margolin K, Longmate J, Baratta T, Synold T, Christensen S, Weber J, Gajewski T, Quirt I, Doroshow JH (2005) CCI-779 in metastatic melanoma: a phase II trial of the California Cancer Consortium. Cancer 104:1045–1048CrossRefPubMedGoogle Scholar
  16. 16.
    Ahima RS (2006) Adipose tissue as an endocrine organ. Obesity (Silver Spring) 14(Suppl 5):242S–249SCrossRefGoogle Scholar
  17. 17.
    Cawthorn WP, Sethi JK (2008) TNF-alpha and adipocyte biology. FEBS Lett 582:117–131CrossRefPubMedGoogle Scholar
  18. 18.
    Egan JJ, Greenberg AS, Chang MK, Wek SA, Moos MC Jr, Londos C (1992) Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet. Proc Natl Acad Sci USA 89:8537–8541CrossRefPubMedGoogle Scholar
  19. 19.
    Wang S, Soni KG, Semache M, Casavant S, Fortier M, Pan L, Mitchell GA (2008) Lipolysis and the integrated physiology of lipid energy metabolism. Mol Genet Metab 95:117–126CrossRefPubMedGoogle Scholar
  20. 20.
    Cohen AW, Razani B, Schubert W, Williams TM, Wang XB, Iyengar P, Brasaemle DL, Scherer PE, Lisanti MP (2004) Role of caveolin-1 in the modulation of lipolysis and lipid droplet formation. Diabetes 53:1261–1270CrossRefPubMedGoogle Scholar
  21. 21.
    Su CL, Sztalryd C, Contreras JA, Holm C, Kimmel AR, Londos C (2003) Mutational analysis of the hormone-sensitive lipase translocation reaction in adipocytes. J Biol Chem 278:43615–43619CrossRefPubMedGoogle Scholar
  22. 22.
    Saha PK, Kojima H, Martinez-Botas J, Sunehag AL, Chan L (2004) Metabolic adaptations in the absence of perilipin: increased beta-oxidation and decreased hepatic glucose production associated with peripheral insulin resistance but normal glucose tolerance in perilipin-null mice. J Biol Chem 279:35150–35158CrossRefPubMedGoogle Scholar
  23. 23.
    Brasaemle DL, Rubin B, Harten IA, Gruia-Gray J, Kimmel AR, Londos C (2000) Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis. J Biol Chem 275:38486–38493CrossRefPubMedGoogle Scholar
  24. 24.
    Martinez-Botas J, Anderson JB, Tessier D, Lapillonne A, Chang BH, Quast MJ, Gorenstein D, Chen KH, Chan L (2000) Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice. Nat Genet 26:474–479CrossRefPubMedGoogle Scholar
  25. 25.
    Aggarwal D, Fernandez ML, Soliman GA (2006) Rapamycin, an mTOR inhibitor, disrupts triglyceride metabolism in guinea pigs. Metabolism 55:794–802CrossRefPubMedGoogle Scholar
  26. 26.
    Burnett PE, Barrow RK, Cohen NA, Snyder SH, Sabatini DM (1998) RAFT1 phosphorylation of the translational regulators p70 S6 kinase and 4E-BP1. Proc Natl Acad Sci USA 95:1432–1437CrossRefPubMedGoogle Scholar
  27. 27.
    Holm C (2003) Molecular mechanisms regulating hormone-sensitive lipase and lipolysis. Biochem Soc Trans 31:1120–1124CrossRefPubMedGoogle Scholar
  28. 28.
    Kishida K, Kuriyama H, Funahashi T, Shimomura I, Kihara S, Ouchi N, Nishida M, Nishizawa H, Matsuda M, Takahashi M, Hotta K, Nakamura T, Yamashita S, Tochino Y, Matsuzawa Y (2000) Aquaporin adipose, a putative glycerol channel in adipocytes. J Biol Chem 275:20896–20902CrossRefPubMedGoogle Scholar
  29. 29.
    Chen JC, Powers T (2006) Coordinate regulation of multiple and distinct biosynthetic pathways by TOR and PKA kinases in S. cerevisiae. Curr Genet 49:281–293CrossRefPubMedGoogle Scholar
  30. 30.
    Manni S, Mauban JH, Ward CW, Bond M (2008) Phosphorylation of the cAMP-dependent protein kinase (PKA) regulatory subunit modulates PKA-AKAP interaction, substrate phosphorylation, and calcium signaling in cardiac cells. J Biol Chem 283:24145–24154CrossRefPubMedGoogle Scholar
  31. 31.
    Budillon A, Cereseto A, Kondrashin A, Nesterova M, Merlo G, Clair T, Cho-Chung YS (1995) Point mutation of the autophosphorylation site or in the nuclear location signal causes protein kinase A RII beta regulatory subunit to lose its ability to revert transformed fibroblasts. Proc Natl Acad Sci USA 92:10634–10638CrossRefPubMedGoogle Scholar
  32. 32.
    Elliott MR, Shanks RA, Khan IU, Brooks JW, Burkett PJ, Nelson BJ, Kyttaris V, Juang YT, Tsokos GC, Kammer GM (2004) Down-regulation of IL-2 production in T lymphocytes by phosphorylated protein kinase A-RIIbeta. J Immunol 172:7804–7812PubMedGoogle Scholar
  33. 33.
    Sztalryd C, Xu G, Dorward H, Tansey JT, Contreras JA, Kimmel AR, Londos C (2003) Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation. J Cell Biol 161:1093–1103CrossRefPubMedGoogle Scholar
  34. 34.
    Garton AJ, Campbell DG, Cohen P, Yeaman SJ (1988) Primary structure of the site on bovine hormone-sensitive lipase phosphorylated by cyclic AMP-dependent protein kinase. FEBS Lett 229:68–72CrossRefPubMedGoogle Scholar
  35. 35.
    Anthonsen MW, Ronnstrand L, Wernstedt C, Degerman E, Holm C (1998) Identification of novel phosphorylation sites in hormone-sensitive lipase that are phosphorylated in response to isoproterenol and govern activation properties in vitro. J Biol Chem 273:215–221CrossRefPubMedGoogle Scholar
  36. 36.
    Shen WJ, Patel S, Natu V, Kraemer FB (1998) Mutational analysis of structural features of rat hormone-sensitive lipase. Biochemistry 37:8973–8979CrossRefPubMedGoogle Scholar
  37. 37.
    Donsmark M, Langfort J, Holm C, Ploug T, Galbo H (2004) Contractions induce phosphorylation of the AMPK site Ser565 in hormone-sensitive lipase in muscle. Biochem Biophys Res Commun 316:867–871CrossRefPubMedGoogle Scholar
  38. 38.
    Donsmark M, Langfort J, Holm C, Ploug T, Galbo H (2004) Regulation and role of hormone-sensitive lipase in rat skeletal muscle. Proc Nutr Soc 63:309–314CrossRefPubMedGoogle Scholar
  39. 39.
    Zechner R, Kienesberger PC, Haemmerle G, Zimmermann R, Lass A (2009) Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores. J Lipid Res 50:3–21CrossRefPubMedGoogle Scholar
  40. 40.
    Wei E, Gao W, Lehner R (2007) Attenuation of adipocyte triacylglycerol hydrolase activity decreases basal fatty acid efflux. J Biol Chem 282:8027–8035CrossRefPubMedGoogle Scholar
  41. 41.
    Zurita-Martinez SA, Cardenas ME (2005) Tor and cyclic AMP-protein kinase A: two parallel pathways regulating expression of genes required for cell growth. Eukaryot Cell 4:63–71CrossRefPubMedGoogle Scholar
  42. 42.
    Soulard A, Cremonesi A, Moes S, Schutz F, Jeno P, Hall MN (2010) The rapamycin-sensitive phosphoproteome reveals that TOR controls protein kinase A toward some but not all substrates. Mol Biol Cell 21:3475–3486CrossRefPubMedGoogle Scholar
  43. 43.
    Chakrabarti P, English T, Shi J, Smas CM, Kandror KV (2010) Mammalian target of rapamycin complex 1 suppresses lipolysis, stimulates lipogenesis, and promotes fat storage. Diabetes 59:775–781CrossRefPubMedGoogle Scholar
  44. 44.
    Laplante M, Sabatini DM (2009) mTOR signaling at a glance. J Cell Sci 122:3589–3594CrossRefPubMedGoogle Scholar
  45. 45.
    Porstmann T, Santos CR, Griffiths B, Cully M, Wu M, Leevers S, Griffiths JR, Chung YL, Schulze A (2008) SREBP activity is regulated by mTORC1 and contributes to Akt-dependent cell growth. Cell Metab 8:224–236CrossRefPubMedGoogle Scholar
  46. 46.
    Kim JE, Chen J (2004) regulation of peroxisome proliferator-activated receptor-gamma activity by mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 53:2748–2756CrossRefPubMedGoogle Scholar
  47. 47.
    Laplante M, Sabatini DM (2010) mTORC1 activates SREBP-1c and uncouples lipogenesis from gluconeogenesis. Proc Natl Acad Sci USA 107:3281–3282CrossRefPubMedGoogle Scholar
  48. 48.
    Leibowitz G, Cerasi E, Ketzinel-Gilad M (2008) The role of mTOR in the adaptation and failure of beta-cells in type 2 diabetes. Diabetes Obes Metab 10(Suppl 4):157–169CrossRefPubMedGoogle Scholar
  49. 49.
    Teleman AA, Chen YW, Cohen SM (2005) Drosophila Melted modulates FOXO and TOR activity. Dev Cell 9:271–281CrossRefPubMedGoogle Scholar
  50. 50.
    Pende M, Kozma SC, Jaquet M, Oorschot V, Burcelin R, Le Marchand-Brustel Y, Klumperman J, Thorens B, Thomas G (2000) Hypoinsulinaemia, glucose intolerance and diminished beta-cell size in S6K1-deficient mice. Nature 408:994–997CrossRefPubMedGoogle Scholar
  51. 51.
    Le Bacquer O, Petroulakis E, Paglialunga S, Poulin F, Richard D, Cianflone K, Sonenberg N (2007) Elevated sensitivity to diet-induced obesity and insulin resistance in mice lacking 4E-BP1 and 4E-BP2. J Clin Invest 117:387–396CrossRefPubMedGoogle Scholar

Copyright information

© AOCS 2010

Authors and Affiliations

  • Ghada A. Soliman
    • 1
    • 3
  • Hugo A. Acosta-Jaquez
    • 2
  • Diane C. Fingar
    • 1
    • 2
  1. 1.Division of Metabolism, Endocrinology, and Diabetes, Department of MedicineUniversity of Michigan Medical SchoolAnn ArborUSA
  2. 2.Department of Cell and Developmental BiologyUniversity of Michigan Medical SchoolAnn ArborUSA
  3. 3.Department of Family and Consumer SciencesWestern Michigan UniversityKalamazooUSA

Personalised recommendations