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mTORC1 Inhibition via Rapamycin Promotes Triacylglycerol Lipolysis and Release of Free Fatty Acids in 3T3-L1 Adipocytes

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Lipids

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.

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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

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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.

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Authors and Affiliations

  1. Division of Metabolism, Endocrinology, and Diabetes, Department of Medicine, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, MI, 48109-2200, USA

    Ghada A. Soliman & Diane C. Fingar

  2. Department of Cell and Developmental Biology, University of Michigan Medical School, 109 Zina Pitcher Place, Ann Arbor, MI, 48109-2200, USA

    Hugo A. Acosta-Jaquez & Diane C. Fingar

  3. Department of Family and Consumer Sciences, Western Michigan University, 1903 W. Michigan Avenue, Kalamazoo, MI, 49008-5322, USA

    Ghada A. Soliman

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  1. Ghada A. Soliman
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Correspondence to Ghada A. Soliman.

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Soliman, G.A., Acosta-Jaquez, H.A. & Fingar, D.C. mTORC1 Inhibition via Rapamycin Promotes Triacylglycerol Lipolysis and Release of Free Fatty Acids in 3T3-L1 Adipocytes. Lipids 45, 1089–1100 (2010). https://doi.org/10.1007/s11745-010-3488-y

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