FoxO1 Controls Insulin-dependent Adipose Triglyceride Lipase (ATGL) Expression and Lipolysis in Adipocytes

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. 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) 2The abbreviations used are: FFA, free fatty acids; ATGL, adipose triglyceride lipase; VLDL, very low density lipoprotein; HSL, hormone-sensitive lipase; PPARγ, peroxisome proliferator-activated receptor-γ; CEBP, CCAAT enhancer-binding proteins; MEF, mouse embryonic fibroblast; DMEM, Dulbecco's modified Eagle's medium; GFP, green fluorescent protein; eGFP, enhanced GFP; PBS, phosphate-buffered saline; PI3, phosphatidylinositol 3-kinase; ChIP, chromatin immunoprecipitation; siRNA, small interfering RNA; Ins, insulin. in the bloodstream (1Fraze E. Donner C.C. Swislocki A.L. Chiou Y.A. Chen Y.D. Reaven G.M. J. Clin. Endocrinol. Metab. 1985; 61: 807-811Crossref PubMed Scopus (195) Google Scholar) 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 (2Stumvoll M. Goldstein B.J. van Haeften T.W. Lancet. 2008; 371: 2153-2156Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 3Wymann M.P. Schneiter R. Nat. Rev. Mol. Cell Biol. 2008; 9: 162-176Crossref PubMed Scopus (960) Google Scholar). 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 (4Zechner R. Kienesberger P.C. Haemmerle G. Zimmermann R. Lass A. J. Lipid Res. 2009; 50: 3-21Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 5Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Annu. Rev. Nutr. 2007; 27: 79-101Crossref PubMed Scopus (644) Google Scholar). Recently discovered adipose triglyceride lipase ATGL (6Zimmermann R. Strauss J.G. Haemmerle G. Schoiswohl G. Birner-Gruenberger R. Riederer M. Lass A. Neuberger G. Eisenhaber F. Hermetter A. Zechner R. Science. 2004; 306: 1383-1386Crossref PubMed Scopus (1518) Google Scholar, 7Villena J.A. Roy S. Sarkadi-Nagy E. Kim K.H. Sul H.S. J. Biol. Chem. 2004; 279: 47066-47075Abstract Full Text Full Text PDF PubMed Scopus (507) Google Scholar, 8Jenkins C.M. Mancuso D.J. Yan W. Sims H.F. Gibson B. Gross R.W. J. Biol. Chem. 2004; 279: 48968-48975Abstract Full Text Full Text PDF PubMed Scopus (681) Google Scholar) is responsible for the bulk of triacylglycerol hydrolase activity in cells and has low affinity to di- and monoacylglycerides (4Zechner R. Kienesberger P.C. Haemmerle G. Zimmermann R. Lass A. J. Lipid Res. 2009; 50: 3-21Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar, 5Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Annu. Rev. Nutr. 2007; 27: 79-101Crossref PubMed Scopus (644) Google Scholar). Importantly, ATGL is now considered the rate-limiting lipolytic enzyme in mammals (9Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. Kratky D. Wagner E.F. Klingenspor M. Hoefler G. Zechner R. Science. 2006; 312: 734-737Crossref PubMed Scopus (1017) Google Scholar), flies (10Gronke S. Mildner A. Fellert S. Tennagels N. Petry S. Muller G. Jackle H. Kuhnlein R.P. Cell Metab. 2005; 1: 323-330Abstract Full Text Full Text PDF PubMed Scopus (411) Google Scholar), and yeast (11Kurat C.F. Natter K. Petschnigg J. Wolinski H. Scheuringer K. Scholz H. Zimmermann R. Leber R. Zechner R. Kohlwein S.D. J. Biol. Chem. 2006; 281: 491-500Abstract Full Text Full Text PDF PubMed Scopus (244) Google Scholar). The major diacylglyceride lipase in adipocytes is hormone-sensitive lipase, or HSL (5Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Annu. Rev. Nutr. 2007; 27: 79-101Crossref PubMed Scopus (644) Google Scholar). ATGL- and HSL-mediated hydrolase activity is controlled by catecholamines primarily via the cAMP-mediated-phosphorylation of perilipin (12Miyoshi H. Perfield II, J.W. Souza S.C. Shen W.J. Zhang H.H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (226) Google Scholar, 13Miyoshi H. Souza S.C. Zhang H.H. Strissel K.J. Christoffolete M.A. Kovsan J. Rudich A. Kraemer F.B. Bianco A.C. Obin M.S. Greenberg A.S. J. Biol. Chem. 2006; 281: 15837-15844Abstract Full Text Full Text PDF PubMed Scopus (231) Google Scholar), the major lipid droplet-forming protein in adipocytes (14Brasaemle D.L. J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (763) Google Scholar). Monoacylglyceride lipase in adipocytes is believed to be hormone-independent (4Zechner R. Kienesberger P.C. Haemmerle G. Zimmermann R. Lass A. J. Lipid Res. 2009; 50: 3-21Abstract Full Text Full Text PDF PubMed Scopus (407) Google Scholar). The effect of insulin on lipolysis is attributed to the stimulation of activity and/or the expression of cyclic nucleotide phosphodiesterases 3B and 4 (15Choi Y.H. Park S. Hockman S. Zmuda-Trzebiatowska E. Svennelid F. Haluzik M. Gavrilova O. Ahmad F. Pepin L. Napolitano M. Taira M. Sundler F. Stenson Holst L. Degerman E. Manganiello V.C. J. Clin. Investig. 2006; 116: 3240-3251Crossref PubMed Scopus (139) Google Scholar, 16Kitamura T. Kitamura Y. Kuroda S. Hino Y. Ando M. Kotani K. Konishi H. Matsuzaki H. Kikkawa U. Ogawa W. Kasuga M. Mol. Cell. Biol. 1999; 19: 6286-6296Crossref PubMed Scopus (310) Google Scholar, 17Oknianska A. Zmuda-Trzebiatowska E. Manganiello V. Degerman E. Biochem. Biophys. Res. Commun. 2007; 353: 1080-1085Crossref PubMed Scopus (13) Google Scholar), which decrease intracellular levels of cAMP and reverse the stimulatory effect of cAMP-dependent protein kinase on lipolysis (5Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Annu. Rev. Nutr. 2007; 27: 79-101Crossref PubMed Scopus (644) Google Scholar). In parallel, insulin may suppress lipolysis in a cAMP-independent fashion by stimulating protein phosphatase 1 and by promoting re-esterification of fatty acids (reviewed in Ref. 5Duncan R.E. Ahmadian M. Jaworski K. Sarkadi-Nagy E. Sul H.S. Annu. Rev. Nutr. 2007; 27: 79-101Crossref PubMed Scopus (644) Google Scholar). It has also been shown that insulin decreases the expression of ATGL at the level of transcription (18Kralisch S. Klein J. Lossner U. Bluher M. Paschke R. Stumvoll M. Fasshauer M. Mol. Cell. Endocrinol. 2005; 240: 43-49Crossref PubMed Scopus (113) Google Scholar, 19Kershaw E.E. Hamm J.K. Verhagen L.A. Peroni O. Katic M. Flier J.S. Diabetes. 2006; 55: 148-157Crossref PubMed Scopus (293) Google Scholar, 20Kim J.Y. Tillison K. Lee J.H. Rearick D.A. Smas C.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 115-127Crossref PubMed Scopus (154) Google Scholar). The molecular mechanism of this effect has not yet been elucidated. Although it has been recently shown that transcription of ATGL is regulated by PPARγ (20Kim J.Y. Tillison K. Lee J.H. Rearick D.A. Smas C.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 115-127Crossref PubMed Scopus (154) Google Scholar, 21Kershaw E.E. Schupp M. Guan H.P. Gardner N.P. Lazar M.A. Flier J.S. Am. J. Physiol. Endocrinol. Metab. 2007; 293: 1736-1745Crossref PubMed Scopus (172) Google Scholar), it is yet not clear whether and to what extent PPARγ is responsible for the regulation of ATGL by insulin. Here, we decided to explore the role of the insulin-responsive transcription factor, FoxO1, in the regulation of ATGL expression and lipolysis. FoxO1 represents a central regulator of metabolism in several cell types and tissues (22Accili D. Arden K.C. Cell. 2004; 117: 421-426Abstract Full Text Full Text PDF PubMed Scopus (1105) Google Scholar, 23Gross D.N. van den Heuvel A.P. Birnbaum M.J. Oncogene. 2008; 27: 2320-2336Crossref PubMed Scopus (429) Google Scholar). Its activity is negatively regulated by insulin and growth factors via Akt-mediated phosphorylation and nuclear exclusion (24Calnan D.R. Brunet A. Oncogene. 2008; 27: 2276-2288Crossref PubMed Scopus (920) Google Scholar). Although FoxO1 is abundant in adipocytes, its biological functions in these cells remain largely unknown. We are showing here that insulin controls the expression of the rate-limiting lipolytic enzyme, ATGL, via FoxO1. Antibodies—Polyclonal antibodies against ATGL and aP2 were kind gifts of Dr. A. Greenberg (Tufts University) and Dr. S. Farmer (Boston University School of Medicine). A rabbit polyclonal antibody against cellugyrin was described previously (25Xu Z. Kandror K.V. J. Biol. Chem. 2002; 277: 47972-47975Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Polyclonal anti-FoxO1, anti-4E-BP, anti-PPARγ, and CEBPα antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal anti-V5 tag antibody and monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase antibody were obtained from Sigma. Monoclonal antibody against RNA polymerase II was from Millipore Corp. (Bedford, MA). Cell Culture—3T3-L1 preadipocytes were cultured, differentiated, and maintained as described previously (25Xu Z. Kandror K.V. J. Biol. Chem. 2002; 277: 47972-47975Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar). Mouse embryonic fibroblasts (MEFs) and 293T cells were cultured in DMEM supplemented with 10% fetal bovine serum in 2 mm l-glutamine, 100 units/ml penicillin, and 100 μg/ml streptomycin. MEFs were infected with pLenti-m1-V5-FoxO1 according to the Invitrogen protocol. Selection of cells was performed with blasticidin (10 μg/ml) for 7–10 days, and pooled clones were used for further experiments. Cell Fractionation—Nuclear and cytoplasmic fractions were isolated from 3T3-L1 adipocytes using NE-PER nuclear and cytoplasmic extraction reagents (Pierce). Quantitative PCR—Total RNA was extracted from differentiated 3T3-L1 cells using the TRIzol reagent (Invitrogen). Reverse transcription of 500 ng of total RNA was performed using random decamers (RETROscript kit; Ambion, Austin, TX), and ATGL mRNA was determined by quantitative PCR with the help of the MX4000 multiplex qPCR system (Stratagene, La Jolla, CA). Reactions were performed in triplicate in the total volume of 25 μl containing 2.5 μl of 1:10 diluted cDNA, 1× SYBR green master mix (Brilliant II SYBR Green qPCR master mix; Stratagene), and gene-specific primers (for ATGL, 5′-AACACCAGCATCCAGTTCAA-3′ and 5′-GGTTCAGTAGGCCATTCCTC-3′; for 36B4, 5′-TCATCCAGCAGGTGTTTGACA-3′ and 5′-GGCACCGAGGCAACAGTT-3′). ATGL expression was normalized by 36B4 expression by the ΔΔCt method. Deoxyribonuclease-treated samples and no-template controls were analyzed in parallel experiments to confirm specificity. Chromatin Immunoprecipitation (ChIP)—ChIP studies were carried out in 3T3-L1 adipocytes using the EZ-chIP kit (Millipore) according to the manufacturer's instructions. Briefly, proteins were cross-linked to DNA with 18.5% formaldehyde, lysed, and sonicated seven times for 15 s. FoxO1 proteins were then immunoprecipitated from precleared lysates. Protein-DNA complexes were eluted and treated with proteinase K. Purified DNA was subjected to PCR using the following primers: 5′-ATCTTTAAAAGGCAATTAAGCTG-3′ and 5′-TAAGTCCAGGTCTTAGAAATGT-3′ to amplify the region between –1004 and –1225 nucleotides of the ATGL promotor. Purified DNA was also analyzed by quantitative PCR using SYBR green reaction (Brilliant II SYBR Green qPCR master mix; Stratagene) with the following primers: 5′-AGGTTTCTAGTTTAGGATTGAAG-3′ and 5′-ACCTGAGCAGTAGTTATATACAT-3′ (–1041 to –1158 nucleotides of the ATGL promotor). For all PCR reactions, 10% input was analyzed along with immunoprecipitated samples. Transient Transfections and Reporter Gene Assays—Transient transfections were performed using Lipofectamine 2000 (Invitrogen) according to the manufacturer's instructions. Briefly, ∼80% confluent 293T cells and ∼50% confluent MEFs were transfected in triplicates with 500 ng of luciferase and 100 ng of eGFP cDNA in a 6-well plate format. After cells were in activity was determined in cell using the luciferase kit and as The expression of eGFP was luciferase was normalized by eGFP to for Knockdown of FoxO1 with against the and for mouse FoxO1 M.S. C. Kim Kim Kim H.S. Park J.Y. Kim Lee Nat. 2006; 9: PubMed Scopus Google Scholar), and were obtained from DNA of was performed with the help of the kit for 3T3-L1 3T3-L1 adipocytes were by with for at II and used for were with in for cells were with PBS, with for with with serum and bovine serum and with antibody and (Invitrogen). with antibody for at and was by with with was used for cells on that were then by using were with the help of the 3T3-L1 adipocytes were in DMEM with fatty bovine serum for 2 at in the or in the of in the was at using the triglyceride reagent against a of glycerol were then with and in and the protein was determined and used to glycerol the experiments were carried out in and were in and to (Millipore) in 25 mm mm After the was with 10% in with for 2 The were with antibodies at 4 and 1 with antibodies at and with an enhanced kit using a was used to the of the of 3T3-L1 cells with insulin for decreases the of basal and isoproterenol-stimulated lipolysis in these cells the of serum from the for the of increases lipolysis The effect of on lipolysis in adipocytes that been for is In with these we that insulin decreases the expression of the rate-limiting lipolytic enzyme, ATGL, at the level of mRNA and protein serum has the effect and also S. Klein J. Lossner U. Bluher M. Paschke R. Stumvoll M. Fasshauer M. Mol. Cell. Endocrinol. 2005; 240: 43-49Crossref PubMed Scopus (113) Google Scholar, 19Kershaw E.E. Hamm J.K. Verhagen L.A. Peroni O. Katic M. Flier J.S. Diabetes. 2006; 55: 148-157Crossref PubMed Scopus (293) Google Scholar, 20Kim J.Y. Tillison K. Lee J.H. Rearick D.A. Smas C.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 115-127Crossref PubMed Scopus (154) Google Scholar). As of 3T3-L1 adipocytes with the kinase insulin on ATGL expression and lipolysis A and we that ATGL be a of FoxO1, which represents the abundant in adipocytes J. Kitamura T. Kitamura Y. Arden K.C. D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) and is regulated by insulin via the M.P. U. S. A. PubMed Scopus Google Scholar). of the region of mouse ATGL using the of at two FoxO1-binding the expression of FoxO1 in 3T3-L1 adipocytes the on of also Ref. J. Kitamura T. Kitamura Y. Arden K.C. D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar) with the of ATGL whether or not FoxO1 the expression of ATGL we 293T cells with the luciferase containing the ATGL (20Kim J.Y. Tillison K. Lee J.H. Rearick D.A. Smas C.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 115-127Crossref PubMed Scopus (154) Google Scholar) with FoxO1 As is shown in FoxO1 increases the expression of luciferase and unphosphorylated FoxO1 mutant activity effect has been further in 3T3-L1 adipocytes and mouse embryonic fibroblasts regulates activity of FoxO1 in several cell types by controlling its nucleo-cytoplasmic M.P. U. S. A. PubMed Scopus Google Scholar). It is yet whether or not this mechanism is in adipose and we that serum to accumulation of endogenous FoxO1 in the insulin cytoplasmic of FoxO1 in 3T3-L1 In parallel, we with the help of the that serum insulin decreases of endogenous FoxO1 with endogenous ATGL and Thus, the effect of insulin on the expression of ATGL (18Kralisch S. Klein J. Lossner U. Bluher M. Paschke R. Stumvoll M. Fasshauer M. Mol. Cell. Endocrinol. 2005; 240: 43-49Crossref PubMed Scopus (113) Google Scholar, 19Kershaw E.E. Hamm J.K. Verhagen L.A. Peroni O. Katic M. Flier J.S. Diabetes. 2006; 55: 148-157Crossref PubMed Scopus (293) Google Scholar, 20Kim J.Y. Tillison K. Lee J.H. Rearick D.A. Smas C.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 115-127Crossref PubMed Scopus (154) Google Scholar) is at in by of FoxO1 confirm the role of FoxO1 in the regulation of ATGL we FoxO1 in 3T3-L1 adipocytes with the help of two As is shown in A and a decrease of FoxO1 expression by a decrease in the expression of ATGL. that of FoxO1 also decreases the expression of is a of FoxO1 O. R. PubMed Scopus Google Scholar, B. O. A. Y. S. O. Zhang W. M.A. J. Biol. Chem. 2007; 282: Full Text Full Text PDF PubMed Scopus Google Scholar), we this effect as a for with FoxO1 functions in preadipocytes has been shown to J. Kitamura T. Kitamura Y. Arden K.C. D. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). of FoxO1 in differentiated cells not the expression of PPARγ, and of Thus, PPARγ is to the expression of ATGL (20Kim J.Y. Tillison K. Lee J.H. Rearick D.A. Smas C.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 115-127Crossref PubMed Scopus (154) Google Scholar, 21Kershaw E.E. Schupp M. Guan H.P. Gardner N.P. Lazar M.A. Flier J.S. Am. J. Physiol. Endocrinol. Metab. 2007; 293: 1736-1745Crossref PubMed Scopus (172) Google Scholar), the effect of FoxO1 in a As decrease in the expression of ATGL attenuates the of lipolysis in adipocytes that this effect not lipolytic such as HSL, and levels of which not of FoxO1 We that FoxO1 controls lipolysis in adipocytes by the expression of the rate-limiting enzyme, ATGL. the is for the insulin regulation of ATGL we used mouse embryonic fibroblasts the expression of FoxO1 is low in the in adipocytes, insulin serum regulates ATGL expression in these cells of FoxO1 with the help of the ATGL expression in MEFs in we a clear effect of insulin on ATGL of serum the expression of ATGL Thus, FoxO1 is largely responsible for the effect of insulin on ATGL in (9Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. Kratky D. Wagner E.F. Klingenspor M. Hoefler G. Zechner R. Science. 2006; 312: 734-737Crossref PubMed Scopus (1017) Google Scholar) and in E.E. Hamm J.K. Verhagen L.A. Peroni O. Katic M. Flier J.S. Diabetes. 2006; 55: 148-157Crossref PubMed Scopus (293) Google Scholar, E. L. W.J. 2006; PubMed Scopus (244) Google Scholar) that the of lipolysis in adipocytes and cells on the levels of ATGL As insulin the expression of ATGL at the level of transcription E.E. Hamm J.K. Verhagen L.A. Peroni O. Katic M. Flier J.S. Diabetes. 2006; 55: 148-157Crossref PubMed Scopus (293) Google Scholar, 20Kim J.Y. Tillison K. Lee J.H. Rearick D.A. Smas C.M. Am. J. Physiol. Endocrinol. Metab. 2006; 291: 115-127Crossref PubMed Scopus (154) Google Scholar), this may help to the of insulin on lipolysis as as its effect on lipid the mechanism of insulin on transcription of ATGL has not been We show here that the expression of ATGL is regulated by FoxO1 and that insulin the expression of ATGL in adipocytes by the nuclear of FoxO1. As of ATGL in the of lipid E. L. W.J. 2006; PubMed Scopus (244) Google Scholar), and K. V. we that are with the by J. 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