2013/04/14 by Anne P.L. Jensen-Urstad, Haowei Song, Irfan J. Lodhi +4 · 1 citation
Biochemistry, Genetics and Molecular Biology · Medicine · #Peroxisome Proliferator-Activated Receptors #Metabolism, Diabetes, and Cancer #Hepatitis C virus research
paper · doi:10.1194/jlr.m036103
Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status. Peroxisome proliferator-activated receptor (PPAR)α is a nuclear receptor that coordinates liver metabolism during fasting. Fatty acid synthase (FAS) is an enzyme that stores excess calories as fat during feeding, but it also activates hepatic PPARα by promoting synthesis of an endogenous ligand. Here we show that the mechanism underlying this paradoxical relationship involves the differential regulation of FAS in at least two distinct subcellular pools: cytoplasmic and membrane-associated. In mouse liver and cultured hepatoma cells, the ratio of cytoplasmic to membrane FAS-specific activity was increased with fasting, indicating higher cytoplasmic FAS activity under conditions associated with PPARα activation. This effect was due to a nutrient-dependent and compartment-selective covalent modification of FAS. Cytoplasmic FAS was preferentially phosphorylated during feeding or insulin treatment at Thr-1029 and Thr-1033, which flank a dehydratase domain catalytic residue. Mutating these sites to alanines promoted PPARα target gene expression. Rapamycin-induced inhibition of mammalian/mechanistic target of rapamycin complex 1 (mTORC1), a mediator of the feeding/insulin signal to induce lipogenesis, reduced FAS phosphorylation, increased cytoplasmic FAS enzyme activity, and increased PPARα target gene expression. Rapamycin-mediated induction of the same gene was abrogated with FAS knockdown. These findings suggest that hepatic FAS channels lipid synthesis through specific subcellular compartments that allow differential gene expression based on nutritional status. Peroxisome proliferator-activated receptor (PPAR)α, one of three known members of a nuclear receptor family targeted to treat lipid disorders, diabetes, and obesity, is highly expressed in the liver. Its induction by fasting promotes lipid uptake, fatty acid β-oxidation, ketogenesis, and gluconeogenesis (1Aoyama T. Peters J.M. Iritani N. Nakajima T. Furihata K. Hashimoto T. Gonzalez F.J. Altered constitutive expression of fatty acid-metabolizing enzymes in mice lacking the peroxisome proliferator-activated receptor alpha (PPARalpha).J. Biol. Chem. 1998; 273: 5678-5684Abstract Full Text Full Text PDF PubMed Scopus (750) Google Scholar, 2Kersten S. Seydoux J. Peters J.M. Gonzalez F.J. Desvergne B. Wahli W. Peroxisome proliferator-activated receptor alpha mediates the adaptive response to fasting.J. Clin. Invest. 1999; 103: 1489-1498Crossref PubMed Scopus (1360) Google Scholar). Ligand binding to PPARα causes it to heterodimerize with retinoid X receptor (RXR)α, allowing activation of gene transcription at peroxisome proliferator response elements (PPRE) (3Kliewer S.A. Umesono K. Noonan D.J. Heyman R.A. Evans R.M. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors.Nature. 1992; 358: 771-774Crossref PubMed Scopus (1520) Google Scholar, 4Palmer C.N. Hsu M.H. Griffin H.J. Johnson E.F. Novel sequence determinants in peroxisome proliferator signaling.J. Biol. Chem. 1995; 270: 16114-16121Abstract Full Text Full Text PDF PubMed Scopus (260) Google Scholar). Synthetic PPARα ligands, such as fibrates, used for human lipid disorders (5Lalloyer F. Staels B. Fibrates, glitazones, and peroxisome proliferator-activated receptors.Arterioscler. Thromb. Vasc. Biol. 2010; 30: 894-899Crossref PubMed Scopus (163) Google Scholar) have been known for decades, but potential endogenous ligands were identified only recently (6Chakravarthy M.V. Lodhi I.J. Yin L. Malapaka R.R. Xu H.E. Turk J. Semenkovich C.F. Identification of a physiologically relevant endogenous ligand for PPARalpha in liver.Cell. 2009; 138: 476-488Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, 7Narala V.R. Adapala R.K. Suresh M.V. Brock T.G. Peters-Golden M. Reddy R.C. Leukotriene B4 is a physiologically relevant endogenous peroxisome proliferator-activated receptor-alpha agonist.J. Biol. Chem. 2010; 285: 22067-22074Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). Mice with liver-specific deletion of the lipogenic enzyme fatty acid synthase (FAS) have impaired PPARα activity (8Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar), and FAS activates PPARα by producing an endogenous phospholipid ligand (6Chakravarthy M.V. Lodhi I.J. Yin L. Malapaka R.R. Xu H.E. Turk J. Semenkovich C.F. Identification of a physiologically relevant endogenous ligand for PPARalpha in liver.Cell. 2009; 138: 476-488Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar). FAS also activates PPARα in brain and macrophages (9Chakravarthy M.V. Zhu Y. Lopez M. Yin L. Wozniak D.F. Coleman T. Hu Z. Wolfgang M. Vidal-Puig A. Lane M.D. et al.Brain fatty acid synthase activates PPARalpha to maintain energy homeostasis.J. Clin. Invest. 2007; 117: 2539-2552Crossref PubMed Scopus (172) Google Scholar, 10Schneider J.G. Yang Z. Chakravarthy M.V. Lodhi I.J. Wei X. Turk J. Semenkovich C.F. Macrophage fatty-acid synthase deficiency decreases diet-induced atherosclerosis.J. Biol. Chem. 2010; 285: 23398-23409Abstract Full Text Full Text PDF PubMed Scopus (54) Google Scholar). Mammalian FAS synthesizes long-chain fatty acids, primarily palmitate, through the activities of seven functional domains: acyl carrier, acyl transferase, β-ketoacyl synthase, β-ketoacyl reductase, β-hydroxyacyl dehydratase, enoyl reductase, and thioesterase (11Stoops J.K. Ross P. Arslanian M.J. Aune K.C. Wakil S.J. Oliver R.M. Physicochemical studies of the rat liver and adipose fatty acid synthetases.J. Biol. Chem. 1979; 254: 7418-7426Abstract Full Text PDF PubMed Google Scholar). Like PPARα, FAS is highly expressed in liver (12Semenkovich C.F. Regulation of fatty acid synthase (FAS).Prog. Lipid Res. 1997; 36: 43-53Crossref PubMed Scopus (203) Google Scholar). In times of nutrient excess, hepatic FAS converts carbohydrate to lipid that is stored in lipid droplets or secreted in the form of VLDL (13Jensen-Urstad A.P. Semenkovich C.F. Fatty acid synthase and liver triglyceride metabolism: Housekeeper or messenger?.Biochim. Biophys. Acta. 2012; 1821: 747-753Crossref PubMed Scopus (224) Google Scholar). Nutrient excess is associated with elevated levels of insulin, known to induce FAS expression. These accepted physiological roles for PPARα and FAS appear to conflict with the observation that inactivation of FAS impairs PPARα activation. How might FAS activate a process stimulated by feeding such as insulin-responsive lipogenesis and also activate a process stimulated by fasting such as the induction of PPARα-dependent gene expression? We hypothesized that distinct subcellular pools of FAS mediate these disparate effects. Compartmentalization would permit regulation of an FAS pool generating lipids for signaling that would be distinct from an FAS pool generating lipids for energy storage. In support of this hypothesis, we demonstrate that FAS at two separate subcellular locations is differentially regulated by nutrients and insulin, that this regulation involves preferential dehydratase domain phosphorylation for the FAS pool that regulates PPARα, and that the effects of the kinase mammalian/mechanistic target of rapamycin complex 1 (mTORC1) on PPARα activity require FAS. Male C57BL/6J mice at eight weeks of age were provided ad libitum access to chow diet (Purina #5053) or fasted for 18 h. All mice were kept on Aspen bedding and had free access to water. Protocols were approved by the Washington University Animal Studies Committee. Using a modification of a previously described assay (14Ullman A.H. White 3rd, H.B. Assay of fatty acid synthase using a bicyclic dione as substrate.Methods Enzymol. 1981; 72: 303-306Crossref PubMed Scopus (8) Google Scholar), 20 µl of sample at 1 µg protein/µl was added to 70 µl of assay buffer [0.14 M potassium phosphate buffer (pH 7.0), 1.4 mM EDTA (pH 8.0), 1.4 mM DTT, 0.24 mM NADPH, 0.1 mM acetyl-CoA]. The rate of NADPH oxidation was monitored at 340 nm at baseline and again after adding 10 µl of 0.85 mg/ml malonyl-CoA (Sigma). The substrate-dependent rate was determined by subtracting the baseline NADPH oxidation rate from the rate after addition of malonyl-CoA. The rate of NADPH oxidation was normalized to FAS protein levels as determined by western blotting and densitometry to determine specific activity. Perfused liver from C57BL/6J mice was homogenized in 20 mM HEPES buffer (pH 7.4) and centrifuged at 100 g for 30 min, and then the pellet was discarded. The supernatant was centrifuged at 500 g for 60 min; 1,200 g for 20 min; 10,000 g for 20 min; 20,000 g for 30 min; 40,000 g for 30 min; 70,000 g for 30 min; 100,000 g for 60 min; and 179,000 g for 75 min. After each spin, the pellet was washed and resuspended, while the supernatant was centrifuged again. All spins were done at 4°C. To obtain crude membrane and cytoplasmic fractions from mouse liver, freshly isolated perfused liver was homogenized in HEPES buffer and centrifuged at 10,000 g for 45 min at 4°C. The resulting pellet was discarded, and the supernatant centrifuged at 179,000 g for 180 min at 4°C. The supernatant (cytoplasm) and pellet (crude membrane) were collected, and the pellet was washed and resuspended in HEPES buffer. To obtain membrane and cytoplasmic extracts from Hepa1-6 cells, a Subcellular Protein Fractionation Kit for Cultured Cells (78840) from Thermo Fisher Scientific was used according to the manufacturer's protocol. Rabbit polyclonal antibodies against FAS (ab22759), PMP70 (ab3421), and phosphothreonine (ab9337) were from Abcam. Mouse against and polyclonal antibodies against and used to for in western blotting were from Rabbit polyclonal antibodies against protein and protein and antibodies against kinase and were from were as previously described X. L. X. F. Z. Y. of the and functional of J. PubMed Scopus Google Scholar) with were isolated from mouse liver by and resuspended in buffer 20 mM HEPES buffer (pH 1 mM and mM The membrane was to treatment with M 0.1 M at or and then centrifuged 30 The resulting and were by western Hepa1-6 and were in to insulin treatment for FAS activity Hepa1-6 were cultured in for h. All insulin were in Hepa1-6 in were in for 30 min. The were then with 500 of After 1 for the were were washed with with and for an or 180 min Cells were and membrane as described FAS was from each were to the was and the to FAS were by were then by was with and using an synthesis was using with an human FAS M. T. et of fatty acid synthase is associated with of and cytoplasmic of in Invest. PubMed Scopus Google Scholar) by was to FAS of the two phosphorylation sites and and two was by and an of the the to and to two The was by using the as a FAS were then and using the two sites to FAS as as of the FAS were by protein FAS was by the FAS from by adding sites for and on the and The was using the and an FAS a mouse FAS was from The and were from at in a were using with µg µg and µg After was and through was added and the used to treat Hepa1-6 After the was and with human FAS after addition of the were with After were and of mouse FAS as as expression of human FAS were To human in 10 were using with µg FAS and µg After were collected, using then was and the was used to treat Hepa1-6 After was and after an were In PPARα target gene expression in the endogenous FAS of Hepa1-6 was to expression of human FAS as described for FAS and or human FAS were as described Hepa1-6 in 10 were with for or FAS for after which the was and with After the was again and with After two of the was with and for 1 h. was also used for Hepa1-6 were with and by The for each 10 of was done as µg of and µg of were added to the of a Cells were by and after adding The was and were washed with The was and were resuspended in and to the by at and of of was added to the were to a and was added to Cells were to for 10 min, then were by washed with three resuspended in and on a from and was then using the Assay according to the manufacturer's activity was as the ratio of to To in hepatic perfused C57BL/6J mouse were homogenized in buffer The was at 10,000 g for 45 min, and the pellet was discarded. FAS was from 10 of the by using a polyclonal were in sample and to The was with the to FAS was and with mM reduced with 20 mM for 1 at washed and with 100 mM for 1 in the at then to of and by in a was with mg/ml or mg/ml for at were from the using in and in in was used to the sample for The sample was with for 1 at were from the in and the sample was with in were then by with a Fisher in were against through to the protein and were also to allow a for protein as as to for To FAS specific to FAS and cytoplasmic membrane and cytoplasmic fractions were isolated from C57BL/6J mice as described FAS was from of membrane and cytoplasmic by using a polyclonal The were then to and as described as of the two were using an was used for two FAS synthesizes palmitate, and FAS deficiency in liver decreases PPARα target the effect of FAS deficiency on PPARα then the of Hepa1-6 with to expression of the PPARα target gene FAS of were the FAS effect was with palmitate, it is that only the of the FAS but also the of synthesis mediates effects. that FAS is a cytoplasmic To determine FAS is also at we mouse liver FAS by FAS with the cytoplasmic but also with for for FAS in Hepa1-6 liver of FAS with and but or FAS appear in the FAS is by insulin and nutrients (12Semenkovich C.F. Regulation of fatty acid synthase (FAS).Prog. Lipid Res. 1997; 36: 43-53Crossref PubMed Scopus (203) Google Scholar). the specific activity of mouse liver cytoplasmic FAS was increased in the insulin levels FAS-specific activity was increased with feeding The activity ratio in liver was increased with fasting, PPARα is In Hepa1-6 cells, a liver insulin cytoplasmic FAS activity an effect that was in the membrane with mouse liver, the activity ratio in Hepa1-6 was increased in the of added insulin a of fasting. To to the that FAS is an of we isolated fractions with FAS by 1 M in the but it was by 0.1 M with or FAS protein These suggest N. T. N. P. of a Biol. 1995; PubMed Scopus Google Scholar, a protein for Biol. Chem. 1997; Full Text Full Text PDF PubMed Scopus Google Scholar, Y. S. of by of to Biol. PubMed Scopus Google Scholar) that FAS a membrane that FAS in the and cytoplasmic compartments that was of protein from one to the of this was in the of FAS membrane and were with insulin the of a a potential to the of mouse and human we the that FAS differential to protein only one of which is of FAS in membrane and to the at the and it identified the FAS protein sequence as This which the of of Protein on Biol. PubMed Scopus Google Scholar), was in membrane and cytoplasmic fractions of the of an All of the FAS protein were in each the that was determined by protein sequence due to a process such as these suggest that the enzyme activities of cytoplasmic and FAS differentially a that appear to be due to of the protein or in To the that differential regulation of cytoplasmic and FAS is by a covalent we hepatic FAS from fasting and and then fractions for the of phosphothreonine by western Cytoplasmic FAS in mice was a modification that was in fasted mice of FAS was under conditions In Hepa1-6 cells, insulin treatment of stimulated phosphorylation of cytoplasmic but FAS of FAS protein from mouse liver by only a that was This modification was at two Thr-1029 and is in liver FAS was cytoplasmic and fractions and to the same the phosphorylated was in the of the in fractions These suggest that the phosphorylated FAS in the with feeding or insulin is at Thr-1029 and These in the dehydratase domain of FAS. The of this domain two catalytic and and a that the of the catalytic S. A. S. two of the dehydratase domain of the fatty acid Biol. 2007; Full Text Full Text PDF PubMed Scopus Google Scholar). The phosphorylated we identified by in in to the catalytic and the by in of the dehydratase from that in addition to of the and by the we identified also as or in and in the of these phosphorylation sites in FAS we and in human FAS to the and in to generating two and and one sites by and the by or human FAS was then expressed in Hepa1-6 of endogenous mouse FAS. with human the had increased levels of the PPARα target gene the show in PPARα target expression of the was associated with increased levels of and To activity in this we a After expression of or FAS and of endogenous mouse were with a three to a activity was increased in the FAS with FAS that effects of the FAS on PPARα target by PPARα activity. of these is that the to FAS FAS enzyme activity to PPARα was recently identified as a physiologically of hepatic PPARα S. M. S. and by 2010; PubMed Scopus Google Scholar). the kinase of is a kinase that preferentially sites with at the S.A. J. Y. Y. et a mechanism of inhibition of PubMed Scopus Google Scholar). the phosphorylated we identified have the highly and at the we a for in FAS Hepa1-6 with the rapamycin for 30 min the in cytoplasmic FAS phosphorylation and was associated with an in cytoplasmic FAS-specific activity of these with 1 at also FAS phosphorylation Hepa1-6 with rapamycin for to a for increased expression of the PPARα target gene These findings in a S. M. S. and by 2010; PubMed Scopus Google Scholar) and that by FAS in the To the and PPARα, FAS was in Hepa1-6 by rapamycin FAS in the of rapamycin expression The induction of levels with rapamycin with FAS expression was with FAS These suggest that in this under these the induction of the PPARα target gene by inhibition of is FAS synthesizes lipid for energy and in the of a lipid ligand in the activation of fatty acid with feeding, and activation of fatty acid oxidation with fasting. To the same enzyme mediates we the that distinct pools of FAS differentially regulated in the liver. We FAS in the but we also FAS to through a membrane FAS-specific activity was higher with feeding/insulin in and higher with fasting in the This effect appear to of FAS compartments or sequence these pools of FAS. this activity was associated with preferential phosphorylation of cytoplasmic membrane) FAS with feeding at sites a catalytic domain of these sites increased endogenous PPARα target gene expression as as activity of a gene with of FAS in the of of with rapamycin FAS phosphorylation, increased FAS enzyme activity, and increased expression of the PPARα target gene an effect that was of these findings is that hepatic FAS in at least two differentially regulated subcellular cytoplasmic and Cytoplasmic FAS is phosphorylated with feeding to PPARα and it is with fasting to PPARα activation. findings and physiological to an observation in Using liver as a and FAS in the and that feeding which was associated with a of enzyme activity R.A. M. of two and of liver fatty acid and their by phosphorylation and Biophys. Res. PubMed Scopus Google Scholar). In treatment with FAS and enzyme activity. The of this a physiological for this covalent and it is known the we in FAS due to the of sequence for this that the phosphorylation of cytoplasmic FAS lipid to gene expression in the and that with nuclear (6Chakravarthy M.V. Lodhi I.J. Yin L. Malapaka R.R. Xu H.E. Turk J. Semenkovich C.F. Identification of a physiologically relevant endogenous ligand for PPARalpha in liver.Cell. 2009; 138: 476-488Abstract Full Text Full Text PDF PubMed Scopus (399) Google Scholar, I.J. Yin L. A.P. K. Coleman T. B. F. et adipose lipogenesis and activation to diet-induced Metab. 2012; Full Text Full Text PDF PubMed Scopus Google Scholar, R.C. et peroxisome proliferator-activated receptor ligands and Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, L. N. of the domain of the protein the of retinoid X in Biol. Chem. 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Full Text Full Text PDF PubMed Scopus Google Scholar), of relevant FAS phosphorylation sites the that phosphorylation at a of lipid signaling from the to the coordinates PPARα activation. is the of the FAS the of were to activate PPARα, would FAS the addition of to liver with FAS deficiency in PPARα-dependent and elevated levels that inactivation of liver FAS in mice impaired activation of PPARα-dependent (8Chakravarthy M.V. Pan Z. Zhu Y. Tordjman K. Schneider J.G. Coleman T. Turk J. Semenkovich C.F. “New” hepatic fat activates PPARalpha to maintain glucose, lipid, and cholesterol homeostasis.Cell Metab. 2005; 1: 309-322Abstract Full Text Full Text PDF PubMed Scopus (398) Google Scholar). by FAS to be a by of preferential phosphorylation on and nutritional is for in of the form of that be from gene expression in the of of which is to effects J. B. of of on the rat brain expression of the PubMed Scopus Google Scholar). is also for in lipid acid from synthesis effects on that from by acid from membrane J. Coleman R.A. complex to insulin 2012; PubMed Scopus Google Scholar). These with In the cytoplasmic FAS is phosphorylated to lipid resulting in PPARα while membrane to phosphorylation, lipids for energy or the of lipid synthesis by from the fasting to the the induction of membrane FAS be through activation by the B. Wakil S.J. Studies on the mechanism of fatty acid The of in the of the fatty acid from Biol. Chem. Full Text PDF PubMed Google Scholar). the activity of FAS in is by insulin and we identified in the dehydratase and is known to PPARα in the liver S. M. S. and by 2010; PubMed Scopus Google Scholar). FAS and appear to in the the physiological effects of FAS inhibition by rapamycin K. Fatty acid synthase energy target of rapamycin complex 1 signaling in the PubMed Scopus Google Scholar), with that inhibition would FAS activity. that hepatic FAS is in the as as associated with These two pools differentially regulated by nutrients and insulin, and differentially to phosphorylation, a for PPARα activation is to the fasting These have of FAS to inhibition of lipid PPARα activation treat fatty liver and disorders associated with nutrient protein mammalian/mechanistic target of rapamycin complex 1 peroxisome proliferator-activated receptor peroxisome proliferator response retinoid X receptor