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Circadian Control of Fatty Acid Elongation by SIRT1 Protein-mediated Deacetylation of Acetyl-coenzyme A Synthetase 1*

Open AccessPublished:January 14, 2014DOI:https://doi.org/10.1074/jbc.M113.537191
      The circadian clock regulates a wide range of physiological and metabolic processes, and its disruption leads to metabolic disorders such as diabetes and obesity. Accumulating evidence reveals that the circadian clock regulates levels of metabolites that, in turn, may regulate the clock. Here we demonstrate that the circadian clock regulates the intracellular levels of acetyl-CoA by modulating the enzymatic activity of acetyl-CoA Synthetase 1 (AceCS1). Acetylation of AceCS1 controls the activity of the enzyme. We show that acetylation of AceCS1 is cyclic and that its rhythmicity requires a functional circadian clock and the NAD+-dependent deacetylase SIRT1. Cyclic acetylation of AceCS1 contributes to the rhythmicity of acetyl-CoA levels both in vivo and in cultured cells. Down-regulation of AceCS1 causes a significant decrease in the cellular acetyl-CoA pool, leading to reduction in circadian changes in fatty acid elongation. Thus, a nontranscriptional, enzymatic loop is governed by the circadian clock to control acetyl-CoA levels and fatty acid synthesis.

      Introduction

      The circadian clock machinery is canonically described as a series of interconnected transcriptional and translational feedback loops (
      • Hastings M.H.
      • Reddy A.B.
      • Maywood E.S.
      A clockwork web: circadian timing in brain and periphery, in health and disease.
      ,
      • Partch C.L.
      • Green C.B.
      • Takahashi J.S.
      Molecular architecture of the mammalian circadian clock.
      ). Additional findings indicate that the clock relies on multiple levels of control, including post-transcriptional (
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      • Canella D.
      • Symul L.
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      • Gilardi F.
      • Liechti R.
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      • Herr W.
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      • Guex N.
      • Hernandez N.
      • Naef F.
      Genome-wide RNA polymerase II profiles and RNA accumulation reveal kinetics of transcription and associated epigenetic changes during diurnal cycles.
      ,
      • Menet J.S.
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      Nascent-Seq reveals novel features of mouse circadian transcriptional regulation.
      ,
      • Kojima S.
      • Shingle D.L.
      • Green C.B.
      Post-transcriptional control of circadian rhythms.
      ), post-translational (
      • Mehra A.
      • Baker C.L.
      • Loros J.J.
      • Dunlap J.C.
      Post-translational modifications in circadian rhythms.
      ), metabolic (
      • Nakahata Y.
      • Sahar S.
      • Astarita G.
      • Kaluzova M.
      • Sassone-Corsi P.
      Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1.
      ,
      • Ramsey K.M.
      • Yoshino J.
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      • Hong H.K.
      • Chong J.L.
      • Buhr E.D.
      • Lee C.
      • Takahashi J.S.
      • Imai S.
      • Bass J.
      Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis.
      ), and transcription-independent pathways (
      • O'Neill J.S.
      • Reddy A.B.
      Circadian clocks in human red blood cells.
      ,
      • O'Neill J.S.
      • van Ooijen G.
      • Dixon L.E.
      • Troein C.
      • Corellou F.
      • Bouget F.Y.
      • Reddy A.B.
      • Millar A.J.
      Circadian rhythms persist without transcription in a eukaryote.
      ). NAD+, a metabolite that acts as a critical coenzyme, has been shown to be an output of the circadian clock (
      • Nakahata Y.
      • Sahar S.
      • Astarita G.
      • Kaluzova M.
      • Sassone-Corsi P.
      Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1.
      ,
      • Ramsey K.M.
      • Yoshino J.
      • Brace C.S.
      • Abrassart D.
      • Kobayashi Y.
      • Marcheva B.
      • Hong H.K.
      • Chong J.L.
      • Buhr E.D.
      • Lee C.
      • Takahashi J.S.
      • Imai S.
      • Bass J.
      Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis.
      ). Moreover, fluctuations in NAD+ can also modulate the clock through NAD+-dependent deacetylation of histones, BMAL1, and PER2 by the SIRT1 deacetylase (
      • Nakahata Y.
      • Kaluzova M.
      • Grimaldi B.
      • Sahar S.
      • Hirayama J.
      • Chen D.
      • Guarente L.P.
      • Sassone-Corsi P.
      The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control.
      ,
      • Hirayama J.
      • Sahar S.
      • Grimaldi B.
      • Tamaru T.
      • Takamatsu K.
      • Nakahata Y.
      • Sassone-Corsi P.
      CLOCK-mediated acetylation of BMAL1 controls circadian function.
      ,
      • Asher G.
      • Gatfield D.
      • Stratmann M.
      • Reinke H.
      • Dibner C.
      • Kreppel F.
      • Mostoslavsky R.
      • Alt F.W.
      • Schibler U.
      SIRT1 regulates circadian clock gene expression through PER2 deacetylation.
      ). SIRT1 has been shown to regulate several metabolic pathways and is implicated in controlling aging and inflammation through its deacetylase activity (
      • Chalkiadaki A.
      • Guarente L.
      Sirtuins mediate mammalian metabolic responses to nutrient availability.
      ,
      • Haigis M.C.
      • Sinclair D.A.
      Mammalian sirtuins: biological insights and disease relevance.
      ). Interestingly, one of the proteins regulated by SIRT1-mediated deacetylation is acetyl-CoA synthetase 1 (AceCS1),
      The abbreviations used are:
      AceCS1
      acetyl-CoA synthetase 1
      ACLY
      ATP-citrate lyase
      LC-MS/MS
      liquid chromatography coupled to tandem mass spectrometry
      MEF
      mouse embryonic fibroblast
      VLCFA
      very long chain fatty acid
      ZT
      Zeitgeber time.
      a central enzyme involved in acetyl-CoA biosynthesis (
      • Hallows W.C.
      • Lee S.
      • Denu J.M.
      Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.
      ). Deacetylation of Lys-661 on AceCS1 by SIRT1 leads to activation of AceCS1 (
      • Hallows W.C.
      • Lee S.
      • Denu J.M.
      Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.
      ). Because SIRT1 activity, and the abundance of its cofactor NAD+, oscillate in a circadian manner (
      • Nakahata Y.
      • Sahar S.
      • Astarita G.
      • Kaluzova M.
      • Sassone-Corsi P.
      Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1.
      ,
      • Nakahata Y.
      • Kaluzova M.
      • Grimaldi B.
      • Sahar S.
      • Hirayama J.
      • Chen D.
      • Guarente L.P.
      • Sassone-Corsi P.
      The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control.
      ), we reasoned that AceCS1 acetylation, and in turn, acetyl-CoA abundance, may display circadian rhythmicity.
      Acetyl-CoA, a metabolite that provides acetyl groups during the acetylation reaction, exists in two separate pools in the cell: a mitochondrial pool and a nuclear/cytosolic pool (
      • Albaugh B.N.
      • Arnold K.M.
      • Denu J.M.
      KAT(ching) metabolism by the tail: insight into the links between lysine acetyltransferases and metabolism.
      ). The mitochondrial pool is derived mainly from the action of the enzyme pyruvate dehydrogenase and from fatty acid oxidation. The nuclear/cytosolic pool, responsible for protein acetylation and fatty acid synthesis, is produced by two enzymes: AceCS1 and ATP-citrate lyase (ACLY). Whereas ACLY uses citrate (produced during the tricarboxylic acid cycle) as a substrate for the production of acetyl-CoA, AceCS1 uses acetate. In mammals, acetate can be produced physiologically by the intestinal flora, alcohol metabolism, prolonged fasting, and histone deacetylation (
      • Shimazu T.
      • Hirschey M.D.
      • Huang J.Y.
      • Ho L.T.
      • Verdin E.
      Acetate metabolism and aging: an emerging connection.
      ). In Saccharomyces cerevisiae, the homolog of AceCS1 (Acs2p), was shown to be the major source of acetyl-CoA (
      • Takahashi H.
      • McCaffery J.M.
      • Irizarry R.A.
      • Boeke J.D.
      Nucleocytosolic acetyl-coenzyme A synthetase is required for histone acetylation and global transcription.
      ). Importantly, Wellen et al. have reported that ACLY and AceCS1 are present in both the cytosol and the nucleus of mammalian cells, and that the loss of either of these proteins leads to a reduction in global histone acetylation (
      • Wellen K.E.
      • Hatzivassiliou G.
      • Sachdeva U.M.
      • Bui T.V.
      • Cross J.R.
      • Thompson C.B.
      ATP-citrate lyase links cellular metabolism to histone acetylation.
      ). Moreover, reduction in histone acetylation upon loss of ACLY can be rescued by supplementing cells with acetate, supporting a critical role for AceCS1 in acetyl-CoA biosynthesis (
      • Wellen K.E.
      • Hatzivassiliou G.
      • Sachdeva U.M.
      • Bui T.V.
      • Cross J.R.
      • Thompson C.B.
      ATP-citrate lyase links cellular metabolism to histone acetylation.
      ). In this study, we demonstrate a novel regulation of the enzymatic activity of AceCS1 by the circadian clock that results in the rhythmicity of fatty acid elongation.

      RESULTS

      To determine whether acetylation of AceCS1 changes with the time of the day, liver extracts were prepared at different zeitgeber times (ZTs) from mice entrained in 12-h light:12-h dark cycle. Using an anti-acetyl-AceCS1 antibody, specific to the acetylated Lys-661 residue (
      • Hallows W.C.
      • Lee S.
      • Denu J.M.
      Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.
      ), we reveal that acetylation of AceCS1 oscillates in a circadian manner in the liver from wild-type (WT) mice (Fig. 1,A and B). The highest level of acetylation was observed at ZT9, whereas AceCS1 was mostly deacetylated at ZT21. Total levels of AceCS1 did not display circadian rhythmicity, either in protein levels (Fig. 1, A–D) or in mRNA levels (Fig. 2, A and B). Interestingly, the phase of oscillation of AceCS1 acetylation parallels that of BMAL1, another clock-related SIRT1 target (
      • Nakahata Y.
      • Kaluzova M.
      • Grimaldi B.
      • Sahar S.
      • Hirayama J.
      • Chen D.
      • Guarente L.P.
      • Sassone-Corsi P.
      The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control.
      ,
      • Hirayama J.
      • Sahar S.
      • Grimaldi B.
      • Tamaru T.
      • Takamatsu K.
      • Nakahata Y.
      • Sassone-Corsi P.
      CLOCK-mediated acetylation of BMAL1 controls circadian function.
      ).
      Figure thumbnail gr1
      FIGURE 1AceCS1 acetylation is regulated by the circadian clock. A–C, mice entrained in 12-h light:12-h dark cycles were sacrificed at indicated times, and their liver was dissected out. Total lysates were prepared and resolved by SDS-PAGE. A, acetylated and total AceCS1 levels were detected by Western blotting using specific antibodies in WT and clock/clock livers. B, acetylated AceCS1 levels (normalized to total AceCS1 levels) were quantified by densitometry. *, p < 0.05 (versus ZT21); n = 4. Error bars, S.E. C, WT and Bmal1−/− MEFs were synchronized by serum shock. Total lysates were prepared at the indicated times after synchronization and resolved by SDS-PAGE followed by Western analysis. Acetylated and total AceCS1, BMAL1, and α-tubulin levels were detected by specific antibodies. D, WT MEFs were pretreated with 50 μm EX527 for 16 h and then synchronized by serum shock. 50 μm EX527 was also added to the medium during and after serum shock. Total lysates were prepared at indicated times, and acetylated and total AceCS1 levels were detected by Western blotting.
      Figure thumbnail gr2
      FIGURE 2AceCS1 mRNA levels do not oscillate. AceCS1 and Dbp (D site of albumin promoter (albumin D-box)-binding protein} gene expressions were quantified by quantitative PCR in WT liver, n = 10 (A) or serum-entrained WT MEFs, n = 3 (B). Gene expression was normalized to 18S ribosomal RNA. Expression at trough was set to 1. ***, p < 0.00001 versus Dbp expression at ZT9. Error bars, S.D. Dbp is a robustly oscillating transcript in most mammalian tissues and hence is used as a positive control for studying oscillation in gene expression.
      To evaluate whether the circadian clock drives AceCS1 acetylation, we used clock/clock (c/c) mutant mice (
      • Vitaterna M.H.
      • King D.P.
      • Chang A.M.
      • Kornhauser J.M.
      • Lowrey P.L.
      • McDonald J.D.
      • Dove W.F.
      • Pinto L.H.
      • Turek F.W.
      • Takahashi J.S.
      Mutagenesis and mapping of a mouse gene, Clock, essential for circadian behavior.
      ) and found that acetylation is indeed drastically reduced in the liver of these mutant mice (Fig. 1A). We further analyzed the oscillation in AceCS1 acetylation in cultured cells by using MEFs. WT and Bmal1−/− MEFs were synchronized by serum shock, and cells were harvested at different time intervals. Acetylated AceCS1 levels were rhythmic in the WT cells with a peak at 18–24 h after synchronization (Fig. 1C), paralleling BMAL1 acetylation profile in MEFs (
      • Nakahata Y.
      • Sahar S.
      • Astarita G.
      • Kaluzova M.
      • Sassone-Corsi P.
      Circadian control of the NAD+ salvage pathway by CLOCK-SIRT1.
      ,
      • Nakahata Y.
      • Kaluzova M.
      • Grimaldi B.
      • Sahar S.
      • Hirayama J.
      • Chen D.
      • Guarente L.P.
      • Sassone-Corsi P.
      The NAD+-dependent deacetylase SIRT1 modulates CLOCK-mediated chromatin remodeling and circadian control.
      ). AceCS1 acetylation levels were almost undetectable in Bmal1−/− MEFs, whereas total protein levels of AceCS1 in both cell types remained unchanged and nonrhythmic. Thus, AceCS1 acetylation oscillates in a circadian manner both in vivo and in cultured cells. Next we validated the role of SIRT1 in circadian deacetylation of AceCS1 by using EX527, a direct pharmacological inhibitor of SIRT1 (
      • Napper A.D.
      • Hixon J.
      • McDonagh T.
      • Keavey K.
      • Pons J.F.
      • Barker J.
      • Yau W.T.
      • Amouzegh P.
      • Flegg A.
      • Hamelin E.
      • Thomas R.J.
      • Kates M.
      • Jones S.
      • Navia M.A.
      • Saunders J.O.
      • DiStefano P.S.
      • Curtis R.
      Discovery of indoles as potent and selective inhibitors of the deacetylase SIRT1.
      ). Indeed, blocking SIRT1 functions leads to elevated and arrhythmic AceCS1 acetylation (Fig. 1D).
      Because the acetylation status of AceCS1 controls its activity (
      • Hallows W.C.
      • Lee S.
      • Denu J.M.
      Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.
      ), we next sought to determine whether total cellular acetyl-CoA levels are also rhythmic. To do so, acetyl-CoA levels were measured by LC-MS/MS by using a modified version of the method described by Hayashi and Satoh (
      • Hayashi O.
      • Satoh K.
      Determination of acetyl-CoA and malonyl-CoA in germinating rice seeds using the LC-MS/MS technique.
      ). We found that acetyl-CoA levels were rhythmic in the liver of WT mice, with highest levels observed at ZT3 (Fig. 3A). This is in keeping with a scenario in which the peak of acetyl-CoA levels (ZT3) follows the peak of deacetylated (and hence, active) AceCS1 (ZT21). Next, we determined whether a functional circadian clock is important for the rhythmicity in the acetyl-CoA levels by analyzing the abundance of acetyl-CoA in the livers from Clock−/− mice. The peripheral tissues of Clock−/− mice have been shown to be arrhythmic (
      • DeBruyne J.P.
      • Weaver D.R.
      • Reppert S.M.
      Peripheral circadian oscillators require CLOCK.
      ). Consistent with a prominent role of the circadian clock machinery in regulating acetyl-CoA levels, there is no oscillation in the abundance of acetyl-CoA in the liver of Clock−/− mice (Fig. 3A). We then measured acetyl-CoA levels in cultured cells by synchronizing MEFs. WT MEFs displayed robust oscillation in the acetyl-CoA levels, with a peak at 12-h post-synchronization and trough at 24 h post-synchronization (Fig. 3B), in agreement with the cylic acetylation of AceCS1 (Fig. 1C). Because acetylation of AceCS1 in Bmal1−/− MEFs is low and noncyclic, we expected high and nonoscillating levels of acetyl-CoA in these cells, and this is in fact the case (Fig. 3B). Also, MEFs treated with EX527 displayed lower and nonoscillating levels of acetyl-CoA compared with untreated cells (Fig. 3C), paralleling the acetylation profile of AceCS1 (Fig. 1D). These results indicate that acetyl-CoA levels are rhythmic in mouse liver and in MEFs, that this rhythmicity is clock-controlled, and that the clock-driven acetylation of AceCS1 contributes to the cyclic abundance of acetyl-CoA.
      Figure thumbnail gr3
      FIGURE 3Circadian oscillation in Acetyl-CoA levels is controlled by the clock. Acetyl-CoA was extracted from mouse liver (A) or MEFs (B and C) harvested at indicated time points and analyzed by LC-MS/MS. A, relative abundance of acetyl-CoA in livers from WT and Clock−/− mice. ***, p = 0.00004 (WT ZT3 versus WT ZT21); **, p < 0.01 (WT ZT3 versus WT ZT 15); *, p < 0.05 (WT ZT3 versus WT ZT9); #, p < 0.05 (ZT3, WT versus CLOCK−/−); n = 4–7. Error bars, S.E. The total amount of acetyl-CoA (2200 pmol/μg DNA) was approximately 30% less in the Clock−/− liver at ZT3, compared with that in the WT liver (3200 pmol/μg DNA). B, acetyl-CoA levels in serum-entrained WT and Bmal1−/− MEFs. *, p < 0.05 (versus corresponding WT samples); **, p < 0.01 (versus WT 12 h); #, p < 0.05 (versus WT 24 h); n = 3. C, acetyl-CoA levels in serum-entrained WT MEFs treated with or without 50 μm EX527. *, p < 0.05 (versus control 12 h); n = 4.
      The relative contribution of ACLY and AceCS1 toward the intracellular abundance of acetyl-CoA is not fully understood. Blocking ACLY in cultured cells, either by RNAi (
      • Hatzivassiliou G.
      • Zhao F.
      • Bauer D.E.
      • Andreadis C.
      • Shaw A.N.
      • Dhanak D.
      • Hingorani S.R.
      • Tuveson D.A.
      • Thompson C.B.
      ATP-citrate lyase inhibition can suppress tumor cell growth.
      ) or by the specific inhibitor SB-204990 (
      • Chu K.Y.
      • Lin Y.
      • Hendel A.
      • Kulpa J.E.
      • Brownsey R.W.
      • Johnson J.D.
      ATP-citrate lyase reduction mediates palmitate-induced apoptosis in pancreatic beta cells.
      ), has been shown to reduce the total cellular acetyl-CoA levels by ∼50%. Moreover, knockdown of ACLY in mouse liver by adenovirus-mediated RNAi caused ∼25% reduction in the hepatic acetyl-CoA levels (
      • Wang Q.
      • Li S.
      • Jiang L.
      • Zhou Y.
      • Li Z.
      • Shao M.
      • Li W.
      • Liu Y.
      Deficiency in hepatic ATP-citrate lyase affects VLDL-triglyceride mobilization and liver fatty acid composition in mice.
      ). To determine the relative contribution of ACLY and AceCS1 on total cellular acetyl-CoA levels, we transiently knocked down ACLY and AceCS1 by siRNAs in cultured cells (Fig. 4A). Our results show that both ACLY and AceCS1 contribute significantly, and in a similar extent, to the total cellular acetyl-CoA pool. We reproducibly observed a reduction of acetyl-CoA levels by 24 or 28% upon the knockdown of ACLY or AceCS1, respectively (Fig. 4A). Furthermore, total acetyl-CoA levels were also reduced by 31% in a cell line where AceCS1 was stably knocked down (
      • Yoshii Y.
      • Furukawa T.
      • Yoshii H.
      • Mori T.
      • Kiyono Y.
      • Waki A.
      • Kobayashi M.
      • Tsujikawa T.
      • Kudo T.
      • Okazawa H.
      • Yonekura Y.
      • Fujibayashi Y.
      Cytosolic acetyl-CoA synthetase affected tumor cell survival under hypoxia: the possible function in tumor acetyl-CoA/acetate metabolism.
      ) (Fig. 4B). AceCS1 mRNA levels were reduced by ∼90% in these cells (Fig. 4C). These results establish that AceCS1 is a major determinant of cellular acetyl-CoA.
      Figure thumbnail gr4
      FIGURE 4Regulation of acetyl-CoA levels by AceCS1. A, WT MEFs were transfected with siRNAs against ACLY, AceCS1, or a nontargeting control. Cells were harvested 3 days after transfection for acetyl-CoA measurement, **, p < 0.01, n = 3. Error bars, S.E. B and C, control and AceCS1-knockdown mammary epithelial carcinoma cell lines were used for acetyl-CoA measurement (B); **, p < 0.01, n = 8; AceCS1 gene expression by quantitative PCR (C); ***, p < 0.00001, n = 9. D, MEFs were infected with lentiviruses expressing shRNA against a nontargeting control or against AceCS1. Several clones were screened, and clone 4 (showing maximum down-regulation of AceCS1 protein levels) was selected for further experiments. E, control (WT) and AceCS1-knockdown MEFs were synchronized by dexamethasone, cells were harvested at indicated time points, and gene expression was analyzed by real-time quantitative PCR. Gene expression was normalized to actin mRNA levels.
      Because acetyl-CoA levels could directly influence histone acetylation and thus, gene expression (
      • Wellen K.E.
      • Hatzivassiliou G.
      • Sachdeva U.M.
      • Bui T.V.
      • Cross J.R.
      • Thompson C.B.
      ATP-citrate lyase links cellular metabolism to histone acetylation.
      ,
      • Cai L.
      • Sutter B.M.
      • Li B.
      • Tu B.P.
      Acetyl-CoA induces cell growth and proliferation by promoting the acetylation of histones at growth genes.
      ,
      • Kaelin Jr., W.G.
      • McKnight S.L.
      Influence of metabolism on epigenetics and disease.
      ), we analyzed changes in circadian gene expression after knocking down AceCS1 in MEFs. Using a lentiviral shRNA against AceCS1, we generated a MEF cell line that expressed significantly lower levels of AceCS1 (Fig. 4D). When synchronized by dexamethasone, both control and AceCS1-knockdown MEFs displayed very similar, robust oscillation of core circadian gene expression (Fig. 4E). These results indicate that AceCS1 is not required for the regulation of circadian gene expression. Because the Km of histone acetyltransferases for acetyl-coA is relatively low (
      • Langer M.R.
      • Fry C.J.
      • Peterson C.L.
      • Denu J.M.
      Modulating acetyl-CoA binding in the GCN5 family of histone acetyltransferases.
      ), it is likely that modest fluctuations in acetyl-CoA levels might not be sufficient to alter histone acetylation and thus affect gene expression.
      Acetyl-CoA is the carbon source for synthesis and elongation of fatty acids. Because AceCS1 is present predominantly in the cytosol (Ref.
      • Wellen K.E.
      • Hatzivassiliou G.
      • Sachdeva U.M.
      • Bui T.V.
      • Cross J.R.
      • Thompson C.B.
      ATP-citrate lyase links cellular metabolism to histone acetylation.
      and Fig. 5A) and the fatty acid synthesis is mostly dependent on cytosolic availability of acetyl-CoA, we explored whether fatty acid synthesis is under circadian control through the AceCS1-mediated oscillation in acetyl-CoA. Acetyl-CoA produced by AceCS1 has been shown to be utilized in lipid synthesis (
      • Hallows W.C.
      • Lee S.
      • Denu J.M.
      Sirtuins deacetylate and activate mammalian acetyl-CoA synthetases.
      ). To validate the role of AceCS1 in lipid synthesis, we measured the incorporation of 14C-labeled acetate into lipids in AceCS1-knockdown and control cell lines. There is a remarkable decrease in 14C incorporation into lipids in AceCS1-knockdown cells compared with the control cells (Fig. 5B).
      Figure thumbnail gr5
      FIGURE 5Regulation of fatty acid synthesis by AceCS1. A, WT mice were sacrificed at indicated time points, and liver was harvested. Cytosolic and nuclear extracts were fractionated. Total AceCS1, α-tubulin, and BMAL1 levels were detected by Western blotting using specific antibodies. B, control and AceCS1-knockdown mammary epithelial carcinoma cell lines were used for measurement of [14C]acetate uptake into lipids; *, p < 0.05, n = 3. C–E, LC-MS analysis of fatty acid levels in control and AceCS1-knockdown mammary epithelial carcinoma cell lines at indicated times after dexamethasone synchronization, n = 6. C, LC-MS analysis of fatty acid levels is shown at indicated times after dexamethasone synchronization. SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. *, p < 0.01 versus corresponding 12-h sample, n = 6. D, long chain fatty acids; E, very long chain fatty acids;*, p < 0.01 versus corresponding 12-h sample, n = 6. Error bars, S.E. F, LC-MS analysis of saturated VLCFAs in WT and c/c MEFs is shown at indicated times after synchronization by serum shock. ***, p < 0.001 compared with the corresponding wild-type sample, n = 6.
      To further understand the role of AceCS1 in lipid metabolism, we used a lipidomics approach. We analyzed the levels of fatty acids of varying length and unsaturation at two time points in synchronized control and the AceCS1-knockdown cultured cells. The overall levels of fatty acids were significantly reduced in the AceCS1-knockdown cells (Fig. 5C). Interestingly, most fatty acids demonstrated a trend where the levels were higher at 12 h post-synchronization compared with the 24-h time point. These fatty acids included saturated and monounsaturated long chain fatty acids (Fig. 5D). This could be because the fatty acids are either oxidized during this time period and/or they are being converted to very long chain fatty acids (VLCFAs). Supporting the latter scenario, we observed that the levels of VLCFAs are higher at the 24-h time point (Fig. 5E). Importantly, the change in VLCFA levels is absent in AceCS1-knockdown cells (Fig. 5E). These results suggest that the reduced level of acetyl-CoA in the AceCS1-knockdown cells leads to reduction in total fatty acid levels and also causes impaired elongation of long chain fatty acids into VLCFAs. To confirm that elongation of fatty acids is regulated by the circadian clock, we measured the levels of fatty acids in WT and c/c MEFs. Although there is a robust oscillation in VLCFAs in the WT MEFs, their levels are significantly lower and nonrhythmic in c/c MEFs (Fig. 5F). Our results demonstrate that the elongation of fatty acids, a process that requires acetyl-CoA, is under the control of the circadian clock machinery. These results also establish AceCS1 as an important contributor to fatty acid elongation.

      DISCUSSION

      Our findings provide evidence that AceCS1 functions as a circadian enzyme, thereby contributing to the cyclic cellular levels of acetyl-CoA. Rhythmicity in AceCS1 acetylation contributes to the oscillation of acetyl-coA levels and, in turn, regulates circadian fatty acid elongation. Acetate could also be converted to acetyl-CoA by the mitochondrial enzyme AceCS2. However, AceCS2 expression is significantly lower compared with AceCS1 in the mouse liver (
      • Fujino T.
      • Kondo J.
      • Ishikawa M.
      • Morikawa K.
      • Yamamoto T.T.
      Acetyl-CoA synthetase 2, a mitochondrial matrix enzyme involved in the oxidation of acetate.
      ) and is almost undetectable in the MEFs (data not shown). Furthermore, in our experiments where cells were treated with [14C]acetate, knocking down AceCS1 is sufficient to reduce the acetate conversion to acetyl-CoA by ∼10-fold (Fig. 5B), confirming the prominent role of AceCS1 in these cells.
      Fatty acid synthesis constitutes a major process that utilizes acetyl-CoA in all cells. We have reported a unique pathway by which the circadian clock regulates the abundance of acetyl-CoA, leading to a clock-driven control of fatty acid elongation. Abolishing the activity of AceCS1 causes a significant decrease in the cellular pool of acetyl-CoA and leads to dampening of oscillations in fatty acid synthesis. This transcription-independent pathway is based solely on cyclic enzymatic function, utilizing the NAD+-dependent SIRT1 deacetylase to control AceCS1 activity, and contributes to modulated biosynthesis of acetyl-CoA. Thus, our study adds another layer to important examples of transcription-independent control by the mammalian circadian clock (
      • O'Neill J.S.
      • Reddy A.B.
      Circadian clocks in human red blood cells.
      ,
      • O'Neill J.S.
      • van Ooijen G.
      • Dixon L.E.
      • Troein C.
      • Corellou F.
      • Bouget F.Y.
      • Reddy A.B.
      • Millar A.J.
      Circadian rhythms persist without transcription in a eukaryote.
      ). These findings underscore that the circadian clock occupies a central position in controlling both NAD+ and acetyl-CoA levels in the cell, linking SIRT1 to fatty acid elongation. Increasing evidence reveals the links between the circadian clock and lipid metabolism (
      • Feng D.
      • Liu T.
      • Sun Z.
      • Bugge A.
      • Mullican S.E.
      • Alenghat T.
      • Liu X.S.
      • Lazar M.A.
      A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism.
      ,
      • Cho H.
      • Zhao X.
      • Hatori M.
      • Yu R.T.
      • Barish G.D.
      • Lam M.T.
      • Chong L.W.
      • DiTacchio L.
      • Atkins A.R.
      • Glass C.K.
      • Liddle C.
      • Auwerx J.
      • Downes M.
      • Panda S.
      • Evans R.M.
      Regulation of circadian behaviour and metabolism by REV-ERBα and REV-ERB-β.
      ,
      • Bugge A.
      • Feng D.
      • Everett L.J.
      • Briggs E.R.
      • Mullican S.E.
      • Wang F.
      • Jager J.
      • Lazar M.A.
      Rev-erbα and Rev-erbβ coordinately protect the circadian clock and normal metabolic function.
      ,
      • Solt L.A.
      • Wang Y.
      • Banerjee S.
      • Hughes T.
      • Kojetin D.J.
      • Lundasen T.
      • Shin Y.
      • Liu J.
      • Cameron M.D.
      • Noel R.
      • Yoo S.H.
      • Takahashi J.S.
      • Butler A.A.
      • Kamenecka T.M.
      • Burris T.P.
      Regulation of circadian behaviour and metabolism by synthetic REV-ERB agonists.
      ). These may involve additional chromatin remodelers, such as HDAC3, whose recruitment to the genome and enzymatic output follow a circadian pattern. As many of the genes regulated by HDAC3 are involved in lipid metabolism, and loss of HDAC3 leads to increased de novo fatty acid synthesis and a fatty liver phenotype (
      • Feng D.
      • Liu T.
      • Sun Z.
      • Bugge A.
      • Mullican S.E.
      • Alenghat T.
      • Liu X.S.
      • Lazar M.A.
      A circadian rhythm orchestrated by histone deacetylase 3 controls hepatic lipid metabolism.
      ), future studies will need to explore its relationship with AceCS1 and the control by SIRT1. Our study has uncovered another level of interplay among the circadian clock, epigenetics, and metabolism. As de novo fatty acid synthesis is known to be increased in cancer (
      • Fritz V.
      • Fajas L.
      Metabolism and proliferation share common regulatory pathways in cancer cells.
      ) and obesity (
      • Strable M.S.
      • Ntambi J.M.
      Genetic control of de novo lipogenesis: role in diet-induced obesity.
      ), our results might pave the way to future strategies for the use of sirtuin modulators (
      • Cen Y.
      • Youn D.Y.
      • Sauve A.A.
      Advances in characterization of human sirtuin isoforms: chemistries, targets and therapeutic applications.
      ) and chronotherapy in their treatment (
      • Sahar S.
      • Sassone-Corsi P.
      Metabolism and cancer: the circadian clock connection.
      ).

      Acknowledgments

      We thank Dr. Kathryn Wellen (University of Pennsylvania) for insightful discussions, Dr. Steven Reppert (University of Massachusetts Medical School) for providing Clock−/− mice, and Dr. Yukie Yoshii and Dr. Yasuhisa Fujibayashi (University of Fukui, Japan) for sharing the AceCS1-knockdown cell line.

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