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Department of Pharmacology, School of Medicine, University of California, Irvine, California 92697Department of Biochemistry and Molecular & Cellular Biology, Georgetown University, Washington, D. C. 20057
* Work in the Sassone-Corsi laboratory was supported by the National Institutes of Health, the Institut de la Sante et de la Recherche Medicale (France), and Sirtris Pharmaceutical Inc. Work in the Imhof laboratory was supported by the Bavarian-Californian Technology Center (BaCaTeC) and the Deutsche Forschungsgemeinschaft. 1 Supported by National Institutes of Health Ruth L. Kirschstein postdoctoral fellowships.
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.
). 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 (
). 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 (
). 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.
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 (
), 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 (
) 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 (
). 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 (
), 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 (
). 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 (
). 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.
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 (
), 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 (
). 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 (
), 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 (
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 (
). 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).
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.
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 (
) 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 (
). 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 (
). 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 (
), 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 (
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.
A clockwork web: circadian timing in brain and periphery, in health and disease.