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Paradoxical activation of transcription factor SREBP1c and de novo lipogenesis by hepatocyte-selective ATP-citrate lyase depletion in obese mice

2022; Elsevier BV; Volume: 298; Issue: 10 Linguagem: Inglês

10.1016/j.jbc.2022.102401

1083-351X

Batuhan Yenilmez, Mark Kelly, Guo‐Fang Zhang, Nicole Wetoska, Olga Ilkayeva, Kyounghee Min, Leslie A. Rowland, Chloe DiMarzio, Wentao He, Naideline Raymond, Lawrence M. Lifshitz, Meixia Pan, Xianlin Han, Jun Xie, Randall H. Friedline, Jason K. Kim, Guangping Gao, Mark A. Herman, Christopher B. Newgard, Michael Czech,

Peroxisome Proliferator-Activated Receptors

Hepatic steatosis associated with high-fat diet, obesity, and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl CoA (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high-fat feeding are unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to the generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, whereas in contrast, ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element–binding protein 1c and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY-depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels because of its increased conversion to malonyl CoA and palmitate. Together, these data indicate that in fat diet–fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or malonyl CoA but rather by activities of DNL enzymes. Hepatic steatosis associated with high-fat diet, obesity, and type 2 diabetes is thought to be the major driver of severe liver inflammation, fibrosis, and cirrhosis. Cytosolic acetyl CoA (AcCoA), a central metabolite and substrate for de novo lipogenesis (DNL), is produced from citrate by ATP-citrate lyase (ACLY) and from acetate through AcCoA synthase short chain family member 2 (ACSS2). However, the relative contributions of these two enzymes to hepatic AcCoA pools and DNL rates in response to high-fat feeding are unknown. We report here that hepatocyte-selective depletion of either ACSS2 or ACLY caused similar 50% decreases in liver AcCoA levels in obese mice, showing that both pathways contribute to the generation of this DNL substrate. Unexpectedly however, the hepatocyte ACLY depletion in obese mice paradoxically increased total DNL flux measured by D2O incorporation into palmitate, whereas in contrast, ACSS2 depletion had no effect. The increase in liver DNL upon ACLY depletion was associated with increased expression of nuclear sterol regulatory element–binding protein 1c and of its target DNL enzymes. This upregulated DNL enzyme expression explains the increased rate of palmitate synthesis in ACLY-depleted livers. Furthermore, this increased flux through DNL may also contribute to the observed depletion of AcCoA levels because of its increased conversion to malonyl CoA and palmitate. Together, these data indicate that in fat diet–fed obese mice, hepatic DNL is not limited by its immediate substrates AcCoA or malonyl CoA but rather by activities of DNL enzymes. One of the debilitating comorbidities of type 2 diabetes in obesity is nonalcoholic steatohepatitis (NASH), characterized by severe liver inflammation and fibrosis that can lead to cirrhosis and the need for liver transplantation (1Younossi Z.M. Koenig A.B. Abdelatif D. Fazel Y. Henry L. Wymer M. Global epidemiology of nonalcoholic fatty liver disease—meta-analytic assessment of prevalence, incidence, and outcomes.Hepatology. 2016; 64: 1388-1389Crossref PubMed Scopus (89) Google Scholar, 2Younossi Z.M. Blissett D. Blissett R. 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Cytosolic AcCoA has two well studied sources—citrate, which yields AcCoA and oxaloacetate when cleaved by ACLY, and acetate, which is converted to AcCoA by ACSS2. Recent studies have demonstrated that fructose supplementation via the drinking water induces hepatic DNL through multiple mechanisms including induction of hepatic lipogenic enzymes, and by metabolism of fructose by the microbiome to generate acetate, which then serves as the substrate for DNL via its conversion to AcCoA by ACSS2 (34Zhao S. Jang C. Liu J. Uehara K. Gilbert M. Izzo L. et al.Dietary fructose feeds hepatic lipogenesis via microbiota-derived acetate.Nature. 2020; 579: 586-591Crossref PubMed Scopus (240) Google Scholar, 35Perry R.J. Peng L. Barry N.A. Cline G.W. Zhang D. Cardone R.L. et al.Acetate mediates a microbiome-brain-β-cell axis to promote metabolic syndrome.Nature. 2016; 534: 213-217Crossref PubMed Scopus (842) Google Scholar, 36Herman M.A. Birnbaum M.J. 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In humans, inhibition of ACLY by the small-molecule drug bempedoic acid decreases blood lipids without decreasing liver fat, but combination therapies that simultaneously target ACLY and ACSS2 have not been reported (37Feng X. Zhang L. Xu S. Shen A.-z. ATP-citrate lyase (ACLY) in lipid metabolism and atherosclerosis: an updated review.Prog. Lipid Res. 2020; 77101006Crossref PubMed Scopus (95) Google Scholar, 38Ference B.A. Ray K.K. Catapano A.L. Ference T.B. Burgess S. Neff D.R. et al.Mendelian randomization study of ACLY and cardiovascular disease.New Engl. J. Med. 2019; 380: 1033-1042Crossref PubMed Scopus (172) Google Scholar, 39Wei J. Leit S. Kuai J. Therrien E. Rafi S. Harwood H.J. et al.An allosteric mechanism for potent inhibition of human ATP-citrate lyase.Nature. 2019; 568: 566-570Crossref PubMed Scopus (82) Google Scholar). Based on these considerations, we have performed a study of the effects of liver-specific suppression of ACLY (ACLY liver KO [LKO]) or ACSS2 (ACSS2 liver knockdown [LKD]) expression alone, or of both genes combined (double), on hepatic DNL and levels of key metabolic intermediates including AcCoA, MalCoA, and acetate in mice fed HFD. Several surprising findings emerged: (1) while hepatocyte AcCoA levels were decreased by depletion of either ACLY or ACSS2 alone, or in combination, MalCoA levels were unchanged in response to any of these maneuvers, indicating that neither ACLY nor ACSS2 are required for maintaining levels of this immediate DNL precursor in HFD-fed obese mice; (2) there is a compensatory increase in nuclear sterol regulatory element–binding protein 1c (SREBP1c) and expression of enzymes in the DNL pathway such as fatty acid synthase (FASN), stearoyl-coenzyme A desaturase 1 (SCD1), and elongation of long-chain fatty acids family member 6 (ELOVL6) when hepatic ACLY is depleted, leading to a paradoxical increase in DNL in the absence of ACLY in HFD-fed obese mice, and (3) the upregulation of DNL enzymes occurring in response to hepatic ACLY depletion is associated with a compensatory increase in ACSS2 and a corresponding decrease in circulating acetate levels. Altogether, these studies reveal unexpected features of hepatic DNL flux in obese mice and provide a framework for understanding mechanisms that link AcCoA producing enzymes to control of more distal enzymes in the DNL pathway. In order to determine the relative contributions of metabolic flux through ACLY versus ACSS2 to form AcCoA and fuel DNL in hepatocytes in vivo, we used mice floxed flanking exon 17 to 19 of the acly gene and injected with either adeno-associated virus (AAV) engineered for hepatocyte-selective Cre expression (pAAV-TBG-PI-Cre) to delete liver ACLY or AAV engineered for hepatocyte-selective expression of an artificial micro-RNA directed against ACSS2 (pAAV-TBG-amiRACSS2) to achieve liver ACSS2 depletion (Fig. 1A). To obtain combined depletion of ACLY and ACSS2, both AAV constructs were injected simultaneously. To establish the utility of these vectors, acly floxed mice fed on chow diet were injected with these AAV constructs or a control AAV and sacrificed 9 weeks later for analysis (Fig. 1B). Immunoblotting analysis of livers from these mice revealed the expected depletion of ACLY, ACSS2, or both proteins dependent on the vectors administered (Fig. 1C). In addition, immunoblots of brown, epididymal, and inguinal adipose tissue showed no decreases in ACLY or ACSS2 expression, confirming the liver specificity of the gene silencing provided by the AAV constructs (Fig. S1). No difference in body weight was noted in response to treatment with any of the AAV vectors (data not shown). Hepatic AcCoA levels did not change significantly in response to suppression of either ACLY or ACSS2 alone but were reduced ∼50% in response to combined depletion of hepatic ACLY plus ACSS2 (Fig. 1D). These data validate the efficacy of the AAV constructs to cause significant depletion of the targeted enzymes and demonstrate that with chow feeding, the mice are able to maintain normal hepatic AcCoA levels by alternate routes when either ACLY levels or ACSS2 levels are diminished but not when both are depleted. Figure 2A depicts the experimental protocol used to address the key questions of our study concerning regulation of DNL in HFD-fed obese mice. Following injection of the AAV constructs, mice were fed chow for 1 week and then switched to a diet containing 60% fat (HFD) for eight additional weeks prior to sacrifice. No changes in body weight (Fig. 2B), glucose tolerance (Fig. S2), or food intake (Fig. 2C) were observed in mice injected with the ACLY or ACCS2 AAV vectors relative to mice injected with control AAV. Treatment of HFD-fed mice with pAAV-TBG-PI-Cre caused near complete suppression of ACLY mRNA (Fig. 2D) and protein (Fig. 2, E and F) levels, both when administered alone or in conjunction with the pAAV-TBG-amiRACSS2 vector that depletes ACSS2. However, in contrast to what was observed in chow-fed mice (Fig. 1), depletion of hepatocyte ACLY caused significant upregulation of ACCS2 expression. Moreover, while injection of the pAAV-TBG-amiRACSS2 vector alone caused a strong depletion of Acss2 mRNA and protein in the obese mice, this suppression was less effective when combined with ACLY depletion, likely because of the compensatory upregulation phenomenon (Figs. 2, D–F and S3A). The levels of ACSS2 protein in double KO mice were similar to those in mice treated with the control AAV vector but were well below levels observed in the ACLY LKO mice. Next, metabolite levels and metabolic flux through DNL were assessed in two separate cohorts of HFD-fed obese mice, and the results of the two studies were combined to provide the data in Figure 3. Under HFD conditions, KD of either ACLY or ACSS2 in hepatocytes led to a significant decrease in AcCoA levels, with a trend for further decline in mice with combined KD of both enzymes (Fig. 3A). Notably, the compensatory increase in ACSS2 expression in the ACLY LKO condition did not restore AcCoA levels in the absence of ACLY. That ACSS2 is active under these conditions is verified by analysis of plasma acetate (Fig. 3B), which was inversely proportional to ACSS2 expression. Notably, plasma acetate levels were also decreased in response to combined suppression of ACLY and ACSS2, suggesting that even though ACSS2 protein was not elevated in this condition compared with mice treated with the control AAV vector, flux through this enzyme and its consumption of acetate may have increased. Remarkably, the fall in AcCoA levels elicited by KD of ACLY, ACSS2, or both enzymes was not accompanied by a decrease in MalCoA levels (Fig. 3C). Also unanticipated, newly synthesized palmitate and total palmitate levels were increased in response to ACLY LKO, either alone or when combined with ACSS2 suppression, whereas ACSS2 LKD caused a modest decrease in these outcomes, despite the decrease in AcCoA levels in response to all these maneuvers (Fig. 3, B D, and E). Total liver TG levels also followed the newly synthesized palmitate and total palmitate trend. Hepatic TG levels were significantly increased by ACLY depletion or double depletion compared with the ACSS2 LKD and control group (Fig. 3F). None of these changes in hepatic lipid biosynthesis were reflected in changes in plasma lipids (Fig. S4). These data indicate that AcCoA levels do not determine MalCoA levels or rates of DNL under HFD-fed conditions in mice. Instead, the metabolite that most strongly correlated, in an inverse fashion, with DNL was plasma acetate, which was reduced in response to ACLY depletion (alone or in concert with ACSS2 KD) and slightly increased in response to ACSS2 LKD alone. We next explored the mechanisms underlying the paradoxical increase in DNL engendered by ACLY LKO, occurring in the face of lowered AcCoA levels and unchanged MalCoA levels. To this end, we measured expression of the genes encoding key enzymes in the DNL pathway as well as transcription factors known to control their expression (Fig. 4). At the mRNA level, three enzymes in the DNL pathway were found to be significantly elevated in ACLY-depleted livers—Fasn, Scd1, and Elovl6 (Fig. 4A). Interestingly, this upregulation has been detected in only HFD-fed obese mice but not in lean chow-fed mice (Fig. S5). In contrast, ACSS2 depletion alone, which had no significant effect on newly synthesized palmitate (Fig. 3D), also had no effect on Fasn, Scd1, or Elovl6 mRNA levels (Fig. 4A). Immunoblot analyses confirmed the increase in FASN and SCD1 at the protein level and also revealed an increase in ACC1, both total amount and its phosphorylated inactivated form in the ACLY LKO condition, resulting in no net change in the inactive to active ACC1 ratio (Fig. 4, B and C). Interestingly, depletion of ACSS2 in addition to ACLY LKO abrogated the increase in DNL enzyme mRNA levels but not protein levels. These data suggest that hepatic DNL in HFD-fed obese mice, but not in chow-fed (Fig. S5) mice, is not limited by or dependent on AcCoA and MalCoA levels but rather is regulated by altered expression and activities of DNL enzymes such as FASN and SCD1. Since it is known that the transcription factors ChREBP and SREBP1c regulate the expression of DNL enzymes in liver, we analyzed expression of these transcription factors in our genetically engineered HFD-fed mice. While no difference in expression levels of these transcription factors at the mRNA level was observed, we found a clear effect on levels of the nuclear-localized form of SREBP1c. Figures 4, B and C and S3B show that hepatic ACLY LKO but not ACSS2 LKD causes increased processing of SREBP1c to the nuclear form, an effect not observed when ACLY LKO was combined with ACSS2 LKD. These data suggest that ACLY depletion causes upregulation of SREBP1c processing to its activated form, associated with upregulation of ACSS2 as well as SCD1 and FASN. Although this effect is not significant when ACLY is depleted in combination with ACSS2 depletion, the trend is still evident and the DNL enzymes are upregulated in this condition (Fig. 4C). We applied correlation analyses of data from individual mice to investigate how Srebp1 processing and DNL enzyme expression may be related to the ACLY and ACSS2 perturbations (Fig. 5). This analysis confirmed the lack of correlation between AcCoA levels and newly synthesized palmitate but showed a significant correlation between plasma acetate levels and DNL (Fig. 5, A and B). Moreover, there were strong correlations between FASN protein expression (Fig. 5C) and SREBP1c processing (Fig. 5C) with DNL flux. Importantly, plasma acetate concentrations correlated inversely with FASN expression (Fig. 5D) and with the nuclear form of SREBP1c (Fig. 5D), consistent with the concept that acetate levels are an indicator of SREBP1c activity, hepatic DNL enzyme expression, and hepatic DNL flux. In addition, lipidomics analysis showed significantly increased diglyceride accumulation (Fig. S6A) as well as fatty acyl chains in TGs in ACLY LKO and double depletion groups compared with ACSS2 LKD and control groups (Fig. 6, A–C). In line with the DNL increase in ACLY-depleted groups (Fig. 3D), the major individual fatty acid species in TGs were also increased in ACLY-depleted groups (Fig. 6, A–C). Interestingly, the increase in newly synthesized palmitate, liver TG, and diglyceride accumulation was inversely correlated with phospholipid biosynthesis, suggesting that DNL is shunted toward TG biosynthesis compared with phospholipid biosynthesis under conditions where ACLY is absent (Fig. S6, B–D). A primary question addressed in this study is the degree to which the two AcCoA synthesis pathways catalyzed by ACLY versus ACSS2 contribute to steady-state levels of AcCoA in livers of HFD-fed obese mice. The importance of this question is reinforced by several perspectives: (1) AcCoA is a major substrate in the pathways of DNL and cholesterol synthesis, both of which are upregulated in obesity to contribute to metabolic disease, (2) AcCoA is a substrate for histone and other protein acetylation reactions that control gene expression and enzyme function, and (3) effects of single and double depletions of hepatocyte ACLY and ACSS2 on levels of key DNL intermediates such as AcCoA, MalCoA, and acetate, and flux through the DNL pathway, have not been investigated in the most commonly used animal model of obesity, the HFD-fed mouse. To perform these experiments in the most rigorous manner, AAV vectors were employed expressing thyroxine-binding globulin (TBG) promoter–based constructs to assure hepatocyte-selective depletion of ACLY and ACSS2 (Fig. 1). The results indicate that ACLY and ACSS2 both contribute significantly to AcCoA levels in hepatocytes of HFD-fed obese mice, as evidenced by significant decreases in content following depletion of each enzyme alone or both in combination (Figs. 2, 3 and S1). In contrast, in mice on chow diet, hepatic AcCoA levels were unchanged in response