Articles https://doi.org/10.1038/s42255-021-00342-6

Murine neonatal ketogenesis preserves mitochondrial energetics by preventing protein hyperacetylation

Yuichiro Arima 1,2,13 ✉ , Yoshiko Nakagawa3,13, Toru Takeo 3,13, Toshifumi Ishida 1, Toshihiro Yamada1, Shinjiro Hino4, Mitsuyoshi Nakao4, Sanshiro Hanada 2, Terumasa Umemoto 2, Toshio Suda2, Tetsushi Sakuma 5, Takashi Yamamoto5, Takehisa Watanabe6, Katsuya Nagaoka6, Yasuhito Tanaka6, Yumiko K. Kawamura7,8, Kazuo Tonami7, Hiroki Kurihara7, Yoshifumi Sato9, Kazuya Yamagata9,10, Taishi Nakamura 1,11, Satoshi Araki1, Eiichiro Yamamoto1, Yasuhiro Izumiya1,12, Kenji Sakamoto1, Koichi Kaikita1, Kenichi Matsushita 1, Koichi Nishiyama2, Naomi Nakagata3 and Kenichi Tsujita1,10

Ketone bodies are generated in the liver and allow for the maintenance of systemic caloric and energy homeostasis during fasting and caloric restriction. It has previously been demonstrated that neonatal ketogenesis is activated independently of starvation. However, the role of ketogenesis during the perinatal period remains unclear. Here, we show that neonatal ketogen- esis plays a protective role in mitochondrial function. We generated a mouse model of insufficient ketogenesis by disrupting the rate-limiting hydroxymethylglutaryl-CoA synthase 2 enzyme gene (Hmgcs2). Hmgcs2 knockout (KO) neonates develop microvesicular steatosis within a few days of birth. Electron microscopic analysis and metabolite profiling indicate a restricted energy production capacity and accumulation of acetyl-CoA in Hmgcs2 KO mice. Furthermore, acetylome analysis of Hmgcs2 KO cells revealed enhanced acetylation of mitochondrial proteins. These findings suggest that neonatal ketogenesis protects the energy-producing capacity of mitochondria by preventing the hyperacetylation of mitochondrial proteins.

etone bodies (beta-hydroxybutyrate, acetoacetate and ace- In adult mice, insufficient ketogenesis attributed to Hmgcs2- tone) are well-known metabolites that help maintain caloric targeted antisense oligonucleotides results in phenotypes resem- Khomeostasis as an alternative fuel1. Their synthesis (ketogen- bling non-alcoholic fatty liver disease10,11. Moreover, the authors esis) and utilization are influenced by several factors, such as the of this study also generated Oxct1/SCOT KO mice lacking the transition from a fed to a fasted state, as well as ischaemia2,3. Ketone rate-limiting enzyme crucial for ketone body oxidation, and the KO bodies are consumed in numerous extrahepatic tissues as an effi- neonates did not survive owing to the development of lethal keto- cient energy source. Furthermore, beta-hydroxybutyrate (βOHB) acidosis within several days of birth11,12. can function as a signalling mediator and an intrinsic histone Previous clinical and basic scientific reports have revealed deacetylase (HDAC) inhibitor4,5, and various roles of ketone bodies changes in ketone body metabolism during the perinatal period9,13,14. have been discovered6. However, details regarding the physiological significance of perina- Ketogenesis is a sequential metabolic pathway that generates tal ketogenesis remain unknown. In this study, we aimed to elucidate ketone bodies from acetyl-CoA. Several enzymes are involved in this the role of perinatal ketogenesis. We generated deletion mutants of reaction; 3-hydroxy-3-methylglutaryl-CoA synthase 2 (HMGCS2) Hmgcs2, established a mouse model of insufficient ketogenesis and is the rate-limiting enzyme of ketogenesis7,8. HMGCS2 is a mito- identified the protective effects of ketogenesis on mitochondria by chondrial protein that synthesizes 3-hydroxy-3-methylglutaryl-CoA modulating post-translational modifications. (HMG-CoA) from acetoacetyl-CoA and acetyl-CoA9. Ketone body utilization is controlled by succinyl-CoA: 3-oxoacid CoA-transferase Results (Oxct1/SCOT), and the tissue-specific expression of this enzyme Ketogenesis is enhanced during the neonatal period. We attempted determines the characteristics of ketogenesis and ketone body to clarify the characteristics of ketogenesis associated with devel- utilization6. opment. Whole-blood concentrations of βOHB, the most stable

1Department of Cardiovascular Medicine, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. 2International Research Center for Medical Sciences (IRCMS), Kumamoto University, Kumamoto, Japan. 3Division of Reproductive Engineering, Center for Animal Resources and Development (CARD), Kumamoto University, Kumamoto, Japan. 4Department of Medical Cell Biology, Institute of Molecular Embryology and Genetics, Kumamoto University, Kumamoto, Japan. 5Division of Integrated Sciences for Life, Graduate School of Integrated Sciences for Life, Hiroshima University, Hiroshima, Japan. 6Department of Gastroenterology and Hepatology, Graduate School of Medical Sciences, Kumamoto University, Kumamoto, Japan. 7Department of Physiological Chemistry and Metabolism, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan. 8Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland. 9Department of Medical Biochemistry, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan. 10Center for Metabolic Regulation of Healthy Aging (CMHA), Kumamoto University, Kumamoto, Japan. 11Department of Medical Information Science and Administration Planning, Kumamoto University Hospital, Kumamoto, Japan. 12Department of Cardiovascular Medicine, Osaka City University Graduate School of Medicine, Osaka, Japan. 13These authors contributed equally: Yuichiro Arima, Yoshiko Nakagawa, Toru Takeo. ✉e-mail: [email protected]

196 Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab NATurE METAbOlISm Articles

a b c βOHB Hmgcs2/18S mtDNA/ncDNA 5 1,500 P = 0.0012 4 P = 0.0002 P < 0.0001 P < 0.0001 4 P < 0.0001 P = 0.0407 3 1,000 3 –1 2 Ratio 2 Copies mmol l 500 1 1

0 0 0 E18.5 P7 Adult E18.5 P7 Adult E18.5 P7 Adult

d e HMGCS2/TP HMGCS2 TP 2.5

P < 0.0001 P < 0.0001 E18.5 P7 Adult 2.0

1.5 a.u. 50 kDa 1.0

0.5

0 E18.5 P7 Adult

Fig. 1 | Ketogenesis is enhanced during the neonatal period. a, βOHB whole-blood concentrations under free-feeding conditions (E18.5, n = 12; P7, n = 17; adult, n = 21). b, Real-time PCR for Hmgcs2 adjusted by 18S (E18.5, n = 6; P7, n = 6; adult, n = 5). c, Mitochondrial copy numbers at each stage (n = 3 each). d, Fluorescence-based western blot analysis of HMGCS2 (green) and total protein (TP; red; n = 3 each). e, Quantitative analysis of HMGCS2 expression was adjusted for TP (n = 3 each). Results are expressed as dot plots with means ± standard deviation (s.d.). One-way analysis of variance (ANOVA) and Holm–Sidak’s multiple-comparisons test were performed (a–e). a.u., arbitrary units. metabolite among ketone bodies, were measured. In a free-feeding system (Fig. 2a). A guide RNA (gRNA) was set in the exon 1 state, βOHB concentrations in embryos and adult mice were low; coding region close to the second methionine (ATG) follow- however, concentrations in neonatal mice were significantly elevated ing the start codon. In vitro transcribed gRNA and recombinant (βOHB concentrations in embryonic day 18.5 (E18.5), postnatal day Cas9 protein were microinjected into C57BL/6 zygotes, and the 7 (P7) and adult mice were 0.7 ± 0.9 mmol l−1, 1.8 ± 0.5 mmol l−1 and resulting zygotes that survived were transferred into pseudopreg- 0.7 ± 0.2 mmol l−1, respectively (P < 0.0001; Fig. 1a). HMGCS2 is the nant ICR strain female mice (Supplementary Table 1). We analysed rate-limiting enzyme in ketogenesis. Real-time PCR revealed that nine founder mice and selected two deletion mutants (Extended hepatic Hmgcs2 transcript levels increased after birth. Furthermore, Data Fig. 1a,b and Supplementary Table 2). Two KO lines were adult Hmgcs2 transcript expression was significantly higher in a established (Fig. 2b and Supplementary Table 3). Western blot free-feeding state than that observed in the neonatal liver (relative analysis revealed a lack of HMGCS2 protein production in Hmgcs2 transcript expression levels in E18.5, P7 and adult mice were the Hmgcs2 homozygous mutants (referred to as KO; Fig. 2c). 0.66 ± 0.14, 2.10 ± 0.36 and 3.27 ± 0.58, respectively (P < 0.0001; Immunohistochemical analysis of HMGCS2 revealed that strong Fig. 1b). As ketogenesis occurs in mitochondria, we measured the expression in hepatocytes was diminished in P7 Hmgcs2 KO copy number of mitochondrial DNA (mtDNA) adjusted by nuclear livers (Fig. 2d). DNA (ncDNA) using droplet-digital PCR (ddPCR). Compared To confirm a state of insufficient ketogenesis, we measured to mtDNA volume in embryos and neonates, mitochondria were βOHB concentrations, which were significantly decreased in P3 most abundant in adults (mtDNA/ncDNA in E18.5, P7 and adult whole blood and P7 serum (P3 whole blood: wild-type (WT), mice were 402 ± 53, 618 ± 156 and 1,141 ± 147 copies, respectively 1.25 ± 0.21 mmol l−1, n = 6; KO, 113 ± 0.08 mmol l−1, n = 8, P < 0.0001; (P = 0.0002; Fig. 1c). Despite the intermediate expression of Hmgcs2 P7 serum: WT, 1.62 ± 0.11 mmol l−1, n = 10; KO, 0.0 ± 0.0 mmol l−1, transcripts and mitochondrial volume, production of βOHB pro- n = 11, P < 0.0001; Fig. 2e,f). Furthermore, gas chromatography– duction was more pronounced in neonates than in adults. We mass spectrometry (GC–MS) analysis of P7 liver samples con- focused on HMGCS2 protein expression and performed quantita- firmed a significant reduction in βOHB concentrations (GC–MS: tive western blots. The fluorescence-based western blotting analysis WT, 2.05 ± 0.46, n = 10; KO, 0.178 ± 0.03, n = 9; P < 0.0001; Fig. 2g). demonstrated the distinct expression of neonatal HMGCS2 protein These data support the successful generation of a functionally insuf- (Fig. 1d). Quantitative analysis confirmed a significant increase in ficient ketogenic model. HMGCS2 protein levels at P7 (relative HMGCS2 protein expression levels in E18.5, P7 and adult mice were 0.69 ± 0.03, 1.65 ± 0.06 and Hepatosteatosis rapidly progresses in Hmgcs2 KO mice after 0.66 ± 0.02, respectively (P < 0.0001; Fig. 1e). These data indicate birth. We assessed the impact of ketogenesis during the prenatal that neonatal ketogenesis is enhanced regardless of starvation. period. Crossed Hmgcs2 heterozygote mutants (Hmgcs2+/−) did not demonstrate a significant reduction in the number of pups, and Generation of Hmgcs2 knockout mice. To clarify the role of keto- genetic inheritance at birth was as expected by the Hardy–Weinberg genesis, we generated Hmgcs2 KO mice by using the CRISPR–Cas9 equilibrium (P = 0.69).

Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab 197 Articles NATurE METAbOlISm

a b +/+ +/– –/– Chr3 161 bp 1 Primer F 151 bp

Hmgcs2 locus Exon1 161 bp 2 Primer R 147 bp

c d WT KO

WT KO 50 kDa HMGCS2

α-Tubulin 50 kDa P7 liver (HMGCS2)

e f g P3 whole blood P7 serum P7 liver 2.0 2.5 3 P < 0.0001 P < 0.0001 P < 0.0001 2.0 ) 1.5 ) –1 –1 2 1.5 1.0 1.0 β OHB (a.u.) 1 β OHB (mmol l β OHB (mmol l 0.5 0.5

0 0 0 WT KO WT KO WT KO

Fig. 2 | Ketogenesis and generation of Hmgcs2 KO mice. a, Design of gRNA for generation of Hmgcs2 loss-of-function alleles. A gRNA was designed to target exon 1. The red box indicates protospacer adjacent motif (PAM) sequences. Arrows denote primer sets for PCR. b, Genotyping of the Hmgcs2 locus. Genotyping revealed 10 bp (1) and 14 bp (2) shorter alleles. c, Western blot analysis of HMGCS2 in liver tissue of Hmgcs2 WT and Hmgcs2 KO mice. d, Immunohistochemical analysis of HMGCS2 in P7 livers. Scale bar: 50 µm. e, Whole-blood concentrations of βOHB at P3 (WT, n = 6; KO, n = 8). f, Serum blood concentrations of βOHB in P7 (WT, n = 10; KO, n = 11). g, GC–MS analysis of P7 liver tissue samples (WT, n = 10; KO, n = 11). Results are expressed as dot plots with means ± s.d. Welch’s two-sided t-test was performed (e–g).

The survival rate of Hmgcs2 KO mice was significantly lower than however, no increase in reactive oxygen species levels was observed that of the control, without demonstrating a lethal phenotype or in Hmgcs2 KO hepatocytes (Extended Data Fig. 2h). sex difference was detected in the neonatal period (Extended Data Biochemical testing of P7 serum revealed no significant differ- Fig. 2a–c). Both 15-week-old Hmgcs2 KO mice and WT mice had ences in serum alanine aminotransferase levels and albumin con- low blood levels of ketone bodies under free feeding, and a signifi- centrations between WT and Hmgcs2 KO mice (Fig. 3j,k). cant difference was observed only in the fasted condition (Extended Serum lipid profiles of P7 neonates showed elevated levels of Data Fig. 2d). The surviving 15-week-old Hmgcs2 KO adult mice total and high-density lipoprotein cholesterol in KO mice (Fig. 3l,n). did not show significant differences in body weight; however, the Furthermore, the concentration of low-density lipoprotein choles- food intake volume and the amount of faeces were significantly terol was significantly decreased in Hmgcs2 KO neonates (Fig. 3m). increased (Extended Data Fig. 2e–g). However, triglyceride levels did not significantly differ between WT Next, we focused on neonatal ketogenesis. The gross appearance and KO mice (Fig. 3o). of the liver did not show a significant difference between WT and We performed real-time PCR for fatty acid metabolism-related KO mice at birth (Fig. 3a,d). However, in Hmgcs2 KO mice, the liver genes. Carnitine palmitoyltransferase 1a (Cpt1a) and carnitine pal- grew rapidly, exhibiting a colour change to white with increasing mitoyltransferase 2 (Cpt2), related to the shuttling of acyl-CoA and size (Fig. 3b,c). beta-oxidation-related genes, long-chain acyl-CoA dehydrogenase Furthermore, section staining revealed a drastic progression of (Acadl), very long-chain acyl-CoA dehydrogenase (Acadvl) and hepatosteatosis. Numerous vacuoles accumulated in the cytoplasm acetyl-coenzyme A acyltransferase 2 (Acaa2) were significantly of P3, P5 and P7 Hmgcs2 KO livers (Fig. 3d). Additionally, Oil Red increased in P7 Hmgcs2 KO livers (Fig. 3p). Furthermore, the O staining also identified severe lipid deposition in the P5 Hmgcs2 expression of peroxisomal acyl-coenzyme A oxidase 1 (Acox1) was KO liver (Fig. 2e). In contrast, Sirius Red staining did not reveal also upregulated (Fig. 3p). significant fibrotic changes (Fig. 3f,g). Immunohistochemistry To assess fatty acid oxidation, we prepared primary hepato- for F4/80 demonstrated no remarkable infiltration of inflamma- cyte cultures from the P2 livers of Hmgcs2 WT or KO neonates. tory cells in the Hmgcs2 KO liver (Fig. 3h,i). We assessed reactive Oxygen consumption rates (OCRs) were measured with or without oxygen species levels in isolated Hmgcs2 WT and KO hepatocytes; BSA-conjugated palmitate. In Hmgcs2 WT hepatocytes, palmitate

198 Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab NATurE METAbOlISm Articles administration enhanced both basal and maximal respiration. KO livers; however, fumarate and malate were significantly reduced Conversely, Hmgcs2 KO hepatocytes did not show an increase in (Extended Data Fig. 4c,d). basal respiration, but the maximal OCR was decreased (Extended The mitochondrial appearance and metabolite profiles suggested Data Fig. 2i). that mitochondrial function was impaired. To further validate this These data indicated that the exogenous fatty acid oxidation observation, we compared the OCR between Hmgcs2 WT and capacity was decreased in Hmgcs2 KO hepatocytes. We speculate KO primary hepatocytes (Fig. 4g). Compared to WT hepatocytes, that steatosis results in enhanced fatty acid oxidation as a compen- Hmgcs2 KO hepatocytes showed a significant reduction in basal res- satory mechanism, but the reserve capacity is reduced in Hmgcs2 piration, ATP production and maximal respiration (Extended Data KO neonates. Fig. 4e). These data indicate that aerobic respiration was impaired To confirm whether the phenotype of the Hmgcs2 KO phenotype in Hmgcs2 KO hepatocytes. was attributable to a lack of ketone synthesis in hepatocytes, we gen- Furthermore, we measured the mitochondrial membrane poten- erated an Hmgcs2flox/flox mouse and crossed it with an Alb-cre mouse tial in isolated hepatocytes (Fig. 4h and Extended Data Fig. 4f). (Fig. 3q, Extended Data Fig. 3a–e and Supplementary Tables 4 and Geometrical mean fluorescence intensity (GeoMFI) of JC-1 green 5). As expected, the P5 livers of Alb-cre;Hmgcs2flox/flox mice showed fluorescence, which reflects mitochondrial mass, did not demon- increased fat deposition when compared with that in Hmgcs2flox/flox strate any significant difference between WT and Hmgcs2 KO hepa- mice (Fig. 3r). These findings suggest that insufficient ketogenesis tocytes (Fig. 4i). However, the GeoMFI of JC-1 red fluorescence, in hepatocytes induces a series of phenotypes, as observed in the indicating mitochondrial membrane potential, and the red/green present study. fluorescence were significantly reduced in Hmgcs2 KO hepatocytes (Fig. 4j,k). These data indicate that the mitochondrial membrane Mitochondrial energy production capacity is impaired in Hmgcs2 potential is decreased in Hmgcs2 KO hepatocytes. KO neonates. Histological analysis of Hmgcs2 KO livers resembled The results of this series of analyses indicate that mitochondrial microvesicular steatosis (Fig. 3d). Electron microscopy analysis of function, especially aerobic respiration, is impaired and that the P3 livers revealed that small lipid droplets filled the hepatocyte cyto- energy-producing capacity of mitochondria is disturbed in Hmgcs2 plasm without nuclei displacement in Hmgcs2 KO livers, and accu- KO livers. mulation patterns were consistent with microvesicular steatosis (Fig. 4a). Hmgcs2 KO hepatocytes contained several swollen mitochon- Acetylation of mitochondrial proteins is increased in Hmgcs2 KO dria, with a sparse mitochondrial matrix (Fig. 4b); in contrast, per- neonates. We quantified glycolysis and TCA cycle-related proteins oxisomes were prominent (Extended Data Fig. 4a). These findings using a method of in vitro proteome-assisted multiple-reaction suggest impaired mitochondrial function in Hmgcs2 KO hepatocytes. monitoring for protein absolute quantification (iMPAQT). Heat To confirm mitochondrial dysfunction and assess the impact map analysis demonstrated that proteins involved in the glycolytic of insufficient neonatal ketogenesis on hepatocyte energy metabo- pathway did not show any significant differences (Extended Data lism, liquid chromatography–mass spectrometry-based metabo- Fig. 5a). However, fold changes (KO/WT) of each protein revealed lome analysis was performed using P3 livers (Fig. 4c). Metabolites that mitochondrial proteins were increased in Hmgcs2 KO mice, in the glycolytic pathway, namely glucose-6-phosphate and with a median fold change of 1.14 (n = 23; 25–75% percentile: 1.02– fructose-6-phosphate, were significantly decreased in Hmgcs2 KO 1.23; Extended Data Fig. 5b). mice, whereas acetyl-CoA levels were increased (Fig. 4c). In P7 Despite mitochondrial dysfunction, mitochondrial proteins were Hmgcs2 KO livers, ELISA analysis confirmed the higher concen- increased in Hmgcs2 KO mice. Next, we investigated the involve- tration of acetyl-CoA in P7 Hmgcs2 KO livers (Fig. 4d). A previ- ment of post-translational modifications. We observed an accumu- ous report revealed the accumulation of acetyl-CoA in isolated lation of acetyl-CoA in the Hmgcs2 KO liver (Fig. 4c,d). Typically, mitochondria from Hmgcs2 antisense oligonucleotide-treated acetylation of mitochondrial proteins reduces enzymatic activity15. adult mice10. We confirmed this finding in the livers of Hmgcs2 Next, mitochondrial protein acetylation in Hmgcs2 KO and WT KO neonates. Real-time PCR revealed enhanced upregulation of neonatal livers was examined. beta-oxidation-related genes (Fig. 3p). These findings indicate that At P7, purified mitochondrial proteins were immunoblotted for fatty acid-derived acetyl-CoA accumulates. acetylated lysine (AcK). Fluorescence-based western blotting and In the tricarboxylic acid cycle (TCA) cycle, we observed that quantitative analysis revealed the enhanced acetylation of mito- succinate, fumarate and malate levels were decreased in Hmgcs2 chondrial proteins in Hmgcs2 KO livers (Fig. 5a,b). An immuno- KO despite the accumulation of acetyl-CoA (Fig. 4c). ATP, ADP precipitation assay with an anti-Ack antibody identified increased and total adenylate (Fig. 4e), as well as NADP+ and NADPH, acetylation of succinyl-CoA ligase (Fig. 5c,d) and long-chain were decreased in the P3 Hmgcs2 KO neonatal livers (Fig. 4f). acyl-CoA dehydrogenase (LCAD) in Hmgcs2 KO mitochondrial Furthermore, we measured the metabolites in P7 neonatal livers by proteins (Extended Data Fig. 5c,d). These data demonstrated GC–MS and normalized them by the sample weight or protein con- increased volume and acetylation of mitochondrial proteins in tent, as we noted that fat accumulation influenced sample weights Hmgcs2 KO neonatal livers. in Hmgcs2 KO neonatal livers (Extended Data Fig. 4b). Both nor- Serial changes in protein acetylation at E18.5, P3, P7, P21 and malization methods gave almost the same results. Compared to the adult stages indicated that protein acetylation was prominent in P7 P3 neonatal livers, citrate was found to accumulate in P7 Hmgcs2 neonates (Extended Data Fig. 5e). These data suggest that neonatal

Fig. 3 | Rapid progression of hepatosteatosis in Hmgcs2 KO neonates. a–c, Gross appearance of livers at P0 (a), P3 (b) and P5 (c). Scale bar: 2 mm. d, Serial liver H&E staining at P0, P3, P5 and P7. Scale bar: 20 µm. e, Oil Red O staining of P5 liver sections. Scale bar: 50 µm. f, Sirius Red staining of P7 liver sections. Scale bar: 200 µm. g, Quantitative analysis of Sirius Red-positive area (WT, n = 7; KO, n = 3). h, Immunohistochemistry for F4/80 on P7 liver sections. Scale bar: 100 µm. i, Quantitative analysis of the F4/80-stained area (WT, n = 7; KO, n = 3). j, Serum examination of alanine aminotransferase (ALT) at P7 (WT, n = 9; KO, n = 11). k, Serum examination of albumin (ALB) at P7 (WT, n = 9; KO, n = 11). l, Serum examination of total cholesterol (T-CHO) at P7 (WT, n = 9; KO, n = 11). m, Serum examination of low-density lipoprotein cholesterol (LDL-C) at P7 (WT, n = 9; KO, n = 11). n, Serum examination of high-density lipoprotein cholesterol (HDL-C) at P7 (WT, n = 9; KO, n = 11). o, Serum examination of triglyceride (TG) levels at P7 (WT, n = 9; KO, n = 11). p, Real-time PCR for Cpt1a, Cpt2, Acadl, Acadvl, Acaa2 and Acox1 in P7 livers (WT, n = 8; KO, n = 8). q, Construct of Hmgcs2-floxed mice. r, H&E staining of P5 livers. Scale bar: 50 µm. Results are expressed as dot plots with means ± s.d. Welch’s two-sided t-test was performed (g and i–p).

Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab 199 Articles NATurE METAbOlISm ketogenesis plays a role in preventing hyperacetylation of mito- (Gcn5l1) and lysine acetyltransferases (KATs; Fig. 5e) to assess chondrial proteins. the acetyltransferase-dependent enzymatic acetylation. However, We performed RNA sequencing of P7 mouse livers (WT, we did not observe any significant differences in transcripts n = 4; KO, n = 4) and compared the transcripts of GCN5 like-1 between WT and KO mouse livers. In particular, GCN5L1 protein

a b c

P0 KO WT P3 KO WT P5 KO WT

d e P0 P3 P5 P7 Oil red (P5) WT WT KO KO

f Sirius red (P7) g h F4/80 (P7) i Area (%) Area (%) P = 0.692 4 P = 0.100 15

3 10 2 5 1

0 0 WT KO WT KO WT KO WT KO

j ALT k ALB l T-CHO m LDL-C n HDL-C o TG 100 2.4 200 30 P = 0.0038 150 300 P = 0.1350 P = 0.9040 P = 0.1741 P < 0.0001 P < 0.0001 80 2.2 150 20 100 200 –1 –1 –1 –1

–1 60 –1 2.0 100 IU l 40 g dl mg dl mg dl mg dl 10 50 mg dl 100 20 1.8 50

0 1.6 0 0 0 0 WT KO WT KO WT KO WT KO WT KO WT KO Fatty acid oxidation

p q 2.5 WT KO LoxP LoxP P < 0.0001 Alb-cre Exon 2 2.0 (hepatocyte specific) Hmgcs2 locus P < 0.0001 P < 0.0001 P = 0.0002 P = 0.0002 P = 0.0083 1.5 r Hmgcs2 flox/flox Alb-cre;Hmgcs2 flox/flox

1.0

Relative mRNA expression 0.5

0 CPT1a CPT2 ACADL ACADVL ACAA2 ACOX1

200 Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab NATurE METAbOlISm Articles expression was decreased in Hmgcs2 KO mice compared to that of To confirm mitochondria-specific acetylation, we focused on WT (Extended Data Fig. 5f). Additionally, extra-mitochondrial oxi- isozymes. Isocitrate dehydrogenase (IDH), malate dehydrogenase dation genes, such as ATP citrate synthase (ACLY), acyl-coenzyme (MDH) and methylenetetrahydrofolate dehydrogenase (MTHFD) A synthetase short-chain family member 2 (ACSS2) and Slc25a present both cytoplasmic and mitochondrial isozymes. We com- regulate the concentration of Acetyl-CoA16,17. However, there pared the acetylation status of both isozymes and found that, com- were no significant changes between Hmgcs2 KO and WT livers pared with the cytoplasmic isozymes (IDH1, MDH1 and MDHFD1), (Extended Data Fig. 5g). These data suggest that enzymatic mito- the mitochondrial isozymes (IDH2, MDH2 and MTHFD1L) exhib- chondrial protein acetylation and acetyl-CoA transport from the ited Ack residues in Hmgcs2 KO mice (Fig. 6b). Next, we focused on mitochondrial matrix to the cytoplasm were not enhanced. mitochondrial respiratory-chain complexes. Among 262 detected Sirtuin 3 (Sirt3) is a key molecule involved in mitochondrial lysine residues in complexes I–IV and ATP synthase, 63 (24.1%) protein deacetylation. A recent report revealed that the effect of were highly acetylated in Hmgcs2 KO mice (Fig. 6c). However, only mitochondrial protein hyperacetylation by Sirt3 KO on mitochon- 11 resides (4.2%) showed decreased acetylation. drial bioenergetics was limited18. Furthermore, previous reports Next, we focused on the nuclear proteins. Bar plot analysis of his- have shown that enhanced ketogenesis promotes Sirt3 protein tone protein acetylation showed several sites presenting decreased expression and mitigates the cognitive phenotypes of Sirt3 haploin- acetylation (Extended Data Fig. 6a). Western blotting of total his- sufficiency19. To clarify the effect of Sirt3 on the hyperacetylation of tone proteins revealed reduced acetylation in Hmgcs2 KO mice mitochondrial proteins caused by insufficient ketogenesis, we intro- (Fig. 6d,e). These results suggested that, unlike mitochondrial duced Sirt3-deficient mice (Sirt3+/−) and Sirt3 overexpressing mice proteins, histone proteins had enhanced deacetylation. To address (RosaCAG-Sirt3; Fig. 5f,g). this issue, we compared HDAC expressions and activities. RNA First, we crossed Sirt3 and Hmgcs2 heterozygotes and assessed sequencing of P7 mouse livers (WT, n = 4; KO, n = 4) did not the acetylation status in P7 neonatal livers. Compared with WT neo- show the significant differences in transcripts of class 1 HDACs nates (Hmgcs2+/+;Sirt3+/+), Hmgcs2 heterozygous mice (Hmgcs2+/−) (HDAC1, HDAC2, HDAC3 and HDAC8; Extended Data Fig. 6b). demonstrated a significant increase in mitochondrial protein Furthermore, protein expression of HDAC1 did not show the sig- acetylation (Fig. 5h). However, Sirt3 heterozygotes did not increase nificant changes (Extended Data Fig. 6c). mitochondrial protein acetylation in Hmgcs2 heterozygotes Therefore, we measured HDAC activity using a fluorometric (Hmgcs2+/−;Sirt3+/−). assay. We prepared nuclear extracts from P7 livers of Hmgcs2 WT Next, we assessed the impact of Sirt3 overexpression. A con- and KO mice and incubated extracts with or without 1 µM tricho- struct of RosaCAG-Sirt3 mice was designed such that Sirt3 and GFP statin A (TSA) for 20 min and relative fluorescence intensity was were linked by the T2A sequence and expressed simultaneously measured 30 times every 2 min. Compared to WT, nuclear extracts (Fig. 5f,g). Functionally, we confirmed a significant reduction in from Hmgcs2 KO mice showed significantly increased fluores- mitochondrial protein acetylation in the adult heart of RosaCAG-Sirt3 cence intensity (Fig. 6f). Furthermore, βOHB treatment suppressed mice (Extended Data Fig. 5h). However, Sirt3 overexpression did HDAC activity in both Hmgcs2 WT and KO nuclear extracts in a not alter protein acetylation in Hmgcs2 KO neonatal livers at P7 concentration-dependent manner (Extended Data Fig. 6d). These (Fig. 5i). These results indicate that the enhanced acetylation of data suggest that insufficient ketogenesis enhances the enzymatic mitochondrial proteins owing to insufficient ketogenesis cannot be activity of nuclear HDACs by the loss of the endogenous HDAC entirely regulated by Sirt3. inhibitor βOHB. Conversely, chromatin immunoprecipitation coupled with quan- Insufficient ketogenesis causes mitochondrial protein-specific titative PCR of acetylated histone 3 and 4 confirmed that the pro- acetylation. Based on the analyses performed so far, Hmgcs2 KO moter regions of fatty acid oxidation were highly acetylated in both mice showed hyperacetylation of mitochondrial proteins. To char- P7 Hmgcs2 WT and KO livers (Extended Data Fig. 6e). A series of acterize protein acetylation in the context of insufficient neonatal results indicate that region-specific acetylation is maintained while ketogenesis, acetylome analysis was performed. overall HDAC activity is enhanced in the Hmgcs2 KO liver. Samples of P7 livers (WT, n = 6; KO, n = 3) were pooled for β-hydroxybutyrylation also regulates gene expression20. The analysis. Liquid chromatography–tandem mass spectrometry iden- expression of Per1, which is known to be upregulated by βOHB, was tified 5,038 modified acetylated sites in 1,634 proteins (Fig. 6a). undoubtedly suppressed in our data. However, other target genes, Among the 1,634 proteins, 810 identified acetylated proteins exhib- including Ppargc1b and Cpt1a, did not demonstrate the same ten- ited more than a twofold difference in expression between Hmgcs2 dency (Extended Data Fig. 6f). WT and KO mice. As expected, ontology analysis revealed that acetylation-related proteins were significantly altered. Next, we Mitochondrial protein acetylation disturbs sequential enzy- tested 369 proteins exhibiting increased acetylation in Hmgcs2 matic reaction in the TCA cycle. Thus far, our analysis revealed KO mice. Terms related to mitochondria (mitochondrion, mito- that the acetylation of mitochondrial proteins was enhanced during chondrial inner membrane and mitochondrial matrix) were highly insufficient ketogenesis. To assess whether mitochondrial protein ranked keywords (Table 1). These findings support the idea that acetylation directly impacts sequential enzymatic reactions in neo- hyperacetylation occurred specifically in the mitochondria of natal hepatocytes, we prepared primary hepatocytes from P1 WT Hmgcs2 KO mice. neonates, followed by treatment with control siRNA or Hmgcs2

Fig. 4 | Mitochondrial dysfunction in Hmgcs2 KO neonatal livers. a, Electron microscopy of P3 hepatocytes. Red arrows represent lipid droplets. Nc, nucleus. Scale bar: 10 µm. b, Electron microscopy of mitochondria in P3 hepatocytes. Red arrows indicate mitochondria. Lip, lipid droplet. Scale bar: 1 µm. c, Metabolome analysis of the glycolytic pathway and the TCA cycle (WT, n = 5; KO, n = 5); *P < 0.05, **P < 0.01, ****P < 0.0001. d, Concentrations of acetyl-CoA measured by ELISA (WT, n = 8; KO, n = 10). e, Concentrations of total adenylate, AMP, ADP and ATP in P3 livers (WT, n = 5; KO, n = 5). f, Concentrations of NAD+, NADH, NADP+ and NADPH in P3 livers (WT, n = 5; KO, n = 5). ND, not detected. g, OCR analysis using hepatocyte primary culture (WT, n = 5; KO, n = 4); ****P < 0.0001. FCCP; carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. h, Histograms of fluorescence intensity of JC-1 green and JC-1 red (Hmgcs2 WT, n = 6; KO, n = 5). i, GeoMFI of JC-1 green in isolated P5 hepatocytes in h. j, GeoMFI of JC-1 red in h. k, The red/green fluorescence ratio in h. Results are expressed as bar plots with means ± s.d. Welch’s two-sided t-test (c–f and i–k) or two-way ANOVA and Holm–Sidak’s multiple-comparisons test were applied (g).

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siRNA for 72 h. Hmgcs2 siRNA partially suppressed Hmgcs2 pro- βOHB, isocitrate, fumarate and malate (Fig. 7a). These findings tein expression and enhanced protein acetylation (Extended Data indicate that the mitochondrial protein acetylation and distur- Fig. 7a–c). Metabolome analysis revealed decreased production of bance of sequential enzymatic reactions in the TCA cycle are not

a b c GLUCOSE

HK2 250 200 ** 150

Glucose-6-phosphate nmol/g 100 50

150 0 GPI WT KO Nc Lip 100 ** nmol/g Nc Nc 50 Fructose-6-phosphate

0 WT KO PFKP 40 Lip 30

20 Fructose-1,6-bisphosphate nmol/g WT KO WT KO 10

0 WT KO

d e Acetyl-CoA 4,000 Dihydroxyacetone 3-phosphoglyceraldehyde P = 0.0372 WT 400 phosphate GAPDH P = 0.0350 KO 3,000 1,3-bisphosphoglycerate

300 –1 –1 2,000 P = 0.1215 PGK1 60 200 Glycerol-3-phosphate 50

nmol g P = 0.0278

P = 0.0482 nmol/g pmol µ l 3-phosphoglycerate 1,000 40 100 800

600 ** 30 WT KO 0 0 400 nmol/g WT KO Total AMP ADP ATP 200 2-phosphoglycerate 0 WT KO ENO1 40 30

f 20 0.008 Phosphoenolpyruvate nmol/g P = 0.0548 WT 10 0 KO PKM2 WT KO 0.006 LDHA Pyruvate Lactate

0.00010 0.004 0.00008 * a.u. P = 0.0071 0.00006 A.U. 0.00004 Acetyl-CoA βOHB 0.00002

2,000 0.002 0.00000 P = 0.0144 WT KO **** 1,500

1,000 nmol/g

ND 500 0.000 0 NAD+ NADH NADP+ NADPH WT KO CS Oxaloacetate 360 Citrate 340 320 g OCR MDH2 ACO2 nmol/g 300 1,500 280

260 ** WT KO 500 WT **** 1,000

Malate nmol/g **** 500 KO **** 400 0 WT KO D-isocitrate 400 FH –1 300 300 ** IDH3A * 200 * nmol/g Fumarate IDH2 IDH3B 100

0 200 WT KO pmol min α-ketoglutarate SDHA 100 SDHB Succinate OGDH DLST 0 DLD Rotenone 900 Base Oligomycin FCCP 800 * Antimycin 700

nmol/g 600

500

400 WT KO

h i j k JC-1 green JC-1 red Red/green 2.0 2.0 2.0 WT P = 0.0105 P = 0.0245

1.5 P = 0.3287 1.5 1.5

KO 1.0 1.0 1.0 Ratio Cell number Cell number

Relative GeoMFI 0.5 0.5 0.5 –100 0 100 103 104 105 –200 0 200 103 104 105 JC-1 green JC-1 red 0 0 0 WT KO WT KO WT KO

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a b c IB: anti-Suclg2 d IB: anti-AcK AcK TP Total IP: anti-AcK Total IP: anti-AcK AcK/TP

2.0 P = 0.0115 250 kDa WT KO WT KO WT KO WT KO 150 kDa 100 kDa 1.5

50 kDa 1.0 a.u.

37 kDa 0.5

WT KO 0 WT KO

e Hmgcs2/Rplp1 KAT/Rplp1 f 10 0.25 Exon 1 CAG Sirt3 t2a EGFP Exon 2

P = 0.9298 Rosa26 locus

8 P = 0.0008 0.20 WT KO g 6 0.15 Sirt3 –/– WT RosaCAG-Sirt3 P = 0.8190 37 kDa P = 0.3432 P = 0.2784 P = 0.6658 4 0.10 P = 0.2634 Fold change

Fold change SIRT3

P = 0.3507 P = 0.3242 2 0.05 P = 0.9459 P = 0.1111 50 kDa P = 0.7538 P = 0.9033 TP

0 0 37 kDa Hmgcs2 Kat1 Kat4 Kat5 Kat7 Kat8 gcn5l1 Kat2a Kat2b Kat3a Kat3b Kat6a Kat6b KATs

h i –/– –/– CAG-Sirt3 AcK/TP Hmgcs2 Hmgcs2 ;Rosa AcK/TP 2.0 P = 0.0261 2.0 P = 0.147 Hmgcs2 +/+ Hmgcs2 +/– Hmgcs2 +/– 75 kDa +/+ +/+ +/– Sirt3 Sirt3 Sirt3 1.5 1.5

AcK 1.0 1.0 a.u. a.u. P = 0.0094 P = 0.6450 AcK 50 kDa 0.5 TP 37 kDa 0.5

0 0 TP Hmgcs2 +/+ +/– +/– Hmgcs2 –/– –/– 25 kDa CAG-Sirt3 – + Sirt3 +/+ +/+ +/– GFP 25 kDa Rosa

Fig. 5 | Acetylation of mitochondrial proteins in Hmgcs2 KO neonatal livers. a, Fluorescence-based western blot analysis for AcK (green) and TP (red; n = 4 each). b, Quantitative analysis of protein acetylation expression adjusted by TP (n = 4 each). Welch’s two-sided t-test was performed. c, Immunoprecipitation assays using P7 livers: IP, anti-Suclg2; IB, anti-Ack. A red arrowhead indicates the Suclg2 protein. d, Immunoprecipitation assays on P7 livers: IP, anti-Ack; IB, anti-Ack. e, Expression of Hmgcs2, GCN5L1 and lysine acetyltransferase family transcripts (Kat), adjusted by Rplp1 in P7 neonatal livers (Hmgcs2: WT, n = 4; KO, n = 4). Welch’s two-sided t-test was performed. f, Construct of RosaCAG-Sirt3. g, Purified proteins of Sirt3-/−, WT and RosaCAG-Sirt3 were blotted using anti-Sirt3. The red arrowhead represents the extrinsic expression AU5-conjugated Sirt3. h, Fluorescence-based western blot analysis for AcK (green) and TP (red) among Hmgcs2+/+;Sirt3+/+, Hmgcs2+/−;Sirt3+/+ and Hmgcs2+/−;Sirt3+/− neonatal livers at P7 (n = 3 each). One-way ANOVA and Tukey’s multiple-comparisons test were applied. i, Fluorescence-based western blot analysis for Ack (green), TP (red) and GFP (green) between Hmgcs2−/− and Hmgcs2–/–;RosaCAG-Sirt3 WT neonatal livers at P7 (n = 4 each). Welch’s two-sided t-test was performed. Results are expressed as means ± s.d. attributed to compensatory machinery, but rather to the primary We co-incubated isolated mitochondria with the 13C-labelled glu- result of insufficient ketogenesis. tamate and fluorocitrate to block the reaction between citrate and Furthermore, we compared protein acetylation between mito- isocitrate to avoid the incorporation of unlabelled metabolites and chondria and peroxisomes. Purified mitochondria demonstrated acetyl-CoA (Fig. 7d). Thereafter, we measured 13C-labelled fuma- substantially increased acetylation in P7 Hmgcs2 KO mice. However, rate (Extended Data Fig. 7d). the peroxisome fraction did not show a prominent difference in We compared isolated mitochondria from Hmgcs2 WT or protein acetylation between WT and KO mice (Fig. 7b,c). These KO mouse livers (Extended Data Fig. 7e). After incubating with data indicate that insufficient ketogenesis causes hyperacetylation 13C-labelled glutamate and fluorocitrate for 120 min, measured in a mitochondria-specific manner. 13C-labelled fumarate was significantly reduced in Hmgcs2 KO liv- Previous reports revealed that acetylation of each enzyme, which ers than in those of WT mice (Extended Data Fig. 7f). This finding is related to the TCA cycle, has an inhibitory effect on its enzy- indicated that intrinsic mitochondrial protein acetylation had an matic activities21–23. However, the impact of mitochondrial acety- inhibitory effect on sequential enzymatic reactions from glutamate lation on sequential enzymatic reactions is not fully understood. to fumarate. To address this issue, we compared the carbon isotope uptake We then examined the effect of inducing the acetylation of in the TCA cycle, using isolated mitochondria. For this, we mitochondrial proteins. We confirmed that incubation of isolated implemented a systematic experiment reported by Zheng et al.24. mitochondria with 1.5 mM acetyl-CoA for 120 min could induce

Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab 203 Articles NATurE METAbOlISm

a b

P7 livers Cytoplasmic Mitochondrial

IDH1 IDH2 K233 K243 K45/K48 K67 K69/K80 K106 Digestion K155 K166 K180 K193 WT × 6 KO × 3 K256 K272 K275 K280

K282 K384 K400 K442

MDH1 MDH2 K118 K298 K91 K185 K239 K296 Immunoprecipitation Analyse eluted K301/K307 K314 K324 peptide fraction by anti-AcK K329/K335 by LC–MS/MS

MTHFD1 MTHFD1L K56 K58 K71 K302 K91 K111 K167/K188 Identified motifs: K309 K473 K553 K866 K310 K351 K515 K595 5,038 acetylated sites in 1,634 proteins K762 K802 K919

Increased Unchanged

c

l ll lll lV ATP

NDUFA2 NDUFS2 SDHA UQCRB COX3 ATP5A1 NDUFA5 NDUFS3 COX4I1 NDUFA6 NDUFS4 UQCRC1 ATP5B NDUFA7 NDUFS6 COX5A NDUFA8 NDUFS7 UQCRC2 COX5B ATP5C NDUFA10 NDUFV1 COX6B1 ATP5D ATP5E NDUFA11 NDUFV2 SDHB ATP5F1 NDUFA12 NUEM UQCRFS1 COX7B NDUFAF6 TYKY UQCRHL COX7C ATP5H NDUFB3 UQCRQ NDUFB4 ATP5I ATP5J ATP5L NDUFB9 ATP5O NDUFB10 NDUFB11 Increased Unchanged Decreased ATP5S ATP8 ATPAF1 ATPAF2

d e f 5 × 107 WT vs KO, P < 0.001

7 WT KO AcK/TP 4 × 10 1.5 P = 0.0476 7 15 kDa 3 × 10 1.0 2 × 107 AcK a.u. 0.5 RFU (counts) 7 15 kDa 1 × 10 0 WT KO TP (histone) 0 0 4 8 12 16 20 24 28 32 36 40 44 48 52 56 60 Time (min) WT KO WT with TSA KO with TSA

Fig. 6 | Acetylome analysis reveals mitochondria-specific acetylation in Hmgcs2 KO neonatal livers. a, Flow chart for the acetylome analysis. b, Comparison of acetylation status between cytoplasmic and mitochondrial isozymes. Red circles represent more than a twofold increase in acetylation in Hmgcs2 KO mice. c, Acetylation status of the mitochondrial respiratory complex. Red circles represent more than a twofold increase in acetylation in Hmgcs2 KO mice. Blue circles represent more than a twofold decrease in acetylation in Hmgcs2 KO mice. I, NADH: ubiquinone oxidoreductase; II, succinate dehydrogenase; III, coenzyme Q: cytochrome c oxidoreductase; IV, cytochrome c oxidase; ATP, ATP synthase. d, Western blot analysis of Ack and TP (histone; n = 4 each). e, Quantitative analysis of protein acetylation expression adjusted for TP (n = 4 each). Welch’s two-sided t-test was performed. f, HDAC fluorometric assay. Relative fluore­ scence intensity with excitation at 360 nm and emission at 450 nm of 50-µg nuclear extracts from P7 mouse livers was measured with or without 1 µM TSA. WT (blue line; n = 6); WT with TSA (blue dotted line; n = 6); Hmgcs2 KO (red line; n = 6); Hmgcs2 KO with TSA (red dotted line; n = 6); RFU, relative fluore­ scence units. All lines represent the mean with standard error. Repeated measures two-way ANOVA and Tukey’s multiple-comparisons test were performed.

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Table 1 | Gene ontology analysis of differentially expressed proteins between wild-type and Hmgcs2 knockout mice (800) Term Count Fold enrichment P value False discovery rate Acetylation 536 4.86 2.68 × 10−275 3.66 × 10−272 Mitochondrion 299 8.01 1.18 × 10−196 1.60 × 10−193 Transit peptide 212 11.78 7.17 × 10−175 9.77 × 10−172 Phosphoprotein 485 1.81 5.76 × 10−57 7.85 × 10−54 Oxidoreductase 121 5.37 2.25 × 10−53 3.07 × 10−50 Mitochondrion inner membrane 75 8.37 5.56 × 10−47 7.57 × 10−44 Fatty acid metabolism 46 10.52 1.16 × 10−33 1.58 × 10−30 Isopeptide bond 117 3.48 1.74 × 10−32 2.37 × 10−29 Methylation 117 3.46 3.43 × 10−32 4.68 × 10−29 Peroxisome 38 10.07 5.13 × 10−27 6.98 × 10−24 Gene ontology analysis of hyperacetylated proteins in Hmgcs2 KO mice (367) GO:0005739 mitochondrion 270 8.41 8.51 × 10−207 1.11 × 10−203 GO:0005743 mitochondrial inner membrane 95 13.15 6.69 × 10−79 8.71 × 10−76 GO:0005759 mitochondrial matrix 64 18.24 3.91 × 10−62 5.09 × 10−59 GO:0005777 peroxisome 30 12.27 3.71 × 10−23 4.83 × 10−20 GO:0070062 extracellular exosome 123 2.46 1.34 × 10−22 1.74 × 10−19 GO:0043209 myelin sheath 34 9.49 1.69 × 10−22 2.19 × 10−19 GO:0070469 respiratory chain 21 19.4 1.40 × 10−20 1.82 × 10−17 GO:0042645 mitochondrial nucleoid 18 20.96 2.78 × 10−18 3.62 × 10−15 GO:0005753 mitochondrial proton-transporting ATP synthase complex 13 38.69 2.41 × 10−17 3.14 × 10−14 GO:0000786 nucleosome 21 10.42 9.87 × 10−15 1.29 × 10−11 the acetylation of mitochondrial proteins (Extended Data Fig. 7g). steatosis within 1 week of birth. The major difference between Using this condition, we co-incubated isolated mitochondria with adult and neonatal livers is the number of mitochondria. The num- 13C-labelled glutamate and fluorocitrate in the presence or absence ber of mitochondria in neonates is lower than that in adults, and of acetyl-CoA for 120 min. At the start of incubation (0 min), there mitochondrial capacities are limited in the neonatal liver. These was no significant difference. However, after 120 min of incubation, immature characteristics might increase the susceptibility to uptake of 13C-labelled fumarate was significantly reduced in mito- carbon stress. chondria with acetyl-CoA (Fig. 7e). These findings indicate that the Perinatal environmental maternal stress results in liver steato- acetylation of mitochondrial proteins leads to reduced efficiency of sis in offspring26. Furthermore, epigenetic modifications and the sequential enzymatic reactions in the TCA cycle. gut microbiome are affected by a maternal high-fat diet27,28. Our study revealed unique metabolic characteristics during the neonatal Discussion period. These features may be related to the pathogenesis of condi- In the present study, we clarified the role of neonatal ketogenesis in tions induced by perinatal environmental stress. mitochondrial energy metabolism. In Hmgcs2 KO mice, insufficient Notably, the accumulation of acetyl-CoA is a characteristic neonatal ketogenesis caused significant hepatosteatosis within sev- finding of this model. However, the TCA cycle metabolites and eral days of birth, with no observed embryonic lethality observed. energy substrates, such as ATP and NADP+/NADPH, were Furthermore, livers isolated from neonatal Hmgcs2 KO mice dem- decreased in Hmgcs2 KO mice. Despite the restricted energy pro- onstrated restricted energy production capacity, with significantly duction capacity, quantitative proteomics verified the increased enhanced mitochondrial protein-specific acetylation (Fig. 7f). protein volume and specific mitochondrial protein acetylation in The perinatal environment dramatically changes owing to the Hmgcs2 KO mice. The localization and dynamics of acetyl-CoA transition from placental nutrition to lactation. Maternal milk should be analysed to clarify the mechanism underlying acetyl-CoA contains various types of fatty acids14, which impart acute carbon accumulation. stress to neonates. Neonatal ketogenesis prevents the accumulation Lysine acetylation modulates protein functions not only in of acetyl-CoA by converting excess acetyl-CoA into ketone bod- histones but also in non-histone proteins, including mitochon- ies. We revealed that insufficient ketogenesis during the neonatal drial proteins29,30. For instance, acetylation of MDH2 and K239 period results in hyperacetylation of mitochondrial proteins, which is enhanced in Hmgcs2 KO mice. A previous report revealed specifically induces mitochondrial dysfunction. Therefore, neonatal that an acetyl mimic protein of MDH2 (K239Q) demonstrated ketogenesis is not safe for mitochondrial energy metabolism. decreased enzymatic activity22. Overall, the acetylation of mito- Previous reports have revealed that a high-fat diet induces hepa- chondrial proteins is presumably a negative-feedback response tosteatosis in the presence of insufficient ketogenesis by utilizing an against the overproduction of acetyl-CoA and is known as an antisense oligonucleotide in adult mice10,11. A noteworthy finding in inhibitory marker15. However, it has been reported that the hyper- our study is the specific phenotypic timing in Hmgcs2 KO neonates. acetylation of mitochondrial proteins has a limited effect on mito- Conventional methods, using a high-fat diet to induce hyperacety- chondrial bioenergetics18. Our findings support an inhibitory lation of mitochondrial proteins, require long-term high-fat diet effect of intrinsic mitochondrial protein acetylation on sequential feeding25. However, Hmgcs2 KO neonates developed microvesicular enzymatic reactions in the TCA cycle. A more comprehensive and

Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab 205 Articles NATurE METAbOlISm

a βOHB Citrate Isocitrate 2KG Fumarate Malate 1.5 P = 0.0339 1.5 1.5 P = 0.0217 1.5 P = 0.0531 1.5 P = 0.0243 1.5 P = 0.0316 NS 1.0 1.0 1.0 1.0 1.0 1.0 a.u. a.u. a.u. a.u. a.u. a.u. 0.5 0.5 0.5 0.5 0.5 0.5

0 0 0 0 0 0 Ctrl Ctr l Ctrl Ctrl Ctrl Ctrl Hmgcs2 Hmgcs2 Hmgcs2 Hmgcs2 Hmgcs2 Hmgcs2 siRNA siRNA siRNA siRNA siRNA siRNA

b Ab 25,000g d Acetyl-CoA 20 min

600g 10 min Oxaloacetate* Citrate* Glutamate*

Mitochondria Nuclei (+debris) Peroxisomes Fluorocitrate

c Malate* Mito Per α-ketoglutarate* WT KO WT KO

Fumarate* Succinyl-CoA*

13 150 kDa Succinate* * C-labelled

e AcK [13C]fumarate 5 × 104 50 kDa AcCoA(–) AcCoA(+) P < 0.0001 37 kDa 4 × 104

3 × 104

4 50 kDa Intensity 2 × 10 HMGCS2

4 75 kDa 1 × 10 P = 0.7633 CATALASE 0 Before After SUCLG2 37 kDa

f Neonatal liver (insufficient ketogenesis)

Milk Milk Glucose Glucose

Glycolysis Cytosol Glycolysis Cytosol Fatty acid Fatty acid

Acetyl-CoA Ketones Acetyl-CoA Ketones Beta-oxidation HMGCS2 Beta-oxidation HMGCS2 Ac Ac Ac Ac Ac Ac mtP Ac Ac Ac mtP Ac Ac Ac Ac Ac mtP Ac mtP mtP TCA mtP TCA Ac mtP mtP Ac Nucleus Mitochondria mtP Mitochondria mtP Nucleus

Fig. 7 | Mitochondrial protein acetylation suppresses sequential enzymatic reactions in the TCA cycle. a, GC–MS-based concentrations of βOHB, citrate, isocitrate, alpha-ketoglutarate (2KG), fumarate and malate in primary hepatocytes treated with control (ctrl) siRNA or Hmgcs2 siRNA (n = 3 each). An unpaired two-sided t-test was performed. Results are expressed as means ± s.d. b, Schema of mitochondria and peroxisome purification. c, Western blot analysis of Ack, Hmgcs2, catalase (peroxisome marker) and Suclg2 (mitochondrial marker) in P7 liver tissues from Hmgcs2 WT and Hmgcs2 KO mice. d, Schema of the experiment; 13C-labelled glutamate was converted to 13C-labelled fumarate by sequential enzymatic reactions in the TCA cycle. e, 13C-labelled fumarate was significantly reduced in acetylated mitochondria (0 min; n = 3 each; 120 min, without acetyl-CoA; n = 4, with acetyl-CoA; n = 6), AcCoA; acetyl-CoA. Two-way ANOVA and Holm–Sidak’s multiple-comparisons test were performed. f, Summarized illustration of this work. NS, not significant. detailed analysis of the effects of acetylation on enzyme sequence NAD+-dependent deacetylases, SIRTs, are well-known enzymes reactions along with energy metabolism will reveal detailed for the deacetylation of mitochondrial proteins31. Among the mechanisms. three mitochondria-localized isoforms (Sirt3, Sirt4 and Sirt5),

206 Nature Metabolism | VOL 3 | February 2021 | 196–210 | www.nature.com/natmetab NATurE METAbOlISm Articles

Sirt3 plays a prominent role in the deacetylation of mitochondrial Collection Tubes (Terumo) and were centrifuged at 3,000g at room temperature proteins22,25,32,33. Our study revealed that both the loss of function (RT) for 10 min. and overexpression of Sirt3 did not significantly affect the hyper- Real-time PCR. Total RNA was prepared using a Qiagen RNeasy Fibrous Mini acetylation of mitochondrial proteins during insufficient keto- Kit according to the manufacturer’s protocol, and complementary DNA was genesis. These data indicate that the regulation of mitochondrial produced with the PrimeScript RT–PCR Kit (Takara). Quantitative real-time PCR acetylation by ketone body formation and Sirt3-dependent deacety- was performed utilizing SsoAdvanced Universal SYBR Green Supermix according lation are regulated by different factors or mechanisms. to the manufacturer’s instructions (Bio-Rad). Transcript expression levels were determined as the number of transcripts relative to those of 18S or 36B4. All Compared to deacetylation, the acetylation mechanisms are specific primers are provided in the Supplementary Table 6. not comprehensively elucidated. Although the enzyme-specific acetylation of mitochondrial proteins has been reported34,35, Droplet-digital PCR. For the quantification of mitochondrial copy number, a non-enzymatic protein acylation mechanisms are also gaining ddPCR assay was used in accordance with a previously described protocol46. Total momentum36,37. The mitochondrial matrix is a favourable location DNA was extracted from liver tissue samples using the QIAamp DNA Mini kit (Qiagen) according to the manufacturer’s instructions. DNA concentrations were for non-enzymatic protein acetylation owing to the alkaline pH measured with a NanoDrop 2000 Spectrophotometer (Thermo Fisher Scientific). environment and mitochondrial acetyl-CoA pool29,38. Furthermore, We prepared 100 ng and 0.1 ng DNA as templates for genomic and mtDNA, the increased acetyl-CoA concentration enhances lysine acetylation respectively. Each PCR reaction contained 10 µl of 2× ddPCR supermix for of mitochondrial proteins both in vitro and in vivo39,40. Acetylation probes without UTP (Bio-Rad), 1 µl of 20× PrimeTime standard quantitative of mitochondrial proteins strongly modulates their function; hence, PCR assay (Integrated DNA Technologies), template DNA, and water was added to 20 µl. Droplets were generated using the QX200 automated droplet generator further functional analysis is warranted. (Bio-Rad). Amplification was performed under the following cycling conditions: Moreover, we detected a paradoxical reduction in histone pro- 95 °C for 10 min; 40 cycles of 94 °C for 30 s followed by 60 °C for 60 s; 98 °C for tein acetylation. βOHB is known as an intrinsic HDAC inhibi- 10 min; and 4 °C hold. The droplets were analysed immediately on the QX200 tor41. Furthermore, citrate concentrations did not differ between droplet reader (Bio-Rad). We used specific primers of ND1 for mtDNA and specific primers of Ago1 for genomic DNA. Primer sequences are provided in WT and Hmgcs2 KO mice despite the significant accumulation of Supplementary Table 6. acetyl-CoA. Conversion of acetyl-CoA and oxaloacetate to citrate is an essential reaction for acetyl-CoA transfer to the cytoplasm and Western blot analysis. Western blot was performed with an SDS–PAGE system. nuclei. Thus, a reduced concentration of βOHB and acetyl-CoA Blotted gels (Bio-Rad, TGX Stain-Free FastCast Acrylamide Solutions) were concentration in the nuclei may be involved in the decreased acety- transferred to PVDF membranes using the Trans-Blot Turbo Transfer System (Bio-Rad). Membranes were immersed in 5% skimmed milk containing blocking lation of histone proteins. solution and reacted with specific primary antibodies. All utilized antibodies are In the current study, we revealed the essential role of ketogenesis listed in Supplementary Table 6. For chemiluminescence western blotting, the in the metabolic adaptation of the neonatal liver. Ketogenesis regu- ECL Prime Western Blotting Detection Reagent (GE Healthcare) was used. For lates the concentration of acetyl-CoA and influences mitochondrial fluorescence-based western blotting, goat anti-rabbit IgG IRDye 800CW (LI-COR protein acetylation. Neonatal ketogenesis exerts protective effects Bioscience Systems) was used as the secondary antibody. Images were obtained by Image analyzer ODYSSEY Fc Imaging System (LI-COR Bioscience Systems). on neonatal hepatocytes by preventing hyperacetylation induced by the carbon stress triggered by breastfeeding. Capillary electrophoresis time-of-flight mass spectrometry analysis. Metabolome measurements were carried out through a facility service at Human Methods Metabolome Technologies. Briefly, frozen P3 liver tissue samples were plunged Experimental animals. Jcl:ICR mice were used to assess neonatal ketogenesis. into 50% acetonitrile/Milli-Q water containing internal standards (304–1002, Hmgcs2 deletion mutants and Hmgcs2-foxed mice were generated in this study. Human Metabolome Technologie) at 0 °C to inactivate enzymes. The tissue was Alb-cre mice were provided by Y.S. and K.Y. RosaCAG-Sirt3 mice were generated and homogenized three times at 1,500 r.p.m. for 120 s with a tissue homogenizer (Micro provided by Y.K.K., K. Tonami and H.K. Heterozygous mutants were maintained Smash MS100R, Tomy Digital Biology), and the homogenate was centrifuged at on a mixed C57BL/6J background. All mice were housed in a controlled 2,300g at 4 °C for 5 min. Subsequently, 800 µl upper aqueous layer was centrifugally environment at 23 °C ± 2 °C, with a relative humidity of 50–60%, and under a filtered through a Millipore 5-kDa cut-off filter at 9,100g and 4 °C for 120 min to 12-h light–dark cycle. CLEA Rodent Diet CE-2 was provided. Genotypes were remove proteins. The filtrate was centrifugally concentrated and resuspended in determined by PCR on tail/fngertip-derived DNA using specifc primers provided 50 µl Milli-Q water for capillary electrophoresis–mass spectrometry analysis. in Supplementary Table 6. Embryonic and neonatal ages were determined by timed mating with the day of the plug being E0.5. All procedures were performed in Gas chromatography–mass spectrometry analysis. A frozen P7 liver tissue accordance with the Kumamoto University animal care guidelines (approval no. sample was plunged into Milli-Q water/methanol/chloroform (at a ratio of 1:2.5:1) M30-040), which conform to the US National Institutes of Health Guide for the containing internal standards (isopropylmalic acid). The sample was homogenized Care and Use of Laboratory Animals (publication no. 85-23, revised 1996). at 2,300 r.p.m. for 30 s with a bead beater-type homogenizer (Beads crusher µT-12, Titec). The homogenized sample was shaken at 1,200 r.p.m. and 37 °C for 30 min Generation of Hmgcs2-floxed mice. We targeted exon 2 of Hmgcs2 as the deletion and centrifuged at 16,000g and 4 °C for 3 min. Subsequently, 225 µl of the upper target. Because Cys166 of exon 2 around the acetyl-CoA binding site is one of the aqueous layer was transferred to a new tube. The remaining sample was mixed critical catalytic residues and it is required for catalysis5,42. with 200 µl Milli-Q water and centrifuged again at 16,000g and 4 °C for 3 min. The Floxed mice were generated using the CRISPR–Cas9 system and upper water layer was mixed with the aqueous layer. The solution was evaporated PITCh (Precise Integration into Target Chromosome) system, which uses using a centrifugal evaporator (DNA120OP230, Thermo Fisher Scientific) and microhomology-mediated end joining-directed plasmid as a donor43–45. lyophilized (FD-1-84A, FST). The sample was derivatized with methoxamine/ The cryopreserved C57BL/6 zygotes were warmed and cultured for 3–3.5 h. pyridine and N-methyl-N-trimethylsilyl-trifluoroacetamide and analysed on We performed microinjection of CRIS-PITCh (v2) donor vector and Cas9 a Shimadzu TQ8050 GC–MS/MS. The chromatograms and mass spectra were ribonucleoprotein containing two gene-specific gRNAs and one generic gRNA analysed utilizing the GC–MS solution software v4.50 (Shimadzu). Compounds targeting the donor vector for generation of 455-bp exon 2 flanked by the loxP were determined with the Smart Metabolites Database v2 (Shimadzu). sequences. After microinjection, the surviving zygotes were transferred to pseudopregnant ICR female mice. We analysed twelve founder mice and selected Histological analysis. H&E and Sirius Red staining were performed using two knock-in mutants. Because we used plasmid donor DNA for microinjection, paraffin-embedded sections (10 µm; K.I. Stainer). For Oil Red staining, we investigated whether the vector backbone was accidentally integrated into the cryosections (10 µm) were stained with Oil Red O solution. Moreover, genome. In the two knock-in founders, described above, PCR amplification of immunohistochemical analysis was performed on paraffin-embedded sections the backbone sequence was performed to detect genomic integrants. In one of the (10 µm). All utilized antibodies are listed in Supplementary Table 6. knock-in founders (5), the vector fragment was amplified; thus, we used the other founder (12) for the establishment of the floxed line and subsequent analyses. Transmission electron microscopy. Transmission electron microscopy analysis was performed by Tokai Electron Microscopy. The samples were fixed with 2% Measurement of beta-hydroxybutyrate. For whole-blood and serum monitoring, paraformaldehyde and 2% glutaraldehyde in 0.1 M phosphate buffer (pH 7.4) at we used FreeStyle Precision Neo and FreeStyle Precision Blood β-ketone test strips 4 °C for 2 h. Fixed samples were dehydrated in graded ethanol solutions (50%, 70%, (Abbott). Whole-blood samples were obtained by tail cutting or by decapitation. 90% and 100%), infiltrated with propylene oxide twice for 30 min each, and placed For serum assessment, whole-blood samples were collected in Capillary Blood into a 70:30 mixture of propylene oxide and resin (Quetol-812; Nisshin EM) for 1 h.

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After infiltration, samples were volatilized overnight, transferred to a fresh 100% Mitochondrial isolation. Mitochondria were isolated from cells using a resin, and polymerized at 60 °C for 48 h. The embedded polymerized samples were Mitochondria Isolation Kit for Tissue (Abcam, ab110168). All procedures were cut into ultra-thin sections at 70 nm with a diamond knife using an ultramicrotome performed at 4 °C. The suspended isolation buffer contained protease inhibitor (Ultracut UCT, Leica), and the sections were mounted on copper grids. They were cocktail (Merck), phenylmethylsulphonyl fluoride (Merck), 1 µM TSA (Fuji Film) stained with 2% uranyl acetate at RT for 15 min and washed with distilled water and 1 mM nicotinamide (Fuji Film). Protein concentration was determined with followed by secondary staining with lead stain solution (Sigma-Aldrich) at RT for the BCA method. 3 min. The grids were observed under a transmission electron microscope (JEM- 1400Plus, JEOL) at an acceleration voltage of 100 kV. Digital images were taken Isolation of mitochondria and peroxisomes. We used the Mitochondria Isolation with a CCD camera (EM-14830RUBY2, JEOL). Kit for mouse tissue (Miltenyi Biotec, 130-096-946). P7 liver samples (50–150 mg) were minced by razor blade and homogenized with five strokes in a Potter Blood panel analysis. Blood panel analysis was carried out through a Kumamoto homogenizer at 1,600 r.p.m. Homogenate was centrifuged at 600g for 10 min at Mouse Clinic service. Whole-blood samples were collected in Capillary Blood 4 °C. Supernatants were incubated with anti-TOM22 microbeads and purified Collection Tubes (Terumo) and centrifuged at 3,000g and RT for 10 min. Serum according to manufacturer’s instructions. As the negative fraction of anti-TOM22 was analysed by BioMajesty JCA-BM6050 (JEOM). microbeads contained peroxisomes, we ultracentrifuged the sample at 25,000g and 4 °C for 20 min. Mitochondrial and peroxisomal pellets were suspended by M-Per Measurement of acetyl-CoA. Measurement of acetyl-CoA concentration was with protease inhibitor (Merck). performed using PicoProbe Acetyl-CoA Fluorometric Assay Kit (BioVision) on frozen P7 liver tissue samples in accordance with the manufacturer’s protocol. Isotope-labelling assay. We implemented the method used previously by Zhang et al.24. The isolated mitochondrial pellet was suspended in a buffer containing Measurement of oxygen consumption rate. Primary hepatocytes were harvested 0.01 M Tris-MOPS, 1% vol/vol EGTA/Tris, 0.2 M sucrose, 0.3 mM NAD, 0.4 mM 13 from P2 or P3 neonatal livers. Pre-perfusion solution (0.5 mM EGTA and 10 mM ADP, 1.0 mM fluorocitrate, 20 mM [ C5]glutamate, with pH adjusted to 7.4. HEPES with HBSS) and collagenase solution (5 mM CaCl2, 10 mM HEPES, 0.5 g Suspended mitochondria were incubated with or without 1.5 mM Acetyl-CoA l−1 collagen type I and 50 µg ml−1 trypsin inhibitor) was perfused from a left at 37 °C for 2 h. Then, samples were immediately frozen in liquid nitrogen and ventricle47. Liver was removed and dissected with scissors. Samples were incubated analysed by GC–MS. at 37 °C for 5 min, then centrifuged through a 100-µm filter at 300g for 5 min. Pellets were resuspended by culture medium (10−6 M dexamethasone, 10% FBS, Poly(A) RNA sequencing. Total RNA from the mouse liver was extracted using NEAA, ITS, penicillin–streptomycin and low-glucose DMEM) and plated type-I the RNeasy Mini Kit (Qiagen). Purification of mRNA was performed using a collagen-coated XF24 Cell Culture Microplates. NEBNext Poly(A) mRNA Magnetic Isolation Module (NEB). A complementary XF24 Extracellular Flux Analyzer (Seahorse Bioscience) was used for the DNA library for sequencing was synthesized using a NEBNext Ultra DNA Library analysis48. During the real-time measurement, inhibitors of respiratory-chain Prep Kit for Illumina (NEB) and was sequenced on a NextSeq 500 sequencer components, oligomycin (1 μg ml−1, the complex V inhibitor), FCCP (1 μM, (Illumina) with 75-bp single-end reads. Obtained Fastq files were subjected to respiratory uncoupler), rotenone and antimycin A (100 nM and 10 μM, respectively; adaptor trimming and quality check using Trim Galore (Babraham Bioinformatics; the complex I and III inhibitors) were administered sequentially. OCR for basal https://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) and then respiration minus proton leak (rate before FCCP injection minus non-mitochondrial mapped to reference mouse genome mm10 using STAR50. Gene expression levels respiration) was determined as ATP consumption. Maximal respiration was were calculated as transcripts per million using RSEM51. determined by the rate during FCCP administration minus the rate after rotenone/ antimycin were added. When fatty acid oxidation was assessed, we mixed 0.23 mM Chromatin immunoprecipitation. Chromatin immunoprecipitation was palmitate-BSA or BSA alone just before the real-time measurement of OCR. performed as previously described with some modifications52. Liver tissue was homogenized in PBS using a Dounce homogenizer followed by fixation in 1% Cell sorting and flow cytometric analysis. We used either a FACS Aria III or formaldehyde. Nuclei were isolated in cell lysis buffer (5 mM PIPES (pH 8.0), FACS CANTO II (both BD Biosciences) for cell sorting and flow cytometric 85 mM KCl and 0.5 % NP-40) and were resuspended in SDS lysis buffer (50 mM analyses49. The mitochondrial membrane potential and mitochondrial superoxide Tris-HCl (pH 8.0), 10 mM EDTA and 1 % SDS). To obtain soluble chromatin level of indicated cells were determined according to the manufacturer’s fragments, nuclei were sonicated using a Sonifier SFX (Branson) and Picoruptor instructions using MitoProbe JC-1 Assay kit and MitoSOX Red Mitochondrial (Diagenode). The soluble chromatin fraction was incubated with anti-acetylated Superoxide Indicator (both Thermo Fisher Scientific), respectively. Cells were histone H3 (Millipore, 06-599), anti-acetylated histone H4 (Millipore, 06-866) stained with 2 µM JC-1 or 5 µM MitoSOX Red for 30 min. After staining, or normal rabbit IgG (Santa Cruz, sc-2027) at 4 °C overnight, and then pulled fluorescence intensity was determined using a flow cytometer. down by Protein A/G Agarose Beads (Millipore). Purified DNA was subjected to quantitative PCR using the primers listed in Supplementary Table 6. Protein absolute quantification assay. To simultaneously perform global analysis of protein expression with absolute quantification, we used in vitro Acetylome analysis. The extent of lysine acetylation was determined using proteome-assisted multiple-reaction monitoring for protein absolute quantification the AcetylScan platform (PTMScan Acetyl-Lysine Motif Antibody, 13416; Cell (iMPAQT; Kyusyu Pro Search). Briefly, frozen tissue samples were crushed by a Signaling Technology). P7 pooled liver tissue samples (WT, n = 6; Hmgcs2 KO, bead shocker (Yasuikikai) and lysed with 150 μl lysis buffer (a solution containing n = 3; final weight of pooled samples was approximately 0.5 g) were used. Obtained 2% SDS, 7 M urea, and 100 mM Tris-HCl (pH 8.8)). The samples were diluted peptides were loaded directly onto a 50 cm × 100 µm PicoFrit capillary column with an equal volume of water, and their protein concentrations were determined packed with C18 reversed-phase resin. The column was developed with a 90-min with BCA assay (Bio-Rad). To block Cys residues, 200 μg of protein samples were linear gradient of acetonitrile in 0.125% formic acid delivered at 280 nl min−1. Mass treated with 5.0 mM Tris(2-carboxyethyl)phosphine hydrochloride (Thermo spectrometry parameter settings were as follows: run time of 108 min, MS1 scan Fisher Scientific) for 30 min at 37 °C, and alkylation with 10 mM 2-iodoacetamide range of 300.0–1500.00 and top 20 MS/MS (minimum signal: 500; isolation width: (Sigma) was performed for 30 min at RT. Subsequently, samples were subjected 2.0; normalized collision energy: 35.0; activation-Q: 0.250; activation time: 20.0; to acetone precipitation. The resulting pellet was resuspended in 100 μl digestion lock mass: 371.101237; charge state rejection: enabled; charge state 1+: rejected; buffer (0.5 M triethylammonium bicarbonate). Each sample was digested with dynamic exclusion: enabled; repeat count: 1; repeat duration: 35.0; exclusion list lysyl-endopeptidase (2 μg; Wako) for 3 h at 37 °C. Next, the samples were further size: 500; exclusion duration: 40.0; exclusion mass width: relative to mass; and digested with trypsin (4 μg; Thermo Fisher Scientific) for 14 h at 37 °C. The exclusion mass width: 10 ppm). Fold change was calculated from the provided resulting cell digests were freeze dried and labelled with the mTRAQ Δ0 overall quantitative results sorted by protein type, and readings above or below (light) reagent. Each sample was spiked with synthetic peptides for internal a twofold change were determined as significant differences. Acetylated proteins standard, causing reductive alkylation and mTRAQ Δ4 (heavy) labelling. were classified by gene ontology term analysis using the DAVID software (http:// Synthetic peptides used human and mouse homology regions from the iMPAQT david.abcc.ncifcrf.gov/) with the options UP_KEYWORDS and CC_DIRECT. database (https://impaqt.jpost.org/iMPAQT/). The samples were subjected to reversed-phase liquid chromatography followed by multiple-reaction Statistics and reproducibility. Experiments were performed more than three monitoring analysis. times. Allele frequencies were determined by direct gene counting, and genotype distributions were checked for departure from the Hardy–Weinberg equilibrium Histone deacetylase fluorometric assay. Nuclear extracts were isolated from using Pearson’s chi-squared test. Data for normally distributed continuous variables neonatal livers using a Nuclear Extract Kit (40010, Active Motif). Next, 50 µg of are expressed as means ± s.d. Three group comparisons were analysed by one-way nuclear extracts was used for measurement of deacetylase activity using CycLex ANOVA and Holm–Sidak’s multiple-comparisons test. Two-group comparisons HDACs Deacetylase Fluorometric Assay Kit v2 (CY-1150V2; CycLex, MBL). were carried out with the Welch’s two-sided t-test. Statistical analyses were Relative fluorescence intensity with excitation at 360 nm and emission at 450 nm performed with Prism 7 and 8 (GraphPad). was detected by SpectraMax i3x (Molecular devices). We adopted a two-step method: after pre-incubation with TSA or beta-hydroxybutyric acid at 37 °C for Reporting Summary. Further information on research design is available in the 20 min, the reaction was initiated and relative fluorescence intensity was measured. Nature Research Reporting Summary linked to this article.

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Data availability 29. Narita, T., Weinert, B. T. & Choudhary, C. Functions and mechanisms of We submitted RNA sequencing data to the Gene Expression Omnibus under non-histone protein acetylation. Nat. Rev. Mol. Cell Biol. 20, 156–174 (2019). accession GSE18853. The dataset of GC–MS analysis and iMAPQ analysis 30. Choudhary, C., Weinert, B. T., Nishida, Y., Verdin, E. & Mann, M. Te and other data that support the findings of this study are available from the growing landscape of lysine acetylation links metabolism and cell signalling. corresponding author upon request. The dataset of acetylome analysis is available Nat. Rev. Mol. Cell Biol. 15, 536–550 (2014). as source data. Source data are provided with this paper. 31. Schwer, B. & Verdin, E. Conserved metabolic regulatory functions of sirtuins. Cell Metab. 7, 104–112 (2008). Received: 27 April 2020; Accepted: 7 January 2021; 32. Newman, J. C., He, W. & Verdin, E. Mitochondrial protein acylation and intermediary metabolism: regulation by sirtuins and implications for Published online: 18 February 2021 metabolic disease. J. Biol. Chem. 287, 42436–42443 (2012). 33. Parodi-Rullan, R. M., Chapa-Dubocq, X. R. & Javadov, S. Acetylation of References mitochondrial proteins in the heart: the role of SIRT3. Front. Physiol. 9, 1. Krebs, H. A. Te regulation of the release of ketone bodies by the liver. 1094 (2018). Adv. Enzyme Regul. 4, 339–354 (1966). 34. Fan, J. et al. Tyr phosphorylation of PDP1 toggles recruitment between 2. Arima, Y. et al. Myocardial ischemia suppresses ketone body utilization. ACAT1 and SIRT3 to regulate the pyruvate dehydrogenase complex. Mol. Cell J. Am. Coll. Cardiol. 73, 246–247 (2019). 53, 534–548 (2014). 3. McGarry, J. D. & Foster, D. W. Regulation of hepatic fatty acid oxidation and 35. Wang, L. et al. GCN5L1 modulates cross-talk between mitochondria and cell ketone body production. Annu. Rev. Biochem. 49, 395–420 (1980). signaling to regulate FoxO1 stability and gluconeogenesis. Nat. Commun. 8, 4. Rojas-Morales, P., Tapia, E. & Pedraza-Chaverri, J. Beta-hydroxybutyrate: a 523 (2017). signaling metabolite in starvation response? Cell. Signal. 28, 917–992 (2016). 36. Paik, W. K., Pearson, D., Lee, H. W. & Kim, S. Nonenzymatic acetylation of 5. Shimazu, T. et al. SIRT3 deacetylates mitochondrial 3-hydroxy- histones with acetyl-CoA. Biochim. Biophys. Acta 213, 513–522 (1970). 3-methylglutaryl CoA synthase 2 and regulates ketone body production. Cell 37. Wagner, G. R. & Hirschey, M. D. Nonenzymatic protein acylation as a carbon Metab. 12, 654–661 (2010). stress regulated by sirtuin deacylases. Mol. Cell 54, 5–16 (2014). 6. Puchalska, P. & Crawford, P. A. Multi-dimensional roles of ketone bodies in 38. Casey, J. R., Grinstein, S. & Orlowski, J. Sensors and regulators of intracellular fuel metabolism, signaling and therapeutics. Cell Metab. 25, 262–284 (2017). pH. Nat. Rev. Mol. Cell Biol. 11, 50–61 (2010). 7. Chapman, M. J., Miller, L. R. & Ontko, J. A. Localization of the enzymes of 39. Davies, M. N. et al. Te acetyl group bufering action of carnitine ketogenesis in rat liver mitochondria. J. Cell Biol. 58, 284–306 (1973). acetyltransferase ofsets macronutrient-induced lysine acetylation of 8. Dashti, N. & Ontko, J. A. Rate-limiting function of 3-hydroxy- mitochondrial proteins. Cell Rep. 14, 243–254 (2016). 3-methylglutaryl coenzyme A synthase in ketogenesis. Biochem. Med. 22, 40. Wagner, G. R. & Payne, R. M. Widespread and enzyme-independent 365–374 (1979). Nε-acetylation and Nε-succinylation of proteins in the chemical conditions of 9. Hegardt, F. G. Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase: a the mitochondrial matrix. J. Biol. Chem. 288, 29036–29045 (2013). control enzyme in ketogenesis. Biochem. J. 338, 569–582 (1999). 41. Shimazu, T. et al. Suppression of oxidative stress by beta-hydroxybutyrate, an 10. d’Avignon, D. A. et al. Hepatic ketogenic insufciency reprograms hepatic endogenous histone deacetylase inhibitor. Science 339, 211–214 (2013). glycogen metabolism and the lipidome. JCI Insight 3, e99762 (2018). 42. Lowe, D. M. & Tubbs, P. K. 3-hydroxy-3-methylglutaryl-coenzyme A synthase 11. Cotter, D. G. et al. Ketogenesis prevents diet-induced fatty liver injury and from ox liver. Purifcation, molecular and catalytic properties. Biochem. J. hyperglycemia. J. Clin. Invest. 124, 5175–5190 (2014). 227, 591–599 (1985). 12. Cotter, D. G., Ercal, B., d’Avignon, D. A., Dietzen, D. J. & Crawford, P. A. 43. Nakade, S. et al. Microhomology-mediated end-joining-dependent integration Impact of peripheral ketolytic defciency on hepatic ketogenesis and of donor DNA in cells and animals using TALENs and CRISPR/Cas9. Nat. gluconeogenesis during the transition to birth. J. Biol. Chem. 288, Commun. 5, 5560 (2014). 19739–19749 (2013). 44. Nakagawa, Y. et al. Culture time of vitrifed/warmed zygotes before 13. Decaux, J. F., Robin, D., Robin, P., Ferre, P. & Girard, J. Intramitochondrial microinjection afects the production efciency of CRISPR–Cas9-mediated factors controlling hepatic fatty acid oxidation at weaning in the rat. FEBS knock-in mice. Biol. Open 6, 706–713 (2017). Lett. 232, 156–158 (1988). 45. Sakuma, T., Nakade, S., Sakane, Y., Suzuki, K. T. & Yamamoto, T. 14. Ward Platt, M. & Deshpande, S. Metabolic adaptation at birth. Semin. Fetal MMEJ-assisted gene knock-in using TALENs and CRISPR–Cas9 with the Neonatal Med. 10, 341–350 (2005). PITCh systems. Nat. Protoc. 11, 118–133 (2016). 15. Baeza, J., Smallegan, M. J. & Denu, J. M. Mechanisms and dynamics of 46. Takihara, Y. et al. High mitochondrial mass is associated with reconstitution protein acetylation in mitochondria. Trends Biochem. Sci. 41, 231–244 (2016). capacity and quiescence of hematopoietic stem cells. Blood Adv. 3, 16. Mews, P. et al. Acetyl-CoA synthetase regulates histone acetylation and 2323–2327 (2019). hippocampal memory. Nature 546, 381–386 (2017). 47. Inoue, T. & Matsumoto, K. Techniques for Illustrated Animal Experiments, Vol. 17. Sivanand, S., Viney, I. & Wellen, K. E. Spatiotemporal control of Acetyl-CoA 24 (Kyoritsu Shuppan, 1981). metabolism in chromatin regulation. Trends Biochem. Sci. 43, 61–74 (2018). 48. Hino, S. et al. FAD-dependent lysine-specifc demethylase-1 regulates cellular 18. Fisher-Wellman, K. H. et al. Respiratory phenomics across multiple models of energy expenditure. Nat. Commun. 3, 758 (2012). 49. Umemoto, T., Hashimoto, M., Matsumura, T., Nakamura-Ishizu, A. & Suda, protein hyperacylation in cardiac mitochondria reveals a marginal impact on 2+ bioenergetics. Cell Rep. 26, 1557–1572 (2019). T. Ca –mitochondria axis drives cell division in hematopoietic stem cells. J. 19. Cheng, A. et al. SIRT3 haploinsufciency aggravates loss of GABAergic Exp. Med. 215, 2097–2113 (2018). interneurons and neuronal network hyperexcitability in an Alzheimer’s 50. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, disease model. J. Neurosci. 40, 694–709 (2020). 15–21 (2013). 20. Xie, Z. et al. Metabolic regulation of gene expression by histone lysine 51. Li, B. & Dewey, C. N. RSEM: accurate transcript quantifcation from beta-hydroxybutyrylation. Mol. Cell 62, 194–206 (2016). RNA-seq data with or without a reference genome. BMC Bioinformatics 12, 21. Finley, L. W. et al. Succinate dehydrogenase is a direct target of sirtuin 3 323 (2011). deacetylase activity. PLoS ONE 6, e23295 (2011). 52. Anan, K. et al. LSD1 mediates metabolic reprogramming by glucocorticoids 22. Hebert, A. S. et al. Calorie restriction and SIRT3 trigger global during myogenic diferentiation. Nucleic Acids Res. 46, 5441–5454 (2018). reprogramming of the mitochondrial protein acetylome. Mol. Cell 49, 186–199 (2013). Acknowledgements 23. Someya, S. et al. Sirt3 mediates reduction of oxidative damage and The authors thank Y. Kimura, M. Nagahiro and S. Tokunaga for their excellent technical prevention of age-related hearing loss under caloric restriction. Cell 143, assistance throughout the experiments. The Kumamoto University School of Medicine 802–812 (2010). Core Laboratory for Medical Research and Education provided support for ddPCR, 24. Zhang, Y. et al. Protein–protein interactions and metabolite channelling in GC–MS and microscopic analysis. Y. Tanoue of the International Research Center for the plant tricarboxylic acid cycle. Nat. Commun. 8, 15212 (2017). Medical Sciences performed the section electron microscopic examination. T. Keida 25. Hirschey, M. D. et al. SIRT3 defciency and mitochondrial protein (Kumamoto Mouse Clinic, Kumamoto University) performed a blood panel analysis. T. hyperacetylation accelerate the development of the metabolic syndrome. Motoyoshi (K.I. Stainer) provided excellent sections for histological analysis. Support Mol. Cell 44, 177–190 (2011). for the iMPAQT analysis was provided by Kyushu Pro Search. M. P. Stokes and K. Abell 26. Brumbaugh, D. E. & Friedman, J. E. Developmental origins of nonalcoholic (Cell Signaling Technology) performed the acetylome analysis. This study was supported fatty liver disease. Pediatr. Res. 75, 140–147 (2014). by a grant-in-aid for Scientific Research (19K08520, 17K16014 and 18K08110) from the 27. Soderborg, T. K. et al. Te gut microbiota in infants of obese mothers Ministry of Education, Culture, Sports, Science and Technology of Japan, the grant for increases infammation and susceptibility to NAFLD. Nat. Commun. 9, Basic Research of the Japanese Circulation Society (2018), a grant from the Sumitomo 4462 (2018). Foundation (2018), a grant from the Takeda Foundation (2019), a grant from the Kanae 28. Ma, J. et al. High-fat maternal diet during pregnancy persistently alters the Foundation (2019), a grant from the Japan Foundation for Applied Enzymology (2019), ofspring microbiome in a primate model. Nat. Commun. 5, 3889 (2014). a grant from the Ono Medical Research Foundation (2019), a Kumamoto University

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Challenging Research Projects (2019) and a grant for Research on Development of New Additional information Drugs (16769865) from the Japan Agency for Medical Research and Development. Extended data is available for this paper at https://doi.org/10.1038/s42255-021-00342-6. Supplementary information The online version contains supplementary material Author contributions available at https://doi.org/10.1038/s42255-021-00342-6. Y.A., Y.N., T.T. and K. Tsujita conceived and designed the experiments. Y.A., Y.N., T.T., T.I., T. Yamada, S. Hanada, S. Hino and T.U. performed the experiments. Y.A., Y.N., Correspondence and requests for materials should be addressed to Y.A. T.T., T.I., T. Yamada, S.H., M.N., T.U., T. Suda, T.W., K. Nagaoka, Y.T., T.N., S.A., E.Y., Peer review information Nature Metabolism thanks Iain Scott, Heng Zhu and the other, Y.I., K.S., K.K., K.M., K. Nishiyama and K. Tsujita analysed the data. Y.A., Y.N., T.T., S. anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Hino, M.N., S. Hanada, T. Sakuma., T.Y., Y.K.K., K. Tonami, H.K., Y.S., K.Y., K.M., K. Handling Editor: George Caputa. Nishiyama and N.N. contributed materials/analysis tools. Y.A., Y.N., T.T., T. Suda., T.N., Reprints and permissions information is available at www.nature.com/reprints. S.A., K.M. and K. Tsujita wrote the paper. Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in Competing interests published maps and institutional affiliations. The authors declare no competing interests. © The Author(s), under exclusive licence to Springer Nature Limited 2021

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Extended Data Fig. 1 | (Related to Fig. 2): The sequencing analyses of Hmgcs2-targeted mice. a, Sanger sequencing results of two lines (#9 and #10) of Hmgcs2 KO heterozygote mice. b, Alignments with the reference sequence of Hmgcs2. Green box represents the first ATG (start codon). Blue box represents the second ATG.

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Extended Data Fig. 2 | See next page for caption.

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Extended Data Fig. 2 | (Related to Fig. 3): Phenotype of Hmgcs2 KO mice. a-c: Kaplan-Meier analysis of Hmgcs2 WT and KO of all (a), male (b), and female (c) mice. d,e: Concentration of β-hydroxybutyrate (d), Body weight (e), food intake (f), and feces (g) of 15 week-old Hmgcs2 WT and KO mice. The body weight of WT and Hmgcs2 KO mice does not differ significantly. However, when fed a normal diet, the food intake and feces volume increase significantly in Hmgcs2 KO mice (WT n = 7, KO n = 7). Two-way ANOVA analysis and Sidak’s multiple comparison test were applied (d). Welch’s two-sided t-tests were performed (e-f). h: Reactive oxygen species (ROS) level in postnatal day 3 (P3) isolated hepatocytes. Wt; Hmgcs2 WT, KO; Hmgcs2 KO, GeoMFI; geometrical mean fluorescence intensity. Welch’s two-sided t-test was performed. i: Oxygen consumption rate with/without palmitate. BSA; bovine serum albumin, FCCP; Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone. Two-way ANOVA analysis and Sidak’s multiple comparison test were applied. All results are expressed as means ± standard deviation (SD).

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Extended Data Fig. 3 | (Related to Fig. 3): Generation of floxed mice at the Hmgcs2 locus. a, Schematic illustration to generate a floxed allele at the Hmgcs2 locus, mediated by the CRIS-PITCh (v2) system. Two gene-specific gRNAs were designed upstream and downstream of exon 2. A PITCh donor plasmid was designed to carry two loxP sites flanking exon 2 and 50-bp homology arms. Arrows indicate the primer sets for PCR. b, Sequence data (sense strand) of region ‘B’. Gray area is the loxP sequence. c, Sequence data (antisense strand) of region ‘C’. Gray area is the loxP sequence. d,e, Genotyping of Hmgcs2+/+, Hmgcs2flox/flox, and Alb-cre;Hmgcs2 flox/+ mice.

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Extended Data Fig. 4 | (Related to Fig. 4): Phenotype in the liver of Hmgcs2 KO. a, Section electron microscopy of postnatal day 3 (P3) hepatocytes. Red arrows represent peroxisomes. Scale bar = 0.5 µm. b, Protein content is represented as weight percent (%). WT; Hmgcs2 WT, n = 7, 13.1 ± 0.82%, KO; Hmgcs2 KO, n = 9, 9.10 ± 0.59%. c, Gas chromatography-mass spectrometry (GC-MS) based concentrations of citrate, alpha-ketoglutarate, fumarate, and malate, adjusted by each sample weight. Hmgcs2 WT (n = 6), Hmgcs2 KO (n = 8). d, GC-MS based concentrations of citrate, alpha-ketoglutarate, fumarate, and malate, adjusted by each protein content. Hmgcs2 WT (n = 6), Hmgcs2 KO (n = 8). e, Basal respiration, ATP production, and maximal respiration of primary hepatocytes from P3 Hmgcs2 WT and KO neonates (Related to Fig. 4g). f, Gating strategy of flow cytometry (Related to Fig. 4h). All results are expressed as means ± standard deviation (SD). Welch’s two-sided t-tests were performed (c-e).

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Extended Data Fig. 5 | See next page for caption.

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Extended Data Fig. 5 | (Related to Fig. 5): Mitochondrial protein acetylation in Hmgcs2 KO livers. a, Heatmap for iMPAQT analysis (WT = 3, KO = 2). Green characters represent mitochondrial proteins. b, Protein fold changes between Hmgcs2 KO and WT mice (Related to Fig. 5a). c, Immunoprecipitation assays on P7 livers: IP, anti-acetylated lysine (AcK); IB, anti-LCAD. Red arrowheads indicate LCAD protein. LCAD, long-chain acyl-CoA dehydrogenase. d, Immunoprecipitation assays on postnatal day 7 (P7) livers: IP, anti-LCAD; IB, anti-acetylated lysine (left), anti-LCAD (right). Red arrowheads indicate LCAD protein. 800 µg purified mitochondrial proteins were used for immunoprecipitation assays. e, Serial changes in protein acetylation in the liver. Whole protein (10 µg) from WT livers (E18.5, P3, P7, P21, and Adult) was blotted using the anti-acetylated lysine antibody. (AcK; acetylated lysine). f, Western blot analysis for GCN5L1, UQCRFS1, and HMGCS2 between P7 Hmgcs2 wild type and knockout livers (n = 4 each). Welch’s two-sided t-test was performed. g, Expressions of Acly, Acss2, and Slc25a1 mRNA, adjusted by Rplp1 in P7 neonatal livers. (Hmgcs2 WT, n = 4, KO, n = 4). Welch’s two-sided t-test was performed. Results are expressed as means ± standard deviation (SD). h, Western blot analysis for AcK and total protein (TP) between RosaCAG-Sirt3 and wild-type adult heart (n = 4 each). Welch’s two-sided t-test was performed.

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Extended Data Fig. 6 | See next page for caption.

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Extended Data Fig. 6 | (Related to Fig. 6): Characteristics of histone protein acetylation in Hmgcs2 KO mice. a, Bar plots of fold changes in histone protein acetylation. b, Expression of Hdac1, Hdac2, Hdac3, and Hdac8 transcripts, adjusted by Rplp1 in P7 neonatal livers. (Hmgcs2 WT, n = 4, KO, n = 4). Welch’s t-test was performed. c, Western blot analysis for HDAC1, HMGCS2, and βACTIN between P7 Hmgcs2 wild type and knockout livers (n = 4 each). Welch’s t-test was performed. d, Histone deacetylases (HDACs) fluorometric assay with β-hydroxybutyrate (βOHB). Relative fluorescent intensity with excitation at 360 nm and emission at 450 nm of 50 µg nuclear extracts from postnatal day 7 mice livers were measured. Before the measurements, nuclear extracts were incubated with 5 mM 3OHB (n = 4), 50 mM 3OHB(n = 4), 500 mM 3OHB(n = 4), or 1 µM trichostatin A (n = 4) for 20 minutes at 37 °C. RFU, relative fluorescent intensity. All lines represent the mean with standard error. After 8 minutes, significant differences were observed in all treatment groups in both WT and KO compared with control (p < 0.0001). Repeated measures two-way ANOVA and Dunnett’s multiple comparisons tests were performed. e, Chip-qPCR analysis of promoter and distal regions in CPT1A and ACADL genes in P7 liver tissue of Hmgcs2 WT and Hmgcs2 KO mice (WT = 5, KO = 5). Two-way ANOVA results are presented. f, Expressions of Per1, Ppargc1b, and Cpt1a mRNA, adjusted by Rplp1 in P7 neonatal livers. (Hmgcs2 WT, n = 4, KO, n = 4). Welch’s two-sided t-test was performed. All results are expressed as means ± standard deviation (SD).

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Extended Data Fig. 7 | See next page for caption.

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Extended Data Fig. 7 | (Related to Fig. 7): In vitro assay for acetylation of mitochondrial proteins. a, Western blot analysis of primary hepatocytes for Hmgcs2, treated with siCtrl or siHmgcs2 (n = 3 each). Welch’s two-sided t-test was performed. b. Immunofluorescent analysis of primary hepatocytes for Hmgcs2, treated with siCtrl or siHmgcs2. c, Western blot analysis of primary hepatocytes for acetylated lysine, treated with siCtrl or siHmgcs2 (n = 3 each). Welch’s two-sided t-test was performed. AcK; acetylated lysine. d, Time-course curve of 13C-labeled fumarate (n = 3 at each time point). Results are expressed as means ± standard deviation (SD). e, Western blot analysis of HMGCS2 in isolated mitochondria from P7 Hmgcs2 WT and Hmgcs2 KO livers. f, 13C-labeled fumarate is significantly reduced in Hmgcs2 KO mitochondria. (0 min; n = 3 each; 120 min; n = 5 each). Two-way ANOVA and Sidak’s multiple comparison test were performed. g, Co-incubation with acetyl-CoA enhances mitochondrial protein acetylation. Western blot analysis of acetylated lysine in isolated mitochondria with/without acetyl-CoA (n = 4, each). *Welch’s two-sided t-test was performed.

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