. 2024 Jan;34(1):13-30.

doi: 10.1038/s41422-023-00864-6. Epub 2024 Jan 2.

Hypoxia induces mitochondrial protein lactylation to limit oxidative phosphorylation

Yunzi Mao^#¹, Jiaojiao Zhang^#¹, Qian Zhou^#¹, Xiadi He^#^{1

2

3}, Zhifang Zheng¹, Yun Wei^{1

2

3}, Kaiqiang Zhou¹, Yan Lin^{1

4

5}, Haowen Yu¹, Haihui Zhang¹, Yineng Zhou¹, Pengcheng Lin⁶, Baixing Wu⁷, Yiyuan Yuan^{1

4}, Jianyuan Zhao^{1

4}, Wei Xu^{8

9

10}, Shimin Zhao^{11

12

13}

Affiliations

¹ The Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Laboratory of Metabolic Remodeling and Health, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital of Fudan University, and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
² Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
³ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
⁴ NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Shanghai, China.
⁵ Shanghai Fifth People's Hospital of Fudan University, Fudan University, Shanghai, China.
⁶ Key Laboratory for Tibet Plateau Phytochemistry of Qinghai Province, College of Pharmacy, Qinghai University for Nationalities, Xining, Qinghai, China.
⁷ Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, RNA Biomedical Institute, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China.
⁸ The Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Laboratory of Metabolic Remodeling and Health, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital of Fudan University, and Institutes of Biomedical Sciences, Fudan University, Shanghai, China. xuwei_0706@fudan.edu.cn.
⁹ NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Shanghai, China. xuwei_0706@fudan.edu.cn.
¹⁰ Shanghai Fifth People's Hospital of Fudan University, Fudan University, Shanghai, China. xuwei_0706@fudan.edu.cn.
¹¹ The Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Laboratory of Metabolic Remodeling and Health, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital of Fudan University, and Institutes of Biomedical Sciences, Fudan University, Shanghai, China. zhaosm@fudan.edu.cn.
¹² NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Shanghai, China. zhaosm@fudan.edu.cn.
¹³ Key Laboratory for Tibet Plateau Phytochemistry of Qinghai Province, College of Pharmacy, Qinghai University for Nationalities, Xining, Qinghai, China. zhaosm@fudan.edu.cn.

^# Contributed equally.

PMID: 38163844
PMCID: PMC10770133
DOI: 10.1038/s41422-023-00864-6

Hypoxia induces mitochondrial protein lactylation to limit oxidative phosphorylation

Yunzi Mao et al. Cell Res. 2024 Jan.

. 2024 Jan;34(1):13-30.

doi: 10.1038/s41422-023-00864-6. Epub 2024 Jan 2.

Authors

Affiliations

¹ The Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Laboratory of Metabolic Remodeling and Health, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital of Fudan University, and Institutes of Biomedical Sciences, Fudan University, Shanghai, China.
² Department of Cancer Biology, Dana-Farber Cancer Institute, Boston, MA, USA.
³ Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA, USA.
⁴ NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Shanghai, China.
⁵ Shanghai Fifth People's Hospital of Fudan University, Fudan University, Shanghai, China.
⁶ Key Laboratory for Tibet Plateau Phytochemistry of Qinghai Province, College of Pharmacy, Qinghai University for Nationalities, Xining, Qinghai, China.
⁷ Guangdong Provincial Key Laboratory of Malignant Tumor Epigenetics and Gene Regulation, Guangdong-Hong Kong Joint Laboratory for RNA Medicine, RNA Biomedical Institute, Medical Research Center, Sun Yat-Sen Memorial Hospital, Sun Yat-Sen University, Guangzhou, Guangdong, China.
⁸ The Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Laboratory of Metabolic Remodeling and Health, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital of Fudan University, and Institutes of Biomedical Sciences, Fudan University, Shanghai, China. xuwei_0706@fudan.edu.cn.
⁹ NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Shanghai, China. xuwei_0706@fudan.edu.cn.
¹⁰ Shanghai Fifth People's Hospital of Fudan University, Fudan University, Shanghai, China. xuwei_0706@fudan.edu.cn.
¹¹ The Obstetrics & Gynecology Hospital of Fudan University, Shanghai Key Laboratory of Metabolic Remodeling and Health, State Key Laboratory of Genetic Engineering, School of Life Sciences, Children's Hospital of Fudan University, and Institutes of Biomedical Sciences, Fudan University, Shanghai, China. zhaosm@fudan.edu.cn.
¹² NHC Key Lab of Reproduction Regulation (Shanghai Institute of Planned Parenthood Research), Shanghai, China. zhaosm@fudan.edu.cn.
¹³ Key Laboratory for Tibet Plateau Phytochemistry of Qinghai Province, College of Pharmacy, Qinghai University for Nationalities, Xining, Qinghai, China. zhaosm@fudan.edu.cn.

^# Contributed equally.

PMID: 38163844
PMCID: PMC10770133
DOI: 10.1038/s41422-023-00864-6

Abstract

Oxidative phosphorylation (OXPHOS) consumes oxygen to produce ATP. However, the mechanism that balances OXPHOS activity and intracellular oxygen availability remains elusive. Here, we report that mitochondrial protein lactylation is induced by intracellular hypoxia to constrain OXPHOS. We show that mitochondrial alanyl-tRNA synthetase (AARS2) is a protein lysine lactyltransferase, whose proteasomal degradation is enhanced by proline 377 hydroxylation catalyzed by the oxygen-sensing hydroxylase PHD2. Hypoxia induces AARS2 accumulation to lactylate PDHA1 lysine 336 in the pyruvate dehydrogenase complex and carnitine palmitoyltransferase 2 (CPT2) lysine 457/8, inactivating both enzymes and inhibiting OXPHOS by limiting acetyl-CoA influx from pyruvate and fatty acid oxidation, respectively. PDHA1 and CPT2 lactylation can be reversed by SIRT3 to activate OXPHOS. In mouse muscle cells, lactylation is induced by lactate oxidation-induced intracellular hypoxia during exercise to constrain high-intensity endurance running exhaustion time, which can be increased or decreased by decreasing or increasing lactylation levels, respectively. Our results reveal that mitochondrial protein lactylation integrates intracellular hypoxia and lactate signals to regulate OXPHOS.

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Conflict of interest statement

The authors declare no competing interests.

Figures

**Fig. 1. Oxygen regulates AARS2 protein levels via PHD2.**
a AARS2 carries a PHD-recognizing sequence. The amino acid (aa) sequence from 369 to 380 of AARS2 and its corresponding AARS1 sequence (aa 343–354) were aligned to the PHD-recognizing sequence. Consensus PHD-recognizing residues are marked in blue. b AARS2 protein levels are regulated by hypoxia. Mitochondrial and cytosolic AARS1, AARS2 and HIF1α levels were detected in C2C12 cells cultured in a hypoxia chamber for the indicated time durations. COXIV and actin were used to demonstrate the successful isolation of mitochondria and cytoplasm, respectively (the same method was employed for subcellular fractionation henceforth). Chamber oxygen levels were monitored. c P377 is required for the regulation of AARS2 protein levels by hypoxia. Stably expressed AARS2^WT and AARS2^P377L protein (P377L) levels in C2C12 cells were measured after the cells were cultured in a hypoxia chamber for the indicated time durations. d, e PHD2 regulates AARS2 protein levels. Mitochondrial and cytosolic AARS1 and AARS2 levels were measured in C2C12 cells or *Phd2* KO C2C12 cells cultured in a hypoxia chamber for the indicated time durations (d) and in C2C12 cells overexpressing PHD2 (e). f–i Hypoxia and PHD2 regulate P377 hydroxylation. P377OH levels were determined via western blot assay in C2C12 cells cultured in a hypoxia chamber for indicated time durations in the presence of MG132 in the culture media to prevent proteasomal degradation (f); in the resting and 30 min running mouse leg skeletal muscles that were intravenously injected with or without 1 mg/kg MG132 twice a week for 4 consecutive weeks (g) (n = 3); in the PHD2 overexpressing C2C12 cells treated with MG132 (h). P377OH levels in PHD2 overexpressing C2C12 cells in the presence and absence of MG132 were quantified by mass spectrometry (i) (n = 3). All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by two-way ANOVA: **P < 0.01; ***P < 0.001.

**Fig. 2. AARS2 acts as a substrate of the PHD2–VHL proteasomal machinery.**
a, b VHL interacts with AARS2. Co-immunoprecipitated, co-expressed (a) and endogenous (b) AARS2 was detected in C2C12 cells ectopically expressing VHL. c P377L mutant shows a weakened affinity for VHL. Interactions between VHL and AARS2, and between VHL and P377L were detected via co-immunoprecipitation when they were co-expressed in C2C12 cells. Densitometric analysis for images was provided. d, e VHL–AARS2 interaction is regulated by hypoxia and PHD2. The amount of AARS2 co-purified VHL was determined under normoxia and 8 h hypoxia (d), as well as with or without PHD2 overexpressing (e) in C2C12 cells. Densitometric analysis for images was provided. f VHL mediates proteasomal degradation. AARS2 ectopically expressed in HEK293T cells was assayed for ubiquitination when expressed alone and when co-expressed with ubiquitin or VHL, or both, under the presence and absence of MG132. g VHL downregulates AARS2 protein levels. Mitochondrial and cytosolic AARS2 levels were measured in C2C12 cells with or without VHL overexpression. h Hypoxia induces AARS2 accumulation in a VHL-dependent manner. Mitochondrial and cytosolic AARS2 levels were measured in C2C12 cells and *Vhl* KO C2C12 cells both cultured in a hypoxia chamber for the indicated time durations. All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test: **P < 0.01.

**Fig. 3. AARS2 inhibits Ac-CoA production and OXOPHOS.**
a, b AARS2 regulates OCR. The effects of overexpressing AARS2 (left, OROBOROS Oxygraph-2K measurements; right, quantitation) (a), and *Aars2* KO using independent sgRNAs (b) on OCR (O₂ influx per volume cells, mV/s) were determined (n = 3). c AARS2 regulates OCR in response to oxygen levels. The effects of re-introducing wide-type AARS2 or P377L into *Aars2*^−/− C2C12 cells were determined in cells under normoxic conditions and cells which were exposed to hypoxia for 8 h (n = 3). d AARS2 regulates metabolite levels. Relative levels (to those of C2C12 cells) of lactate, pyruvate, and Ac-CoA in C2C12 cells and C2C12 cells overexpressing AARS2 (n = 3) were evaluated. e, f AARS2 regulates PDHA1-specific activity. Relative specific activities (to those from C2C12 cells) of PDHA1 purified from C2C12 cells overexpressing AARS2 (e) and *Aars2* KO C2C12 cells (f) were determined (n = 3). g AARS2 regulates CPT2-mediated Ac-CoA production. The percentages of unlabeled (M + 0), single labeled (M + 1), and double-labeled (M + 2) Ac-CoA from ¹³C-palmitate in C2C12 cells and C2C12 cells in which *Cpt2* had been knocked out using independent sgRNAs, were determined with or without AARS2 overexpression; 100 μM ¹³C-palmitate chasing was performed for 12 h (n = 3). The M + 2 percentages in C2C12 cells with and without AARS2 overexpression were compared for significance. h, i AARS2 regulates CPT2-specific activity. The relative specific activity (to those from C2C12 cells) of CPT2, isolated from both AARS2-overexpressing (h) and *Aars2* KO C2C12 cells (i) was determined (n = 3). j Running induces HIFαs and AARS2 expression in mouse leg skeletal muscles. HIF1α, HIF2α, HIF3α, AARS1, and AARS2 levels in mouse leg skeletal muscles were determined before and after running for the indicated durations (n = 6). k, l Running inactivates PDC and CPT2 in vivo. The relative (to those of resting mice) specific activities of PDC (k) and CPT2 (l) in mouse leg skeletal muscles were determined after mice started running for indicated durations (n = 6). m Endurance running-induced lactate accumulation and Ac-CoA reduction. The lactate, pyruvate, and Ac-CoA levels in mouse leg skeletal muscles were determined after mice were allowed to run for the indicated durations (n = 6). n AARS2 inactivates PDC via a mechanism other than phosphorylation. The relative PDC activities (to protein amount) of wild-type and *Aars2*^−/− mouse leg skeletal muscles sampled at different time points after starting to run with or without dephosphorylation induced by λ phosphatase, were determined (n = 6). The activities at time 0 were set as 100%. All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test and two-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance.

**Fig. 4. Lactate inactivates PDHA1 and CPT2 dependent on AARS2.**
a Lactate treatments inactivate cellular PDHA1 and CPT2. C2C12 cells were treated with 10 mM lactate. The relative (to those from untreated C2C12 cells) specific activities of PDHA1 and CPT2 were determined (n = 3). b Me-Lac treatments decrease cellular OCRs. OCRs of C2C12 cells that were untreated or treated with 2 mM or 10 mM Me-Lac for 4 h (n = 3) were determined. c, d Me-Lac treatments inactivate cellular PDHA1 and CPT2. Relative specific activities (to those of untreated C2C12 cells) of PDHA1 (c) and CPT2 (d) in C2C12 cells that were untreated or treated with 2 mM or 10 mM Me-Lac (n = 3) were determined. e–h Lactate treatments decrease Ac-CoA influx from glycolysis and FAO. Relative ¹³C-Ac-CoA levels in ¹³C-glucose (e, f) and ¹³C-palmitate (g, h) (to untreated C2C12 cells) and ¹³C-Ac-CoA (M + 2 only)-treated C2C12 cells, *Aars2* KO C2C12 cells (e, g) and HL-1 cells, *Aars2* KO HL-1 cells (f, h) were detected before and after being treated with 10 mM Me-Lac (n = 3). The chasing time for 10 mM ¹³C-glucose and 100 μM ¹³C-palmitate was 1 h and 12 h, respectively. All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test and two-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance.

**Fig. 5. AARS2 inactivates PDHA1 and CPT2 by lactylating these proteins.**
a Lactic acid, β-alanine, and alanine are analogs. Alignment of chemical structures of alanine, lactic acid, and β-alanine. b AARS2 lactylates PDHA1 K336 and CPT2 K457/8 peptides in vitro. MS-based detection of lactylated synthetic PDHA1 K336 and CPT2 K457/8 peptides catalyzed by AARS2 and AARS2^N104Y are summarized (×: no product detected, √: product detected, : less product detected; see Supplementary information, Fig. S6b for MS results). c AARS2 is an efficient lactyltransferase. The K_m and K_cat of recombinant AARS2 toward lactate were determined when the synthetic K336-containing PDHA1 peptide was kept at 0.5 mg/mL (n = 3). d, e *AARS2* KO abolishes lactate-linked induction of Lac-K336 and Lac-K457/8. Lac-K336 levels (d) and Lac-K457/8 levels (e) were detected for PDHA1 and CPT2 purified from C2C12 cells and C2C12 cells in which *Aars2* had been knocked out using independent sgRNAs, with or without 10 mM Me-Lac in the culture media. f–h Inhibiting the import of lactate into mitochondria decreases Lac-K336 and Lac-K457/8 levels. C2C12 cells that were either untreated, treated with 5 mM α-CHCA for 2 h, or with 10 mM lactate for 4 h. Mitochondrial lactate (f) (n = 3), Lac-K336 (g) and Lac-K457/8 (h) were detected. i Running proportionally increases lactate levels in mitochondria. Mitochondrial and lysate lactate levels in mouse leg skeletal muscles were detected before and after 30 min of running (n = 6). Success mitochondria extraction isolation was confirmed by staining both mitochondria marker COXIV and cytosolic marker GAPDH (here in after). j Running induces Lac-K336 and Lac-K457/8. Lac-K336 and Lac-K457/8 levels in purified PDHA1 and CPT2 of mouse leg skeletal muscles were detected after running for indicated time durations (n = 3). k, l Hypoxia-induced Lac-K336 and Lac-K457/8. Lac-K336 (k) and Lac-K457/8 (l) levels of PDHA1 and CPT2 in C2C12 cells were detected before and after they were exposed to hypoxia for 8 h. m Combined AARS2 and lactate treatments increased Lac-K336 and Lac-K457/8 levels. Lac-K336 and Lac-K457/8 levels in the leg skeletal muscles of resting mice and AARS2-overexpressing mice given lactate gastrocnemius muscle injections were estimated using MS (n = 3). n, o Running and hypoxia increased Lac-K336 and Lac-K457/8 levels. Lac-K336 and Lac-K457/8 levels in the mouse leg skeletal muscles before and after 30 min of running (n) and perfused with Krebs Ringer solution aerated with or without O₂ in a 1200 A Isolated Intact Muscle Test System for 30 min (o) were estimated using MS (n = 3). p, q AARS2 inactivates PDHA1 and CPT2 by inducing lactylation. The effects of AARS2 overexpression on lactylation levels and specific activities (n = 3) of PDHA1 (p) and CPT2 (q) and their respective lactylation site mutants were detected. r, s Lactate inactivates PDHA1 and CPT2 by inducing lactylation. The effects of 10 mM Me-Lac on Lac-K336 and the specific activities of PDHA1 and its lactylation site mutants (r), and the effects of Me-Lac on Lac-K457/8 and the specific activities of CPT2 and its lactylation site mutants (s) were detected (n = 3). All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test and two-way ANOVA: **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance.

formula image — **Fig. 5. AARS2 inactivates PDHA1 and CPT2 by lactylating these proteins.**
a Lactic acid, β-alanine, and alanine are analogs. Alignment of chemical structures of alanine, lactic acid, and β-alanine. b AARS2 lactylates PDHA1 K336 and CPT2 K457/8 peptides in vitro. MS-based detection of lactylated synthetic PDHA1 K336 and CPT2 K457/8 peptides catalyzed by AARS2 and AARS2^N104Y are summarized (×: no product detected, √: product detected, : less product detected; see Supplementary information, Fig. S6b for MS results). c AARS2 is an efficient lactyltransferase. The K_m and K_cat of recombinant AARS2 toward lactate were determined when the synthetic K336-containing PDHA1 peptide was kept at 0.5 mg/mL (n = 3). d, e *AARS2* KO abolishes lactate-linked induction of Lac-K336 and Lac-K457/8. Lac-K336 levels (d) and Lac-K457/8 levels (e) were detected for PDHA1 and CPT2 purified from C2C12 cells and C2C12 cells in which *Aars2* had been knocked out using independent sgRNAs, with or without 10 mM Me-Lac in the culture media. f–h Inhibiting the import of lactate into mitochondria decreases Lac-K336 and Lac-K457/8 levels. C2C12 cells that were either untreated, treated with 5 mM α-CHCA for 2 h, or with 10 mM lactate for 4 h. Mitochondrial lactate (f) (n = 3), Lac-K336 (g) and Lac-K457/8 (h) were detected. i Running proportionally increases lactate levels in mitochondria. Mitochondrial and lysate lactate levels in mouse leg skeletal muscles were detected before and after 30 min of running (n = 6). Success mitochondria extraction isolation was confirmed by staining both mitochondria marker COXIV and cytosolic marker GAPDH (here in after). j Running induces Lac-K336 and Lac-K457/8. Lac-K336 and Lac-K457/8 levels in purified PDHA1 and CPT2 of mouse leg skeletal muscles were detected after running for indicated time durations (n = 3). k, l Hypoxia-induced Lac-K336 and Lac-K457/8. Lac-K336 (k) and Lac-K457/8 (l) levels of PDHA1 and CPT2 in C2C12 cells were detected before and after they were exposed to hypoxia for 8 h. m Combined AARS2 and lactate treatments increased Lac-K336 and Lac-K457/8 levels. Lac-K336 and Lac-K457/8 levels in the leg skeletal muscles of resting mice and AARS2-overexpressing mice given lactate gastrocnemius muscle injections were estimated using MS (n = 3). n, o Running and hypoxia increased Lac-K336 and Lac-K457/8 levels. Lac-K336 and Lac-K457/8 levels in the mouse leg skeletal muscles before and after 30 min of running (n) and perfused with Krebs Ringer solution aerated with or without O₂ in a 1200 A Isolated Intact Muscle Test System for 30 min (o) were estimated using MS (n = 3). p, q AARS2 inactivates PDHA1 and CPT2 by inducing lactylation. The effects of AARS2 overexpression on lactylation levels and specific activities (n = 3) of PDHA1 (p) and CPT2 (q) and their respective lactylation site mutants were detected. r, s Lactate inactivates PDHA1 and CPT2 by inducing lactylation. The effects of 10 mM Me-Lac on Lac-K336 and the specific activities of PDHA1 and its lactylation site mutants (r), and the effects of Me-Lac on Lac-K457/8 and the specific activities of CPT2 and its lactylation site mutants (s) were detected (n = 3). All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test and two-way ANOVA: **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance.

**Fig. 6. SIRT3 reverses Lac-K336 and Lac-K457/8.**
a NAM treatments increase Lac-K336 and Lac-K457/8. Lac-K336 and Lac-K457/8 levels of PDHA1 and CPT2 purified from C2C12 cells and NAM-treated C2C12 cells were detected. Cells were examined 3 h after 5 mM NAM treatment. b SIRT3 delactylates Lac-K336 and Lac-K457/8. The ability of SIRT3, SIRT4 catalytic domain and SIRT5 to delactylate synthetic Lac-K336- and Lac-K457/8-containing peptides was analyzed via MS to detect the formation of lactylated species. c–f SIRT3 decreases Lac-K336 and Lac-K457/8 levels. The Lac-K336 and Lac-K457/8 levels in purified PDHA1 (c) and CPT2 (d) were detected before and after being delactylated with SIRT3 or catalytic dead SIRT3^H248A, and the SIRT3 expression effects on wild-type, lactylation-null PDHA1 (e), and CPT2 (f) were detected in C2C12 cells. g Ablation of *Sirt3* increases Lac-K336 and Lac-K457/8 levels in vivo. Lac-K336 and Lac-K457/8 levels of wild-type and *Sirt3*^−/− mouse leg skeletal muscles were detected (n = 5). h, i SIRT3 regulates PDHA1 and CPT2 activities. Relative activities of PDHA1 and CPT2 that were purified from C2C12 cells and SIRT3-overexpressing (h) or *Sirt3* KO (i) C2C12 cells were compared (n = 3). j–m SIRT3 regulates Ac-CoA production from glycolysis and FAO. The M + 0, M + 1, and M + 2 fractions of Ac-CoA from ¹³C-glucose (j, k) and ¹³C-palmitate (l, m) were detected in C2C12 cells either overexpressing SIRT3 (j, l) or in which *Sirt3* had been knocked out using independent sgRNAs (k, m) (n = 3). The chasing time for 10 mM ¹³C-glucose and 100 μM ¹³C-palmitate were 1 h and 12 h, respectively. n–q SIRT3 regulates lactate and free fatty acids levels. Lactate (n, p) and free fatty acid levels (o, q) in C2C12 cells overexpressing SIRT3 (n, o) or C2C12 cells in which *Sirt3* had been knocked out via independent sgRNAs (p, q) were detected (n = 3). All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test and two-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001.

**Fig. 7. OXPHOS drives lactylation to feedback limit OXPHOS and endurance running ability.**
a OXPHOS induces intracellular hypoxia. HIF1α and AARS2 levels of C2C12 cells in DMEM base and glucose- (25 mM), palmitate- (200 μM) and Me-lac- (2 mM) supplemented DMEM media were tested in the presence or absence of 1 μM rotenone which inhibits OXPHOS. b Lactate injection induces mouse muscle hypoxia. HIF1α and AARS2 levels of mouse hind leg thighs were detected before and after 2 g/kg lactate was injected into gastrocnemius muscle (n = 3). c Lactate production is essential for inducing hypoxia and lactylation in mouse leg muscles. The HIF1α, AARS2, and lactylation levels of mouse hind leg thighs of untreated mice and mice pre-treated with FX-11 were assessed when they started to run. d Running leads to the accumulation of lactate in mouse leg skeletal muscles. Relative lactate levels in type I and type II mouse skeletal muscles were determined before and after 30 min of running (n = 6). e Running induces more pronounced lactylation in type I muscle. HIF1α and AARS2, as well as Lac-K336 and Lac-K457/8 levels in type I and type II mice skeletal muscles, were determined when started to run. f Drop of ATP in type I skeletal muscle is correlated with an increase in Lac-K336 and Lac-K457/8. ATP levels in type I and II mouse leg skeletal muscles were monitored after running for indicated times (n = 6). Changes in Lac-K336 and Lac-K457/8 are shown (Fig. 5j). g Basal AARS2 levels in mouse leg skeletal muscle are inversely correlated with their running exhaustion time. High-intensity running exhaustion time and resting leg muscle AARS2 levels were correlated (n = 30). h Lac-K336 and Lac-K457/8 inhibit the influx of mouse skeletal muscle Ac-CoA. The ¹³C-Ac-CoA levels in wild-type, AARS2 overexpressing and *Sirt3*^−/− mouse leg skeletal muscle were detected 1 h after each mouse received a ¹³C-glucose injection via the tail vein (n = 6). i, j Lactylation reduces mouse skeletal muscle OCRs and ATP production. OCR (i) and ATP levels (j) in mitochondria in the leg skeletal muscles of resting, AARS2 overexpressing, and *Sirt3*^−/− mice, were analyzed (n = 6). k Lac-K336 and Lac-K457/8 inhibit mouse running exhaustion time. Mouse high-intensity running exhaustion time was measured in wild-type, muscle AARS2-overexpressing, and *Sirt3*^−/− mice (n = 9). l Lactate and β-alanine inversely regulate mouse running exhaustion time. High-intensity running exhaustion times of untreated mice or treated with lactate or β-alanine via gastrocnemius muscle injection were measured, respectively (n = 9). m β-alanine prolongs mice exhaustion time in an AARS2-dependent manner. High-intensity running exhaustion times of wild-type, AARS2 overexpressing, and *Aars2*^−/− mice with or without gastrocnemius muscle β-alanine injection (n = 10) were measured. n, o Loss of lactylation results in higher ROS damage. ROS levels (n) and induced malondialdehyde levels (o) of wild-type and *Aars2*^−/− mice at the time of running exhaustion (n = 6) were compared. p Diagram showing that exercise promotes lactate oxidation through OXPHOS, which promotes intracellular hypoxia that induces AARS2 and Lac-K336 and Lac-K457/8 to feedback inhibit Ac-CoA influxes from lactate/glycolysis and FAO and OXPHOS. The mechanisms revealed in this study are marked by red arrows. All data are reported as mean ± SEM of three independent experiments. Statistical significance was assessed by unpaired two-tailed Student’s t-test and two-way ANOVA: *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns no significance.

See this image and copyright information in PMC

Comment in

Lactylation constrains OXPHOS under hypoxia.
Ren H, Zhang D. Ren H, et al. Cell Res. 2024 Feb;34(2):91-92. doi: 10.1038/s41422-023-00872-6. Cell Res. 2024. PMID: 38163845 No abstract available.

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Dong Q, Zhang Q, Yang X, Nai S, Du X, Chen L. Dong Q, et al. Int J Mol Sci. 2024 Feb 26;25(5):2692. doi: 10.3390/ijms25052692. Int J Mol Sci. 2024. PMID: 38473939 Free PMC article.

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Hypoxia induces mitochondrial protein lactylation to limit oxidative phosphorylation

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Hypoxia induces mitochondrial protein lactylation to limit oxidative phosphorylation

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