Abstract
Mitochondrial proteases are emerging as key regulators of mitochondrial plasticity and acting as both protein quality surveillance and regulatory enzymes by performing highly regulated proteolytic reactions. However, it remains unclear whether the regulated mitochondrial proteolysis is mechanistically linked to cell identity switching. Here we report that cold-responsive mitochondrial proteolysis is a prerequisite for white-to-beige adipocyte cell fate programming during adipocyte thermogenic remodelling. Thermogenic stimulation selectively promotes mitochondrial proteostasis in mature white adipocytes via the mitochondrial protease LONP1. Disruption of LONP1-dependent proteolysis substantially impairs cold- or β3 adrenergic agonist-induced white-to-beige identity switching of mature adipocytes. Mechanistically, LONP1 selectively degrades succinate dehydrogenase complex iron sulfur subunit B and ensures adequate intracellular succinate levels. This alters the histone methylation status on thermogenic genes and thereby enables adipocyte cell fate programming. Finally, augmented LONP1 expression raises succinate levels and corrects ageing-related impairments in white-to-beige adipocyte conversion and adipocyte thermogenic capacity. Together, these findings reveal that LONP1 links proteolytic surveillance to mitochondrial metabolic rewiring and directs cell identity conversion during adipocyte thermogenic remodelling.
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Data availability
The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD031197 and the project name ‘TurboID-based proximity labeling to identify LONP1-targeting proteins’. The ChIP-seq data have been deposited in the NCBI Gene Expression Omnibus and are accessible through the accession number GSE222490. Source data are provided with this paper. All of the other data supporting the findings of this study are available from the corresponding authors upon reasonable request.
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Acknowledgements
We thank Z. Yan (University of Virginia) for providing the MitoTimer reporter mice and Y. Liu (Wuhan University), L. Rui (University of Michigan) and Y. Qiu (Peking University) for critical reading of the manuscript. We also thank F. Zhang (Shanghai Jiao Tong University) for technical assistance with the lineage-tracing experiments. This work was supported by grants from the National Natural Science Foundation of China (91857105, 31922033, 32071136, 32100922 and 82170891 to Z.G., T.F. and M.S.); Ministry of Science and Technology of China (National Key R&D Program of China 2018YFA0800700 and 2022YFA0806000) and Natural Science Foundation of Jiangsu Province (BK20170014 and SWYY-002) (to Z.G.); Fundamental Research Funds for the Central Universities (090314380036 and 021414380517 to T.F. and 021414380511, 021414380529, 021414380533 and 021414380524 to Z.G.); and Shanghai Pujiang Program (21PJ1414600) and Shanghai Municipal Science and Technology Major Project (2019SHZDZX02) (to M.S.).
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T.F., W.S., J.X. and Z.Z. designed and performed most of the experiments, analysed the data and wrote the manuscript. W.W., Q.G., X.C., D.Z., Z.X., L.L., L.X., Y.M., L.Y., Y.Y., X.-N.Z. and Q.W. participated in collection and analysis of the data. B.L., Y.C., M.-S.Z., P.E.S., L.F. and H.-L.P. contributed reagents and provided scientific insight and discussion. M.S. and Z.G. conceptualized the study, interpreted the experiments and wrote the manuscript. Z.G. supervised the work. All authors reviewed and contributed to the manuscript.
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Extended data
Extended Data Fig. 1 WAT-selective upregulation of mitochondrial protein turnover and mitochondrial proteostasis regulators.
(a, b) 5-week-old WT mice exposed to 8˚C or 30˚C for 7 days. (a) Body temperature and iWAT mass measurements. (b) Representative UCP1 staining and H&E staining of iWAT section after exposure to cold or thermoneutrality. Data shown are representative of 6 (a) or 5 (b) mice/group. (c) iWAT and BAT MitoTimer Red:Green ratio of Adipoq-Cre/MitoTimer mice exposed to 8°C or 30°C. > 38 images counted from 7 mice/group. (d, e) 4-week-old WT mice were injected with CL316,243 for 5 days. (d) Oxygen consumption measurements before and after injection. n = 8 mice/genotype. (e) Representative UCP1 staining and H&E staining of iWAT section. n = 8 mice/genotype. Scale bar, 50 μm. (f) iWAT MitoTimer Red:Green ratio of MitoTimer mice at indicated time points of CL316,243-treatment. >48 images counted from 5 mice/group. (g) BAT MitoTimer fluorescence images at indicated time points of CL316,243-treatment and quantification (h). Scale bar, 10 μm. Representative images are shown. >30 images counted from 5 mice/group. (i) LONP1/Tubulin and CLPP/Tubulin protein ratios in iWAT and BAT of WT mice exposed to 8°C or 30°C. n = 8 mice/group. (j, k) Immunoblot analysis of proteins in EWAT isolated from mice after thermogenic stimulations. n = 4 mice/group. (l) Immunoblot analysis of proteins in iWAT of WT mice exposed to 8°C or 30°C for 7 days. n = 4 mice/group. (m) iWAT Mitochondrial DNA levels of WT mice exposed to cold or thermoneutrality. n30˚C = 10; n8˚C = 12 mice. (n) Thermogenic gene expression levels in differentiated beige adipocytes. n = 5 experiments. (o) MitoTimer Red:Green ratio in adipocytes transfected with Lonp1 or Clpp siRNA. >77 images counted from 5 experiments. (p) MitoTimer Red:Green ratio in WT and LONP1 AKO iWAT adipocytes differentiated with or without Rosi. >59 images counted from 6 experiments. All data are shown as mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test (a, c, i-o) or one-way ANOVA (f, h) or two-way ANOVA (d, p). Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 2 Thermogenic stimulus specifically promotes mitochondrial proteostasis in WAT but not in BAT.
(a-c) Body weight (nWT = 7; nAKO = 6), mice picture (8-week-old) and oxygen consumption (nWT = 10; nAKO = 6) of WT and LONP1 AKO mice. (d) Representative electron micrographs of iWAT mitochondria in sections from WT and LONP1 AKO mice at the age of 4 weeks. n = 3 mice per group. Scale bar, 500 nm. (e) Quantitative PCR measurement of mitochondrial DNA levels in iWAT of indicated mice at 4 weeks of age using primers for NADH dehydrogenase (Nd1, mitochondria-encoded) and lipoprotein lipase (Lpl, nuclear-encoded). Nd1 levels were normalized to Lpl DNA content and expressed relative to WT ( = 1.0) iWAT. nWT = 12; nAKO = 8 mice per group. (f-i) Immunoblot analysis of LONP1 and Tubulin expression in EWAT (f, g) and BAT (h, i) of WT and LONP1 AKO mice following CL316,243 treatment or cold exposure. Data shown are representative examples of 4 mice per group. All data are shown as the mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test (e) or two-way ANOVA (a, c, f-i). Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 3 Adipocyte LONP1 is required for white-to-beige adipocyte cell fate programming.
(a) (Left) Immunoblot analysis on the indicated proteins in iWAT from 5-week-age WT and LONP1 AKO mice after being kept in cold (8˚C) or thermoneutral (30˚C) environment for 7 consecutive days. (Right) Quantification of UCP1/Tubulin protein ratios. n = 8 mice per group. (b) Quantification of MitoTimer Red:Green ratio in iWAT of vehicle or CL316,243 treated WT and LONP1 AKO mice in Fig. 2b. n = 6 mice per group. >39 images were counted for each condition. (c) MitoTimer fluorescence images of iWAT depots from 5-week-old WT and LONP1 AKO mice after being kept in cold (8˚C) or thermoneutral (30˚C) environment for 7 consecutive days. Images are representative of 5 mice per group. Scale bar, 10 μm. (d) Quantification of MitoTimer Red:Green ratios as shown in (c). n = 5 mice per group. >37 images were counted for each condition. (e, f) Measurements of iWAT mitochondrial respiration rates in mice of the indicated genotypes and treatments. (e) (Left) n = 5 mice per group; (Right) nWT 30˚C = 6; nWT 8˚C = 5; nAKO 30˚C = 6; nAKO 8˚C = 5 mice. (f) n = 5 mice per group. (g, h) Representative confocal images (g) of iWAT in 4-week-old mt-Keima-TG mice and LONP1 AKO/mt-Keima mice following CL316,243 treatment for 5 consecutive days. Scale bar, 10 μm. The emission signal obtained after excitation with the 458-nm laser is shown in green, and that obtained after excitation with the 561-nm laser is shown in red. Quantification of the red/green mt-Keima signal ratios (h) normalized (= 1.0) to the WT controls is shown. nWT Con = 7; nWT CL = 6; nAKO Con = 4; nAKO CL = 5 mice. >40 images were counted for each condition. All data are shown as the mean ± SEM. P value was calculated by two-way ANOVA. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 4 Evaluation of the effects by LONP1 inactivation on creatine- or SERCA-mediated thermogenic pathways.
(a) Immunoblot analysis on the indicated proteins in BAT from 5-week-age WT and Lonp1f/f/UCP1-Cre mice. n = 6 mice per group. (b) H&E staining of BAT sections from 5-week-old WT and Lonp1f/f/UCP1-Cre mice. Images are representative of 5 mice per group. (c, d) Representative UCP1 and H&E staining of iWAT sections from 5-week-old WT and Lonp1f/f/UCP1-Cre mice subjected to cold exposure (c: nWT 30˚C = 4; nWT 8˚C = 5; nUCP1-Cre 30˚C = 5; nUCP1-Cre 8˚C = 4 mice) or β3-AR agonist CL316,243 treatment (d: nWT Con = 5; nWT CL = 5; nUCP1-Cre Con = 4; nUCP1-Cre CL = 5 mice). Scale bar, 50 μm. (e) Body temperature of 5-week-old WT and Lonp1f/f/UCP1-Cre mice exposed to cold (4˚C) for 6 hours. nWT = 5; nUCP1-Cre = 6 mice per group. (f) RT-qPCR analysis of expression of the genes involved in creatine- and SERCA-mediated thermogenesis in WT and LONP1 AKO adipocytes, n = 5 independent experiments. (g) Immunoblot analysis of CKB, GAMT, CKMT1A and LONP1 protein levels (left) and quantification of CKB/Tubulin, GAMT/Tubulin and CKMT1A/Tubulin protein ratios (right) in WT and LONP1 AKO adipocytes. n = 6 independent experiments. (h) Creatine and PCr to creatine (Cr) ratio in WT and LONP1 AKO adipocytes. n = 5 independent experiments. (i, j) RT-qPCR analysis of expression of the genes involved in creatine- and SERCA-mediated thermogenesis in iWAT (i: nWT 30˚C = 7; nWT 8˚C = 7; nAKO 30˚C = 5; nAKO 8˚C = 7 mice) and BAT (j: nWT 30˚C = 8; nAKO 30˚C = 9; nWT 8˚C = 8; nAKO 8˚C = 8 mice) of WT and LONP1 AKO mice exposed to cold or thermoneutrality. All data are shown as the mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test (f, g, h) or two-way ANOVA (e, i, j). Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 5 Identification of LONP1 interacting proteins by TurboID-mediated proximity labeling.
(a) Heat map showing species of fatty acids and carnitines identified by LC-MS system in in vitro-differentiated WT and LONP1 AKO beige adipocytes. n = 5 per group. (b, c) The levels of indicated species of carnitine (b) and fatty acid (c) in in vitro-differentiated WT and LONP1 AKO beige adipocytes. n = 5 per group. (d) Schematic of LONP1-TurboID reconstitution using the proximity-dependent biotinylation catalyzed by promiscuous biotin ligases. LONP1-N-TurboID constructs (mitochondrial targeted sequence; substrate binding domain of LONP1; full-length TurboID; HA-tag), or the reference constructs TurboID (mitochondrial targeted sequence; full-length TurboID; HA-tag). (e) Immunoblot analysis of total biotinylated proteins in 293 T cells using Streptavidin-HRP antibody. (f) The percentage of mitochondrial proteins within all identified biotinylated proteins using MitoCarta2.0. (g) Functional protein association networks analysis of identified mitochondrial proteins. (h, i) Blue native polyacrylamide gel electrophoresis (BN-PAGE) analyses of the mitochondrial respiratory chain complexes in WT and LONP1 AKO adipocytes followed by immunoblot analysis (h) using antibodies against complex I (anti-NDUFS1), complex II (anti-SDHA), complex III (anti-UQCRC2), complex IV (anti-MTCO1) and complex V (anti-ATP5A) or in-gel activity of complex I (i). Data shown are representative examples of 3 independent experiments. All data are shown as the mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test. Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 6 SDH-mediated succinate metabolism is critical for LONP1-regulated cell fate programming of mature adipocytes.
(a) UCP1 staining of BAT sections from WT mice treated with vehicle or CL, fed with or without 1.5% sodium succinate for 2 weeks. Scale bar, 100 μm. Images represent 4 mice/group. (b) UCP1/Tubulin signal ratios normalized to WT vehicle-treated mice as described in Fig. 6b. nCon = 6; nCL = 6; nCon+Succinate = 6; nCL+Succinate = 7 mice (c) Schematic illustration of AAV-mediated SDHB overexpression experiment. UCP1 and H&E staining of iWAT sections after treatments. Representative images are shown. nGFP Con = 10; nGFP CL+succinate = 9; nSDHB Con = 9; nSDHB CL+Succinate = 8 mice. Scale bar, 50 μm. (d) Immunoblot analysis of proteins in iWAT. n = 8 mice/group. (e) iWAT mitochondrial respiration rates. nGFP Con = 9; nGFP CL+succinate = 10; nSDHB Con = 9; nSDHB CL+Succinate = 10 mice. (f) Malonate inhibition of SDH experiment schematic. UCP1 and H&E staining of iWAT sections. Representative images are shown. nCon PBS = 10; nCon CL = 11; nMalo PBS = 5; nMalo CL = 7 mice. Scale bar, 50 μm. (g-i) Immunoblot analysis (g) (nCon PBS = 10; nCon CL = 10; nMalo PBS = 9; nMalo CL = 10 mice), mitochondrial respiration rates (h) (nCon PBS = 8; nCon CL = 9; nMalo PBS = 8; nMalo CL = 9 mice) and succinate content (i) (nCon PBS = 7; nCon CL = 10; nMalo PBS = 7; nMalo CL = 10 mice) in iWAT from WT mice of indicated treatments. (j) AdipoChaser mice treated with doxycycline-diet for 10 days, followed by CDDO alone and in combination with malonate-mediated SDH inhibition for 12 days. (k) Representative image of iWAT sections stained with anti-GFP (green) and anti-PERILIPIN (red) antibodies and counterstained with DAPI (blue). nPluse = 4; nControl = 6; nCL = 4; nCDDO+CL = 5; nCDDO+CL+Succinate = 6 mice. Scale bar, 20 μm. (l) Percentage of multilocular adipocytes from labeled cells (expressing mGFP), nPluse = 4; nControl = 6; nCL = 4; nCDDO+CL = 5; nCDDO+CL+Succinate = 6 mice. >400 adipocytes were counted for each condition. All data are shown as mean ± SEM. P value was calculated by two-way ANOVA (b, d, e, g-i) or one-way ANOVA (l). Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 7 The metabolic response of LONP1 AKO mice to dietary succinate supplementation.
(a) iWAT succinate content in mice subjected to dietary succinate supplementation. 4-week-old WT and LONP1 AKO mice fed with or without 1.5% sodium succinate drinking for 2 weeks. n = 5 mice per group. (b) Quantification of the UCP1/Tubulin signal ratios from iWAT of vehicle or CL316,243 treated WT and LONP1 AKO mice fed with or without sodium succinate in Fig. 6f. n = 5 mice per group. (c) Body composition measured to determine fat mass and lean tissue mass of 4-week-old WT and LONP1 AKO mice fed with or without 2 weeks sodium succinate water. nWT Con = 9; nWT Succinate = 5; n AKO Con = 5; nAKO Succinate = 5 mice. (d) Growth curve of WT and LONP1 AKO mice. nWT = 14; n AKO = 12 mice. (e) Fat mass and lean tissue mass of indicated mice at 4 months of age, nWT = 9; n AKO = 6 mice. (f) Bodyweight of 4-month-old WT and LONP1 AKO mice fed with or without sodium succinate water for 10 weeks. nWT Con = 7; nWT Succinate = 6; n AKO Con = 7; nAKO Succinate = 6 mice. (g-k) Metabolic evaluation of WT and LONP1 AKO mice using a comprehensive laboratory animal monitoring system (CLAMS). nWT Con = 11; nWT Succinate = 6; n AKO Con = 8; nAKO Succinate = 6 independent mice. (g) Food consumption. (h) Locomotor activity. (i) Energy expenditure. (j) Oxygen consumption. (k) Carbon dioxide production. (l) Left: Glucose tolerance test (GTT). Right: Area under the curve for GTT is shown. (m) Left: Insulin tolerance test (ITT). Right: Area under the curve for ITT is shown. (l, m) nWT Con = 6; nWT Succinate = 7; n AKO Con = 7; nAKO Succinate = 7 mice. All data are shown as the mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test (e), or two-way ANOVA (a-d, f-m). Source numerical data are available in source data.
Extended Data Fig. 8 ChIP-Seq analysis to characterize genome-wide H3K4me1 status in LONP1 AKO adipocytes.
(a) Left: Diagram showing protoporphyrin and heme synthesis pathway. Right: Expression of the genes involved in protoporphyrin and heme synthesis in WT and LONP1 AKO adipocytes determined by RT-qPCR. n = 5 independent experiments. (b) Intracellular heme content in WT and LONP1 AKO adipocytes. n = 6 independent experiments. (c) Signal intensity plot of H3K4me1 ChIP-Seq signal in WT or LONP1 AKO adipocytes are shown. The peak distribution of H3K4me1 binding in WT adipocytes is shown in red, whereas the peak distribution in the LONP1 AKO is shown in blue. (d, e) H3K4me1 ChIP-Seq signals at the Cox7a1, Cidea, Kcnk3, Elovl3 and Eva1b loci in WT and LONP1 AKO adipocytes. H3K4me1 binding peaks were mapped onto the mm10 mouse genome and visualized using the Integrative Genomics Viewer tool. (f) Expression levels of beige thermogenic genes determined by RT-qPCR in iWAT from WT and LONP1 AKO mice fed with or without 1.5% sodium succinate drinking for 2 weeks, following vehicle or CL316,243 treatment. nWT Con = 7; nWT CL = 7; nAKO Con = 6; nAKO CL = 7; nAKO CL+Succinate = 7 mice. All data are shown as the mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test (a, b) or two-way ANOVA (f). Source numerical data are available in source data.
Extended Data Fig. 9 Metabolic phenotyping of the LONP1 AOE mice.
(a, b) 5-week-age WT and LONP1 AOE mice treated with CL316,243 for 5 days. (a) UCP1 staining and H&E staining of iWAT sections. Images are representative of 4 mice per genotype. Scale bar, 50 μm. (b) Immunoblot analysis of the indicated protein levels in iWAT. n = 4 mice per group. (c) Adipose tissues weight. n = 6 mice per group. (d-h) Metabolic evaluation of WT and LONP1 AOE mice using CLAMS. n = 7 mice per group. (d) Food intake. (e) Locomotor activity. (f, g) Oxygen consumption (f) and carbon dioxide production (g) of 10-month-old WT and LONP1 AOE mice. Graphs on the left side of each panel represent absolute value. The regression plots on the right side of each panel depict the respective metabolic parameter as a function of mouse weight, in the dark and light cycles. (h) Measurements of mitochondrial respiration rates in iWAT of 10-month-old WT and LONP1 AOE following CL316,243 treatment. nWT Con = 6; nWT CL = 7; nAOE Con = 6; nAOE CL = 6 mice). (i) Measurements of mitochondrial respiration rates in iWAT from 2-month-old and 7-month-old WT mice with the indicated treatments. n2-month Con = 5; n2-month CL = 6; n7-month Con = 6; n7-month CL = 7; n7-month CL+Succinate = 7 mice (j) iWAT succinate content in 2-month-old and 7-month-old mice subjected to dietary succinate supplementation for 2 weeks. n = 5 mice per group. All data are shown as the mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test (c, d) or two-way ANOVA (e, h-j). Source numerical data and unprocessed blots are available in source data.
Extended Data Fig. 10 Metabolically beneficial effects of succinate supplementation in aged mice.
Metabolic phenotyping of 11-month-old WT mice fed with or without 1.5% sodium succinate water for 7 weeks. (a) Bodyweight. (b) Body composition measured to determine fat mass and lean tissue mass. (c-f) Metabolic evaluation of mice using CLAMS. (c) Food consumption. (d) Oxygen consumption. (e) Carbon dioxide production. (f) Energy expenditure. Graphs on the left side of each panel represent absolute value. The regression plots on the right side of each panel depict the respective metabolic parameter as a function of mouse weight, in the dark and light cycles. (g) Liver weight. (h) Representative H&E and oil red stain of livers. Scale bar, 50 μm. (i) Liver triglyceride levels. (a, g, h) nCon = 6; nSuccinate = 8 mice; (b-f) nCon = 8; nSuccinate = 7 mice; (i) nCon = 7; nCon+Succinate = 7 mice. All data are shown as the mean ± SEM. P value was calculated by unpaired two-sided Student’s t-test (b, c, g, i) or two-way ANOVA (a, d-f). Source numerical data are available in source data.
Supplementary information
Supplementary Tables
Supplementary Table 1. List of LC-MS/MS data from this study. LC-MS/MS analysis of WT and LONP1 KO beige adipocytes. Statistical significance was determined by unpaired two-sided Student’s t-test. Supplementary Table 2. List of CE-MS data from this study. CE-MS analysis of WT and LONP1 KO beige adipocytes. Statistical significance was determined by unpaired two-sided Student’s t-test. Supplementary Table 3. Primers. Supplementary Table 4. Information on antibodies.
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Fu, T., Sun, W., Xue, J. et al. Proteolytic rewiring of mitochondria by LONP1 directs cell identity switching of adipocytes. Nat Cell Biol (2023). https://doi.org/10.1038/s41556-023-01155-3
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DOI: https://doi.org/10.1038/s41556-023-01155-3
