Abstract
Cholesterol is an essential lipid and its synthesis is nutritionally and energetically costly1,2. In mammals, cholesterol biosynthesis increases after feeding and is inhibited under fasting conditions3. However, the regulatory mechanisms of cholesterol biosynthesis at the fasting–feeding transition remain poorly understood. Here we show that the deubiquitylase ubiquitin-specific peptidase 20 (USP20) stabilizes HMG-CoA reductase (HMGCR), the rate-limiting enzyme in the cholesterol biosynthetic pathway, in the feeding state. The post-prandial increase in insulin and glucose concentration stimulates mTORC1 to phosphorylate USP20 at S132 and S134; USP20 is recruited to the HMGCR complex and antagonizes its degradation. The feeding-induced stabilization of HMGCR is abolished in mice with liver-specific Usp20 deletion and in USP20(S132A/S134A) knock-in mice. Genetic deletion or pharmacological inhibition of USP20 markedly decreases diet-induced body weight gain, reduces lipid levels in the serum and liver, improves insulin sensitivity and increases energy expenditure. These metabolic changes are reversed by expression of the constitutively stable HMGCR(K248R). This study reveals an unexpected regulatory axis from mTORC1 to HMGCR via USP20 phosphorylation and suggests that inhibitors of USP20 could be used to lower cholesterol levels to treat metabolic diseases including hyperlipidaemia, liver steatosis, obesity and diabetes.
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All source data for immunoblotting are shown in Supplementary Fig. 1. Source data are provided with this paper.
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Acknowledgements
We thank D. Liang and B.-Y. Xiang for technical assistance; Y. Qiu and S. Fu for reagents; and X. Zhou and L. Deng for bomb calorimetry. This work was supported by grants from the National Natural Science Foundation China (91957103, 91954203, 31690102 and 32021003) and Ministry of Science and Technology China (2016YFA0500100). B.-L.S. acknowledges the support from the Tencent Foundation through the Xplorer Prize.
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Contributions
X.-Y.L. and X.-J.S. carried out the overall experiments and analysed the data. A.H., Y.D., and W.J. performed the screening of the DUB expression library. A.H. performed the in vitro kinase assay and in vitro deubiquitination assay. J.-Q.W. performed experiments in UCP1-knockout mice and HMGCR(K248R) knock-in mice. Y.D. and M.S. assisted with the animal and cell experiments. X.Z. performed metabolites analysis and analysed the data. B.-L.S. conceived the project and directed the research. B.-L.S., X.-Y.L., X.-J.S., W.Q. and J.L. wrote the paper with input from the other authors.
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Extended data figures and tables
Extended Data Fig. 1 In vitro ubiquitination assay of HMGCR.
a, Quantification of the proteins in Fig. 1a. The signals of HMGCR, FDFT1, LSS and DHCR24 were normalized to that of GAPDH. The amount of each protein in fasted state was defined as 1. b, Relative amounts of mRNAs in the livers of the mice in Fig. 1a measured by qPCR. c, Immunoblot analysis of HMGCR, FDFT1, LSS and DHCR24 in the livers of mice fasted (F) for 12 h, or fasted for 12 h and then refed (R) with a high-carbohydrate/low-fat diet for 3 h, 6 h, or 12 h (n = 3 per group) respectively. d, Quantification of proteins in (c). e, The mRNA levels of indicated genes in mouse liver (n = 4 per group) treated as in (c). f, Schematic representation of the experimental design for in vitro ubiquitination assays. Membrane fractions were prepared from the sterol-depleted CHO-7 cells to provide un-ubiquitinated HMGCR and E3 complex. The liver cytosols were prepared from mice under fasted or refed conditions. The membrane fractions were incubated with E1, UBE2G2, ATP, FLAG-Ubiquitin, the indicated cytosol and 25-hydroxycholesterol (25-HC) at 37 °C for 30 min. Samples were solubilized and HMGCR was immunoprecipitated with polyclonal anti-HMGCR antibodies. Immunoblotting was carried out with monoclonal anti-FLAG or monoclonal anti-HMGCR antibodies. g, In vitro ubiquitination of HMGCR as described in (f). Experiments were performed as indicated three times with similar results. All values are presented as mean ± SEM. Data were analysed by unpaired two-tailed Student’s t-test (a, b), or one-way ANOVA with Tukey’s multiple comparisons test (d, e).
Extended Data Fig. 2 The screening of the DUB expression library.
Experiments were performed as indicated three times with similar results. a–p, CHO-7 cells were set up for experiments on day 0 at 4 × 105 cells per 60-mm dish in DMEM/F12 supplemented with 5% FBS. On day 1, cells were transfected in 3 ml of DMEM/F12 supplemented with 5% FBS containing 1 μg of pCMV-HMGCR-T7, 30 ng of pCMV-Insig-1-Myc and 0.3 μg indicated Dub. The total DNA was adjusted to 2 μg dish-1 using pcDNA3 mock vector. Eight hours after transfection, cells were incubated in 3 ml of DMEM/F12 supplemented with 5% FBS. On day 2, cells were washed with phosphate-buffered saline (PBS) and then switched to DMEM/F12 containing 5% lipoprotein-deficient serum (LPDS), 1 μM lovastatin, and 10 μM mevalonate. After incubation for 16 h at 37 °C, the cells were treated with 1 μg ml-1 25-HC plus 10 mM mevalonate as indicated. After 5 h at 37 °C, cells from 2 dishes were pooled and collected, lysed, and subjected to immunoblotting. Immunoblot analysis was carried out with anti-T7 IgG (against HMGCR) and anti-FLAG IgG (against DUBs) as described. Mev., mevalonate.
Extended Data Fig. 3 Characterization of the deubiquitylase USP20.
a, In vitro activities of USP20 variants indicated by the hydrolysis of a fluorogenic substrate Ubiquitin-7-amino-4-trifluoromethylcoumarin (Ub-AFC). The WT USP20 and USP20(C154S) proteins were purified from HEK 293T cells. Negative control (NC) means no protein added. Inset western blotting shows the similar amount of the proteins. b, Hydrolysis of various linked di-ubiquitin (Ub2) by USP20 in vitro. c, USP20 decreases sterol-induced ubiquitination of HMGCR. CHO-7 cells were transfected with the indicated plasmids, depleted of sterols as in Extended Data Fig. 2 and then treated with 25-HC plus 10 μM MG132 for 2 h. Cells lysates were immunoprecipitated with anti-T7 beads. The pellet was immunoblotted with anti-HA antibody and anti-HMGCR. The input was immunoblotted with anti-FLAG. d, GSK2643943A inhibits USP20-mediated deubiquitination of HMGCR. CHO-7 cells were transfected with plasmids and depleted of sterols. Then the cells were treated with 1 μg ml-1 25-HC in the presence of MG132 for 2 h. Cell lysates were immunoprecipitated with anti-T7 beads. The pellet was immunoblotted with anti-HA and anti-HMGCR antibodies. The input was immunoblotted with anti-FLAG. e, GSK2643943A inhibits USP20-mediated HMGCR deubiquitination in vitro. The sterol-depleted CHO-7 cells were treated with 25-HC plus MG132. Cells lysates were immunoprecipitated with anti-HMGCR antibody-coupled agarose. The pellet samples were then incubated with recombinant USP20 protein for 30 min at 37 °C. *: non-specific band. USP20 was inactivated by boiling for 10 min as a control. Experiments in (a–e) were performed as indicated three times with similar results. f–h, Knockdown of Usp20 accelerated sterol-induced degradation of HMGCR. Huh7 cells were transfected with scrambled (negative control, NC) or USP20-targeting siRNA, treated as in Fig. 1d. The experiments were repeated for three times. i, Quantification of HMGCR in (f–h). Data are mean ± SEM and were analysed by two-way ANOVA.
Extended Data Fig. 4 Characterization of L-Usp20−/− mice and the regulation of hepatic HMGCR by glucose and insulin.
a, Tissue distribution of mouse USP20 protein. b, Schematic of L-Usp20−/− gene-targeting strategy. c, Immunoblotting analysis of different tissues of WT and L-Usp20−/− mice. d-f, Body weight (d), food intake (e) and serum AST (f) of male WT and L-Usp20−/− (n = 5 per group) mice fed chow diet ad libitum. g, Incorporation of tritium-labelled H2O into sterol in kidney of the mice (n = 4 per group) as in Fig. 1f. h, Eight-week-old male L-Usp20−/− and their male WT littermates were fed chow diet and subjected to fasting and refeeding treatment. The metabolic parameters were measured. Each value represents mean ± SEM of data from five mice. *P < 0.05, **P < 0.01, *** P < 0.001 for the level of statistical significance (unpaired two-tailed Student’s t-test) between WT and L-Usp20−/− mice under the same condition. The “a” means refed WT mice versus fasted WT mice; “b” means refed L-Usp20−/− versus fasted L-Usp20−/−; “c” means fasted L-Usp20−/− versus fasted WT mice; “d” means refed L-Usp20−/− versus refed WT mice. i, Relative amounts of mRNAs in the livers of the mice treated as in (h). Each value represents mean of data from three mice. j, Immunoblotting analysis of hepatic HMGCR protein upon glucose and insulin stimulation. Eight-week-old male WT mice were fasted overnight and intraperitoneally injected with 2 mg g-1 glucose or 0.75 mU g-1 insulin or both. After 3 h, liver samples were subjected to immunoblotting. k, qPCR analysis of the mRNAs in mice livers (n = 3 per group) treated as in (j). l, WT mouse primary hepatocytes were untreated (-) or treated (+) with 25.5 mM glucose or 10 nM insulin for 3 h or 5 h. m, qPCR analysis the mouse primary hepatocytes (n = 3 per group) treated as in (l). n, Inhibition of mTOR signalling antagonizes insulin-induced stabilization of HMGCR. WT primary hepatocytes were grown in M199 medium. On day 1, cells were transfected plasmids and depleted of cholesterol by incubating in M199 medium containing 5% LPDS, 1 μM lovastatin and 10 μM mevalonate for 16 h. Then, the cells were switched to the same medium with indicated inhibitor (Wort. wortmannin, 0.2 μM; AKTi, AKT1/2 kinase inhibitor 10 μM; Rap. Rapamycin, 0.1 μM). After 2 h, cells were treated with or without 1 μg ml-1 25-HC plus 10 mM mevalonate in the absence (-) or presence (+) of insulin and the indicated inhibitors for 5 h. Immunoblots were performed with the indicated antibodies. o, Effect of AMPK inhibition Dorsomorphin. WT primary hepatocytes were transfected, depleted of sterol, treated with 1 μM Dorsomorphin, 5.5 mM (-) or 25.5 mM (+) glucose combined with 1 μg ml-1 25-HC plus 10 mM mevalonate for 5 h. p, Effect of AMPK activation A-769662. WT primary hepatocytes were transfected, depleted of sterol, treated with 1 μM AMPK activator A-769662, 25.5 mM (+) glucose combined with 1 μg ml-1 25-HC plus 10 mM mevalonate for 5 h. Experiments in (a, c, l–p) were performed as indicated twice with similar results. Statistical significance was determined using unpaired two-tailed Student’s t-test (d–g, k, m). Bars represent mean ± SEM.
Extended Data Fig. 5 Analysis the phosphorylation sites of USP20 and its interaction with gp78.
a, Procedure to identify the phosphorylated sites of USP20. b, The top 10 of phosphorylated USP20 peptides. H: high glucose; L: low glucose. c, Mass spectrum showing that USP20 was phosphorylated at S132 and S134. d, Domain organization of USP20. zf-UBP: zinc-finger ubiquitin binding domain; DUSP: domain present in ubiquitin-specific protease. e, Validation of the home-made phospho-specific antibody recognizing p-S132/p-S134 of USP20 (p-USP20). USP20 was immunoprecipitated from HEK 293T cells, treated with or without 1U μl-1 λ-phosphatase and 5U ml-1 CIP (PPase), eluted with FLAG peptide and subjected to immunoblotting. f, Rapamycin attenuates USP20 phosphorylation. WT primary hepatocytes were transfected with the indicated plasmid, pre-treated with 100 nM rapamycin for 30 min, and then stimulated with 25.5 mM glucose plus 10 nM insulin for additional 1 h. Cells were lysed with 0.5% NP-40 containing protease and phosphatase inhibitors and immunoprecipitated with anti-FLAG agarose. g, Eight-week-old male WT mice were subjected to fasting and refeeding. Vehicle or rapamycin (5 mg kg-1) were intraperitoneally injected 1 h before fasting or refeeding respectively. Immunoblot analysis of liver samples was performed. F: fasted; R: refed; V: vehicle; Rapa.: rapamycin. h, i, Serum total cholesterol (h) and triglyceride (i) of the mice in (g) (n = 5 per group). j, k, Liver total cholesterol (j) and triglyceride (k) of the mice in (g) (n = 5 per group). l, In vitro activities of USP20 variants. The inset demonstrated equal amounts of USP20 proteins. m, Immunoblot analysis of endogenous Insig-1 and 2 in WT and gp78-KO CHO cells. n, o, Overexpression of Insig-1 (n) or Insig-2 (o) does not block HMGCR-USP20 interaction. p, Mapping the region of gp78 that interacts with USP20. q, The interaction of gp78 and USP20 is unresponsive to 25-HC and mevalonate. Experiments in (e, f, l–q) were performed as indicated twice with similar results. All values are presented as mean ± SEM. Data were analysed by unpaired two-tailed Student’s t-test (h–k).
Extended Data Fig. 6 Characterization of the Usp20KI/KI mice and analysis the metabolism of L-Usp20−/− and Usp20KI/KI mice on HFHS.
a, Strategy to generate Usp20KI/KI mice expressing USP20(S132A/S134A). b–f, Eight-week-old male Usp20KI/KI mice and their male WT littermates under chow diet were subjected to fasting and refeeding (n = 5 per group). TC (b), TG (c), free FA (d), insulin (e) and glucose (f) in the serum were measured. g, Relative amounts of mRNAs in the livers of WT and Usp20KI/KI mice subjected to fasting and refeeding treatments measured by qPCR. Each value represents mean of data from five mice. h, i, In vitro HMGCR deubiquitination assay performed as depicted in Fig. 1b. j–n, Eight-week-old male L-Usp20−/− mice and their male WT littermates were randomly grouped (n = 6 per group) and allowed ad libitum access to water and the HFHS diet for 23 weeks as in Fig. 3a. j, Representative image of the mice after 23-week of HFHS diet. k, Cumulative food intake (n = 6 per group). l, Measurement of fecal energy by bomb calorimetry (n = 5 per group). m, n, Respiratory exchange ratio (RER) (m) and movement (n) as determined by metabolic cages (n = 6 per group). o–u, Eight-week-old male Usp20KI/KI mice and their male WT littermates were randomly grouped (n = 5 per group) and allowed ad libitum access to water and the HFHS Diet for 14 weeks. o, Representative images after 14-week of HFHS diet. p, Body weight (n = 5 per group). q, r, Whole-body composition (n = 5 per group). s, Liver weight (n = 5 per group). t, GTT (n = 5 per group). u, ITT (n = 5 per group). v, w, Effect of chronic HFHS diet. Eight-week-old male L-Usp20−/− mice and their male WT littermates were randomly grouped and allowed ad libitum access to water and normal chow diet or HFHS diet for 8 weeks. v, Mice were fasted for 4 h and then sacrificed. w, Mice were subjected to fasting and refeeding treatment as in Fig. 1a. Liver samples were analysed by western blotting. CD: chow diet; HFHS: high-fat and high sucrose diet; F: fasted; R: refed. Each value represents mean ± SEM. Experiments in (h, i, v, w) were performed as indicated twice with similar results. Statistical significance was determined using unpaired two-tailed Student’s t-test (b–f, l, m, q–s); or two-way ANOVA (n, p, t, u).
Extended Data Fig. 7 Expression of HMGCR(K248R) by AAV or knock-in reverses the phenotypes of L-Usp20−/− mice.
a, HMGCR(K248R) was resistant to sterol-induced degradation. +: 0.3 μg ml-1 25-HC and 3 mM Mev; ++: 1 μg ml-1 25-HC and 10 mM Mev. The experiments were carried out as described in Fig. 1d. b, USP20 did not increase the level of HMGCR(K248R). 1 μg ml-1 25-HC and 10 mM Mev were used. c-j, Eight-week-old male L-Usp20−/− mice and their male WT littermates were injected with 1 × 1010 viral genome (v.g.) of AAV-HMGCR(K248R) or AAV-control (Ctr). After HFHS diet for 8 weeks, the mice were analysed. c, Immunoblotting analysis of liver samples. d, Body weight (n = 3 for WT injected with AAV-Ctr, n = 4 for other groups). e, Total cholesterol in serum (n = 3 for WT injected with AAV-Ctr, n = 4 for other groups). f, Triglyceride in serum (n = 3 for WT injected with AAV-Ctr, n = 4 for other groups). g, h, Whole-body oxygen consumption of mice over dark and light cycles (n = 3 for WT injected with AAV-Ctr, n = 4 for other groups). i, GTT (n = 3 for WT injected with AAV-Ctr, n = 4 for other groups). j, Strategy to generate HmgcrKI/KI mice expressing HMGCR(K248R). k–p, Eight-week-old male mice were fed the HFHS diet for 7 weeks and then subjected to fasting-refeeding treatment. k, western blotting analysis of liver samples. l, Total cholesterol in serum (n = 3 per group). m, Triglyceride in serum (n = 3 per group). n, o, Whole-body oxygen consumption of mice (n = 4 per group). p, GTT (n = 5 per group). Each value represents mean ± SEM. Experiments in (a–c, k) were performed as indicated twice with similar results. Statistical significance was determined using unpaired two-tailed Student’s t-test (d–f, h, i (right), l, m, o, p (right)); or two-way ANOVA (g, i (left), n, p (left)). F: fasted; R: refed.
Extended Data Fig. 8 Level of tricarboxylic acid metabolites and some factors.
a, Proposed link between cholesterol biosynthetic pathway and succinate. b, Levels of the metabolites in liver. The male WT and L-Usp20−/− littermates (n = 4 per group) were fasted for 12 h and then refed for 6 h. The metabolites were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). c, Changes in the metabolites associated with the tricarboxylic acid cycle in liver. The male WT and L-Usp20−/− littermates (n = 4 per group) were fasted for 12 h and then refed for 6 h. Liver metabolites were measured by LC-MS/MS. The fold change was calculated through dividing the L-Usp20−/− mice value by the WT mice mean value. *P < 0.05 for the level of statistical significance (unpaired two-tailed Student’s t-test) between WT and L-Usp20−/− mice. d–l, The epinephrine (d), norepinephrine (e), triiodothyronine (T3) (f), thyroxine (T4) (g), secretin (h), thyroid-stimulating hormone (TSH) (i), FGF21 (j), ALT (k) and AST (l) of L-Usp20−/− and WT mice (n = 6 per group). The data were analysed by unpaired two-tailed Student’s t-test (b, d–l). The values present as mean ± SEM.
Extended Data Fig. 9 Analysis of the mice receiving GSK2643943A.
a, Chemical structure of GSK2643943A. b, In vitro deubiquitylase activity of USP20 inhibited by 1, 3, or 10 μM GSK2643943. NC: no USP20 control. c–i, The mice were treated as in Fig. 4a. TC (c) and TG (d) in serum of mice treated with vehicle (V) or GSK2643943A (G) were measured after fasting and refeeding (n = 4 per group). e, f, GTT (n = 6 per group). g, Quantification of whole-body oxygen consumption of mice (n = 6 per group) in Fig. 4e. h, i, Serum ALT (h) and AST (i) of the mice (n = 6 per group). j-y, The mice were treated as in Fig. 4f. j, Food intake (n = 6 per group). k, Measurement of fecal energy (n = 5 per group) by bomb calorimetry. Fat mass (l), lean mass (m) of the mice (n = 6 per group) measured at the indicated time. Liver weight (n), BAT weight (o), heart weight (p) and kidney weight (q) of the mice (n = 6 per group) measured on Day 14. r, On Day 14 shown in Fig. 4f, the mice were fasted for 4 h and then sacrificed. The tissue samples were analysed by western blotting. Experiments in (r) were performed as indicated twice with similar results. s, t, Whole-body oxygen consumption of the mice (n = 6 per group) measured by metabolic cages. u–w, AST (u), ALT (v) and creatinine (w) in serum of the mice (n = 6 per group). x, Serum TNFα (n = 5 per group). y, Expression of the genes including macrophage markers (CD68, F4/80 and Arg1) and inflammatory markers (TNFα and IL6) of the mice (n = 6 per group) determined by qPCR. All values are expressed as means ± SEM. Data were analysed by two-way ANOVA (e, l, s), or unpaired two-tailed Student’s t-test (c, d, f–k, n–q, t–y).
Extended Data Fig. 10 Analysis the effects of GSK2643943A on Ucp1 KO or L-Usp20−/− mice.
a–l, Eight-week-old male Ucp1 KO mice and their male WT littermates were randomly grouped, fed the HFHS diet and gavaged with vehicle or 30 mg kg-1 GSK2643943A daily for 13 days. GTT and metabolic cage analysis were performed on day 7 and 10, respectively. a, Immunoblotting analysis. *: non-specific band. b, Body weight per mouse (n = 3 per group) during the experiments. c, Cumulative food intake for each treatment. Each value represents mean of data from three mice. d, e, Whole-body oxygen consumption of the mice (n = 3 per group) measured by metabolic cages. f, Respiratory exchange ratio (RER) over dark and light cycles (n = 3 per group). g, h, GTT analysis (n = 3 per group). i–l, TC (i), TG (j), AST (k), and ALT (l) in the serum (n = 3 per group). m–s, Eight-week-old male L-Usp20−/− mice and their male WT littermates were randomly grouped, fed the HFHS diet and gavaged with vehicle or 30 mg kg-1 GSK2643943A daily for 13 days. GTT and metabolic cage analysis were performed on day 7 and 10, respectively. m, Immunoblotting analysis. n, o, Whole-body oxygen consumption of the mice (n = 4 per group) measured by metabolic cages. p, q, GTT analysis (n = 4 per group). r, Total cholesterol in serum (n = 4 per group). s, Triglyceride in serum (n = 4 per group). All values are expressed as means ± SEM. Experiments in (a, m) were performed as indicated twice with similar results. Data were analysed by unpaired two-tailed Student’s t-test (e, f, h–l, o, q–s), or two-way ANOVA (b, d, g, n, p).
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Lu, XY., Shi, XJ., Hu, A. et al. Feeding induces cholesterol biosynthesis via the mTORC1–USP20–HMGCR axis. Nature 588, 479–484 (2020). https://doi.org/10.1038/s41586-020-2928-y
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DOI: https://doi.org/10.1038/s41586-020-2928-y
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