Abstract
Non-alcoholic fatty liver disease (NAFLD), the most prevalent liver pathology worldwide, is intimately linked with obesity and type 2 diabetes. Liver inflammation is a hallmark of NAFLD and is thought to contribute to tissue fibrosis and disease pathogenesis. Uncoupling protein 1 (UCP1) is exclusively expressed in brown and beige adipocytes, and has been extensively studied for its capacity to elevate thermogenesis and reverse obesity. Here we identify an endocrine pathway regulated by UCP1 that antagonizes liver inflammation and pathology, independent of effects on obesity. We show that, without UCP1, brown and beige fat exhibit a diminished capacity to clear succinate from the circulation. Moreover, UCP1KO mice exhibit elevated extracellular succinate in liver tissue that drives inflammation through ligation of its cognate receptor succinate receptor 1 (SUCNR1) in liver-resident stellate cell and macrophage populations. Conversely, increasing brown and beige adipocyte content in mice antagonizes SUCNR1-dependent inflammatory signalling in the liver. We show that this UCP1-succinate–SUCNR1 axis is necessary to regulate liver immune cell infiltration and pathology, and systemic glucose intolerance in an obesogenic environment. As such, the therapeutic use of brown and beige adipocytes and UCP1 extends beyond thermogenesis and may be leveraged to antagonize NAFLD and SUCNR1-dependent liver inflammation.
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Data availability
Source data for all mouse experiments have been provided. Mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the dataset identifier PXD024717. All other data are available from the corresponding author upon request. Source data are provided with this paper.
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Acknowledgements
This work was supported by the Claudia Adams Barr Program, the Lavine Family Fund and NIH grant no. DK123095 (E.T.C), NIH grant no. DK123321 (E.L.M.), the National Cancer Center (H.X.), grant no. R01DK078081 (N.N.D.) and the Juvenile Diabetes Research Foundation (A.F.). We thank B. Spiegelman, P. Puigserver, K. Sharabi, E. Rosen, S. Patel and R. Bronson for discussions, the Nikon Imaging Center at Harvard Medical School and the Harvard Center for Biological Imaging for assistance with microscopy, Dana-Farber/Harvard Cancer Center Rodent Histopathology Core (grant no. NIH-5-P30-CA06516) for preparing histology slides and the Harvard Digestive Disease Center, Core D for assistance with bomb calorimetry. Cartoon illustrations in Figs. 1f, 3a, 3h, 4c were created with BioRender.com.
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E.L.M. designed research, carried out experiments and analysed data. H.X., M.P.J., J.S., G.A.B. and A.F. carried out and analysed data from mass spectrometry experiments. R.G. and N.V.T. assisted with animal physiology experiments. C.H. and H.P. assisted with flow cytometry experiments. A.R. assisted with imaging experiments. S.P.G. and N.N.D. oversaw mass spectrometry experiments. L.L. oversaw flow cytometry experiments. E.T.C. directed research and cowrote the paper with assistance from the other authors.
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Extended data
Extended Data Fig. 1 UCP1KO depletes the UCP1 catabolic circuit in BAT.
a, Protein abundance differences between WT and UCP1KO BAT of mitochondrial respiratory chain proteins (WT n = 5; UCP1KO n = 4). b, Depiction of the UCP1 catabolic circuit that includes UCP1 and the mitochondrial respiratory chain components that generate mitochondrial membrane potential, which is subsequently dissipated by UCP1. KO of UCP1 depletes the entire circuit in BAT, as shown in (a). See source data for precise p values.
Extended Data Fig. 2 Validation of western diet as a model of NAFLD, food intake during WD, chow diet liver assessment and lymphoid immune populations.
a, b, Relative gene expression in liver of mice following 14 weeks on chow or western diet (n = 4 except Cd11b chow n = 3, Cd11c WD n = 3). c, Levels of ALT (left) and AST (right) in plasma following 14 weeks chow or WD feeding (n = 4). d, Calories consumed during 14 weeks WD feeding comparing WT and UCP1KO mice (n = 10). e-i, Protein abundance differences of annotated pathways proteins or HSC activation proteins between WT and UCP1KO liver following 14 weeks chow feeding. (WT n = 4, UCP1KO n = 5). Data represent fold over WT. j, Fraction of CD45+ cells for each indicated population from livers of WT and UCP1KO mice following 14 weeks on WD (CD3, CD8 WT n = 10, UCP1KO n = 13; CD4 WT n = 10, UCP1KO n = 12; CD19 WT n = 13, UCP1KO n = 16). *P < 0.05, **P < 0.01, ***P < 0.001 (two-tailed Student’s t-test for pairwise comparisons). Data are mean ± s.e.m. See source data for precise p values.
Extended Data Fig. 3 The UCP1 catabolic circuit controls liver extracellular succinate levels.
a, Extracellular fluid extraction protocol. b, Liver tissue metabolomics: All annotated metabolites (grey), metabolites significantly changed (black), succinate (red) following 14 weeks on WD (n = 10). c, Absolute succinate concentration in liver and epi EF following 14 weeks on WD (Liver EF: WT, UCP1KO n = 8; SUCNR1/UCP1KO n = 9; Epi EF: WT n = 5; UCP1KO, SUCNR1/UCP1KO n = 9). d, e, BAT succinate catabolism determines as % of 2 min (m + 4) 13C-succinate and downstream (m + 4) 13C-TCA cycle metabolites remaining at 30 mins in BAT (c) and subQ (d) following 10 days daily injection with vehicle or i.p. β-adrenoreceptor agonism with CL-316,243 (1 mg/kg) and subsequent bolus i.v. 13C-succinate (100 mg/kg) for the indicated times (n = 5, except vehicle 2 min n = 4 in d). f, Abundance of succinate in TCA metabolites in BAT (left) and SubQ (right) following either 29 °C housing or 2 weeks 4 °C exposure (n = 5). g, (m + 4) 13C-succinate and downstream (m + 4) TCA cycle metabolite abundance following pre-treatment with vehicle or i.p. β-adrenoreceptor agonism with CL-316,243 (1 mg/kg; 30 min) and subsequent bolus i.v. 13C-succinate (100 mg/kg) for the indicated times. (Fumarate: 0 min n = 11, 2 min veh n = 11, 5 min veh n = 13, 30 min veh n = 12, 2 min CL n = 11, 5 min CL n = 13, 30 min CL n = 12; malate: 0 min n = 10, 2 min veh n = 11, 5 min veh n = 13, 30 min veh n = 12, 2 min CL n = 11, 5 min CL n = 13, 30 min CL n = 12; succinate: 0 min n = 10, 2 min veh n = 11, 5 min veh n = 13, 30 min veh n = 12, 2 min CL n = 11, 5 min CL n = 12, 30 min CL n = 11). *P < 0.05, **P < 0.01, ***P < 0.001. (two-tailed Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons involving independent variable, two-way ANOVA for multiple comparisons involving two independent variables). Data are mean ± s.e.m. See source data for precise p values.
Extended Data Fig. 4 Assessment of UCP1/SUCNR1KO energy expenditure, caloric absorption and caloric intake during WD.
a-c, Whole body energy expenditure of mice during 7 days WD feeding for WT, UCP1KO, and UCP1/SUCNR1KO mice (n = 8 except a WT n = 7) as determined by indirect calorimetry. d, e, Caloric absorption (d) and energy assimilation (e) during 7 days WD feeding. Proportion of energy assimilated from diet was determined by subtracting the total calories remaining in mouse feces from the total calories consumed in the same period (n = 8 from one assessment). f, Calories consumed during 14 weeks WD feeding (n = 10). One-way ANOVA for multiple comparisons involving independent variable, two-way ANOVA for multiple comparisons involving two independent variables, ANCOVA for b, c. Data are mean ± s.e.m. Assessments of UCP1/SUCNR1KO mice were performed simultaneously with WT and UCP1KO as depicted in Extended Data Fig. 2, so WT and UCP1KO presentations in this figure are from the same underlying data reported in Figure Extended Data Fig. 2. See source data for precise p values.
Extended Data Fig. 5 Assessment of UCP1/SUCNR1KO during WD and succinate drinking water experiments.
a, Representative cytofluorimetric dot plots for indicated immune cell populations from livers of WT, UCP1KO and UCP1/SUCNR1KO mice following 14 weeks on WD. b, Relative gene expression in WT, UCP1KO, and UCP1/SUCNR1KO livers following 14 weeks WD feeding (n = 10 except Il6, Nos2 n = 9 in WT and Nos2 n = 9 in UCP1/SUCNR1KO). c, Fraction of CD45+ cells for each indicated gated cell population from livers of WT, UCP1KO and UCP1/SUCNR1KO mice following 14 weeks on WD (n = 9 except CD8 n = 7 in UCP1/SUCNR1KO). d-h, Protein abundance differences of annotated pathways proteins or HSC activation proteins between vehicle and 1.5% succinate-treated UCP1KO liver following 6 weeks HFD (n = 3). Data represent fold over 0%. i, Sirius red staining observed in liver harvested from mice following 14 weeks WD feeding (upper panels 20x magnification, scale bars 100 μm; middle panels, 10x magnification, scale bars 200 μm, lower panels, 4x magnification, scale bars 200 μm; n = 4 biological replicates/genotype imaged). j, Relative abundance of hydroxyproline in plasma following 14 weeks on WD WT (n = 10). *P < 0.05, **P < 0.01, ***P < 0.001. (two-tailed Student’s t-test for pairwise comparisons, one-way ANOVA for multiple comparisons involving independent variable). Data are mean ± s.e.m. Assessments of UCP1/SUCNR1KO mice were performed simultaneously with WT and UCP1KO as depicted in Fig. 2 and Extended Data Fig. 2, so WT and UCP1KO presentations in this figure are from the same underlying data reported in Fig. 2 and Extended Data Fig. 2. See source data for precise p values.
Extended Data Fig. 6 Physiological assessment of WT and SUCNR1KO mice upon WD feeding.
a, b, Change in body mass (a) and final body weight (b) during WD feeding (22 °C n = 18, 29 °C n = 26). c, Body composition of mice following 14 weeks WD feeding (22 °C n = 18, 29 °C n = 26). d, Relative Ucp1 gene expression in BAT following 14 weeks on WD (n = 8). e, f, Change in body mass (e) and final body weight (f) of SUCNR1KO mice during 14 weeks WD feeding (22 °C n = 27, 29 °C n = 30). g, Body composition of mice following 14 weeks WD feeding (22 °C n = 27, 29 °C n = 30). h, Relative Ucp1 gene expression in BAT following 14 weeks on western diet (n = 8). i, ANCOVA analysis of energy expenditure following 14 weeks WD feeding. (WT 22 °C n = 18, WT 29 °C n = 27, SUCNR1KO 22 °C n = 27, SUCNR1KO 29 °C n = 30). j, Calories consumed during 14 weeks WD feeding. (WT 22 °C n = 19, WT 29 °C n = 26, SUCNR1KO 22 °C n = 27, SUCNR1KO 29 °C n = 31). **P < 0.01, ***P < 0.001. (two-tailed Student’s t-test for pairwise comparisons, ANCOVA for i). Data are mean ± s.e.m. See source data for precise p values.
Extended Data Fig. 7 SUCNR1 ablation is protective against liver dysfunction initiated by thermoneutral housing.
a, Protein abundance differences of annotated pathway proteins between WT and SUCNR1KO liver following 14 weeks WD feeding at thermoneutrality. Top pathways enriched in proteins exhibiting > 30% decrease between groups highlighted; (WT n = 7, SUCNR1KO n = 8). b-e, Protein abundance differences of top enriched pathways between WT and SUCNR1KO liver following 14 weeks WD feeding at thermoneutrality. (WT n = 7, SUCNR1KO n = 8). Data represent fold over WT. For (e, j): 1. Innate immune response in mucosa; 2. MyD88-dependent toll-like receptor signalling pathway; 3. Positive regulation of cell-matrix adhesion; 4. Interleukin-12-mediated signaling pathway; 5. Transforming growth factor beta receptor signaling pathway. 6. Additional inflammatory proteins not annotated in pathways. f, Protein abundance differences of annotated pathway proteins between WT and SUCNR1KO liver following 14 weeks WD feeding at room temperature. Top pathways found to be enriched in proteins exhibiting > 30% decrease between WT and SUCNR1KO at thermoneutrality in (a) are highlighted; (WT n = 7, SUCNR1KO n = 8). g-j, Protein abundance differences of annotated pathways proteins between WT and SUCNR1KO liver following 14 weeks WD feeding at room temperature. (WT n = 7, SUCNR1KO n = 8). Data represent fold over WT. k, Protein abundance differences of annotated pathway proteins between WT and SUCNR1KO liver following 14 weeks WD feeding at room temperature. Top pathways found to be enriched in proteins exhibiting > 30% decrease between WT and SUCNR1KO at room temperature are highlighted; (WT n = 7, SUCNR1KO n = 8). l, Protein abundance differences of annotated pathway proteins between WT and SUCNR1KO liver following 14 weeks WD feeding at room temperature. (WT n = 7, SUCNR1KO n = 8). Data represent fold over WT. *P < 0.05, **P < 0.01, ***P < 0.001. (two-tailed Student’s t-test for pairwise comparisons). Data are mean ± s.e.m. See source data for precise p values.
Extended Data Fig. 8 SUCNR1 ablation limits WD-induced inflammatory and ECM remodelling protein abundance at thermoneutrality but not at room temperature.
a-h, Protein abundance differences of annotated pathways proteins between WT and SUCNR1KO liver following 14 weeks WD feeding at thermoneutrality (a, c, e, g) or room temperature (b, d, f, h). (WT n = 10-11, SUCNR1KO n = 10-12; except A4, GAS6, ICAM2, LAMA1, LOXL2, ITA4, MMP14, WT n = 5–7, SUCNR1KO n = 8). Data represent fold over WT at each temperature. Illustrated proteins are from top pathways found to be enriched in proteins exhibiting > 50% increase between WT and UCP1KO in Fig. 2. i, 13C4-succinate uptake into brown adipocytes, KC and HSCs following 2 min incubation with 13C4-succinate (100 μM). Data represent relative signal normalized to cell number (n = 3). Data represent fold over brown adipocytes vehicle. j, UCP1 protein abundance (sum signal to noise ratio) in BAT, KC and HSCs (BAT n = 5; KC, HSC n = 3). k, Relative Ucp1 expression in BAT, KC and HSCs (BAT n = 5; KC, HSC n = 3). Data represent fold over BAT. *P < 0.05, **P < 0.01, ***P < 0.001. (two-tailed Student’s t-test for pairwise comparisons). Data are mean ± s.e.m. See source data for precise p values.
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Mills, E.L., Harmon, C., Jedrychowski, M.P. et al. UCP1 governs liver extracellular succinate and inflammatory pathogenesis. Nat Metab 3, 604–617 (2021). https://doi.org/10.1038/s42255-021-00389-5
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DOI: https://doi.org/10.1038/s42255-021-00389-5
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