Abstract
Obesity occurs when excess energy accumulates in white adipose tissue (WAT), whereas brown adipose tissue (BAT), specialized for energy expenditure through thermogenesis, potently counteracts obesity. Factors that induce brown adipocyte commitment and energy expenditure would be a promising defence against adiposity. Here, we show that Lgr4 homozygous mutant (Lgr4m/m) mice show reduced adiposity and resist dietary and leptin mutant-induced obesity with improved glucose metabolism. Lgr4m/m mice show a striking increase in energy expenditure, and exhibit brown-like adipocytes in WAT depots with higher expression of BAT and beige cell markers. Furthermore, Lgr4 ablation potentiates brown adipocyte differentiation from the stromal vascular fraction of epididymal WAT, partially through retinoblastoma 1 gene (Rb1) reduction. A functional low-frequency human LGR4 variant (A750T) has been associated with body mass index in a Chinese obese-versus-control study. Our results identify an important role for LGR4 in energy balance and body weight control through regulating the white-to-brown fat transition.
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Acknowledgements
This work was supported by grants from the National Natural Science Foundation of China (nos 81030011, 30725037, 81100601, 81100634 and 30890043), the Sector Funds of Ministry of Health (no. 201002002) and the National Key New Drug Creation and Manufacturing Program of the Ministry of Science and Technology (2012ZX09303006-001). We thank S. Lai (Johns Hopkins School of Medicine) and D. Cai (Albert Einstein College of Medicine) for revision of the manuscript. We thank N. Fan and F. Li for their technical assistance in immunostaining and animal experiments.
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J.W. and G.N. conceived the project and designed the experiments, and J.W. carried out most of the experiments. J.W., R.L. and G.N. wrote the paper. F.W. carried out a subset of in vitro experiments. J.H., J.S., B.C., W.G., Y.Z. and W.W. recruited the obese patients and normal individuals and contributed with the human study. X.L. assisted with statistical analysis. R.L. carried out SVF related experiments. M.C., Y.K. and X.Z. contributed with genotyping and animal experiments. Q.M. and R.W. carried out DNA isolation and sequencing. Z.Z. contributed with fat content scanning. X.X. contributed comments and advice on the manuscript. M.L. contributed with Lgr4m/m mice and valuable materials. All authors were involved in editing the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Improved metabolic parameters in Lgr4m/m mice fed CD or HFD and Lgr4/leptin double mutant mice.
(a) Fat mass and lean mass content normalized to body weight in wild-type and Lgr4m/m mice. (b) Blood glucose levels of CD-fed 8-week and 40-week wild-type and Lgr4m/m mice fasted for 16h (n = 8–12 for each group). (c) Blood glucose levels of CD-fed 8-week wild-type and Lgr4m/m mice fasted for 12 h, 36 h and 48 h (n = 10-17 for each group). (d) Plasma insulin levels of CD-fed wild-type and Lgr4m/m mice at 0, 30 and 60 min after glucose injection (n = 8-9 for each group). (e, f) Plasma TG levels (e) and total cholesterol levels (f) of wild-type and Lgr4m/m mice fed CD (n = 9 for each group). (g) Blood glucose levels of HFD-fed 12-week wild-type and Lgr4m/m mice fasted for 16h (n = 10-13 for each group). (h) Plasma insulin levels of HFD-fed wild-type and Lgr4m/m mice at 0, 30 and 60 min after glucose injection (n = 8-11 for each group). (i) Fat mass and lean mass content normalized to body weight in ob/ob and m/m;Ob mice. (j, k) Glucose tolerance test (j) and insulin tolerance test (k) in ob/ob and m/m;Ob mice fed CD (n = 6 for each group). (l, m) Plasma TG levels (l) and total cholesterol levels (m) of ob/ob and m/m;Ob mice fed CD (n = 8 for each group). WT, wild type; m/m, Lgr4m/m; Ob, ob/ob mice; m/m;Ob, Lgr4/leptin double mutant mice. TG, triglycerides; TC, total cholesterol. *,p<0.05;**,p<0.01;***,p<0.001. Error bars, s.e.m.
Supplementary Figure 2 Increased energy expenditure in Lgr4/leptin double mutant mice.
(a-d) Food intake (a, b) and physical activity (c, d) of the two groups was measured in a 24 h period (a, c, 24 h period; b, d, the average quantification for 12 h light and 12 h dark in a and c, respectively) (n = 8 for each group). (e, f) 24 h energy expenditure was compared between the two groups (e, energy expenditure per mouse was plotted against lean mass; f, the adjusted means of energy expenditure in the two groups analyzed by ANCOVA with fat mass and lean mass as two covariates, n = 8, P = 0.1). (g-j) O2 consumption (g, h) and CO2 production (i, j) was recorded during a 24 h period (g, i, 24 h period; h, j, average of light and dark time, respectively) (n = 8 for each group). WT, wild type; m/m, Lgr4m/m; Ob, ob/ob mice; m/m;Ob, Lgr4/leptin double mutant mice. *,p<0.05;**,p<0.01;***,p<0.001. Error bars, s.e.m.
Supplementary Figure 3 Unchanged BAT phenotypes in Lgr4m/m mice.
Ablation of Lgr4 induces white-to-brown fat transition but does not affect the interscapular BAT. (a) Hematoxylin and eosin staining of BAT in wild-type and Lgr4m/m mice. Scale bar, 100 μm. (b) Electron microscopy of BAT in wild-type and Lgr4m/m mice. Scale bar, 1,000 nm. (c, d) Mitochondrion number of BAT between wild-type and Lgr4m/m mice determined by the normalized 16s rRNA level to hexokinase 2 level (c) and the normalized cytochrome b level to gapdh level (d). Hexo, hexokinase 2 gene; Cyt b, cytochrome b gene. (e) Protein levels of BAT-related genes in wild-type and Lgr4m/m mice under CD (left panel) or HFD (right panel). Uncropped data are depicted in Supplementary Fig. S7b. WT, wild-type mice; m/m, Lgr4m/m mice. Error bars, s.e.m.
Supplementary Figure 4 Ablation of Lgr4 induces white-to-brown fat transition.
(a) Electron microscopy of eWAT in wild-type and Lgr4m/m mice. Wild-type BAT was used as the reference. Scale bar, 10 μm. (b) Hematoxylin and eosin staining of eWAT in wild-type and Lgr4m/m mice; 4-month-old wild-type and mutant mice were treated under normal conditions or cold room for 7 days, or isoprenaline injection (0.75 mg/kg body weight) for 10 days. Scale bar, 100 μm. (c, d) Representative haematoxylin and eosin staining (scale bar, 100 μm) (c) and electron microscopy (scale bar, 5 μm). (d) of eWAT in Ob and m/m;Ob mice under normal conditions. (e) Immunohistochemical staining of CD137 and TMEM26 in eWAT of wild-type and Lgr4m/m mice under isoprenaline treatment for 10 days (scale bar, 50 μm). (f) Protein levels of BAT-related genes in eWAT and iWAT of wild-type and Lgr4m/m mice under cold room stress. eWAT, left panel, iWAT, right panel. Uncropped data are depicted in Supplementary Fig. S7c. (g-l) Plasma levels of thyroid hormones (g, h) and principal catecholamines (i, j), and 12 h urinary catecholamine excretion normalized to body weight (k, l) in wild-type and Lgr4m/m mice (n = 6-12 for each group). WT, wild-type mice; m/m, Lgr4m/m mice; Ob, ob/ob mice; m/m;Ob, Lgr4/leptin double mutant mice. *,p<0.05;**,p<0.01;***,p<0.001. Error bars, s.e.m.
Supplementary Figure 5 Ablation of Lgr4 promotes the differentiation of eWAT SVF to brown adipocyte in vitro.
(a, b) Representative phase (a) and Oil Red O staining images (b) of the fully differentiated eWAT SVF with brown adipocyte induction under microscopy. Scale bar, 50 μm. (c) Relative mRNA levels of beige adipocyte related genes in the differentiated SVF cells from the eWAT of wild-type and Lgr4m/m mice (n = 3-5). Source data are provided in Supplementary Table S5. (d) Gene set enrichment analysis (GSEA). Black columns indicated 214 genes significantly down-regulated in the fully differentiated Lgr4m/m SVF, which were involved in cell adhesion, angiogenesis, cell cycle, cell-matrix adhesion, negative regulation of cell proliferation, actin filament bundle assembly, collagen fibril organization, cell shape regulation, vascular endothelial growth factor receptor signaling pathway. Differentially expressed genes were ranked according to the folds of Lgr4m/m adipocytes vs. wild-type adipocytes. d also referred to Supplementary Table S2. (e) Basal and insulin-stimulated 2-deoxyglucose uptake of the differentiated SVF cells (n = 3-4 for each group). (f) Relative mRNA levels of WAT selective markers in the differentiated SVF cells from wild-type and Lgr4m/m mice after eight-day differentiation with classical white adipocyte induction cocktails (n = 3-4 for each group). (g) Relative mRNA levels of BAT related genes in the differentiated SVF cells from the eWAT of wild-type and Lgr4m/m mice after isoprenaline injection. Wild-type and Lgr4m/m mice were treated with isoprenaline (0.75mg/kg body weight) for 10 days before isolating the SVF, which were then differentiated into brown adipocytes. (n = 6 for wild type and n = 3 for m/m). For e-g, source data are provided in Supplementary Table S5. (h) Isolation of the PDGFRα+ cells from the eWAT SVF of wild-type and Lgr4m/m mice by FACS. Isotype IgG was used as a negative control. WT, wild-type mice; m/m, Lgr4m/m mice. *,p<0.05;**,p<0.01;***,p<0.001. Error bars, s.e.m.
Supplementary Figure 6 LGR4 regulates Rb expression.
(a) Relative mRNA levels of the indicated genes in eWAT SVF of wild-type and Lgr4m/m mice (n = 5-7 for wild type and n = 4 for m/m). Source data are provided in Supplementary Table S5. (b) Rb mRNA levels in eWAT of wild-type and Lgr4m/m mice under cold room stimulation and isoprenaline treatment for 7 days and 10 days respectively. (c) Relative Rb mRNA levels after knockdown of Lgr4 in C3H10T1/2 cells using its SiRNA (n = 4). Source data are provided in Supplementary Table S5. (d) The transcriptional activity of the indicated truncated Rb promoters was compared (n = 3). (e) Rb mRNA levels in wild-type SVF and Lgr4 mutant SVF infected with lentiviral vector or lentiviral Rb construct (n = 3). (f) The Lgr4 mRNA levels in wild-type SVF and Lgr4 mutant SVF infected with lentiviral vector or lentiviral LGR4 construct (n = 3). For d-f, source data are provided in Supplementary Table S5. (g) The protein levels of cytoplasmic and nuclear β-catenin in eWAT of wild-type and Lgr4m/m mice. α-tubulin was used as the loading control for cytoplasmic proteins; Lamin B was used as the loading control for nuclear proteins. Uncropped data are depicted in Supplementary Fig. S7e. WT, wild-type mice; m/m, Lgr4m/m mice. Trc, Truncated; mRb, mouse Rb; Ctrl, control. LV, lentiviral vector. *,p<0.05;**,p<0.01;***,p<0.001. Error bars, s.e.m.
Supplementary Figure 7 Full scans of western immunoblotting.
(a) Related to Fig. 3i, (b) Related to Supplementary Fig. 3e, (c) Related to Supplementary Fig. 4f, (d) Related to Fig. 5b (e) Related to Supplementary Fig. 6g. NC, normal condition, CR, cold room stimulation, ISO, isoprenaline treatment, HFD, high-fat diet. WT, wild-type, m/m, Lgr4 mutant.
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Wang, J., Liu, R., Wang, F. et al. Ablation of LGR4 promotes energy expenditure by driving white-to-brown fat switch. Nat Cell Biol 15, 1455–1463 (2013). https://doi.org/10.1038/ncb2867
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DOI: https://doi.org/10.1038/ncb2867
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