Fig 1: Circulating RSPO2 leads to unhealthy expansion of adipose tissue and insulin resistance in vivo.a–o, RSPO2 overexpression in mice by tail-vein delivery of pAAV–CAG–Rspo2. Representative immunoblots (a) and quantification of RSPO2 and HSP90 in liver (b), circulation (c), ingWAT (d) and visWAT (e) in RSPO2-overexpression mice. Body weight curve (f), lean mass and fat mass (g) and ingWAT and visWAT tissue weight (h) of AAV-infected mice. Representative H&E staining images (i), average of adipocytes size (µm2) and adipocyte size frequency distribution of ingWAT. Blood glucose normalized to initial blood glucose after insulin injection (l) in ITT and area under the curve (AUC) was quantified as shown in m. Fasting blood glucose (n) and triglycerides (o) in AAV-injected mice. Data are shown as mean ± s.e.m., n = 6 mice. Data were analyzed using a two-tailed Student’s t-test. Scale bar, 100 µm. p–r, RSPO2 overexpression by injection into ingWAT. Adipocyte size frequency distribution (p) and representative H&E staining of ingWAT. Data are shown as mean ± s.e.m. Glucose levels in blood in ITT and glucose was normalized to time point 0 (r). Data are shown as mean ± s.d. Comparison of AUC (r, right) in ITT. Data are shown as mean ± s.e.m., n = 5 mice (CAG–GFP), n = 6 mice (CAG–Rspo2). Data analysis was performed using a two-tailed Student’s t-test. s, Circulating RSPO2 levels in insulin-sensitive and insulin-resistant individuals. Data are shown as mean ± s.d., n = 11 (male, insulin sensitive), n = 10 (male, insulin resistant), n = 18 (female, insulin sensitive), n = 21 (male, insulin resistant). Data analysis was performed using a two-tailed Student’s t-test. t–v, Spearman correlation coefficient analysis of circulating RSPO2 and glucose infusion rate (t), visceral fat area (u) and max adipocyte volume (v). P values are corrected by two-stage step-up method of Benjamini, Krieger and Yekutieli with an FDR = 0.05.Source data
Fig 2: Rspo2 inhibits transition of eP1 cells to eP2 cells.a–f, Experimental scheme (a) for transplantation of tdTomato+ eP1 cells into inguinal adipose tissue of wild-type (WT) mice. FACS analysis (b) of VAP1 and CD142 expression in tdTomato+ eP1 cells 10 d after transplantation. Expression of P1 marker genes (c) (Cd55, Dpp4, Pi16 and Psck6), P2 marker genes (d) (Vap1, Icam1, Col4a1 and Sparcl1), Pparg and Cebpa (e) and P3 marker genes (f) (Cd142, Gdf10, Clec11a and Igfbp3) in eP1 cells (from donor mice), eP2 cells (from donor mice), eP3 cells (from donor mice), VAP1+ cells (derived from implanted eP1 cells) and VAP1- cells (derived from implanted eP1 cells). Data are shown as mean ± s.e.m., n = 4 biological replicates. g–m, Experimental scheme (g) for injection of AAVs into ingWAT for overexpression of RSPO2. Western blot images (h) and quantification (i) of RSPO2 protein and Rspo2 mRNA (j) in ingWAT. FACS analysis of eP1/SVF (k), eP2/SVF (l), CD55+VAP1+ (m) in ingWAT. Data are shown as mean ± s.e.m., n = 6 mice (h,i), n = 5–6 mice (j), n = 5 mice (k–m). Data were analyzed using two-tailed Student’s t-test. n,o, Experimental scheme (n) for transplantation of tdTomato+ eP1 cells into RSPO2 overexpression mice. FACS analysis of (VAP1+:tdTomato+) cells in tdTomato+ eP1 cells (o). Data are shown as mean ± s.e.m., n = 5 biological replicates. Data were analyzed using a two-tailed paired Student’s t-test.Source data
Fig 3: pAAV–CAG-GFP and pAAV-CAG-Rspo2 infection mice on HFD.a–j) Overexpression of RSPO2 by tail vein delivery of AAVs in mice (a). Images of ingWAT and visWAT (b), food intake per day (c), Time-resolved oxygen consumption (d), quantification of triglyceride per gram of liver (e), representative images of H&E staining of liver (f) from pAAV-CAG-GFP and pAAV-CAG-Rspo2 infection mice. Blood glucose (g) and area under curve (AUC) (h) shown by intraperitoneal pyruvate tolerance test. Triglyceride levels in blood (i) and hepatic triglyceride secretion rate (j) after tyloxapol injection. Data shown as mean ± SD (i, j), n = 6 mice. Data analysis was performed using two-tailed Student’s t-test. k–p) Experimental scheme (k) for overexpression of RSPO2 in ingWAT by injection AAV into ingWAT. Quantification of RSPO2 in ingWAT by western blot (l) and in circulation (m). Body weight (n), tissue weight (o), and time-resolved oxygen consumption (p) of pAAV-CAG-GFP and pAAV-CAG-Rspo2 ingWAT infection mice. Data shown as mean ± SD, n = 5 mice (CAG-GFP); n = 6 mice (CAG-Rspo2). Data analysis was performed using two-tailed Student’s t-test. q–r) Spearman correlation of serum RSPO2 level with HOMA-IR (q), mean adipocyte volume (r) in male and female subjects. The correlation coefficient was calculated using a Spearman’s Correlation Test. This figure is related to Fig. 6. Source data
Fig 4: Rspo2 inhibits adipogenesis of eP1 cells in vivo.a–f, Experimental scheme (a) for cell transplantation in Matrigel. Rspo2 expression in eP1 Matrigel plugs and in eP2 Matrigel plugs (b). Quantification of adipocytes and cell number in eP1 Matrigel plugs (c) and eP2 Matrigel plugs (e). Representative hematoxylin and eosin (H&E) staining of eP1 Matrigel plugs (d) and eP2 Matrigel plugs (f). Data show mean ± s.e.m., n = 3 biological replicates (b), n = 5 biological replicates (c,e). Data analysis was performed using a two-tailed Student’s t-test. Scale bar, 100 µm. g–k, Experimental scheme for overexpression of RSPO2 in AdipoCre-NucRed mice fed with HFD or chow diet. Western blot images (h) and quantification (i) of RSPO2 protein in liver and ingWAT; HSP90 bands were used as loading control. Quantification of adipocyte numbers in ingWAT (j) and visWAT (k) of mice shown in g. Data are shown as mean ± s.d., n = 6 mice. Data analysis was performed by two-tailed Student’s t-test (i) and one-way ANOVA (j,k). In j, Total cell number, F(3,20) = 14.4, P < 0.0001; adipocyte, F(3,20) = 15.50, P < 0.0001; non-adipocyte, F(3,20) = 14.1, P < 0.0001. In k, total cell number, F(3,20) = 14.4, P < 0.0001; adipocyte, F(3,20) = 15.50, P < 0.0001; non-adipocyte, F(3,20) = 14.1, P < 0.0001. l–o, Experimental scheme (l) for overexpression of RSPO2 in ingWAT by injection of AAV into ingWAT of AdipoCre-NucRed mice. Western blot images (m) and quantification (n) of RSPO2 protein in ingWAT of mice shown in l. HSP90 bands were used as loading control. Quantification of cell numbers by quantitative PCR in ingWAT (o). Data shows mean ± s.d., n = 5–6 mice. Data were analyzed using a two-tailed Student’s t-test.Source data
Fig 5: snRNA-seq reveals Rspo2 reducing adipocytes number in vivo.a, Integrated analysis of snRNA-seq, including 14,303 nuclei from ingWAT in mice fed on HFD with chronic expression of GFP or RSPO2 by AAV, yielding 2,218 genes (median). Unsupervised clustering shown as a UMAP plot, seven populations were identified, including adipocytes (adipo) (red), pre-adipocytes (PreA) (blue), macrophages (macro) (green) and natural killer (NK) cells (orange). b, Dot plots for representative markers of each cluster. Expression level (indicated by red color) refers to the log normalized ratio of gene expression reads, normalized to the sum of all reads within each nucleus. Percent expressed refers to the ratio of cells within each cluster that express the genes listed in x axis. c, Cluster compositions in CAG–GFP (n = 7,190 nuclei) and CAG–Rspo2 (n = 7,143 nuclei) conditions. d, Violin plots for Acss2, Nkain2, Sntg1, S100a6, Mrc1 and Gpx1, which are differentially expressed between CAG–GFP and CAG–Rspo2 conditions. e, Subclustering analysis of preadipocyte populations. Unsupervised subclustering of 6,411 preadipocyte nuclei from ingWAT, yielding 2,577 (median) genes. Five subpopulations of preadipocytes (PA-1–PA-5) were identified. f, Feature plots for Dpp4, Pparg and Fmo2, shown as separated plots by conditions. g,h, Pre-adipocyte cluster compositions in CAG–GFP (n = 3,539 nuclei) and CAG–Rspo2 (n = 2,872 nuclei) conditions.
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