Human RSPO1 Mutation Represses Beige Adipocyte Thermogenesis and Contributes to Diet‐Induced Adiposity

Abstract Recent genetic evidence has linked WNT downstream mutations to fat distribution. However, the roles of WNTs in human obesity remain unclear. Here, the authors screen all Wnt‐related paracrine factors in 1994 obese cases and 2161 controls using whole‐exome sequencing (WES) and identify that 12 obese patients harbor the same mutations in RSPO1 (p.R219W/Q) predisposing to human obesity. RSPO1 is predominantly expressed in visceral fat, primarily in the fibroblast cluster, and is increased with adiposity. Mice overexpressing human RSPO1 in adipose tissues develop obesity under a high‐fat diet (HFD) due to reduced brown/beige fat thermogenesis. In contrast, Rspo1 ablation resists HFD‐induced adiposity by increasing thermogenesis. Mechanistically, RSPO1 overexpression or administration significantly inhibits adipocyte mitochondrial respiration and thermogenesis via LGR4–Wnt/β‐catenin signaling pathway. Importantly, humanized knockin mice carrying the hotspot mutation (p.R219W) display suppressed thermogenesis and recapitulate the adiposity feature of obese carriers. The mutation disrupts RSPO1's electrostatic interaction with the extracellular matrix, leading to excessive RSPO1 release that activates LGR4–Wnt/β‐catenin signaling and attenuates thermogenic capacity in differentiated beige adipocytes. Therefore, these findings identify that gain‐of‐function mutations and excessive expression of RSPO1, acting as a paracrine Wnt activator, suppress fat thermogenesis and contribute to obesity in humans.

The frequency of RSPO1 mutations (p.R219W/Q) in lean subjects (controls, n = 2,161) versus young subjects with severe obesity (cases, n = 1,944).  fed with one-week HFD (n = 8 per group). The hourly measurements were assessed by two-way ANOVA model to evaluate the interaction between genotype and time, and pairwised t-test with Benjamini-Hochberg correction was used as post-hoc test to evaluate the differences between genotypes in each hour.
K) The heatmap of genes differentially expressed in BAT between hRSPO1 Tg and WT mice fed HFD (n = 3 per group). Representative genes related to mitochondrial biogenesis and function were labeled.
L) The most significant changed pathways revealed by GSEA analysis based on GO:BP database in BAT of hRSPO1 Tg mice.
Data are shown as the mean ± sem, and statistical significances between genotypes were assessed by unpaired Student's t test (B-G). FDR below 0.05 was considered as the criteria for evaluating differential expressed genes and pathways between genotypes (K-L). *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant (p > 0.05).

Figure S4. Human RSPO1 overexpression represses thermogenic capacities of brown/beige fat in vivo.
A) The mRNA expression of thermogenic and mitochondrial genes in BAT of hRSPO1 Tg and WT mice exposed to cold (n = 7-8 per group).
B) The heatmap of genes differentially expressed in iWAT between hRSPO1 Tg and WT mice exposed to prolonged cold stimulation (n = 3 per group).
C) The top-upregulated pathways (FDR < 0.05) were revealed by GSEA analysis based on GO:BP database in iWAT of hRSPO1 Tg versus WT mice exposed to prolonged cold stimulation.
D) The volcano plot of genes differentially expressed in eWAT of hRSPO1 Tg versus WT mice exposed to prolonged cold stimulation. Data are shown as the mean ± sem, and statistical significance between genotypes was assessed by unpaired Student's t test (A, G, J, and M). FDR below 0.05 were considered as the criteria for evaluating differential expressed genes and pathway between genotypes (B-D). *p < 0.05; **p < 0.01; ***p < 0.001. Figure S5. Ablation of endogenous Rspo1 reduces adiposity and enhances energy expenditure.
. Figure S6. Ablation of Rspo1 promotes browning program of white adipose tissue.
A) Rectal temperature changes of WT and Rspo1 -/mice in response to acute cold stimulation (4 °C) (n = 8-10 per group).
B) The heatmap of genes differentially expressed in eWAT between Rspo1 -/versus WT mice under prolonged cold stimulation (4 °C) for 10 days (n = 3 per group).
C Data are shown as the mean ± sem, and statistical significance between genotypes was assessed by unpaired Student's t test (A, G, J, and M). FDR below 0.05 was considered as the criteria for evaluating differential expressed genes and pathways between genotypes (B-D). *p < 0.05; **p < 0.01; ***p < 0.001.

Figure S7. RSPO1 inhibits thermogenic gene expression in induced beige adipocytes.
A) Schematic of experimental design for the induction of beige adipocytes derived from SVF cells of iWAT.
D-F) Cumulative food intake (D) and daily fecal calorie loss (E) (n =14 per group), and physical activities (n = 12 per group) (F) of female WT and Rspo1 R219W mice fed HFD. The repeated measurements of physical 23 activities were assessed by two-way ANOVA model to evaluate the interaction between genotype and time, and pairwised t-test with Benjamini-Hochberg correction was used as post-hoc test for evaluating the differences between genotypes in each hour.
G) Representative images of UCP1 immunohistochemical staining in iWAT of female WT and Rspo1 R219W mice exposed to cold stimulus (4 C°) for 10 days (n = 3 per group). Scale bars were indicated in the panels.
H-K) Representative images of H&E staining (H), UCP1 immunofluorescent staining (I), the immunohistochemical staining and Western blotting of UCP1 protein (J), and electron microscopic images (K) in eWAT of female WT and Rspo1 R219W mice under CL316,243 treatment (n = 3 per group). UCP1 (red) and Perilipin protein (green) were used to mark beige adipocytes and lipid droplets, respectively. Scale bars were indicated in the panels.
Data are shown as the mean ± sem, and statistical significance between groups was assessed by unpaired Student's t-test (A-E). *p < 0.05; **p < 0.01; ***p < 0.001; ns, no significant (p >0.05). (hRSPO1 Tg mice) also well mimic the effect of excessive RSPO1 expression in the fat tissue of obese cases.
C) In an intervention model, Rspo1 deletion or neutralization disinhibits its effects on the mitochondrial abundance and thermogenic capacity of brown/beige adipocytes, and consequently reduces and eventually combats adiposity.