Deubiquitinating enzyme USP2 regulates brown adipose tissue thermogenesis via controlling EBF2 stabilization

Figure 1. Identification of adipose Usp2 as a potential thermogenic regulator.A. A volcano plot of significantly upregulated (magenta) and downregulated (blue) DUBs in BAT (n = 70; 7/3 upregulated/downregulated differentially expressed genes (DEGs)) in 8-week-old C57BL/6 male mice at RT (22 °C) or after 168 h of 4 °C cold exposure, determined by mRNA-seq. Statistically significant DEGs were defined as P < 0.05 and |log2 Foldchange| >1. n = 3 animals per temperature condition were analyzed. B. A volcano plot of significantly upregulated (magenta) and downregulated (blue) DUBs in iWAT (n = 72; 3/0 upregulated/downregulated DEGs) in 16-week-old C57BL/6 male mice at RT (22 °C) or after 168 h of 4 °C cold exposure, determined by mRNA-seq. Statistically significant DEGs were defined as P < 0.05 and |log2 Foldchange| >1. n = 2 animals per temperature condition were analyzed. C. The bar graph of upregulated (blue) and downregulated (magenta) DUBs in BAT in C57BL/6 male mice fed normal diet or high-fat diet for 20 weeks, determined by mRNA-seq. n = 3 animals per dietary condition were analyzed. D. Quantitative PCR analysis of Usp2 in BAT of 8-week-old C57BL/6 male mice at RT (22 °C) (n = 3) or…

Figure 2. Usp2 BKD mice exhibit defective energy expenditure and thermogenesis.A. Schematic diagram of the in-situ injection of AAV-shUsp2 into BAT for the establishment of the Usp2BKD model. Image created with BioRender.com. B. Rectal temperature of Usp2BKD vs control mice during acute cold exposure. Usp2BKD vs control, (n = 10 vs 8). C. Representative infrared images of Usp2BKD and control mice following 4 h of acute cold exposure. D. Adipose tissue weights of Usp2BKD and control mice. Usp2BKD vs control, (n = 4 vs 6). E. Representative photographs of BAT of Usp2BKD and control mice. F. Representative H&E staining and UCP1 immunostaining of BAT (Scale bar, 100 μm). G-I. Oxygen consumption rate (G), carbon dioxide production rate (H) and heat production rate (I) of Usp2BKD and control mice over 24-hour period monitored at cold (4 °C). Usp2BKD vs control, (n = 4 vs 5). J-L. Average oxygen consumption rate (J), average carbon dioxide production rate (K) and average heat production rate (L) at day time or night time during the 24-hour of monitoring as in (G-I). M. Quantitative PCR analysis of Usp2 and thermogenic genes in BAT of Usp2BKD and control mice exposed to cold (4 °C) for 3 days (n =…

Figure 3. Pharmacological inhibition of USP2 suppresses thermogenesis in vivo and in vitro.A. Schematic diagram of intraperitoneal injection of ML364, followed by cold exposure for 3 days. B. Body weight of mice (n = 6). C. Tissue weight of the indicated tissue in mice treated with ML364 or vehicle (n = 6). D. Representative photographs of adipose tissue in mice treated with ML364 or vehicle. E. Representative H&E staining of adipose tissue in mice treated with ML364 or vehicle. F. Rectal temperature of mice treated with ML364 or vehicle during acute cold exposure (n = 10). G. Representative infrared images of mice treated with ML364 or vehicle following 4 h of acute cold exposure. H. Quantitative PCR analysis of thermogenic and adipogenic genes of BAT in mice treated with ML364 or vehicle (n = 5). I. Quantitative PCR analysis of thermogenic and adipogenic genes of iWAT in mice treated with ML364 or vehicle (n = 6). J. Immunoblot analysis of UCP1 of BAT in mice treated with ML364 or vehicle (n = 6). K. Immunoblot analysis of UCP1 of iWAT in mice treated with ML364 or vehicle (n = 6). L. Mitochondrial DNA levels of BAT in mice treated with ML364 or vehicle (n = 4). M. Mitochondrial DNA…

Figure 4. Depletion of USP2 cell-autonomously inhibits adipocyte thermogenic metabolism. A. Schematic diagram of transcriptional profiling analysis in mature brown primary adipocytes transfected with shUsp2 lentivirus. Image created with BioRender.com. B. Top significantly downregulated KEGG pathways, on the basis of RNA-seq data (n = 3). C. Profile of transcript levels of differentially expressed BAT-specific genes in Usp2 knockdown or control adipocytes. D. Quantitative PCR analysis of thermogenic and adipogenic genes in Usp2 knockdown or control adipocytes (n = 4). E. Immunoblot analysis of UCP1 in Usp2 knockdown or control adipocytes (n = 3). F. Quantitative PCR analysis of mitochondrial genes in Usp2 knockdown or control adipocytes (n = 3). G. Representative fluorescent images of MitoTracker staining of Usp2 knockdown or control adipocytes (Scale bar, 400 μm). H. Mitochondrial DNA levels of Usp2 knockdown or control adipocytes (n = 3). I. Oxygen consumption rates of Usp2 knockdown or control adipocytes (n = 6). J. Bar graphs represent quantification of basal and maximal oxygen consumption of the OCRs in I. K. Representative TEM images of mitochondria in Usp2 knockdown or control…

Figure 5. Overexpression of Usp2 in BAT ameliorates HFD-induced obesity and metabolic dysfunction.A. Schematic diagram of the in-situ injection of AAV-OeUsp2 into BAT for the establishment of the Usp2BOE model, followed by HFD feeding. B. Body weight of Usp2BOG and control mice housed at RT (22 °C). Usp2BOE vs control, (n = 5 vs 6). C. Lean mass and fat mass. Usp2BOE vs control, (n = 7 vs 9). D. The weight of indicated tissues (n = 5). E. Rectal temperature of Usp2BOE and control mice during acute cold exposure (n = 8). F. Representative infrared images of Usp2BOE and control mice following 4 h of acute cold exposure. G. Representative H&E staining of adipose tissue and liver in Usp2BOE and control mice (Scale bar, 100 μm). H. Quantitative PCR analysis of thermogenic genes in BAT of Usp2BOE and control mice. Usp2BOE vs control, (n = 4 vs 5). I. Representative Ucp1 immunostaining of BAT in Usp2BOE and control mice (Scale bar, 100 μm). J-L. Oxygen consumption rate (J), carbon dioxide production rate (K) and heat production rate (L) of Usp2BOE and control mice over 24-hour period monitored at RT (22 °C) (n = 4). M-O. Average oxygen consumption rate (M), average carbon dioxide production rate…

Figure 6. USP2 stabilizes EBF2 by removing K63-linked polyubiquitination at the K321 site of EBF2.A. Schematic illustration of a quantitative proteomic screen to identify proteins binding to USP2 in brown adipose. B. Heatmaps representing the upregulated and downregulated genes in BAT, assessed by quantitative proteomics (n = 4) (Fold change>2.0 and P value < 0.05). C. Top significantly downregulated KEGG pathways, on the basis of quantitative proteomic data. D. Venn diagram showing proteins consistently present in control group while consistently absent in USP2 knockdown group. E. Quantitative PCR analysis of EBF2 in USP2 knockdown BAT (n = 4). F. Immunoblot analysis of EBF2 protein expression in USP2 knockdown BAT (n = 3). G. Immunoblot analysis of EBF2 protein expression in primary brown adipocytes treated with varying doses of ML364. H. Immunoblot analysis of EBF2 protein expression in primary brown adipocytes pretreated with ML364 (2.5 μM) for 24 h, followed by cycloheximide (25 μg/mL) treatment for the indicated time. I-J. Co-IP analysis of association of USP2 with EBF2 in HEK293T cells co-transfected with Flag-labeled USP2 and HA-labeled EBF2 plasmids. K. Representative…

Figure 7. EBF2 overexpression attenuates USP2 deficiency-potentiated thermogenic dysfunction.A. Schematic illustration of the establishment of three groups: AAV-NC + Adv-GFP vs AAV-shUsp2 + Adv-GFP vs AAV-shUsp2 + Adv-EBF2, n = 6 vs 5 vs 5. Image created with BioRender.com. B. Tissue weight of BAT. C. Tissue weight of iWAT and eWAT. D. Representative H&E staining of BAT. E. Representative UCP1 immunostaining of BAT. F. Immunoblot analysis of UCP1. G. Quantitative PCR analysis of thermogenic genes of BAT (n = 5 vs 5 vs 5). H-J. Oxygen consumption rate (H), carbon dioxide production rate (I) and heat production rate (J) of mice over 24-hour period monitored at RT (22 °C) (n = 4 vs 4 vs 4). K-M. Average oxygen consumption rate (K), average carbon dioxide production rate (L) and average heat production rate (M) at day time or night time during the 24-hour of monitoring as in (H–J). For statistical analysis, two-way ANOVA was performed in H-J. Unpaired, two-tailed t-tests were performed in B, C, G and K-M. (∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001). Each point represents a biological replicate. Data were presented as the mean ± S.E.M.