Ctnnb1/β-catenin inactivation in UCP1-positive adipocytes augments the browning of white adipose tissue

Figures

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  Figure 1. Ablation of Ctnnb1 in UCP1-positive adipocytes promotes the inguinal WAT “browning” under cold exposure (A) The schematic diagram for 7-day chronic coldness exposure. (B–E) Body weight (B), body temperature (C), body composition (D), and tissue weight (E) of UBKO and control male mice (n = 8 for each group). (F) H&E staining and immunolabeling for β-catenin and UCP1 of sections of iWAT depots from control and UBKO mice. Scale bars, 50 μm (n = 3 for each group). (G and H) mRNA levels of thermogenic genes in BAT (G) and iWAT (H) of UBKO and control male mice. (n = 8 for each group). (I) Expression of UCP1 protein in the iWAT of UBKO and control male mice (20 μg of total protein from tissue lysate was loaded onto gel). WT BAT lysate (1 μg) was loaded as a control. (n = 3 for each group). For B–I, 8-week-old UBKO and control male mice were placed under 4°C for 7 days. Data are shown as means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figures S1 and S2.

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  Figure 2. UBKO mice exhibit enhanced “browning” of WAT under the β3-adrenergic receptor agonist CL-316,243 administration (A) The schematic diagram for the 5-day CL-316,243 treatment. (B) Body weight of UBKO and control male mice (n = 4–5 for each group). (C and D) H&E staining and immunolabeling for β-catenin and UCP1 of sections of eWAT (C) and iWAT (D) depots from control and UBKO mice. Scale bars, 50 μm (n = 3 for each group). (E and F) mRNA levels of thermogenic (E) and mitochondrial function-associated (F) genes in the eWAT of UBKO and control male mice. (n = 4–5 for each group). (H and I) mRNA level of thermogenic (H) and mitochondrial function-associated (I) genes in the iWAT of UBKO and control male mice. (n = 4–5 for each group). (G and J) Expression of UCP1 protein in the eWAT (G) and iWAT (J) of UBKO and control male mice (20 μg of total protein from tissue lysate was loaded onto gel). WT 0.1 μg BAT lysate and 1 μg BAT lysate were loaded as control of eWAT and iWAT, respectively. (n = 3 for each group). For B–J, 8-week-old UBKO and control male mice were injected with CL-316,243 for 5 days. Data are shown as means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S3.

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  Figure 3. UBKO male mice exhibit increased whole-body energy expenditure at baseline (A and B) Oxygen consumption was measured in metabolic cages (A) and analyzed during light and dark periods (B) for UBKO and control 8-week-old male mice. (C and D) Carbon dioxide production was measured (C) and analyzed during the light and dark periods (D). (E and F) Energy expenditure was measured (E) and calculated during the light and dark period (F). (G) Energy expenditure was plotted in relation to lean mass using ANCOVA. n = 8 for each group. Data are shown as means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S4.

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  Figure 4. β-catenin inhibits the expression of thermogenic and mitochondria-related genes in vitro (A) mRNA levels of thermogenic and mitochondrial function-associated genes in adenovirus-mediated overexpression of β-catenin in iWAT preadipocytes differentiated for 8 days into beige adipocytes. (n = 6 for each group). (B) Protein levels of total β-catenin, flag-β-catenin (S33Y/S37F/T41A), UCP1, and PGC1α in induced beige adipocytes from iWAT (15 μg of total protein from cell lysate was loaded onto gel). WT BAT lysate (1 μg) was loaded as a control. (n = 3 for each group). (C) mRNA levels of thermogenic and mitochondrial function-associated genes in shRNA-mediated knockdown of β-catenin in iWAT preadipocytes differentiated for 8 days into beige adipocytes. (n = 6 for each group). (D) Protein levels of total β-catenin, UCP1, and PGC1α in induced beige adipocytes from iWAT as in C (15 μg of total protein from cell lysate was loaded onto gel). WT BAT lysate (1 μg) was loaded as a control. (n = 2 for each group). (E and F) Luciferase reporter assay was performed in 293T cells transfected with the indicated plasmids. pRL-TK (expressing Renilla luciferase) was used as the normalized control. (n =…

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  Figure 5. Bulk RNA-seq reveals enhanced mitochondrial functions and decreased inflammation in UBKO mice (A) Principal component analysis (PCA) of the transcriptome profiles of eWAT of control (blue) and UBKO (orange) mice. (B) Volcano plot of differentially expressed genes between control and UBKO samples (p < 0.05, |log2(FC)|>0.58). (C) The heatmap of the top 50 (up and down: top 25 each) genes significantly highly expressed in control or UBKO samples. (D) Upregulated KEGG pathways between eWAT of control and UBKO group. (E) Upregulated GO pathways between eWAT of control and UBKO group. (F) The heatmap of thermogenic genes significantly highly expressed in UBKO samples. (G) The heatmap of genes involves metabolic pathways that are significantly highly expressed in UBKO samples. (H) Downregulated KEGG pathways between eWAT of control and UBKO group. (I) Downregulated GO pathways between eWAT of control and UBKO group. (J) The heatmap of lysosome-related genes in eWAT of two groups. (K) The heatmap of the top 50 genes involved in the immune system process in eWAT of two groups. 8-week-old UBKO and control male mice were injected with CL-316,243 for 5 Days n = 4–5 for each group.

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  Figure 6. Ctnnb1 deficiency in UCP1-positive adipocytes improves metabolic performance in 50-week-old mice (A–C) Body weight (A), tissue weight (B), and percentage of tissue weight (C) of 50-week-old UBKO and control male mice. (D and E) Glucose tolerance test after 16-h fasting (D) and calculation of area under the curve (AUC) of 10-month-old UBKO and control male mice (E). (F and G) Insulin tolerance test after 6-h fasting (F) and calculation of AUC (G) of 10-month-old UBKO and control male mice. (H–J) mRNA levels of thermogenic genes in BAT (H), iWAT (I), and eWAT (J) of 50-week-old UBKO and control male mice. (K and L) mRNA levels of genes related to inflammation in iWAT (K) and eWAT (L) of 50-week-old UBKO and control male mice. For A–G, n = 25 for the control group, and n = 19 for the UBKO group. For H–L, n = 8 for each group. Data are shown as means ± SEM. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001. See also Figure S6.

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  Figure 7. CTNNB1 expression inversely correlates with thermogenic genes in human adipocytes (A) Correlation analysis of UCP1, PRDM16, CIDEA, BMI, WHR, and CTNNB1 expression in subcutaneous adipose from GSE70353 database (n = 770). (B) Correlation analysis of PRDM16, CIDEA, BMI, and CTNNB1 expression in purified adipocytes of subcutaneous adipose from GSE174475 database (n = 43).