TSH promotes adiposity by inhibiting the browning of white fat

Figures & data

Figure 1. TSH promotes adiposity and its metabolic consequences. SCH: subclinical hypothyroidism mice; NC: normal control mice; TPO-Tshr: TPO-Cre/Tshr^flox/flox mice; WT: wild-type littermates. AUC: area under curve; ACC: area above curve. (a) Body weight of the control and SCH mice (n = 10). (b) Body weight evaluation of the TPO-Tshr mice treated with TSH or vehicle for 2 weeks (n = 6). (c) Weight of eWAT, iWAT and BAT normalized to the body weights of the control and SCH mice (n = 8). (d) Weight of eWAT, iWAT and BAT normalized to the body weights of TPO-Tshr mice injected with TSH or vehicle (n = 6). (e, f) Results of the OGTT (f) and ITT (g) in the control and SCH mice (n = 8). The values represent the means ± s.e.m. Error bars represent s.e.m. Significant differences in SCH compared with NC mice and TPO-Tshr mice treated with TSH compared with vehicle controls are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test or 2-way ANOVA with repeated measures and Bonferroni post-hoc tests)

Figure 2. Elevated TSH levels reduces energy consumption

SCH: subclinical hypothyroidism mice; NC: normal control mice; TPO-Tshr: TPO-Cre/Tshr^flox/flox mice; WT: wild-type littermates; LM: lean mass. (A) The 24 h food intake of the control and SCH mice (n = 5–8). (B) Physical activity of the control and SCH mice over a 24 h recording period (n = 5–8). (C) Rectal temperature of the SCH mice and their controls measured at 16:00 (n = 5–8). (D-G) O₂ consumption (D, E) and CO₂ production (F, G) normalized to lean mass of the SCH and control mice were recorded in metabolic cages for 24 h (D, F: 24 h period; E, G: average of light and dark periods, respectively) (n = 5–8 for each group). (H, I) 24 h energy expenditure was compared between the control and SCH mice (H, energy expenditure per mouse was plotted against lean mass; I, the adjusted means of energy expenditure in the two groups analysed by ANCOVA with fat mass and lean mass as two covariates, P = 0.046). (J) The 24 h food intake of TPO-Tshr mice that had been treated with TSH or vehicle for 2 weeks (n = 5–8). (K) The physical activity of TPO-Tshr mice that had been treated with TSH or vehicle for 2 weeks was recorded for 24 h (n = 5). (L-O) O₂ consumption (L, M) and CO₂ production (N, O) normalized to lean mass of the TPO-Tshr mice treated with TSH or vehicle for 2 weeks were recorded in metabolic cages for 24 h (L and N: 24 h period; M and O: average of light and dark periods, respectively) (n = 5–7 for each group). (P,Q) 24 h energy expenditure was compared between the TPO-Tshr mice treated with TSH and vehicle (P, energy expenditure per mouse was plotted against lean mass; I, the adjusted means of energy expenditure in the two groups analysed by ANCOVA with fat mass and lean mass as two covariates, P = 0.049).Values represent the means ± s.e.m. Error bars represent s.e.m. Significant differences in SCH mice compared with NC or TPO-Tshr mice treated with TSH compared with vehicle controls are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test or analysis of covariance (ANCOVA)).

Figure 3. Tshr-knockout mice resist adiposity and its metabolic complications

FABP4-Tshr: FABP4-Cre/Tshr^flox/flox mice; WT: wild-type littermates; Tshr^−/-: global Tshr-knockout mice; Tshr^+/+: the wild-type littermates of Tshr^−/- mice. AUC: area under curve; ACC: area above curve. (A) Body weights of the FABP4-Tshr mice and their wild-type littermates fed the CD were recorded for 11 weeks (n = 6 for each group). (B) Weights of the eWAT, iWAT and BAT normalized to body weights of the FABP4-Tshr mice and the wild-type fed the CD (n = 6 for each group). (C, D) Results of the OGTT (C) and ITT (D) for FABP4-Tshr mice and wild-type mice fed the CD (n = 6 for each group). (E) Serum triglyceride levels in FABP4-Tshr mice and wild-type fed the CD. (n = 10) (F) The body weights of the Tshr^−/- and wild-type mice fed the CD were recorded for 11 weeks (n = 6 for each group). (G) Weights of eWAT, iWAT and BAT normalized to the body weights of the Tshr^{−/ –} and wild-type mice fed the CD (n = 8 for each group). (H, I) Results of the OGTT (H) and ITT (I) for Tshr^−/- and wild-type mice fed the CD (n = 6 for each group). (J) Serum triglyceride levels in the Tshr^−/- and wild-type mice fed the CD (n = 9). (K) Weight gain of the Tshr^−/- mice and their wild-type littermates fed a high-fat diet (HFD) for 16 weeks (n = 7–8 for each group). (L) Weights of eWAT, iWAT and BAT normalized to the body weights of the Tshr^−/- and wild-type mice fed the HFD (n = 7–8 for each group). (M, N) Results of the OGTT (M) and ITT (N) for the Tshr^−/- and wild-type mice fed the HFD (n = 7–8 for each group). (O, P) Representative photograph of 12-week-old Tshr^−/- mice, FABP4-Tshr mice and their wild-type littermates. Values represent the means±s.e.m. Error bars represent s.e.m. Significant differences in FABP4-Tshr mice compared with their wild-type littermates or Tshr^−/- mice compared with Tshr^+/+ are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test or 2-way ANOVA with repeated measures and Bonferroni post-hoc tests).

Figure 4. Tshr knockout increases energy expenditure

FABP4-Tshr: FABP4-Cre/Tshr^flox/flox mice; WT: wild-type littermates; Tshr^−/-: global Tshr-knockout mice; Tshr^+/+: the wild-type littermates of Tshr^−/- mice; LM: lean mass. (A) The 24 h food intake was measured in both FABP4-Tshr mice and their wild-type littermates (n = 7–8 for each group). (B) Physical activity was recorded for the FABP4-Tshr mice and their wild-type littermates over 24 h (n = 7–8). (C) Rectal temperatures of the FABP4-Tshrmice and their wild-type littermates (n = 6–8). (D-G) O₂ consumption (D, E) and CO₂ production (F, G) normalized to lean mass of the FABP4-Tshrmice and their wild-type littermates were recorded in metabolic cages for 24 h (D and F: 24 h period; E and G: average of light and dark periods) (n = 7–8). (H,I) 24 h energy expenditure was compared between FABP4-Tshr mice and their wild-type littermates (H, energy expenditure per mouse was plotted against lean mass; I, the adjusted means of energy expenditure in the two groups analysed by ANCOVA with fat mass and lean mass as two covariates, P = 0.015). (J-M) O₂ consumption (J, K) and CO₂ production (L, M) normalized to lean mass of the Tshr^−/- mice and their wild-type littermates during a 24 h period (J and L: 24 h period; K and M:average of light and dark periods) (n = 6–8 for each group). (N,O) 24 h energy expenditure was compared between the Tshr^−/- mice and their wild-type littermates (N, energy expenditure per mouse was plotted against lean mass; O, the adjusted means of energy expenditure in the two groups analysed by ANCOVA with fat mass and lean mass as two covariates, P = 0.006). (P) Comparison of the body temperatures of the Tshr^−/- and wild-type mice (n = 7). (Q) The 24 h food intake of the Tshr^−/- and the wild-type mice (n = 6). (R) Physical activity of the Tshr^−/- mice and their wild-type littermates in a 24 h recording period (n = 6). Values represent the means±s.e.m. Error bars represent s.e.m. Significant differences in FABP4-Tshrmice compared with their wild-type littermates or Tshr^−/- mice compared with Tshr^+/+ are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test or analysis of covariance (ANCOVA)).

Figure 5. Tshr knockout induces the white-to-brown fat transition in Tshr^−/- mice

Tshr^−/-: global Tshr-knockout mice; Tshr^+/+: the wild-type littermates of Tshr^−/- mice. (A) Representative images of the BAT, eWAT and iWAT depots in wild-type and Tshr^−/- mice. (B, C) Representative HE staining of eWAT and iWAT of Tshr^−/- and wild-type mice housed at room temperature and in a cold room (4°C) for 4 weeks, respectively (scale bar, 20 μm). (D) Electron microscopy of eWAT in wild-type and Tshr^−/- mice under normal conditions and after four weeks of cold stress (scale bar 1 μm), the arrows refer to lipid droplets and mitochondria. (E, F) Immunohistochemical staining of UCP1 in eWAT and iWAT of wild-type and Tshr^−/- mice housed at room temperature or 4°C for 4 weeks, respectively (scale bar 20 μm). (G, H) Real-time PCR analysis of the expression of thermogenic and related genes is shown in eWAT and iWAT of Tshr^−/-and their wild-type littermates (n = 6). (I) Representative western blots showing the key protein levels of UCP1 and PGC-1α in the eWAT and iWAT of wild-type and Tshr^−/- mice housed under different conditions. Actin was used as the loading control. Values represent the means±s.e.m. Error bars represent the s.e.m. Significant differences in Tshr^−/- mice compared with Tshr^+/+ are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test).

Figure 6. Tshr knockout induces the white-to-brown fat transition in FABP4-Tshr mice

FABP4-Tshr: FABP4-Cre/Tshr^flox/flox mice; WT: wild-type littermates. (A, B) Electron microscopy of eWAT (A) and iWAT (B) in FABP4-Tshr mice and their wild-type littermates under normal conditions and after four weeks of cold stress (scale bar 2 μm); the arrows refer to lipid droplets and mitochondria. (C, D) Immunohistochemical staining of UCP1 in eWAT (C) and iWAT (D) of FABP4-Tshr mice and their wild-type littermates housed at room temperature or 4°C for 4 weeks (scale bar 20 μm). (E, F) Real-time PCR analysis of the expression of thermogenic and related genes in eWAT (E) and iWAT (F) of FABP4-Tshr and wild-type mice (n = 6). (G, H) Representative western blots showing the key protein levels of UCP1 in the eWAT and iWAT of FABP4-Tshr and wild-type mice housed under normal conditions. (I, J) Immunofluorescence staining of CD137 in eWAT and iWAT of FABP4-Tshr mice and their wild-type littermates housed at room temperature (q) and 4°C (r) for 4 weeks, respectively (scale bar 20 μm). Values represent the means±s.e.m. Error bars represent the s.e.m. Significant differences in FABP4-Tshr mice compared with their wild-type littermates are indicated by *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test).

Figure 7. TSH may affect the browning of eWAT and iWAT through the AMPK/PRDM16/PGC1α pathway

FABP4-Tshr: FABP4-Cre/Tshr^flox/flox mice; WT: wild-type littermates; Tshr^−/-: global TSHR knockout mice; Tshr^+/+: the wild type littermates of Tshr^−/- mice. (A, B) Expression profiles of genes related to the browning of WAT in the eWAT and iWAT of wild-type and Tshr^−/- mice (n = 6. (C, D) Gene expression profiles related to the browning of WAT in the eWAT and iWAT of wild-type and FABP4-Tshr mice(n = 6). (E, F) Levels of the PRDM16 protein in the eWAT and iWAT of wild-type and Tshr^−/- mice. (G, H) Levels of the AMPK protein in the eWAT and iWAT of Tshr^−/- and wild-type mice housed under normal conditions. (I) The dose-dependent effects of TSH on PGC1α and UCP1 protein expression in brown-differentiated 3T3-L1 cells. (J-L) Brown-differentiated 3T3-L1 cells and primary SVF cells were transfected with non-targeting or Tshr siRNA. After 24 h, the cells were incubated with or without TSH (10 mu/ml) for 24 h. Total AMPK, p-AMPK, PRDM16, PGC1α and UCP1 expression were detected by western blotting. (M, N) Brown-differentiated 3T3-L1 cells were incubated with or without AICAR/Dorsomorphin and TSH (10 mu/ml) for 24 h. Total AMPK, p-AMPK, PRDM16, PGC1α and UCP1 expression were detected by western blotting. Values represent the means±s.e.m. Error bars represent s.e.m. Significant differences in FABP4-Tshr mice compared with their wild-type littermates, Tshr^−/- mice compared with Tshr^+/+,TSH(1, 10, 100MU/ml) compared with TSH (0MU/ml), non-TSH group compared with TSH group. TSH(10mU/ml, 100mU/ml) compared with TSH(1mU/ml) were indicated by ^#P < 0.05, ^##P < 0.01 and ^###P < 0.001.