Nuclear corepressor SMRT is a strong regulator of body weight independently of its ability to regulate thyroid hormone action

Abstract

Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) and the nuclear receptor co-repressor1 (NCoR1) are paralogs and regulate nuclear receptor (NR) function through the recruitment of a multiprotein complex that includes histone deacetylase activity. Previous genetic strategies which deleted SMRT in a specific tissue or which altered the interaction between SMRT and NRs have suggested that it may regulate adiposity and insulin sensitivity. However, the full role of SMRT in adult mice has been difficult to establish because its complete deletion during embryogenesis is lethal. To elucidate the specific roles of SMRT in mouse target tissues especially in the context of thyroid hormone (TH) signaling, we used a tamoxifen-inducible post-natal disruption strategy. We found that global SMRT deletion causes dramatic obesity even though mice were fed a standard chow diet and exhibited normal food intake. This weight gain was associated with a decrease in energy expenditure. Interestingly, the deletion of SMRT had no effect on TH action in any tissue but did regulate retinoic acid receptor (RAR) function in the liver. We also demonstrate that the deletion of SMRT leads to profound hepatic steatosis in the setting of obesity. This is unlike NCoR1 deletion, which results in hepatic steatosis due to the upregulation of lipogenic gene expression. Taken together, our data demonstrate that SMRT plays a unique and CoR specific role in the regulation of body weight and has no role in TH action. This raises the possibility that additional role of CoRs besides NCoR1 and SMRT may exist to regulate TH action.

Fig 1. Global SMRT deletion leads to obesity, glucose intolerance, and lower energy expenditure.

(A) We used qPCR analyses to determine the level of Smrt exon 11 expression in the liver, heart, muscle, PGWAT, and BAT at 12 weeks after tamoxifen treatment. Also Western blot analysis was used to determine the levels of SMRT protein using a SMRT specific antibody in the 1^st cohort of mice. Three samples were run in control mice (gray) and UBC-SKO mice (black). Blots were stripped and re-probed with anti-Pol-II as control. (B) Body weights in the 1^st cohort of mice starting at Day0 of tamoxifen administration through 12 weeks. (C) Food intake and body temperature were measured weekly (2^nd cohort). (D) We calculated the correlation between the levels of SMRT expression in the liver and body weight (2^nd cohort). (E) Serum glucose, insulin, glucose tolerance tests, serum triglyceride, total cholesterol, and leptin were measured at 12 weeks after tamoxifen treatment (2^nd cohort). (F) The 2^nd cohort mice were placed in a CLAMS apparatus and monitored for oxygen consumption (VO₂), carbon dioxide consumption (VCO₂), and Respiratory-expiratory ratio (RER) over 72 hours. All data were normalized to body weight. (G) The weights of liver, PGWAT, SCWAT, and quadriceps were normalized to body weight in the 2^nd cohort of mice. For qPCR data in panels A, and panels E-G (except the figure of the glucose tolerance test in panel E), the data were analyzed by unpaired t-test. In the analyses for BW (panel B), Food consumption (panel C), Body temperature (panel C), and glucose tolerance (panel E), Repeated Measures Two-way ANOVA was used. Results are shown as the mean±SEM, the p-value was shown as; ****, p< 0.0001; ***, p< 0.001; **, p< 0.01; *, p< 0.05. Panel D was analyzed by Pearson’s correlation coefficient analysis, and the p-value was shown as p = 0.022; *, p< 0.05. 1^st cohort included n = 6–7 mice/group, and 2^nd cohort included n = 6–8 mice/group.

Fig 2. Global SMRT deletion does not affect the HPT axis or thyroid hormone action.

(A) Serum total T4 and TSH were measured, and gene expression of Cga, Tshβ subunit, and Trhr in the pituitary were quantified by qPCR in control and UBC-SKO male mice 12 weeks after tamoxifen treatment (1^st and 2^nd cohort). (B) mRNA expression levels of T3-target genes in the liver, heart, skeletal muscle, and brown adipose tissue (BAT taken from 2^nd cohort mice) were quantified by qPCR. (C) Heart rate (HR) was measured weekly by cardiac echocardiography in the 1^st cohort mice weekly. For panels A and B, the data were analyzed by unpaired t-test. Panel C was analyzed by Repeated-Measures Two-way ANOVA. Results are shown as the mean±SEM, and the p-values as; ****, p< 0.001; **, p< 0.01; *, p< 0.05. 1^st cohort included n = 6–7 mice/group, and 2^nd cohort included n = 6–8 mice/group.

Fig 3. SMRT deletion actively enhances hepatic lipogenesis.

(A) Hepatic triglyceride and cholesterol levels were measured in the 1^st cohort mice 12th weeks after tamoxifen treatment. Images of control and UBC-SKO mice the livers are shown. (B) The mRNA expression of lipogenic and gluconeogenic-related genes in the liver, PGWAT, and muscle (1^st cohort of mice) (C) mRNA expression of NCoR1 in the liver, skeletal muscle, and PGWAT were quantified by qPCR. Protein expression of NCoR1 in the liver and heart were assessed by Western blot, using a NCoR1 specific antibody (n = 3–4 animals per group). Control samples are indicated by gray lanes and UBC-SKO mice are indicated by black lanes. For panel A and all qPCR analyses in panels B, and C, the data were analyzed using an unpaired t-test. These results are shown as the mean±SEM, and the p-values as; ****, p< 0.001; ***, p< 0.001; **, p< 0.01; *, p< 0.05 (1^st cohort of mice, n = 6–7 mice/group).

Fig 4. Insulin resistance and fatty liver in UBC-SKO mouse occur secondary to the progression of obesity.

(A) Body weight of control and UBC-SKO mice were measured weekly for 3 weeks after tamoxifen treatment in the 3^rd male cohort. (B) Liver weights in control and UBC-SKO of male mice (3^rd cohort). (C) Body composition was measured via MRI in control and UBC-SKO male mice (3^rd cohort). (D) Hepatic triglyceride and cholesterol levels were measured in both control and UBC-SKO male mice (3^rd cohort). (E) Glucose tolerance and insulin tolerance tests were performed in male mice 4 weeks after tamoxifen treatment (3^rd cohort). (F) Serum leptin levels were measured in both control and UBC-SKO male mice (3^rd cohort). All results are shown as the mean±SEM. For panels A and E, Two-way Repeated-Measures ANOVA was used. For panels B, C, D, and F, the data were analyzed by unpaired t-test (3^rd male cohort n = 7–8 mice/group).

Fig 5. Global deletion of SMRT predominantly activates RAR signaling in the liver.

(A) mRNA expression levels of the RAR signaling target genes in the liver of control and UBC-SKO mice 12 weeks after tamoxifen treatment were quantified by qPCR. (B) mRNA expression levels of RAR signaling target genes in PGWAT of control and UBC-SKO mouse were assessed by qPCR. (C) mRNA expression levels of RAR signaling target genes in the liver of control and global NCoR mutant (UBC-NCoR1ΔID) mice were assessed by qPCR. For panels A, B, and C, all qPCR data were analyzed by an unpaired t-test. These results are shown as the mean±SEM. ****, p< 0.0001; ***, p< 0.001; **, p< 0.01. The 1^st cohort mice (n = 6–7 mice/group) were used in panels A and B, and UBC-NCoR1ΔID mice (n = 6–8 mice/group) were used in panel C.