PPARγ deacetylation dissociates thiazolidinedione's metabolic benefits from its adverse effects

Abstract

Thiazolidinediones (TZDs) are PPARγ agonists with potent insulin-sensitizing effects. However, their use has been curtailed by substantial adverse effects on weight, bone, heart, and hemodynamic balance. TZDs induce the deacetylation of PPARγ on K268 and K293 to cause the browning of white adipocytes. Here, we show that targeted PPARγ mutations resulting in constitutive deacetylation (K268R/K293R, 2KR) increased energy expenditure and protected from visceral adiposity and diet-induced obesity by augmenting brown remodeling of white adipose tissues. Strikingly, when 2KR mice were treated with rosiglitazone, they maintained the insulin-sensitizing, glucose-lowering response to TZDs, while displaying little, if any, adverse effects on fat deposition, bone density, fluid retention, and cardiac hypertrophy. Thus, deacetylation appears to fulfill the goal of dissociating the metabolic benefits of PPARγ activation from its adverse effects. Strategies to leverage PPARγ deacetylation may lead to the design of safer, more effective agonists of this nuclear receptor in the treatment of metabolic diseases.

Keywords: Adipose tissue; Diabetes; Metabolism; Obesity; Therapeutics.

Figure 1. Increased energy wasting in deacetylation-mimetic 2KR mice.

(A) cDNA sequencing of mRNA from adipose tissue confirmed the replacement of PPARγ WT allele by 2KR mutant allele. The mutated sites were highlighted. (B) BW of 4-month-old male mice fed a chow diet. n = 12 WT; n = 12 2KR. (C–G) Calorimetric analyses of above mice in B at ambient temperature. (C) Total activity counts within 1 light/dark cycle. *P < 0.05. (D) Food intake on 2 constitutive days. (E) RER. (F) Oxygen consumption and (G) heat production. In D–G, *P < 0.05 by t test at multiple detection points. n = 11 WT; n = 12 2KR (1 chamber out of order). Data represent mean ± SEM. Student’s t test was used for statistical analyses.

Figure 2. PPARγ deacetylation promotes thermogenic responses.

(A–D) Calorimetric analyses of 4-month-old male mice fed a chow diet during acute cold exposure (day 1) and after chronic cold exposure (day 5). (A) Food intake. (B) RER. (C) Oxygen consumption and (D) heat production. *P < 0.05 at multiple detection points. n = 9 WT; n = 11 2KR (2 WT and 1 mutant mice were hypothermic at the cold exposure and terminated). (E and F) qPCR analyses of gene expression in iWAT (E) and BAT (F) of 5- to 6-month-old chow-fed male mice after 5-day cold exposure. *P < 0.05; **P < 0.01 for WT vs. 2KR. n = 6 WT; n = 6 2KR. (G) qPCR analyses of gene expression in iWAT primary adipocytes after 4-hour forskolin (5 μM) treatment. *P < 0.05; **P < 0.01 for WT vs. 2KR. n = 5 WT; n = 5 2KR. Data represent mean ± SEM. Student’s t test was used for statistical analyses.

Figure 3. Protection against visceral obesity in 2KR mice.

(A) BW of chow-fed young (3 months old) and aged (12 to 13 months old) male mice. *P < 0.05. n = 8 WT (young); n = 8 2KR (young); n = 9 WT (aged); n = 9 2KR (aged). (B and C) WAT fat pad sizes in young (B) or aged (C) male mice sacrificed at ad libitum feeding. *P < 0.05; **P < 0.01. n = 8 WT (young); n = 8 2KR (young); n = 9 WT (aged); n = 9 2KR (aged). (D) Representative H&E staining of visceral epididymal fat (eWAT) and quantification of adipocyte size in aged male mice. Original magnification, ×100. (E) Isolation of Tregs (CD4⁺CD25⁺) from eWAT of aged male WT mice. (F) Confirmation of the presence of fat Treg marker Foxp3 in isolated T cells. (G) Proportion of Treg (CD25⁺) cells among spleen and eWAT CD4⁺ T cells. **P < 0.001. n = 5 WT; n = 5 2KR. This experiment was repeated twice. (H) Mean fluorescence intensity (MFI) of Foxp3 in isolated T cells. *P < 0.05. n = 5 WT; n = 5 2KR. (I) Fat Treg signature gene expression profiling by RNA-seq. Spleen Tregs were used as control. Data represent mean ± SEM. Student’s t test was used for statistical analyses.

Figure 4. 2KR mice are protected from DIO.

(A) BW curve of mice that started HFD feeding at 6 weeks old. *P < 0.05 for WT vs. 2KR. n = 16 male (m) WT; n = 15 male 2KR; n = 14 female (f) WT; n = 13 female 2KR. (B) Body composition of male mice after 8 weeks of HFD feeding. *P < 0.05; **P < 0.01 for WT vs. 2KR. n = 10 WT; n = 9 2KR. (C and D) i.p. GTT (C) and AUC (D) in male mice at 12 weeks of HFD feeding. *P < 0.05 for WT vs. 2KR. n = 10 WT; n = 9 2KR. (E) PTT in male mice at 12 weeks of HFD feeding. *P < 0.05; **P < 0.01 for WT vs. 2KR. n = 10 WT; n = 5 2KR. (F and G) Male mice started HFD feeding at 6 weeks old and were sacrificed after 24 weeks on HFD feeding after overnight fasting, followed by 4 to 6 hours of refeeding. (F) Improved obesity-associated hepatic steatosis in 2KR mice indicated by histological analysis of the livers by H&E and oil red O staining. Original magnification, ×100. (G) qPCR analysis of hepatic lipogenic gene expression. *P < 0.05; **P < 0.01 for WT vs. 2KR. n = 7 WT; n = 5 2KR. Data represent mean ± SEM. Student’s t test was used for statistical analyses.

Figure 5. 2KR mice respond to TZD treatment.

(A) BW curve on Rosi treatment. Mice were rendered insulin resistant after 16 weeks of HFD feeding (started at 6 weeks old), then switched to a HFD containing Rosi. *P < 0.05 for female WT vs. female 2KR by 2-tailed t test. n = 8 male WT; n = 8 male 2KR; n = 7 female WT; n = 8 female 2KR. (B) Body composition of male mice on Rosi treatment for 10 weeks. Rosi treatment started at 8 weeks of HFD feeding. *P < 0.05; **P < 0.01 for WT vs. 2KR by 2-tailed t test. n = 8 WT; n = 8 2KR. (C) WAT fat pad sizes in male mice on 15-week Rosi treatment. Mice were sacrificed after overnight fasting followed by 4 hours of refeeding. *P < 0.05 for WT vs. 2KR by 2-tailed t test. n = 8 WT; n = 8 2KR. (D and E) ipGTT (D) and AUC (E) before and after 2 weeks of Rosi treatment in BW-matched DIO male mice. n = 7 WT; n = 6 2KR. (F) Fasting blood glucose levels in male mice after 16 weeks on Rosi treatment. n = 7 WT (vehicle [veh]); n = 7 2KR (vehicle); n = 8 WT (Rosi); n = 8 2KR (Rosi). (G and H) ITT (G) and AUC (H) in male mice at 18 weeks on Rosi treatment. n = 7 WT (vehicle); n = 7 2KR (vehicle); n = 8 WT (Rosi); n = 8 2KR (Rosi). (I) Plasma insulin levels in male mice after 18 weeks on Rosi treatment. n = 7 WT (vehicle); n = 7 2KR (vehicle); n = 8 WT (Rosi); n = 8 2KR (Rosi). (J and L) Liver size (J), histological analysis by H&E staining (K), and hepatic TG content (L) in male mice after 24 weeks of Rosi treatment. Mice were sacrificed after overnight fasting. Original magnification, ×100. n = 7 WT (vehicle); n = 7 2KR (vehicle); n = 8 WT (Rosi); n = 8 2KR (Rosi). In D–L, effects of 2KR (*P < 0.05; **P < 0.01, WT vs. 2KR) and Rosi (^#P < 0.05; ^##P < 0.01, vehicle vs. Rosi) by 2-way ANOVA. In F–L, n = 7 WT (vehicle); n = 7 2KR (vehicle); n = 8 WT (Rosi); n = 8 2KR (Rosi). Data represent mean ± SEM.

Figure 6. 2KR inhibits TZD-induced bone loss and bone marrow adiposity.

(A) Bone density determined by DEXA scanning in 12-month-old male mice exposed to HFD for 4 weeks and then treated with Rosi for 5 weeks. **P < 0.01. n = 7 WT (vehicle); n = 6 2KR (vehicle); n = 6 WT (Rosi); n = 7 2KR (Rosi). (B–F) Bone mineral density (B), intratrabecular (intra.) bone mineral density (BMD) (C), cortical bone mineral density (D), representative images of a cross-section of mid-shaft femurs (E), and relative cortex volume (F) determined by μCT scanning in the femur of male mice on HFD feeding (starting at 6 weeks of age) for 16 weeks, then treated with Rosi for 24 weeks. *P < 0.05. n = 7 WT (vehicle); n = 7 2KR (vehicle); n = 7 WT (Rosi); n = 8 2KR (Rosi). (G) Gross morphology of femur indicating inhibition of bone marrow adiposity in Rosi-treated 2KR mice. (H) Representative osmium tetroxide staining of bone marrow adipocytes assessed by μCT scanning in the femur of male mice after 24 weeks of Rosi treatment. (I) Quantification of bone marrow lipid volume in the femurs of male mice by μCT scanning. **P < 0.01. n = 12 WT (chow); n = 12 2KR (chow); n = 7 WT (HFD); n = 7 2KR (HFD); n = 7 WT (Rosi); n = 8 2KR (Rosi). Data represent mean ± SEM. Student’s t test was used for statistical analyses. (J) In Pparg^–/– MEFs, reconstitution of PPARγ2-2KR, but not PPARγ1-2KR, fully rescued adipogenesis as indicated by oil red O staining. Original magnification, ×100.

Figure 7. Protection from TZD’s cardiovascular side effects in 2KR mice.

(A–D) Heart weight normalized to femur length (A), wall thickness (B and C), and FS (D) determined by echocardiography in male mice that were on HFD feeding for 16 weeks, then were treated with Rosi for 24 weeks. *P < 0.05; **P < 0.01. n = 7 WT (vehicle); n = 7 2KR (vehicle); n = 7 WT (Rosi); n = 8 2KR (Rosi). (Please note that 1 in WT Rosi group died during echocardiographical measurement). (E) Representative M-mode echocardiographic images of left ventricular dimensions. (F) qPCR measurement of cardiac lipid-handling gene expression in above male mice. ^#P < 0.05; ^##P < 0.01 for WT vehicle vs. WT Rosi mice. *P < 0.05; **P < 0.01 for WT vs. 2KR mice under the same treatment. n = 6 WT (vehicle); n = 6 2KR (vehicle); n = 6 WT (Rosi); n = 6 2KR (Rosi). (G) Fluid body composition changes in male mice during early treatment of Rosi. Rosi treatment was started at 8 weeks of HFD feeding. *P < 0.05; **P < 0.01. n = 8 WT; n = 8 2KR. (H) Hematocrit determined by an automated blood cell counter in male mice after 24 week of Rosi treatment. **P < 0.01. n = 7 WT (vehicle); n = 7 2KR (vehicle); n = 7 WT (Rosi); n = 8 2KR (Rosi). Data represent mean ± SEM. Student’s t test was used for statistical analyses.