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. 2011 Nov 11;147(4):815-26.
doi: 10.1016/j.cell.2011.09.050.

Adipocyte NCoR knockout decreases PPARγ phosphorylation and enhances PPARγ activity and insulin sensitivity

Pingping Li  1 , Wuqiang Fan, Jianfeng Xu, Min Lu, Hiroyasu Yamamoto, Johan Auwerx, Dorothy D Sears, Saswata Talukdar, DaYoung Oh, Ai Chen, Gautam Bandyopadhyay, Miriam Scadeng, Jachelle M Ofrecio, Sarah Nalbandian, Jerrold M Olefsky
Affiliations

Adipocyte NCoR knockout decreases PPARγ phosphorylation and enhances PPARγ activity and insulin sensitivity

Pingping Li et al. Cell. .

Abstract

Insulin resistance, tissue inflammation, and adipose tissue dysfunction are features of obesity and Type 2 diabetes. We generated adipocyte-specific Nuclear Receptor Corepressor (NCoR) knockout (AKO) mice to investigate the function of NCoR in adipocyte biology, glucose and insulin homeostasis. Despite increased obesity, glucose tolerance was improved in AKO mice, and clamp studies demonstrated enhanced insulin sensitivity in liver, muscle, and fat. Adipose tissue macrophage infiltration and inflammation were also decreased. PPARγ response genes were upregulated in adipose tissue from AKO mice and CDK5-mediated PPARγ ser-273 phosphorylation was reduced, creating a constitutively active PPARγ state. This identifies NCoR as an adaptor protein that enhances the ability of CDK5 to associate with and phosphorylate PPARγ. The dominant function of adipocyte NCoR is to transrepress PPARγ and promote PPARγ ser-273 phosphorylation, such that NCoR deletion leads to adipogenesis, reduced inflammation, and enhanced systemic insulin sensitivity, phenocopying the TZD-treated state.

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Figures

Figure 1
Figure 1. NCoR targeting strategy and adipocyte-specific deletion
(A) Shown (top to bottom) are wild-type, floxed, and deleted NCoR gene loci. Primers used to distinguish WT and floxed alleles and sizes of the expected PCR products are indicated. (B) Genotyping results of wild type +/+, f/+, and f/f mice. (C) Relative messenger RNA levels of NCoR in adipose tissue and macrophages. Relative NCoR (D) and SMRT (E) mRNA levels in various tissues. Values are fold induction of gene expression normalized to the housekeeping gene Gapdh and expressed as mean ± SEM, n=8-10 in C- E, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2.
Figure 2
Figure 2. Obese phenotype of adipocyte specific NCoR KO (AKO) mice
(A) Photograph, (B) Body weight, (C) Food intake, (D) Coronal section views of 3D MRI scan, (E) Subcutaneous fat mass, (F) Visceral fat mass, (G) Epi-WAT weight, and (H) Adipogenic gene expression levels in Epi-WAT. Values are expressed as mean ± SEM, n=8-10 in B, C, E- H, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2 and Figure S1.
Figure 3
Figure 3. Improved glucose metabolism and insulin sensitivity in AKO mice
(A) Fasting blood glucose levels (B) Fasting blood insulin levels. (C) Insulin tolerance tests. (D) Glucose tolerance tests. (E) Glucose infusion rate (GIR) during hyperinsulinmic euglycemic clamp. (F) Glucose disposal rate (GDR). (G) insulin-stimulated glucose disposal rate (IS-GDR). (H) Basal hepatic glucose production (HGP). (I) Percent suppression of HGP by insulin (HGP suppression). (J) Percent suppression of free fatty acid levels (FFA suppression). (K) Insulin-stimulated phospho- Akt (Ser473) in liver, (L) muscle, and (M) adipose tissue. Values are expressed as mean ± SEM, n=8-10 in A- D, n=6 in E-J, * P<0.05, ** P<0.01 for AKO versus WT. See also Figure S1 and S2.
Figure 4
Figure 4. Increased adipogenesis and reduced adipocyte cell death in AKO mice
(A) Adipose tissue was stained for caveolin and adipocyte diameter was quantified. (B) Relative mRNA levels of PPARγ, (C) Cathepsin D activity, (D) Perilipin staining, (E) Tunnel staining, and (F) Circulating adipokine (leptin, high molecular weight-adiponectin, resistin and PAI-1) levels in control and AKO mice. (G) Blood free fatty acid (FFA) levels. (H) Basal and insulin stimulated 2-DOG glucose uptake in primary adipocytes. Values are expressed as mean ± SEM, n=8-10 in B, C, F and G. n=5 in H, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2 and Figure S3.
Figure 5
Figure 5. Decreased adipose tissue inflammation in AKO mice
(A) FACS analysis of F4/80+/CD11b+ cells in SVF. (B) Relative mRNA levels of the macrophage marker F4/80 in Epi-WAT. (C) F4/80 immuno staining in epi-WAT. (D) FACS analysis of F4/80+/CD11b+/CD11c+ cells in SVF. (E) Relative mRNA levels of the proinflammatory M1- like macrophage marker CD11c in Epi-WAT. (F) Relative mRNA levels of inflammatory and anti-inflammatory cytokines in Epi-WAT. (G) Effect of conditioned medium (CM) from WT and AKO primary adipocytes on macrophage chemotaxis. (H) In vivo PKH26 flourescently labeled macrophage tracking in WT and AKO mice on HFD. (I) Subpopulations of recruited PKH26+ macrophages. Values are expressed as mean ± SEM, n=6 in A, B, D and E, n=8 in F, n=5-6 in G-I, * P<0.05, ** P<0.01 for AKO versus WT. See also Table S2 and Figure S5.
Figure 6
Figure 6. Effects of Rosiglitazone treatment and PPARγ serine273 phosphorylation
(A) IS-GDR in mice with or without Rosiglitazone treatment. (B) Percent suppression of HGP (HGP suppression). (C) Percent suppression of free fatty acid levels (FFA suppression). (D) Phospho- PPARγ (Ser273) and phospho-CDK5 (Tyr15) levels in epi-WAT. (E) Phospho-PPARγ levels in primary adipocytes. (F) Phospho-PPARγ and phospho-CDK5 levels in epi-WAT with or without Rosiglitazone treatment. (G) Phospho-Rb levels in epi-WAT. (H) TNFα–induced PPARγ ser 273 phosphorylation in primary adipocytes. (I) Mammalian two-hybrid assays in HEK293T cells using Gal4-PPARγ and VP16-NCoR-N (N-terminal 1-740 aa), VP16-NCoR-M (middle 742-1798 aa) or VP16-NCoR-C (C-terminal 1803-2439 aa). Also shown is the effect of Rosiglitazone on the PPARγ-NCoR-C interaction. (J) co-immunoprecipitation of CDK5 with PPARγ in epi-WAT. (K) Effects of NCoR, or the NCoR C-terminal domain, on TNFα -induced PPARγ ser 273 phosphorylation. (L) Mammalian two-hybrid assays using Gal4-CDK5 and VP16-PPARγ. (M) Effect of NCoR and Rosiglitazone on the PPARγ-CDK5 interaction. Values are expressed as mean ± SEM, n=6 in A- C, n=4-5 in D- H, and J * P<0.05, ** P<0.01 for AKO versus WT. See also Figure S4.
Figure 7
Figure 7. mRNA levels of PPARγ target genes in epi-WAT
(A) mRNA levels of up-regulated genes in epi-WAT. (B) mRNA level of a down-regulated gene (RGS2). (C) SRC3 occupancy on PPARγ responsive elements (PPRE) in the Pepck/Gpd1 promoters. Values are expressed as mean ± SEM, n=6-8 in A and B, * P<0.05 for AKO versus WT. See also Table S1 and S2.
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