The depot-specific and essential roles of CBP/p300 in regulating adipose plasticity

in Journal of Endocrinology
Correspondence should be addressed to L Qiang: lq2123@cumc.columbia.edu

*(M Namwanje and L Liu contributed equally to this work)

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Fat remodeling has been extensively explored through protein deacetylation, but not yet acetylation, as a viable therapeutic approach in the management of obesity and related metabolic disorders. Here, we investigated the functions of key acetyltransferases CBP/p300 in adipose remodeling and their physiological effects by generating adipose-specific deletion of CBP (Cbp-AKO), p300 (p300-AKO) and double-knockout (Cbp/p300-AKO) models. We demonstrated that Cbp-AKO exhibited marked brown remodeling of inguinal WAT (iWAT) but not epididymal WAT (eWAT) after cold exposure and that this pattern was exaggerated in diet-induced obesity (DIO). Despite this striking browning phenotype, loss of Cbp was insufficient to impact body weight or glucose tolerance. In contrast, ablation of p300 in adipose tissues had minimal effects on fat remodeling and adiposity. Surprisingly, double-knockout mice (Cbp/p300-AKO) developed severe lipodystrophy along with marked hepatic steatosis, hyperglycemia and hyperlipidemia. Furthermore, we demonstrated that pharmacological inhibition of Cbp and p300 activity suppressed adipogenesis. Collectively, these data suggest that (i) CBP, but not p300, has distinct functions in regulating fat remodeling and that this occurs in a depot-selective manner; (ii) brown remodeling occurs independently of the improvements in glucose metabolism and obesity and (iii) the combined roles of CBP and p300 are indispensable for normal adipose development.

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  • Figure S1. cDNA sequencing validation of Cbp-AKO and p300-AKO mice. (A) Cloning Cbp cDNA from fat of control mice and Cbp-AKO mice; (B) sequencing of Cbp WT or KO allele, showing Exon 9 which encodes the catalytic domain and deleted region in the KO allele. (C) Cloning p300 cDNA from fat of control mice and p300-AKO mice; (B) sequencing of p300 WT or KO allele, showing Exon 9 which encodes the catalytic domain and deleted region in the KO allele. The WT bands in Cbp-AKO and p300-AKO arose from non-adipocytes in adipose tissues. These cells don’t express Adipoq-Cre and account for ~50% of total adipose tissue cells.
  • Figure S2. (A-C) qPCR analysis of gene expression in BAT of Cbp-AKO and control mice on HFD feeding. n=6, 6, *: p<0.05.
  • Figure S3.FPLC fractionation of lipoprotein cholesterol in pooled sera from Cbp/p300 double knockouts and control mice fed on chow diet.
  • Supplemental Table 1.Expected and observed Mendelian ratios at weaning from an intercrosses between (A) CbpF/F, p300F/F and CbpF/+ p300F/+ Adipoq-Cre+ breeding pairs to generate Cbp/p300-AKO (double knockout) mice (Chi square =19.11, Degrees of Freedom =7, p = 0.0078); (B) CbpF/F, p300F/F and CbpF/+ p300F/F Adipoq-Cre+ breeding pairs to generate Cbp/p300-AKO (double knockout) mice (Chi square =19.37, Degrees of Freedom =3, p =0.0002); (C) CbpF/F, p300F/F and CbpF/F p300F/+ Adipoq-Cre+ breeding pairs to generate Cbp/p300-AKO (double knockout) mice (Chi square =13.48 , Degrees of Freedom =3, p =0.0037).

 

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    Depot-specific changes of Cbp and p300 expression during fat remodeling. Male WT C57BL/6 mice were either induced obesity by HFD feeding (HFD-RT) or induced brown remodeling by exposure to chronic cold (4°C for 4 days). The expression of Cbp (A) and p300 (B) in different depots was analyzed by qPCR. Data are presented as mean ± s.e.m., *P < 0.05 and **P < 0.01 vs control group (Chow-RT), n = 6/group.

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    Adipose loss of Cbp or p300 has no effect on adiposity or glucose metabolism. Six-month-old mice maintained on regular chow. (A, B, C, D and E) in Cbp-AKO and control mice, (A) body weight, (B and C) body fat composition in males and females (N = 8, 7 males, 14, 15 females), (E) glucose tolerance test and (F) insulin tolerance test. (F, G and H) In p300-AKO and p300F/F mice, (F) body weight, (G and H) body fat composition in males and females (N = 5, 4 males; 4, 6 females). Data are presented as mean ± s.e.m.

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    Loss of Cbp in adipose induces brown remodeling selectively in subcutaneous WAT after cold exposure. 6-month-old male Cbp-AKO and CbpF/F control mice maintained on regular chow and housed at 4°C for 4 days. (A) Core body temperature; (B) body weight; (C) adipose mass; real-time qPCR analysis of browning genes and regulators (D, F and H) and adipogenic genes (E, G and I) from iWAT, eWAT and BAT respectively of Cbp-AKO compared to CbpF/F controls (N = 6, 6). Data are presented as mean ± s.e.m. and * denotes a statistical significance at a P value ≤0.05.

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    Loss of p300 has minimal effect on cold-induced brown remodeling. Six-month-old male p300-AKO and p300F/F controls mice maintained on regular chow and housed at 4°C for 4 days. (A) Core body temperature; (B) body weight; (C) adipose mass; real-time qPCR analysis of browning genes and regulators (D, F and H) and adipogenic genes (E, G and I) from iWAT, eWAT and BAT respectively of p300-AKO compared to p300F/F controls (N = 4, 6). Data are presented as mean ± s.e.m. and *denotes a statistical significance at a P value ≤0.05.

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    Adipose deficiency of Cbp reduces adiposity and induces brown remodeling in subcutaneous WAT after diet-induced obesity. Male Cbp-AKO and CbpF/F mice were maintained on HFD for 12 weeks. (A) Body weight; (B) body composition; (C) eWAT and iWAT adipose mass; (D) quantification of the average adipocyte size from eWAT and iWAT (N = 6, 6 males). (E) Representative histological sections of eWAT, iWAT and BAT stained with hematoxylin and eosin (H&E). (F) Intraperitoneal glucose tolerance test from Cbp-AKO and CbpF/F (N = 7, 14). (G, H, I and J) Real-time qPCR analysis of browning genes and regulators (G and I) and adipogenic genes (H and J) from iWAT and eWAT respectively of Cbp-AKO and CbpF/F(N = 6, 6). Data are presented as mean ± s.e.m. and * denotes a statistical significance at a P value ≤0.05. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0361.

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    Adipose deficiency of p300 does not affect adiposity and metabolism in diet-induced obesity. Male p300-AKO and control mice were maintained on HFD for 8 weeks. (A) Body weight curve during HFD feeding; (B) body composition; (C) eWAT and iWAT adipose mass; (D) representative histological sections of eWAT, iWAT and BAT stained with hematoxylin and eosin. (E) Intraperitoneal glucose tolerance test; (F, G, H and I) Real-time qPCR analysis of browning genes and regulators (F and H) and adipogenic genes (G and I) from iWAT and eWAT respectively(N = 5, 5). Data are presented as mean ± s.e.m. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0361.

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    Loss of Cbp and p300 leads to severe lipodystrophy. In chow-fed Cbp/p300-AKO and CbpF/F/p300F/F controls, (A) body weight, (B, C, D and E) blood glucose, serum triglycerides (TG), free fatty acids and total cholesterol measurements (N = 5, 4); (F) Liver sizes; (G) anatomic comparison of adipose tissues and liver; (H) histological analysis of livers by H&E staining; (I) ELISA measurement of serum leptin levels; (J) Western blotting analyses of serum adipsin and adiponectin. (K) Anatomic analyses of Cbp/p300-AKO and CbpF/F/p300F/F mice in diet-induced obesity. Data are presented as mean ± s.e.m. and * denotes a statistical significance at a P-value ≤0.05. A full colour version of this figure is available at https://doi.org/10.1530/JOE-18-0361.

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    Inhibition of Cbp and p300 activity suppresses adipogenesis. (A) 3T3-L1 preadipocytes were treated with A486, a mimic (Control) or with the inhibitor (A485) at the induction of adipogenesis, Oil Red O (red) and BODIPY (green) counterstained with DAPI (blue) to assess differentiation efficiency by lipid accumulation. (B) Real-time qPCR analysis of genes involved in adipocyte differentiation upon treatment with or without inhibitor during induction. (C and D) Fully differentiated mature adipocytes were treated with A485 since Day 9 of differentiation for 3 days, (C) brown adipocyte genes and regulators and (D) pan-adipocyte genes by qPCR analysis. Data are presented as mean ± s.e.m. and * denotes a statistical significance at a P value ≤0.05.

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