Adipocyte SIRT1 knockout promotes PPARγ activity, adipogenesis and insulin sensitivity in chronic-HFD and obesity

Abstract

Objective: Adipose tissue is the primary site for lipid deposition that protects the organisms in cases of nutrient excess during obesogenic diets. The histone deacetylase Sirtuin 1 (SIRT1) inhibits adipocyte differentiation by targeting the transcription factor peroxisome proliferator activated-receptor gamma (PPARγ).

Methods: To assess the specific role of SIRT1 in adipocytes, we generated Sirt1 adipocyte-specific knockout mice (ATKO) driven by aP2 promoter onto C57BL/6 background. Sirt1 (flx/flx) aP2Cre (+) (ATKO) and Sirt1 (flx/flx) aP2Cre (-) (WT) mice were fed high-fat diet for 5 weeks (short-term) or 15 weeks (chronic-term). Metabolic studies were combined with gene expression analysis and phosphorylation/acetylation patterns in adipose tissue.

Results: On standard chow, ATKO mice exhibit low-grade chronic inflammation in adipose tissue, along with glucose intolerance and insulin resistance compared with control fed mice. On short-term HFD, ATKO mice become more glucose intolerant, hyperinsulinemic, insulin resistant and display increased inflammation. During chronic HFD, WT mice developed a metabolic dysfunction, higher than ATKO mice, and thereby, knockout mice are more glucose tolerant, insulin sensitive and less inflamed relative to control mice. SIRT1 attenuates adipogenesis through PPARγ repressive acetylation and, in the ATKO mice adipocyte PPARγ was hyperacetylated. This high acetylation was associated with a decrease in Ser273-PPARγ phosphorylation. Dephosphorylated PPARγ is constitutively active and results in higher expression of genes associated with increased insulin sensitivity.

Conclusion: Together, these data establish that SIRT1 downregulation in adipose tissue plays a previously unknown role in long-term inflammation resolution mediated by PPARγ activation. Therefore, in the context of obesity, the development of new therapeutics that activate PPARγ by targeting SIRT1 may provide novel approaches to the treatment of T2DM.

Keywords: Glucose homeostasis; Insulin resistance; Obesity; PPAR03B3; Phosphorylation; SIRT1.

Graphical abstract

Figure 1

Quantification of adipocyte-specific deletion of Sirt1 in mice (ATKO). A: DNA genotyping. Upper panel, Sirt1^flx/flx (floxed-ATKO) and Sirt1^+/+ (WT) DNA expression. Lower panel, Cre-Recombinase (all mice are Sirt1^flx/flx). C+, Cre positive control; C- Cre negative control. B: Semi-qPCR showing the Sirt1 floxed exon 4 (Δ4 ATKO) in several adipose depots and other different tissues from WT and ATKO mice. 36b4 (Rplp0) expression was used as housekeeping gene. C: Sirt1 mRNA expression in intraperitoneal macrophages and epididymal white adipose tissue (eWAT). D: Semi-qPCR showing Sirt1 mRNA expression in adipocytes or Stromal vascular fraction (SVF) from WT and ATKO eWAT. E: Protein expression of SIRT1 determined by Western Blot in adipocytes isolated from eWAT. F: Acetylation levels of SIRT1 target protein p65 (NF-κB) in adipocytes from ATKO or WT mice. Hsp90 expression was used as loading control. The Western blots shown are representative of three independent experiments (**P ≤ 0.01 vs. WT). G: Relative mRNA expression of Sirt2 and Sirt3 in different adipose tissues from WT and ATKO mice after 15 weeks of HFD.

Figure 2

Adipocyte Sirt1 deficiency causes insulin resistance on standard chow diet during aging. A: Body weight of WT and ATKO mice on standard chow diet during 60 weeks (NCD) (n = 10–15 per group). B: sqWAT, eWAT, BAT and liver weight of WT and ATKO mice on NCD. Values are expressed as means ± SEM (*P ≤ 0.05 **P ≤ 0.01 ***P < 0.001 vs. WT; n = 10/15). C: Mice body weight during the GTTs and ITTs. D: Glucose stimulated insulin secretion (GSIS). Plasma insulin concentration from WT and ATKO mice during IP-GTTs at the indicated time points. E: Intraperitoneal glucose tolerance tests (IP-GTT; 1 g/kg) and Area under curve (AUC) at 8 or 19 weeks of age (n = 8 per group). F: Intraperitoneal insulin tolerance tests (IP-ITTs; 0.6 U/kg) at 21 weeks of age (n = 8 per group). All of these studies were performed in the same cohort of mice. G: Glucose uptake of primary adipocytes from 15 weeks old mice, WT or ATKO, fed with NCD. Values are expressed as means ± SEM (*P ≤ 0.05, **P ≤ 0.01 vs. WT at the same time point).

Figure 3

Body composition of Sirt1 ATKO mice during HFD. A: Body weight of WT and Sirt1 ATKO mice fed with 60% high fat diet (HFD) (n = 25–30 per group). B: sqWAT, eWAT, BAT and liver weight of WT and ATKO mice on short term or chronic HFD (S, Short term; C, Chronic). C: Food intake of Sirt1 WT and ATKO mice at 5 weeks of HFD. D: Dual energy X-ray absorptiometry (DEXA). Body composition was assessed using DEXA scan (PIXImus2; Lunar, Madison, WI) and analyzed with PIXImus software (2.10; GE/Lunar) after 15 weeks of HFD. BW, body weight. TBA, Total body area. TTM, Total tissue mass. LBM, Lean body mass. Fat mass and percentage of fat. Length and bone area of the mice. BMC, Bone mineral content. BMD, Bone mineral density. Values are expressed as means ± SEM (*P < 0.05 vs. WT; n = 5/6 mice per group).

Figure 4

Dual effect of Sirt1 deficiency in adipose tissue during HFD. Short term vs. chronic effects. A, C: Intraperitoneal glucose tolerance tests (IP-GTT; 1 g/kg) and intraperitoneal insulin tolerance tests (IP-ITT; 0.6 U/kg) during short term HFD feeding (S, 5 wks; n = 10–12 per group) and chronic HFD feeding (C, 15 wks; n = 10–12 per group). B, D: Area under curve (AUC) and basal insulin levels from previous GTTs. Values are expressed as means ± SEM (*P ≤ 0.05, **P ≤ 0.01 and ***P ≤ 0.001 vs. WT at the same time point). E: Hyperinsulinemic-euglycemic clamp study in chronic HFD-fed mice (15 weeks). BW, Body weight. WT and ATKO matched body weights of mice during clamp studies. GIR, glucose infusion rate during hyperinsulinemic-euglycemic clamp. GDR, Glucose disposal rate. IS-GDR, insulin-stimulated glucose disposal rate. Basal-HGP, basal hepatic glucose production rate. HGP-Suppression, percent suppression of HGP. FFA, Free fatty acid suppression. Values are expressed as means ± SEM (*P ≤ 0.05 vs. WT; n = 5/6 mice per group). F: Western blot showing acute insulin-stimulated phosphorylation of AKT in liver, muscle and eWAT from chronic HFD-fed mice. Hsp90 expression was used as loading control. The western blot shown is representative of four independent experiments. G: Densitometry analysis and ratio of phospho-Ser473-Akt/total Akt from F. Values are expressed as means ± SEM (*P ≤ 0.05 **P ≤ 0.001 vs. WT basal. ⁺P ≤ 0.05; ⁺⁺P ≤ 0.001 vs. ATKO basal).

Figure 5

ATKO mice show reduced inflammation and infiltration in eWAT after prolonged HFD. A–B: Plasma protein and mRNA levels of inflammatory cytokines and chemokines measured by Milliplex during short term (S) or chronic HFD (C) (n = 8 per group). C: mRNA levels of brown fat-selective genes (Ucp1, PGC-1α, Cox7a1 and Cidea), anlayzed by qPCR. D-E: Immunofluorescence of eWAT from short term or chronic HFD-fed mice. Adipose tissue was stained with Caveolin as adipocyte membrane marker and F4/80 as macrophage cell marker. Scale bar is 100 μm (n = 4/5 per group). F: Gene expression of adipose tissue infiltration markers during HFD, analyzed by qPCR. Values are expressed as means ± SEM. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. WT at the same point, WT; n = 4/5 per group in qPCR and n = 10/12 per group for plasmatic cytokines).

Figure 6

ATKO mice show higher hyperplasia during chronic HFD. A: Immunofluorescence of eWAT from short term HFD feeding. Adipose tissue was stained with Caveolin (blue) as adipocyte membrane marker. Scale bar is 200 μm. The images below are 4x magnification corresponding to the white box on the upper images. B-C: Quantification, area and adipocyte size frequency measurement from eWAT inmunofluorescence images. D-F: Same analysis in WT and ATKO eWAT from chronic HFD feeding mice. Values are expressed as means ± SEM. (n = 4 mice/group and 2 images/mouse were analyzed). G: Hematoxylin & Eosin of eWAT, sqWAT, and BAT from WT and ATKO mice from chronic HFD-fed mice. Fat tissues were fixed with 4% PFA, embedded in paraffin blocks and sectioned in the UCSD Mouse Phenotype Service Core (Moores Cancer Center, UCSD). Bright field photographs were taken using a Zeiss Observer.Z1 microscope. Scale bar is 100 μm. (n = 4 mice/group and 2 images/mouse were analyzed).

Figure 7

ATKO mice show reduced phosphorylation of PPARγ-Ser273 after chronic HFD feeding. A: Immunoprecipitation of PPARγ and western blot with antibodies to acetylated lysine (Ac-K) or PPARγ in eWAT from short term (S) or chronic (C) HFD-fed mice. β-actin was used as a loading control, and densitometry analysis is shown in the graph. One representative western blot selected from four independent experiments is shown. B: mRNA levels of genes know to be repressed by PPARγ (angiotensinogen [agt], Pank3, resistin [retn], chemerin and Wdnm1-like) analyzed by qPCR. C: CDK5 and PPARγ phosphorylation status in WT and ATKO mice fed with NCD or HFD measured by western blot at the indicated times. Hsp90 expression was used as loading control. D: Densitometry analysis and ratio of phospho-Y15-CDK5/Hsp90 and phospho-S273-PPARγ/PPARγ from Figure 7C (n = 4 per group). E-G: qPCR analysis of p-S273-PPARγ repressed genes in NCD-fed mice and short term or chronic HFD-fed mice (n = 4/5 per group). Values are expressed as means ± SEM. (*P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 vs. WT).