TGF-β antagonism synergizes with PPARγ agonism to reduce fibrosis and enhance beige adipogenesis

Abstract

Objectives: Adipose tissue depots vary markedly in their ability to store and metabolize triglycerides, undergo beige adipogenesis and susceptibility to metabolic disease. The molecular mechanisms that underlie such heterogeneity are not entirely clear. Previously, we showed that TGF-β signaling suppresses beige adipogenesis via repressing the recruitment of dedicated beige progenitors. Here, we find that TGF-β signals dynamically regulate the balance between adipose tissue fibrosis and beige adipogenesis.

Methods: We investigated adipose tissue depot-specific differences in activation of TGF-β signaling in response to dietary challenge. RNA-seq and fluorescence activated cell sorting was performed to identify and characterize cells responding to changes in TGF-β signaling status. Mouse models, pharmacological strategies and human adipose tissue analyses were performed to further define the influence of TGF-β signaling on fibrosis and functional beige adipogenesis.

Results: Elevated basal and high-fat diet inducible activation of TGF-β/Smad3 signaling was observed in the visceral adipose tissue depot. Activation of TGF-β/Smad3 signaling was associated with increased adipose tissue fibrosis. RNA-seq combined with fluorescence-activated cell sorting of stromal vascular fraction of epididymal white adipose tissue depot resulted in identification of TGF-β/Smad3 regulated ITGA5+ fibrogenic progenitors. TGF-β/Smad3 signal inhibition, genetically or pharmacologically, reduced fibrosis and increased functional beige adipogenesis. TGF-β/Smad3 antagonized the beneficial effects of PPARγ whereas TGF-β receptor 1 inhibition synergized with actions of rosiglitazone, a PPARγ agonist, to dampen fibrosis and promote beige adipogenesis. Positive correlation between TGF-β activation and ITGA5 was observed in human adipose tissue, with visceral adipose tissue depots exhibiting higher fibrosis potential than subcutaneous or brown adipose tissue depots.

Conclusions: Basal and high-fat diet inducible activation of TGF-β underlies the heterogeneity of adipose tissue depots. TGF-β/Smad3 activation promotes adipose tissue fibrosis and suppresses beige progenitors. Together, these dual mechanisms preclude functional beige adipogenesis. Controlled inhibition of TβRI signaling and concomitant PPARγ stimulation can suppress adipose tissue fibrosis and promote beige adipogenesis to improve metabolism.

Keywords: Beige adipogenesis; Fibrosis; Metabolism; PPARγ; Rosiglitazone; Smad3; TGF-β.

Graphical abstract

Figure 1

(A, B) Protein expression (A) and mRNA expression (B) of indicated markers in inguinal fat (iWAT), epididymal fat (eWAT), or brown fat (BAT) from 8 weeks of high fat diet (HFD)-fed mice or age matched normal chow diet (CD)-fed mice (n = 3 mice each group). (C, D) Protein expression (C) and mRNA expression (D) of indicated markers in primary preadipocytes of stromal vascular fraction (SVF-PA) isolated from eWAT of 8 weeks of HFD-fed or age matched CD-fed mice (n = 3 mice each group). (E) Protein expression of αSma in SVF-PA isolated from eWAT in response to TGF-β (2 ng/ml) or SB431542 (10 μM) treatment for 3 days. (F) Immunofluorescence of SVF-PA treated with TGF-β or SB431542 for αSma (green), DAPI (blue). Scale bars: 20 μm. (G, H) mRNA expression of fibrosis related genes and Ppargc1a (Pgc1a) in primary preadipocytes (G) or differentiated adipocytes (H) in response to TGF-β and SB431542 treatment. Data are represented as mean ± SEM. Two-tailed unpaired Student's t test were used for statistical analysis. ∗When comparing with between CD and HFD or between Control-treated (Ctrl) and TGF-β treated, #when comparing same doses between Ctrl and SB431542 treated. ∗P < 0.05; ∗∗,##P < 0.01; ∗∗∗,###P < 0.001. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 2

(A–C) Analyses of RNA-seq data generated using SVF-PA isolated from eWAT of 8 weeks of HFD-fed or age matched CD-fed mice (n = 3 each group), SVF-PA isolated from eWAT and subsequently treated with TGF-β (2 ng/ml) with/without SB431542 (10 μM) (n = 3 each group). (A) Venn diagram showing the overlap of differentially expressed genes (DEGs; P < 0.05) comparing SVF-PA from HFD fed mice (HFD-SVF-PA) vs from CD fed mice (CD-SVF-PA) with TGF-β-treated SVF-PA (TGF-β-SVF-PA) vs TGF-β & SB431542-treated SVF-PA (T/SB-SVF-PA); (B) GO analysis of Co-upregulated 395 overlapping genes; (C) Heatmap for cell adhesion genes (DEGs, P < 0.05) from 395 genes co-upregulated in both HFD-SVF-PA and TGF-β-SVF-PA. (D, E) Histogram depicting the frequency of CD45-/CD31-/CD34+/Itga5+ population in (D) SVF-PA from eWAT of 12 weeks of HFD fed mice or age matched CD fed mice (2–4 mice pooled per sample) and (E) SVF-PA treated with TGF-β (2 ng/ml) and SB431542 (10 μM). Un-stained: No antibodies. (F, G) Itga5 high- (Itga5^High) or Itga5 low- (Itga5^Low) expressing cells isolated from eWAT of 12 weeks of HFD-fed mice (n = 3 per group) by FACS sorter. (F) Immunofluorescence of differentiated Itga5^High or Itga5^Low cells staining for lipid droplets (LipidTOX, green), UCP1 (red), and nuclei (DAPI, blue). Scale bars: 200 μm (upper) and 100 μm (lower) from white boxes; (G) mRNA expression of Acta2 in Itga5^High or Itga5^Low cells. Data are normalized to Itga5^High cells and expressed as mean ± SEM and were analyzed by 2-tailed unpaired Student's t test ∗P < 0.05. (H) Fold change (Log₂ FC) of beige differentiation related genes from RNA-seq analyses of differentiated Itga5^High or Itga5^Low cells isolated from eWAT of 12 weeks of HFD-fed mice (n = 3 per group). (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)

Figure 3

(A) H&E stained (upper panel) and Mason's trichrome stained (lower panel) section of eWAT from TβRI ^AdWT (WT) or TβRI ^AdKO (KO) mice fed high fat diet for 16 weeks. Scale bars: 50 μm. (B, C) mRNA expression of (B) Acta2, Itga5, Serpine1 and (C) fibrosis genes in eWAT of 16 weeks of HFD fed WT and KO mice (N = 3–4 each group.). (D) Protein expression in SVF from eWAT of 16 weeks of HFD fed WT and KO mice (N = 3–4 each group). (E) Histogram depicting frequency of CD45-/CD31-/CD34+/Itga5+ population in eWAT of 16 weeks of HFD fed WT and KO mice (2–4 mice pooled per sample). Un-stained: No antibodies. (F) Oxygen Consumption Ratio (OCR) test on differentiated adipocytes from 16 weeks of HFD fed WT and KO mice. Data are represented as mean ± SEM. Two-tailed unpaired Student's t test were used for statistical analysis comparing with between WT and KO. ∗P < 0.05; ∗∗P < 0.01.

Figure 4

(A, B) Acta2 mRNA (A) and αSma protein (B) expression regulated by rosiglitazone (R) with/without TGF-β (T) in SVF-PA from eWAT of wild type mice (2–4 mice pooled per sample). 1 μM rosiglitazone were added into SVF-PA for 30 min before 2 ng/ml TGF-β for 3 consecutive days. (N = 2–3 each group). (C and D) mRNA expression of Acta2 and Itga5 upon treatment of SVF-PA from eWAT of wild type mice with mock or rosiglitazone and TGF-β or SB431542 (SB) (N = 3–4 each group). (E) Histogram depicting frequency of CD45-/CD31-/CD34+/Itga5+ population in rosiglitazone and/or SB431542 treated SVF-PA from eWAT of wild type mice (2–4 mice pooled per sample). Un-stained: No antibodies. (F) mRNA expression of PGC-1α upon treatment of SVF-PA from eWAT of wild type mice with mock or rosiglitazone and TGF-β or SB431542 (SB) (N = 3–4 each group). (G–I) mRNA (G and H) and protein (I) expression in differentiated adipocytes (N = 3 each group) upon treatment by rosiglitazone (Rosi) with/without TGF-β (T) or SB431542 (SB). (J) Oxygen Consumption Ratio (OCR) test in differentiated adipocytes treated with rosiglitazone (Rosi) with/without TGF-β and/or SB431542 (SB). Data are represented as mean ± SEM. Two-tailed unpaired Student's t test were used for statistical analysis. ∗P < 0.05; ∗∗P < 0.01; ∗∗∗P < 0.001.

Figure 5

(A, B) Immunoblotting (A) of indicated proteins and quantification of pSMAD3/total SMAD3 (B) in human omental fat (OM), subcutaneous fat (SQ) and supraclavicular fat (SCLV) tissue samples (N = 5–6 each group). (C) Relative mRNA expression levels ITGA5 in the three adipose tissue depots from human samples (OM, n = 9; SQ, n = 7; SCLV n = 6). (D) Scatterplots depicting correlation of mRNA levels of SERPINE1 with ITGA5 in the three adipose tissue depots (OM, red circles, n = 9; SQ, blue circles, n = 7; SCLV, black circles, n = 6). Circles in scatterplots denote gene expression levels of all individuals. Data are represented as mean ± SEM. Mixed-effect model and Spearman correlation coefficient (r) were used for statistical analysis. ∗P < 0.05; ∗∗P < 0.01. (For interpretation of the references to color/colour in this figure legend, the reader is referred to the Web version of this article.)