TET3 plays a critical role in white adipose development and diet-induced remodeling

Abstract

Maintaining healthy adipose tissue is crucial for metabolic health, requiring a deeper understanding of adipocyte development and response to high-calorie diets. This study highlights the importance of TET3 during white adipose tissue (WAT) development and expansion. Selective depletion of Tet3 in adipose precursor cells (APCs) reduces adipogenesis, protects against diet-induced adipose expansion, and enhances whole-body metabolism. Transcriptomic analysis of wild-type and Tet3 knockout (KO) APCs unveiled TET3 target genes, including Pparg and several genes linked to the extracellular matrix, pivotal for adipogenesis and remodeling. DNA methylation profiling and functional studies underscore the importance of DNA demethylation in gene regulation. Remarkably, targeted DNA demethylation at the Pparg promoter restored its transcription. In conclusion, TET3 significantly governs adipogenesis and diet-induced adipose expansion by regulating key target genes in APCs.

Keywords: CP: Metabolism; CP: Molecular biology; DNA demethylation; TET3; adipocyte expansion; adipocyte precursor cells; adipocytes; adipogenesis; epigenetics.

Figure 1.. TET3 is necessary for full adipogenesis in vitro and ex vivo

(A) Cartoon of breeding strategy to generate APC-selective Tet3 KO (Pdgfra-Tet3 KO) mice. (B) KD efficiency of Tet3 in Lin⁻/PDGFRα⁺ APCs from iWAT of WT and KO mice (n = 4), determined using qPCR. (C and D) Adipogenic potential was estimated by oil red O (ORO) staining (C) and mRNA expression analysis (D) in primary WT and Tet3-KO APCs at day 6 of adipogenesis (n = 3). (E and I) Dot blot analysis showing global 5hmC levels in 3T3-L1 transduced with scramble (Scr), shTet3 (Tet3 KD), GFP, or TET3 (TET3 OE) plasmids. Methylene blue staining was used as a loading control. (F–H and J–L) Protein analysis (F and J), ORO staining (G and K), and mRNA expression analysis (H and L) following adipogenic differentiation of cells from (E) and (I) (n = 6 for H; n = 3 for L). Data are presented as mean ± standard error of the mean (SEM). The scale bars in (C), (G), and (K) represent 50 μm. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed Student’s t test (B, D, H, and L).

Figure 2.. Pdgfra-Tet3 KO male mice show reduced adiposity on a chow diet

(A–C) BW (A), body composition (B), and tissue weight (C) of 8-week-old WT and Pdgfra-Tet3 KO male mice on a chow diet (n = 11 for A and B; n = 7 for C). Quad, quadriceps muscle. (D–I) H&E staining (D and G), adipocyte size distribution (E and H), and total adipocyte number (F and I) of iWAT and eWAT from WT and Pdgfra-Tet3 KO mice on a chow diet (n = 3 for E, F, H, and I). The scale bar represents 100 μm. (J and K) Gene expression analysis in WAT from WT and Pdgfra-Tet3 KO mice on a chow diet (n = 5). (L and M) Immunoblotting analysis showing PPARγ protein levels in WAT from WT and Pdgfra-Tet3 KO mice on a chow (n = 3). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed Student’s t test (A–C, E, F, H–K, and M).

Figure 3.. Pdgfra-Tet3 KO male mice on an HFD are protected from unhealthy adipose expansion and remodelling

(A–E) BW (A), whole-body and WAT photographs (B and C), body composition (D), and adipose tissue weight (E) of WT and Pdgfra-Tet3 KO male mice on an HFD. pWAT, perirenal WAT; mWAT, mesenteric WAT; GA, gastrocnemius; EDL, extensor digitorum longus (n = 11 for A and D; n = 10 for adipose tissues and liver; quad, spleen, GA, EDL, and soleus n = 4 [WT] or 3 [KO] in E). (F–K) H&E staining (F and I), frequency distribution of adipocyte size (G and J), and total adipocyte number (H and K) of WATs from WT and Pdgfra-Tet3 KO mice on an HFD (n = 6). (L–N) mRNA expression analysis (L and M) and PPARγ protein levels and quantification (N) in WAT of WT and Pdgfra-Tet3 KO mice on an HFD (n = 5 for L and M; n = 6 for N). (O–Q) Serum leptin levels (O), immunofluorescence staining of Mac2 (P), and quantification of hydroxyproline (Q) in WAT from WT and KO mice on an HFD (n = 10 for O; n = 3 for Q). The scale bar represents 100 μm. Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed Student’s t test (A, D, E, G, H, J–O, and Q).

Figure 4.. Pdgfra-Tet3 KO male mice display improved whole-body metabolism on an HFD

(A–I) GTT (A, B, E, and F), ITT (C, D, G, and H), and HOMA-IR (I) from Pdgfra-Tet3 KO and WT male mice reared on a chow at 8 weeks of age after being fed an HFD for 2 months (n = 5 mice for A–D; n = 8 mice for E, F, and I, n = 12 mice for G and H). AOC, area over the curve. Data are presented as mean ± SEM. *p < 0.05, using two-tailed Student’s t test (A–G). (J) H&E staining of liver sections of Pdgfra-Tet3 KO and WT male mice on a chow and fed an HFD. The scale bar represents 50 μm (K) Quantification of triglyceride (TG) in Pdgfra-Tet3 KO and WT male mice fed an HFD (n = 6). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed Student’s t test (A–I).

Figure 5.. TET3 regulates the critical target genes involved in adipogenesis and remodeling in APCs

(A) Overall study design of transcriptomic and DNA methylome analysis of Pdgfra-Tet3 KO vs. WT APCs. (B) Volcano plot depicting up- and downregulated genes in Pdgfra-Tet3 KO vs. WT iWAT APCs on an HFD. (C) Top Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways enriched in downregulated gene sets. ECM-related pathways are depicted in bold. (D) qPCR validation of the downregulated genes in KO APCs (n = 3). (E) mRNA expression of Cilp in confluent 3T3-L1 preadipocytes transduced with Scr or shTet3 (n = 6). (F and G) mRNA expression of Cilp during 3T3-L1 adipogenesis (F) and WATs from lean vs. obese WT C57BL/6J mice (G) (n = 3 for F; n = 6 for G). (H and I) ORO staining (H) and gene expression analysis (I) in 3T3-L1 cells treated with vehicle (control [Cont]) and recombinant CILP (25 or 75 ng/mL) from adipogenic days 0–4. The scale bar represents 50 μm (n = 3 for I). Data are presented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001 using two-tailed Student’s t test (D–G and I).

Figure 6.. TET3 modifies the DNA methylation profile of key target genes in APCs

(A) Correlation map depicting relative CpG density and methylation levels between WT and Tet3 KO APCs. (B) Pie chart of hyper- and hypo-DMR counts in Tet3 KO APCs. (C) Pie chart of downregulated gene counts linked with hyper-DMRs in Tet3 KO APCs. (D) KEGG pathway most enriched in genes linked to hyper-DMRs in Tet3 KO APCs. ECM-related pathways are depicted in bold. (E–G) Snapshots of WGBS track showing hypermethylated regions (boxed region) near the TET3 target genes. The height of the gray and red bars represents the degree of CpG methylation in WT and Tet3 KO APCs, respectively. (H) HEK-293T cells were transfected with methylated (mCpG) and unmethylated (CpG) versions of pCpGL reporter plasmids containing target regions with differential methylation or control sequences (basic and CMV/EF1 were used as a negative and positive controls, respectively). These cells were then assayed for luciferase reporter activity. The C1qtnf7 DMR appeared on the left side of the track that was tested (n = 3). (I–K) TET3 ChIP-qPCR analysis with WT iWAT at hypermethylated regions in Tet3 KO APCs (n = 3). (L) The overrepresented transcription factor motifs predicted by motif analysis using HOMER (v.4.11) on the hyper-DMRs in Tet3 KO APCs. (M and N) Pparg2 mRNA expression (M) and 5mC enrichment at the Pparg2 promoter (N) in 3T3-L1 preadipocytes expressing pINDUCER-dCas9-Tet1CD (dCas9-Tet1CD) or pINDUCER-dCas9-Tet1CDM (dCas9-Tet1CDM) compared with Pparg gRNAs (n = 3 for M and N). Data are presented as mean ± SEM. **p < 0.01, ***p < 0.001 using two-tailed Student’s t test (H–K, M, and N).