Liver TET1 promotes metabolic dysfunction-associated steatotic liver disease

Abstract

Global hepatic DNA methylation change has been linked to human patients with metabolic dysfunction-associated steatotic liver disease (MASLD). DNA demethylation is regulated by the TET family proteins, whose enzymatic activities require 2-oxoglutarate (2-OG) and iron that both are elevated in human MASLD patients. We aimed to investigate liver TET1 in MASLD progression. Depleting TET1 using two different strategies substantially alleviated MASLD progression. Knockout (KO) of TET1 slightly improved diet induced obesity and glucose homeostasis. Intriguingly, hepatic cholesterols, triglycerides, and CD36 were significantly decreased upon TET1 depletion. Consistently, liver specific TET1 KO led to improvement of MASLD progression. Mechanistically, TET1 promoted CD36 expression through transcriptional upregulation via DNA demethylation control. Overexpression of CD36 reversed the impacts of TET1 downregulation on fatty acid uptake in hepatocytes. More importantly, targeting TET1 with a small molecule inhibitor significantly suppressed MASLD progression. Conclusively, liver TET1 plays a deleterious role in MASLD, suggesting the potential of targeting TET1 in hepatocytes to suppress MASLD.

Keywords: 5-Hydroxymethylcytosine; Alcoholic Liver Disease; Epigenetics; Fatty Liver; Nonalcoholic Fatty Liver Disease.

Figure 1. Hepatic TET1 and TET3 are downregulated in MASLD.

(A) Body weight, liver weight over body weight (LW/BW), and visceral fat weight over BW (FW/BW) were examined in male C57/BL6 mice fed with either a normal chow (NC) or high-fat diet (HFD) for 16 weeks, n = 5 in NC; n = 5 in HFD. The representative (B) gross images, (C) 100X H&E, 400X H&E, and 400X Oil Red O staining images of NC and HFD fed mice. (D) 5mC dot blot and methylene blue staining were done using the genomic DNA of liver samples derived from NC and HFD mice. (E) Quantification results of 5mC/methylene blue, n = 5 in NC; n = 5 in HFD (F) TET1, TET2, and TET3 protein expression levels were determined in NC and HFD treated mice. Bottom panel: The relative ratios of TET1, TET2, TET3 over GAPDH, n = 6 in NC; n = 7 in HFD Data are presented as mean ± S.D. in (A, E), and (F). P values were determined using unpaired two-tailed Student’s T test. Exact p values were as indicated. Source data are available online for this figure.

Figure 2. Ubiquitously knocking out TET1 using two strategies suppressed MASLD progression.

(A) Body weight was measured in male TET1^wt/wt and TET1^−/− mice fed with an HFD for 16 weeks starting at 8 weeks old, n = 11 in TET1^wt/wt; n = 7 in TET1^−/−. (B) The representative liver images of TET1^wt/wt and TET1^−/− mice fed with an HFD. (C) LW/BW, (D) FW/BW, (E) ALT, and (F) AST were determined in these mice. For (C) and (D), n = 11 in TET1^wt/wt; n = 7 in TET1^−/−. For (E) and (F), n = 5 in TET1^wt/wt; n = 7 in TET1^−/−. (G) The gross liver images of male TET1^L/L and Actb^Cre/- TET1^L/L mice fed with an HFD for 16 weeks starting at 8 weeks old. (H) BW and (I) LW/BW were determined in these mice, n = 4 in TET1^L/L; n = 3 in Actb^Cre/-TET1^L/L Data are presented as mean ± S.D. in (A, C–F, H), and (I). P values were determined using unpaired two-tailed Student’s T test. Exact p values were as indicated. Source data are available online for this figure.

Figure 3. TET1 KO suppressed hepatic lipid contents through targeting hepatic lipid metabolism.

(A) The representative H&E and Oil Red O staining images of TET1^wt/wt and TET1^−/− mice suggested decreased lipid deposition in TET1 KO mice. (B) Hepatic cholesterols, (C) hepatic triglycerides (TG), (D) serum cholesterols, and (E) serum TG were measured in male TET1^wt/wt and TET1^−/− mice fed with an HFD, n = 5 in TET1^wt/wt; n = 7 in TET1^−/− By using RNA sequencing, hepatic differentially expressed genes (TET1^−/− versus TET1^wt/wt) were determined in the liver samples derived from male TET1^wt/wt and TET1^−/− mice fed with an HFD for 16 weeks. (F) A volcano plot of DEGs exhibits significantly downregulated genes which include CD36 in TET1 KO mice on the left-hand side. (G) Top altered 50 genes including CD36 and Igfbp2 were listed in a heat map. The gene set enrichment analysis using GO term revealed significantly (H) upregulated and (I) downregulated pathways in TET1^wt/wt mice. Pathways involved in lipid metabolism were highly downregulated in TET1^−/− mice. Data are presented as mean ± S.D. in (B–D), and (E). P values were determined using unpaired two-tailed Student’s T test. Exact p values were as indicated. Source data are available online for this figure.

Figure 4. CD36 is involved in the TET1-mediated free fatty acid uptake.

(A) IGFBP2 and (B) CD36 genes were examined in the liver samples of TET1^wt/wt and TET1^−/− mice fed with an HFD, n = 3 in WT; n = 4 in TET1 KO for (A) and (B, C) TET1 mRNA, (D) protein, (E) 5hmC formation, (F) methylene blue, (G) IGFBP2, (H) CD36 were determined in shRNA-luciferase (shLuc), shRNA-TET1#23 (shTET1#23), and shRNA-TET1#26 (shTET1#26) treated human hepatocytes, n = 4 in shLuc, shTET1#23, and shTET1#26 for (C, G), and (H). (I) The representative Oil Red O staining images of shLuc, shTET1#23, and shTET1#26 human hepatocytes treated with 100 µM oleic acid (OA) for 24 h. All scale bar indicate 50 µM. (J) Semi-quantification results of Oil Red O staining, n = 6 for all groups in (J, K) The representative flow cytometry images of free fatty acid uptake were shown for shLuc, shTET1#23, and shTET1#26 human hepatocytes. (L) Quantification results of (K), n = 6 for all groups in (L, M) The representative images of free fatty acid uptake in human hepatocytes treated with 200 µM 2-oxoglutarate or 5 µM UC514321 for 48 h. (N) Quantification results of (M), n = 3 for all groups in (N, O) The protein expression levels of TET1, CD36, and α-tubulin were determined in human hepatocytes treated with shLuc + empty vector, shLuc + CD36, shTET1#23 + empty vector, shTET1#23 + CD36. (P) The representative images and (Q) quantification results of free fatty acid uptake were shown for these cells, n = 4 for all groups in (Q) Data are presented as mean ± S.D. in (A, B, C, G, H, J, L, N), and (Q). P values were determined using unpaired two-tailed Student’s T test. Exact p values were as indicated. Source data are available online for this figure.

Figure 5. CD36 is transcriptionally regulated by TET1 through DNA methylation control.

(A) The representative histograms of TET1 ChIP bindings to the CD36 gene in three datasets, including GSE26832, GSE140999, and GSM611192. (B) Summary of the TET1 binding sites on CD36 promoter (P) and enhancers. (C) The TET1 binding peaks were reduced in enhancer 3 (E3) and E5 of TET1 KO samples derived from the GSE26832 dataset. (D) The TET1 binding peaks of E5 and E7 were decreased in TET1 knockdown (KD) cells compared to the control (CTRL) ones derived from the GSM611192 dataset. (E) The relative luciferase activities of CD36-P, E2, E3, E4, E5, and E7 were determined in human cells treated as indicated for 24 h, n = 3 for all groups (F) Relative luciferase activities of CD36-P, E3, E5, and E7 were examined in human cells transfected with pIRESII-GFP control vector, pIRESII-GFP-TET1, pIRESII-GFP-TET1-mut (with catalytic domain mutation), pIRESII-GFP-TET1-CD (with TET1 catalytic domain only) for 24 h, n = 3 for all groups. (G) Relative CD36 mRNA expression levels were determined in human hepatocytes manipulated with shLuc, shTET1#23, and shTET1#26 in the presence of DMSO or 10 μM 5-Azacytidine (5-Aza) for 48 h, n = 4 for all groups. Data are presented as mean ± S.D. in (E, F), and (G). P values were determined using unpaired two-tailed Student’s T test. Exact p values were as indicated. Source data are available online for this figure.

Figure 6. Liver specific KO inhibited MASLD progression.

(A) The weekly BW of male TET1^L/L and Alb^Cre/- TET1^L/L mice fed with an HFD for 16 weeks. (B) The representative gross liver images of these experimental mice. (C) LW, (D) LW/BW, and (E) ALT were determined in these mice. (F) The representative H&E images of the liver samples derived from these experimental mice. Left panel: Scale bars indicate 100 µm. Right panel: Scale bars indicate 50 µm. (G) Serum TG, (H) serum cholesterols, (I) hepatic cholesterols, and (J) hepatic TG were examined in these mice. Relative hepatic mRNA expression levels of (K) CD36, IGFBP2, (L) TET1, TET2, and TET3 were determined in these mice. Data are presented as mean ± S.D. in (A, C, D, E, G–K), and (L). P values were determined using unpaired two-tailed Student’s T test, n = 5 in TET1^L/L; n = 15 in Alb^Cre/-TET1^L/L for (A, C, D, E, G–K), and (L). Exact p values were as indicated. Source data are available online for this figure.

Figure 7. Targeting TET1 with a small molecule inhibitor ameliorated MASLD progression.

(A) The experimental scheme of TET1 inhibitor (TET1 i) treatment. (B) BW gains were measured in male C57BL/6J mice fed with an HFD and treated with a TET1 i (2.5 mg/kg UC514321) for 8 weeks. (C) LW, (D) LW/BW, (E) FW, (F) and FW/BW were determined in these mice. (G) The representative liver gross images of these experimental mice. Left panel: Scale bars indicate 100 µm, right panel: Scale bars indicate 50 µm. (H) Serum cholesterols, (I) serum TG, (J) hepatic cholesterol, and (K) hepatic TG in these mice. (L) The CD36 mRNA levels were examined in these mice. (M) TET1, CD36, and a-tubulin protein levels were examined in these mice. (N) Semi-quantification of TET1 and CD36 protein expression. Data are presented as mean ± S.D. in (B–F, H–L), and (N). P values were determined using unpaired two-tailed Student’s T test, n = 6 in Veh; n = 7 in TET1 i for (B–F, H–L), and (N). Exact p values were as indicated. Source data are available online for this figure.