LDHA- Mediated Histone Lactylation Promotes the Nonalcoholic Fatty Liver Disease Progression Through Targeting The METTL3/ YTHDF1/SCD1 m6A Axis

Abstract

Nonalcoholic fatty liver disease (NAFLD) is characterized by elevated hepatic lipids caused by nonalcoholic factors, where histone lactylation is lately discovered as a modification driving disease progression. This research aimed to explore the role of histone 3 lysine 18 lactylation (H3K18lac) in NAFLD progression using a high-fat diet (HFD)-treated mouse model and free fatty acids (FFA)-treated L-02 cell lines. Lipids accumulation was screened via Oil Red O staining, real-time quantitative polymerase chain reaction (RT-qPCR), western blotting, and commercially available kits. Similarly, molecular mechanism was analyzed using immunoprecipitation (IP), dual-luciferase reporter assay, and RNA decay assay. Results indicated that FFA upregulated lactate dehydrogenase A (LDHA) and H3K18lac levels in L-02 cells. Besides, LDHA-mediated H3K18lac was enriched on the proximal promoter of methyltransferase 3 (METTL3), translating into an increased expression. Moreover, METTL3 or LDHA knockdown relieved lipid accumulation, decreased total cholesterol (TC) and triglyceride (TG) levels, and downregulated lipogenesis-related proteins in FFA-treated L-02 cell lines, in addition to enhancing the m6A and mRNA levels of stearoyl-coenzyme A desaturase 1 (SCD1). The m6A modification of SCD1 was recognized by YTH N6-methyladenosine RNA binding protein F1 (YTHDF1), resulting in enhanced mRNA stability. LDHA was found to be highly expressed in HFD-treated mice, where knocking down LDHA attenuated HFD-induced hepatic steatosis. These findings demonstrated that LDHA-induced H3K18lac promoted NAFLD progression, where LDHA-induced H3K18lac in METTL3 promoter elevated METTL3 expression, thereby promoting m6A methylation and stabilizing SCD1 via a YTHDF1-dependent manner. Keywords: Nonalcoholic fatty liver disease, LDHA, METTL3, YTHDF1, Histone lactylation.

Fig. 1

LDHA and H3K18lac were upregulated in FFA-treated cells. (A) lactate and (B) LDHA levels in L-02 cells treated with FFA for different times. (C) The H3K18lac and LDHA levels in the FFA-treated L-02 cells. *p<0.05, **p<0.01, ***p<0.001.

Fig. 2

Knockdown of LDHA prevented lipids accumulation in vitro. (A–B) Transfection efficiency of shLDHA, after shLDHA transfection. (C) The lactate levels and (D) H3K18lac in the FFA-treated L-02 cells were tested. (E) Oil Red O staining of the FFA-treated L-02 cells was performed. Scale bar: 200 μm. (F) The TG and (G) TC levels in the FFA-treated L-02 cells were assessed. (H) The protein levels of FAS, ACC and SREBP1 in the FFA-treated L-02 cells were detected. **p<0.01, ***p<0.001.

Fig. 3

Overexpression of LDHA promoted lipids accumulation in vitro. (A–B) Transfection efficiency of oeLDHA. The FFA-treated L-02 cells were transfected with oeLDHA. Then (C) the lactate levels, (D) H3K18lac, (E) Oil Red O staining (scale bar: 200 μm), (F) TG and (G) TC levels, and (H) the protein levels of FAS, ACC and SREBP1 were analyzed. **p<0.01, ***p<0.001.

Fig. 4

LDHA upregulated METTL3 levels through H3K18 lactylation. (A) M6A levels in the FFA-treated L-02 cells. (B) The expression levels of m6A methylation-related enzymes in the FFA-treated L-02 cells. (C) The METTL3 and METTL4 mRNA and (D) protein levels in the FFA-treated and shLDHA transfected L-02 cells. (E–F) CHIP assay was conducted to analyze the H3K18 enrichment on the METTL3 promoter. (G) The lactate levels, (H) H3K18lac levels and (I) H3K18 enrichment on the METTL3 promoter in the FFA, shLDHA and lactate-treated L-02 cells. *p<0.05, **p<0.01, ***p<0.001.

Fig. 5

Knockdown of METTL3 prevented lipids accumulation in vitro. (A–B) Transfection efficiency of shMETTL3. The FFA-treated L-02 cells were transfected with shMETTL3. Then, (C) Oil Red O staining (Scale bar: 200 μm), (D) TG and (E) TC levels, and (F) the protein levels of FAS, ACC and SREBP1 were analyzed. **p<0.01, ***p<0.001.

Fig. 6

METTL3 promoted the m6A and mRNA levels of SCD1. (A) The relationship between METTL3 and SCD1, FAS, ACC or SREBP1 was validated using RIP assay. (B) The m6A expression of SCD1 was assessed after METTL3 knockdown and overexpression. (C) The bioinformatic analysis of the methylation binding sites of SCD1. (D) The Luciferase activity of SCD1-wt and SCD1-mut were assessed after METTL3 knockdown and overexpression. (E) The mRNA expression of SCD1 was assessed after METTL3 knockdown and overexpression. **p<0.01, ***p<0.001.

Fig. 7

YTHDF1 elevated the SCD1 mRNA stability in an m6A-dependent manner. (A) Validation of transfection efficiency. (B) SCD1 levels were assessed after the knockdown of m6A readers. (C) The YTHDF1 levels and (D) SCD1 levels were determined after YTHDF1 overexpression. (E) The Luciferase activity of SCD1-wt and SCD1-mut was assessed after YTHDF1 knockdown and overexpression. (F) The stability of SCD1 was assessed after YTHDF1 knockdown and overexpression. (G) The mRNA and (H) protein levels of SCD1 were determined after oeMETTL3 and shYTHDF1 transfection. *p<0.05, **p<0.01, ***p<0.001.

Fig. 8

The establishment of the NAFLD model in vivo. (A) The FFA, (B) TG, (C) TC, and (D) protein levels of FAS, ACC and SREBP1 in the HFD-treated mice were analyzed. The (E) mRNA and (F) protein levels of LDHA in the HFD-treated mice were analyzed. ***p<0.001.

Fig. 9

LDHA knockdown relieved the NAFLD progression in vivo. The (A) body weight, (B) liver weight, and (C) adipose tissue weight of mice in each group were quantified. The (D) FFA, (E) TG and (F) TC levels in the HFD-treated mice after LDHA knockdown were analyzed. (G) Represent images of Oil Red O, H&E, Masson trichrome staining of the liver in the HDF-treated mice. The scale bar for Oil Red O staining is 50 μm, and those for H&E and Masson trichrome staining is 200 μm. (H–I) The protein levels of FAS, ACC, SREBP1, METTL3 and SCD1 in the livers of the HDF-treated mice after LDHA knockdown were analyzed. **p<0.01, ***p<0.001.