Cth/Mpst double ablation results in early onset fatty liver disease in lean mice

Abstract

Metabolic dysfunction-associated fatty liver disease (MAFLD), a condition that stems from hepatic lipid accumulation in the absence of liver damage and overt inflammation, has become the most common hepatic disorder worldwide. Hydrogen sulfide (H₂S), a gasotrasmitter, endogenously generated mainly by cystathionine-γ lyase (CTH), cystathionine-β synthase (CBS) and 3-mercaptopyruvate sulfurtransferase (MPST) enzymes, exhibits protective effect in steatosis. Herein, we have demonstrated that CTH and MPST play a central role in MAFLD pathogenesis. Young Cth/Mpst knockout (Cth/Mpst^-/-) mice, fed a normal diet, had increased liver mass caused by enhanced hepatic lipid accumulation. Decreased insulin and glucose sensitivity was observed in CTH/MPST-deficient mice. At the cellular level, CTH/MPST inhibition resulted in increased lipid deposition and glucose uptake in hepatocytes. Transcriptome analysis revealed significant upregulation of cholesterol biosynthesis and SREBP-related genes in the liver of Cth/Mpst^-/- mice. Transcription factor enrichment analysis of differentially expressed genes between two genotypes, revealed a major impact of LXR, RXR and PPARA in the observed phenotype. Sulfide donor (SG1002) treatment attenuated the fatty liver disease of CTH/MPST-deficient mice. Our findings underline the importance of endogenously produced H₂S in the pathogenesis of MAFLD and introduce the Cth/Mpst^-/- mouse as a new animal model of early onset hepatic steatosis.

Keywords: CTH; Fatty liver; MAFLD; MPST; Steatosis; Sulfide.

Graphical abstract

Fig. 1

Cth/Mpst double ablation leads to an early steatosis phenotype. Representative western blots and quantification of (A) CTH, MPST, CBS and (B) ETHE1, TST, SQRDL in liver of WT and Cth/Mpst^−/− mice. (C) H₂S production capacity of liver extracts from WT or Cth/Mpst^−/− mice in the presence of l-cysteine (L-cys) and l-homocysteine (L-hys). Data were obtained using the lead acetate method. Liver mass and representative photomicrographs from H&E− and Oil Red O- stained sections of the liver tissue of (D, E) male and (F, G) female WT and Cth/Mpst-deficient mice. Protein expression is presented as ratio over WT group (A, B). Sulfide levels are presented as ratio over WT group (C). Arrows indicate areas of increased lipid deposition. Data are presented as means ± SEM, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, N = 6–7 mice per group. Scale bar: 100 μm.

Fig. 2

Cth/Mpst^−/− mice are characterized by increased glucose, insulin and pyruvate tolerance. (A) Body weight, (B) glucose (GTT), (C) insulin (ITT) and (D) pyruvate (PTT) tolerance tests in WT and Cth/Mpst^−/− mice (AUC = area under the curve). (E) Photomicrographs and quantitation of lipid accumulation in hepatocytes in the presence or absence of a CTH inhibitor (PAG) and an MPST inhibitor (IMST-3). (F) Glucose levels in the supernatants of hepatocytes following the PAG and IMST-3 treatment. (G)CTH and MPST were knocked down in hepatocytes using silencing RNA for 48h. The reduction of enzyme expression after transfection was confirmed by real-time PCR. (H) Lipid accumulation was determined using Oil Red O in hepatocytes transfected with a control siRNA (siNEG) or a combination of siRNA targeting both the CTH and MPST genes. Optical density is presented as ratio over vehicle (E) or siNEG (H) group. mRNA levels are presented as ratio over siNEG group. (H) Data are presented as means ± SEM, ∗p < 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001, (A) N = 6–7, (B, D) N = 5, (C) N = 8–10 mice per group, (E, F) N = 5–6, (G, H) N = 6–8 independent cell cultures. Scale bar: 200 μm (E) or (H) 150 μm.

Fig. 3

Dysregulated cholesterol homeostasis in liver of Cth/Mpst^−/− mice. (A) Volcano plot depicting the Differentially Expressed Genes (DEGs) in Cth/Mpst^−/− as compared to wild-type mice. (Cut-offs for DEGs: log₂FC>0.58, log₂FC<-0.58, p-adjusted<0.05) and (B) Gene ontology (GO) enrichment analysis based on Biological Process of upregulated DEGs in liver of WT and mutant mice. (C) Heatmap depicting expression levels (z score) of the genes related to cholesterol biosynthesis pathway. (D) GO enrichment analysis based on the Reactome database of the upregulated DEGs in liver of Cth/Mpst^−/− mice. (E) Representative western blots and quantitation of SREBP1 and SREBP2 proteins in the liver of WT and Cth/Mpst-double ablated mice. (F) Enrichment analysis regarding Transcription Factor Targets for upregulated genes performed using ChEA database from Enrichr webtool. (G) Heatmap illustrating changes in gene expression levels (z-score) of LXR-,RXR and PPARA-target genes in liver of WT vs. Cth/Mpst^−/−. (H) Protein-protein interaction network of enriched TFs in liver of Cth/Mpst-deficient mice. Data were obtained from RNA-seq (A-D, F, G). Protein expression is presented as ratio over WT group (E). (A-D, F, G). Data are presented as means ± SEM (E), ∗p ≤ 0.05, (A-D, F, G) N = 3, (E) N = 6–8 mice per group.

Fig. 4

Therapeutic administration of a sulfide donor reduces Cth/Mpst-deficient-induced steatosis.Cth/Mpst^−/− mice were fed a drug-free chow diet (CD) or a CD containing the sulfide donor SG1002 for 4 weeks. Based on daily chow consumption, mice received 80 mg/kg SG1002. (A) Liver mass, (B) representative photomicrographs of liver sections stained with H&E, (C) body weight and (D) ALT serum levels in CD mice with or without SG1002 treatment. Data are presented as means ± SEM, ∗p < 0.05, ∗∗∗p ≤ 0.001, N = 4–6 mice per group. Scale bar: 100 μm.