Dietary and genetic disruption of hepatic methionine metabolism induce acid sphingomyelinase to promote steatohepatitis

Abstract

Alcoholic (ASH) and nonalcoholic. (NASH).steatohepatitis are advanced.stages.of.fatty.liver.disease.Methionine adenosyltransferase 1A (MAT1A) plays a key role in hepatic methionine metabolism and germline Mat1a deletion in mice promotes NASH. Acid sphingomyelinase (ASMase) triggers hepatocellular apoptosis and liver fibrosis and has been shown to downregulate MAT1A expression in the context of fulminant liver failure. Given the role of ASMase in steatohepatitis development, we investigated the status of ASMase in Mat1a^-/- mice and the regulation of ASMase by SAM/SAH. Consistent with its role in NASH, Mat1a^-/- mice fed a choline-deficient (CD) diet exhibited macrosteatosis, inflammation, fibrosis and liver injury as well as reduced total and mitochondrial GSH levels. Our data uncovered an increased basal expression and activity of ASMase but not neutral SMase in Mat1a^-/- mice, which further increased upon CD feeding. Interestingly, adenovirus-mediated shRNA expression targeting ASMase reduced ASMase activity and protected Mat1a^-/- mice against CD diet-induced NASH. Similar results were observed in CD fed Mat1a^-/- mice by pharmacological inhibition of ASMase with amitriptyline. Moreover, Mat1a/ASMase double knockout mice were resistant to CD-induced NASH. ASMase knockdown protected wild type mice against NASH induced by feeding a diet deficient in methionine and choline. Furthermore, Mat1a^-/- mice developed acute-on-chronic ASH and this outcome was ameliorated by amitriptyline treatment. In vitro data in primary mouse hepatocytes revealed that decreased SAM/SAH ratio increased ASMase mRNA level and activity. MAT1A and ASMase mRNA levels exhibited an inverse correlation in liver samples from patients with ASH and NASH. Thus, disruption of methionine metabolism sensitizes to steatohepatitis by ASMase activation via decreased SAM/SAH. These findings imply that MAT1A deletion and ASMase activation engage in a self-sustained loop of relevance for steatohepatitis.

Keywords: Alcoholic steatohepatitis; Amitriptyline; Ceramide; Methionine; Nonalcoholic steatohepatitis.

Graphical abstract

Fig. 1

ASMase knockdown protects Mat1a^−/− mice against CD diet induced NASH. A, ASMase activity in Mat1a^−/− mice fed RD or CD diet for 14 days with or without treatment with control SH and ASMSH adenovirus. B, Body weight and liver to body weight change in Mat1a^−/− mice fed RD or CD diet for 14 days with or without treatment with control SH and ASMSH adenovirus. C, serum ALT and AST in Mat1a^−/− mice fed RD or CD diet for 14 days with or without treatment with control SH and ASMSH adenovirus. D, liver triglyceride content Mat1a^−/− mice fed RD or CD diet for 14 days with or without treatment with control SH and ASMSH adenovirus. E-F, representative images of H&E, and oil red staining of liver sections. G Sirius red staining of liver samples from Mat1a^−/− mice fed RD or CD diet for 14 days with or without treatment with control SH and ASMSH adenovirus, with the percentage of stained areas being 2.5 ± 0.3; 3.0 ± 0.4; 12.5 ± 1.7 and 2.8 ± 0.3, respectively. H, Liver hydroxyproline content and col1a1 and α-sma mRNA levels from Mat1a^−/− mice fed RD or CD diet for 14 days with or without treatment with control SH and ASMSH adenovirus. I, Tnfa, Ccl2 and Il1b mRNA levels of Mat1a^−/− mice fed RD or CD diet for 14 days with or without treatment with control SH and ASMSH adenovirus. Results are mean ± SEM of N = 5–7 mice per group. *p < 0.05 Mat1a^−/− mice fed CD diet treated with control SH vs ASMSH treatment. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 2

Pharmacological ASMase inhibition protects Mat1a^−/− mice against CD diet induced NASH. A, ASMase activity in Mat1a^−/− mice fed RD or CD diet for 14 days with or without amitriptyline (AMI) treatment to block ASMase. B, Body weight and liver to body weight change in Mat1a^−/− mice fed RD or CD diet for 14 days with or without AMI. C, serum ALT and AST of Mat1a^−/− mice fed RD or CD diet for 14 days with or without AMI treatment. D-E, representative images of H&E and oil red staining of liver sections. F, Sirius red staining of liver samples from Mat1a^−/− mice fed RD or CD diet for 14 days with or without AMI treatment, with the percentage of stained areas being 2.6 ± 0.4; 3.2 ± 0.5; 18.4 ± 2.1 and 4.1 ± 0.5, respectively. G, Col1a1 mRNA levels and liver hydroxyproline from Mat1a^−/− mice fed RD or CD diet for 14 days with or without AMI treatment. H, Tnfa, Ccl2 and Il1b mRNA levels of Mat1a^−/− mice fed RD or CD diet for 14 days with or without AMI treatment. Results are mean ± SEM of N = 5–7 mice per group. *p < 0.05 Mat1a^−/− mice fed CD diet treated with AMI vs control. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

Mat1a^−/−/ASMase^−/− double knockout mice are protected against CD diet induced NASH. A, ASMase activity and body weight and liver to body weight changes from Mat1a^−/− and Mat1a^−/−/ASMase^−/− mice fed RD or CD diet for 7 days. B–C, H&E and oil red staining of liver samples from Mat1a^−/− and Mat1a^−/−/ASMase^−/− mice fed RD or CD diet for 7 days. D, liver triglycerides levels from Mat1a^−/− and Mat1a^−/−/ASMase^−/− mice fed RD or CD diet for 7 days. E, Sirius red staining of liver samples from Mat1a^−/− and Mat1a^−/−/ASMase^−/− mice fed RD or CD diet for 7 days, with the percentage of stained areas being 2.8 ± 0.4; 3.1 ± 0.4; 13.6 ± 1.6 and 3.5 ± 0.4, respectively. F, Liver hydroxyproline levels from Mat1a^−/− and Mat1a^−/−/ASMase^−/− mice fed RD or CD diet for 7 days. G, Col1a1 and Pdgfr mRNA levels of liver samples from Mat1a^−/−/ASMase^−/− mice fed RD or CD diet for 7 days. H, Il1b and Ccl2 mRNA levels of liver samples from Mat1a^−/−/ASMase^−/− mice fed RD or CD diet for 7 days. Results are mean ± SEM of N = 5–7 mice per group. *p < 0.05 Mat1a^−/−/ASMase^−/− mice vs. Mat1a^−/− fed CD diet. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 4

Amitriptyline of ASMase protects WT mice against MCD-induced NASH. A, ASMase activity and body weight change of WT mice fed RD or MCD for 14 days with or without amitriptyline (AMI) treatment. B, serum ALT and AST of WT mice fed RD or CD diet for 14 days with or without AMI treatment. C, H&E, oil red and Sirius red staining of liver samples from WT mice fed RD or CD diet for 14 days with or without AMI treatment. D, Hepatic triglyceride content of WT mice fed RD or CD diet for 14 days with or without AMI treatment. E-F, Oil red and Sirius red staining of liver samples from WT mice fed RD or CD diet for 14 days with or without AMI treatment, with the percentage of stained areas being 2.5 ± 0.3; 2.7 ± 0.3; 15.2 ± 1.57 and 2.9 ± 0.4, respectively. G, Hydroxyproline and α-sma mRNA levels of WT mice fed RD or CD diet for 14 days with or without AMI treatment. H, Ccl2 and Il1b mRNA of WT mice fed RD or CD diet for 14 days with or without AMI treatment. Results are mean ± SEM of N = 5–7 mice per group. *p < 0.05 WT mice fed MCD diet vs. AMI and WT mice fed CD diet. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 5

Heatmap of lipidome profile of WT, Mat1a^−/− and Mat1a^−/−/ASMase mice fed RD and CD diet. A-C, Heatmap representing individual lipidomic classes obtained from the comparison of the genetic background of mice (A), diet RD vs CD (B) and treatment to target ASMase (C). Color codes are presented as log2 (fold-change) and paired Student's t-test p-values indicated at the bottom of figure. N = 5 mice per group and comparison were used for the lipidomic analyses. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 6

ASMase knockdown protects Mat1a^−/− mice against alcohol induced liver disease. A, ASMase activity from Mat1a^−/− mice fed acute-on-chronic alcohol with or without treatment with control SH and ASMSH adenovirus. B, serum ALT and AST of Mat1a^−/− mice fed acute-on-chronic alcohol with or without treatment with control SH and ASMSH adenovirus. C-D, representative images of H&E and Oil red staining of liver sections. F, Sirius red staining for liver samples from Mat1a^−/− mice fed acute-on-chronic alcohol with or without treatment with control SH and ASMSH adenovirus, with the percentage of stained areas being 2.4 ± 0.3; 12.1 ± 1.4 and 2.5 ± 0.3, respectively. F, serum ethanol levels of Mat1a^−/− mice fed acute-on-chronic alcohol with or without treatment with control SH and ASMSH adenovirus. G, Hepatic hydroxyproline levels and Tfgb and Col1a1 mRNA levels of Mat1a^−/− mice fed acute-on-chronic alcohol with or without treatment with control SH and ASMSH adenovirus. H, Tnfa, Ccl2 and Il6 mRNA levels from Mat1a^−/− mice fed acute-on-chronic alcohol with or without treatment with control SH and ASMSH adenovirus. Results are mean ± SEM of N = 5–7 mice per group. *p < 0.05 vs. Mat1a^−/− mice fed pair-fed diet or ethanol diet plus ASMSH treatment. . (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7

Low SAM/SAH and increased HCY induce the expression of ASMase.Primary isolated hepatocytes were isolated and incubated with SAM (1 mM), SAH (0.2–5 mM) and homocysteine (5–10 mM) as indicated. Cells extracts were used for the determination of ASMase mRNA levels and activity as described in Methods. Results are mean ± SEM of N = 5–12 PMH preparations. *p < 0.05 vs. control PMH.

Fig. 8

MAT1A and ASMase mRNA levels from liver samples of patients with ASH and NASH. A-B, Liver samples from control subjects and patients with ASH and NASH were used to determine the mRNA of MAT1A and ASMase. C-E, Correlation between the mRNA of ASMase and MAT1A in the cohort of patients with ASH and NASH. Clinical characteristics of patients are shown in Supplementary Table 1. *p < 0.05 vs. control subjects.