Dysregulated Hepatic Methionine Metabolism Drives Homocysteine Elevation in Diet-Induced Nonalcoholic Fatty Liver Disease

Abstract

Methionine metabolism plays a central role in methylation reactions, production of glutathione and methylarginines, and modulating homocysteine levels. The mechanisms by which these are affected in NAFLD are not fully understood. The aim is to perform a metabolomic, molecular and epigenetic analyses of hepatic methionine metabolism in diet-induced NAFLD. Female 129S1/SvlmJ;C57Bl/6J mice were fed a chow (n = 6) or high-fat high-cholesterol (HFHC) diet (n = 8) for 52 weeks. Metabolomic study, enzymatic expression and DNA methylation analyses were performed. HFHC diet led to weight gain, marked steatosis and extensive fibrosis. In the methionine cycle, hepatic methionine was depleted (30%, p< 0.01) while s-adenosylmethionine (SAM)/methionine ratio (p< 0.05), s-adenosylhomocysteine (SAH) (35%, p< 0.01) and homocysteine (25%, p< 0.01) were increased significantly. SAH hydrolase protein levels decreased significantly (p <0.01). Serine, a substrate for both homocysteine remethylation and transsulfuration, was depleted (45%, p< 0.01). In the transsulfuration pathway, cystathionine and cysteine trended upward while glutathione decreased significantly (p< 0.05). In the transmethylation pathway, levels of glycine N-methyltransferase (GNMT), the most abundant methyltransferase in the liver, decreased. The phosphatidylcholine (PC)/ phosphatidylethanolamine (PE) ratio increased significantly (p< 0.01), indicative of increased phosphatidylethanolamine methyltransferase (PEMT) activity. The protein levels of protein arginine methytransferase 1 (PRMT1) increased significantly, but its products, monomethylarginine (MMA) and asymmetric dimethylarginine (ADMA), decreased significantly. Circulating ADMA increased and approached significance (p< 0.06). Protein expression of methionine adenosyltransferase 1A, cystathionine β-synthase, γ-glutamylcysteine synthetase, betaine-homocysteine methyltransferase, and methionine synthase remained unchanged. Although gene expression of the DNA methyltransferase Dnmt3a decreased, the global DNA methylation was unaltered. Among individual genes, only HMG-CoA reductase (Hmgcr) was hypermethylated, and no methylation changes were observed in fatty acid synthase (Fasn), nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfκb1), c-Jun, B-cell lymphoma 2 (Bcl-2) and Caspase 3. NAFLD was associated with hepatic methionine deficiency and homocysteine elevation, resulting mainly from impaired homocysteine remethylation, and aberrancy in methyltransferase reactions. Despite increased PRMT1 expression, hepatic ADMA was depleted while circulating ADMA was increased, suggesting increased export to circulation.

Fig 1. Methionine cycle: hepatic methionine depletion and homocysteine accumulation in diet-induced NAFLD.

(A) HFHC diet for 52 weeks resulted in methionine (met) depletion (p< 0.01) and increased downstream products s-adenosylmethionine (SAM), s-adenosylhomocysteine (SAH) (p< 0.01) and homocysteine (Hcy) (p< 0.01) and (B) increased SAM/methionine ratio (p< 0.05), which is indicative of increased methionine utilization. Methionine sulfoxide (Met So) concentration remained unchanged. (C) The gene expression of methionine adenosyltransferase Mat1a and Mat2a was decreased (p< 0.01). However, the protein levels of MAT I/III, expressed solely by hepatic Mat1a, were relatively unchanged. Although SAH hydrolase (Ahcy) mRNA levels did not change, SAHH protein expression was decreased significantly (p< 0.01) which correlated with the increase in SAH levels but not the excess in Hcy. (D) The gene expression of betaine-homocysteine methyltransferase (Bhmt) and protein levels of BHMT and methionine synthase (MS), enzymes responsible for conversion of homocysteine to methionine, did not change. (E) Serine, the main one-carbon donor in the folate cycle needed for homocysteine remethylation, was depleted (p< 0.01). Whereas betaine levels trended down modestly, the decrease in dimethylglycine (DMG) almost approached significance (p = 0.07) that further indicates impairment of methionine reformation. Together, these findings suggest that homocysteine accumulation mainly results from impaired homocysteine remethylation to methionine. Data are represented as mean ± SEM.

Fig 2. Transsulfuration pathway: depletion of serine limits the ability to replete glutathione in diet-induced NAFLD.

(A) HFHC diet for 52 weeks resulted only in modest non-significant increase in cystathionine (CST) and cysteine (Cys) levels in spite of homocysteine accumulation and decrease in glutathione levels. (B) mRNA expression of cystathionine β-synthase (Cbs) and γ-glutamylcysteine synthetase (γ-Gcs) decreased but the protein levels of CBS and GCS did not significantly change. (C) Glutathione (GSH) was depleted (p< 0.05), likely as a result of oxidative stress, while cysteinyl-glycine, a catabolic product of GSH, remained unchanged (C). Data are represented as mean ± SEM.

Fig 3. Transmethylation pathway: aberrancy in methyltransferase reactions in diet-induced NAFLD.

(A) Heat map representation of substrates and methylated products of major SAM-derived transmethylation reactions (PEMT, GNMT, GAMT and PRMT) with HFHC diet for 52 weeks. (B) Levels of glycine, a substrate for GNMT, were decreased (p< 0.01) while the product sarcosine did not change between chow and HFHC group. (C) The gene expression of Gnmt, the most abundant methyltranserase in the liver, decreased significantly but only approached significance at the protein level (p = 0.06). (D) Levels of PE, the substrate for PEMT, was significantly reduced (p< 0.01) while PC trended down, resulting in a significant PC/PE ratio (p< 0.01), which suggests increase in PEMT activity There were no significant changes in the concentrations of guanidinoacetate and creatine, the substrate and product for GAMT, respectively. (E) The methylated products of PRMT activity, MMA and ADMA, were decreased significantly (p< 0.01 and p< 0.05) while the decrease in SDMA approached significance (p = 0.07). (F) The gene expression of Prmt1, mainly responsible for the methylation of MMA and ADMA, decreased (p< 0.01) but the protein levels of PRMT1 increased significantly (p< 0.05). (G) Circulating ADMA was increased toward significance (p = 0.06), suggesting increased export to the circulation. Data are represented as mean ± SEM. Legend: arginine (Arg); asymmetric dimethylarginine (ADMA); creatine (Crea); glycine (Gly); glycine N-methyltransferase (GNMT); guanidinoacetate (GAA); guanidinoacetate methyltransferase (GAMT); homocysteine (Hcy); monomethylarginine (MMA); phosphatidylcholine (PC); phosphatidylethanolamine (PE); phosphatidylethanolamine methyltransferase (PEMT); protein arginine methyltransferase (PRMT); s-adenosylhomocysteine (SAH); s-adenosylmethionine (SAM); sarcosine (Sar); symmetric dimethylarginine (SDMA).

Fig 4. Stable global DNA methylation and hydroxymethylation and HMG-CoA reductase DNA hypermethylation in diet-induced NAFLD.

(A-B) Although the gene expression for Dnmt1 and Dnmt3a tended to decrease and decreased, respectively, the percent methyldeoxycytidine did not change between chow and HFHC group. (C) The relative 5hmdC concentrations also did not change. (D) Among the individual genes involved in the pathogenesis of NAFLD, Hmgcr was hypermethylated (p< 0.01) but there were no methylation changes for Fasn, Nfκb1, c-Jun, Bcl-2, and Caspase 3in HFHC group. Data are represented as mean ± SEM. Legend: B-cell lymphoma 2 (Bcl-2); DNA methyltransferase 1 and 3a (Dnmt 1 and Dnmt3a); fatty acid synthase (Fasn); 5-hydroxymethyl-2’-deoxycytidine (5hmdC); 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMG-CoA reductase, Hmgcr); nuclear factor of kappa light polypeptide gene enhancer in B-cells 1 (Nfkb1).

Fig 5. Summary of changes in the methionine cycle, transsulfuration pathway and methyltransferase reactions in advance NAFLD.

HFHC diet for 52 weeks resulted in methionine depletion, excess homocysteine and aberrancy in transmethylation pathway. The decrease in the substrate serine impairs homocysteine remethylation and limits the ability to replete glutathione in the transsulfuration pathway. Legend: cystathionine β-synthase (CBS); dimethylglycine (DMG); γ-glutamylcysteine synthetase (GCS); glutathione (GSH); 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR); methionine adenosyltransferase (MAT); phosphatidylcholine (PC); phosphatidylethanolamine (PE); protein arginine methytransferas 1 (PRMT1); s-adenosylmethionine (SAM); s-adenosylhomocysteine (SAH); s-adenosylhomocysteine hydrolase (SAHH).