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.

MCD diet-induced steatohepatitis is associated with alterations in asymmetric dimethylarginine (ADMA) and its transporters

“…The hepatic ADMA depletion documented in the present study after a 4-week MCD diet also occurred in mice after a 52-week HFHC diet; otherwise, in the HFCF model, circulating ADMA increased concomitantly with a partial reduction in hepatic methionine (30 % less than in controls) [25]. The MCD diet also induces a marked decrease in serum levels of glucose, cholesterol and triglycerides as previously reported, which is further supported in this study [26].…”

“…A similar trend was recently found in another model of diet-induced NAFLD: the hepatic levels of the PRMT-1 protein increased significantly in mice after a 52-week high-fat high-cholesterol (HFHC) diet [25]. The hepatic ADMA depletion documented in the present study after a 4-week MCD diet also occurred in mice after a 52-week HFHC diet; otherwise, in the HFCF model, circulating ADMA increased concomitantly with a partial reduction in hepatic methionine (30 % less than in controls) [25].…”

High levels of serum homocysteine in non-alcoholic steatohepatitis

“…Decreased GSH in patients with NASH and the negative correlation between GSH and Hcy may indicate an impairment of the transsulfuration pathway. These findings are consistent with the data published by Pacana et al [6] and Bravo et al [10], which showed that high fat diet-induced NAFLD in rats is associated with increased plasma Hcy levels caused by down-regulation of hepatic cystathionine-β-synthase (CBS) and cystathionine gamma-lyase (CGL) activities.…”

“…To support this assumption they refer inappropriately to data of Pastore et al [5] obtained by investigating children with NAFLD; on the one hand, it is well known that the histological pattern of pediatric NAFLD is frequently different from that observed in adults (so the comparison is improper) and on the other hand, Pastore et al have observed increased levels of cysteine and Hcy, also. Contrary to Polyzos et al speculation, the metabolomic analysis of hepatic methionine metabolism in mice with diet-induced NAFDL [6] showed an increased hepatic Hcy level accompanied by the depletion of methionine and serine. The serine depletion is expected to impair both the transfer of methyl groups from methyltetrahydrofolate [7] and thus the remethylation and the transsulfuration pathways and thus, the synthesis of glutathione, an important antioxidant.…”

Cardiovascular Risk in Patients with Non-alcoholic Fatty Liver Disease

“…The Korean Journal of Gastroenterology 34,35 Portal hypertension affecting the systemic vascular resistance Metalloproteinase 38 Decreased elastic fiber and increased collagen in media of the large arteries increased arterial stiffness VEGF [39][40][41][42] Instability of atheroma 43,44 HMGB-1 45 Involving in Systemic inflammation and angiogenesis Dysfunction of endothelial cells ADMA 47 Increased arterial wall stiffness EMPs 48,49 Increased EMPs which are products of endothelial destruction Endothelial progenitor cell 48,49 Decreased endothelial progenitor cells which indicating endothelial repair function Inflammation and cytokine IL-1b 50 , IL-6 51,52 hs-CRP, TNF-a, CCL3, sICAM-1 51,53 Inflammation markers increased in NAFLD Homocysteine and inflammation Hyperhomocysteinemia [58][59][60] Oxidative stress, Decreased NO synthesis, Involving hepatic fibrosis Hepatokine and insulin resistance FetA 65 Increased inflammatory cytokine and insulin resistance and decreased adiponectin 65 Decreased vascular calcification by inhibiting TGF-β1signal pathway 65 FGF-21 [66][67][68] Increased level of FGF-21 for compensating insulin resistance in NAFLD SeP 69,70 Involving in Insulin resistance, hs-CRP, increased intimal thickness of carotid artery Lipid profiles TG, LDL, small dense LDL particles (LDL3, LDL4) 73 Increased in NAFLD, contributing to atherogenesis HDL 72 A defending factor of atherogenesis, but decrease in NAFLD. oxLDL 77 Contributing to oxidative stress in NASH and hepatic fibrosis Coagulation factors Factor 8, 9, 10, 12 78 Increased in NAFLD PAI-1 79,80 Increa...…”