Hepatic S-adenosylmethionine Levels Research Articles

Acute ethanol-induced liver injury is prevented by betaine administration.

Binge drinking is the most common form of excessive alcohol use. Repeated episodes of binge drinking cause multiple organ injuries, including liver damage. We previously demonstrated that chronic ethanol administration causes a decline in the intrahepatic ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH). This decline causes impairments in essential methylation reactions that result in alcohol-induced fatty liver (steatosis) and other features of alcohol-associated liver disease (ALD). Co-treatment with betaine during chronic ethanol feeding, normalizes hepatocellular SAM:SAH ratio and alleviates many features of liver damage including steatosis. Here, we sought to examine whether betaine treatment similarly protects against liver injury in an alcohol binge-drinking model. We hypothesized that ethanol binge with prior or simultaneous betaine administration would prevent or attenuate acute alcohol-induced liver damage. Male C57Bl/6 mice were gavaged twice, 12 h apart, with either 6 g ethanol/kg BW or with an equal volume/kg BW of 0.9% NaCl. Two separate groups of mice (n = 5/group) were gavaged with 4 g betaine/kg BW, either 2 h before or simultaneously with the ethanol or saline gavages. All mice were sacrificed 8 h after the last gavage and serum and liver parameters were quantified. Ethanol binges caused a 50% decrease in hepatic SAM:SAH ratio and a >3-fold rise in liver triglycerides (p ≤ 0.05). These latter changes were accompanied by elevated serum AST and ALT activities and blood alcohol concentrations (BAC) that were ∼three-times higher than the legal limit of intoxication in humans. Mice that were treated with betaine 2 h before or simultaneously with the ethanol binges exhibited similar BAC as in mice given ethanol-alone. Both betaine treatments significantly elevated hepatic SAM levels thereby normalizing the SAM:SAH ratio and attenuating hepatic steatosis and other injury parameters, compared with mice given ethanol alone. Simultaneous betaine co-administration with ethanol was more effective in preventing or attenuating liver injury than betaine given before ethanol gavage. Our findings confirm the potential therapeutic value of betaine administration in preventing liver injury after binge drinking in an animal model.

Methionine adenosyltransferases in liver cancer.

Methionine adenosyltransferases (MATs) are essential enzymes for life as they produce S-adenosylmethionine (SAMe), the biological methyl donor required for a plethora of reactions within the cell. Mammalian systems express two genes, MAT1A and MAT2A, which encode for MATα1 and MATα2, the catalytic subunits of the MAT isoenzymes, respectively. A third gene MAT2B, encodes a regulatory subunit known as MATβ which controls the activity of MATα2. MAT1A, which is mainly expressed in hepatocytes, maintains the differentiated state of these cells, whilst MAT2A and MAT2B are expressed in extrahepatic tissues as well as non-parenchymal cells of the liver (e.g., hepatic stellate and Kupffer cells). The biosynthesis of SAMe is impaired in patients with chronic liver disease and liver cancer due to decreased expression and inactivation of MATα1. A switch from MAT1A to MAT2A/MAT2B occurs in multiple liver diseases and during liver growth and dedifferentiation, but this change in the expression pattern of MATs results in reduced hepatic SAMe level. Decades of study have utilized the Mat1a-knockout (KO) mouse that spontaneously develops non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma (HCC) to elucidate a variety of mechanisms by which MAT proteins dysregulation contributes to liver carcinogenesis. An increasing volume of work indicates that MATs have SAMe-independent functions, distinct interactomes and multiple subcellular localizations. Here we aim to provide an overview of MAT biology including genes, isoenzymes and their regulation to provide the context for understanding consequences of their dysregulation. We will highlight recent breakthroughs in the field and underscore the importance of MAT’s in liver tumorigenesis as well as their potential as targets for cancer therapy.

Metabolism of Sulfur-Containing Amino Acids in the Liver: A Link between Hepatic Injury and Recovery.

Methionine is an essential sulfur-containing amino acid that is metabolized mainly in the liver, where it is converted to S-adenosylmethionine (SAM) by methionine adenosyltransferase. Importantly, SAM is a metabolically pleiotropic molecule that participates in three types of biochemical reactions; transmethylation, transsulfuration (which results in the transfer of sulfur from methionine to serine to form cysteine), and amino propylation (to synthesize polyamines). Critical roles of SAM in the liver have been extensively studied using transgenic animals with chronically reduced or increased hepatic SAM levels. Interestingly, both models with abnormal hepatic SAM concentrations develop liver disease suggesting that SAM homeostasis plays a pivotal role in liver disease. The transsulfuration pathway is connected to the production of glutathione (GSH), which has potent antioxidant capacity in the liver. Accumulating data show that GSH depletion renders the liver vulnerable to oxidative stress and prone to progression of liver disease. In this review, we highlight the importance of homeostasis in the metabolism of sulfur-containing amino acids with a particular focus on the transsulfuration pathway which could be a promising therapeutic target in liver injury.

S-Adenosylmethionine increases circulating very-low density lipoprotein clearance in non-alcoholic fatty liver disease

Very-low-density lipoproteins (VLDLs) export lipids from the liver to peripheral tissues and are the precursors of low-density-lipoproteins. Low levels of hepatic S-adenosylmethionine (SAMe) decrease triglyceride (TG) secretion in VLDLs, contributing to hepatosteatosis in methionine adenosyltransferase 1A knockout mice but nothing is known about the effect of SAMe on the circulating VLDL metabolism. We wanted to investigate whether excess SAMe could disrupt VLDL plasma metabolism and unravel the mechanisms involved. Glycine N-methyltransferase (GNMT) knockout (KO) mice, GNMT and perilipin-2 (PLIN2) double KO (GNMT-PLIN2-KO) and their respective wild type (WT) controls were used. A high fat diet (HFD) or a methionine deficient diet (MDD) was administrated to exacerbate or recover VLDL metabolism, respectively. Finally, 33 patients with non-alcoholic fatty-liver disease (NAFLD); 11 with hypertriglyceridemia and 22 with normal lipidemia were used in this study. We found that excess SAMe increases the turnover of hepatic TG stores for secretion in VLDL in GNMT-KO mice, a model of NAFLD with high SAMe levels. The disrupted VLDL assembly resulted in the secretion of enlarged, phosphatidylethanolamine-poor, TG- and apoE-enriched VLDL-particles; special features that lead to increased VLDL clearance and decreased serum TG levels. Re-establishing normal SAMe levels restored VLDL secretion, features and metabolism. In NAFLD patients, serum TG levels were lower when hepatic GNMT-protein expression was decreased. Excess hepatic SAMe levels disrupt VLDL assembly and features and increase circulating VLDL clearance, which will cause increased VLDL-lipid supply to tissues and might contribute to the extrahepatic complications of NAFLD.

S-adenosyl methionine regulates ubiquitin-conjugating enzyme 9 protein expression and sumoylation in murine liver and human cancers

Ubiquitin-conjugating enzyme 9 (Ubc9) is required for sumoylation and is overexpressed in several malignancies, but its expression in hepatocellular carcinoma (HCC) is unknown. Hepatic S-adenosyl methionine (SAMe) levels decrease in methionine adenosyltransferase 1A (Mat1a) knockout (KO) mice, which develop HCC, and in ethanol-fed mice. We examined the regulation of Ubc9 by SAMe in murine liver and human HCC, breast, and colon carcinoma cell lines and specimens. Real-time polymerase chain reaction and western blotting measured gene and protein expression, respectively. Immunoprecipitation followed by western blotting examined protein-protein interactions. Ubc9 expression increased in HCC and when hepatic SAMe levels decreased. SAMe treatment in Mat1a KO mice reduced Ubc9 protein, but not messenger RNA (mRNA) levels, and lowered sumoylation. Similarly, treatment of liver cancer cell lines HepG2 and Huh7, colon cancer cell line RKO, and breast cancer cell line MCF-7 with SAMe or its metabolite 5'-methylthioadenosine (MTA) reduced only Ubc9 protein level. Ubc9 posttranslational regulation is unknown. Ubc9 sequence predicted a possible phosphorylation site by cell division cycle 2 (Cdc2), which directly phosphorylated recombinant Ubc9. Mat1a KO mice had higher phosphorylated (phospho)-Ubc9 levels, which normalized after SAMe treatment. SAMe and MTA treatment lowered Cdc2 mRNA and protein levels, as well as phospho-Ubc9 and protein sumoylation in liver, colon, and breast cancer cells. Serine 71 of Ubc9 was required for phosphorylation, interaction with Cdc2, and protein stability. Cdc2, Ubc9, and phospho-Ubc9 levels increased in human liver, breast, and colon cancers. Cdc2 expression is increased and Ubc9 is hyperphosphorylated in several cancers, and this represents a novel mechanism to maintain high Ubc9 protein expression that can be inhibited by SAMe and MTA.

S24 * S-ADENOSYLMETHIONINE IN THE PATHOGENESIS AND TREATMENT OF ALCOHOLIC LIVER DISEASE * S24.1 * THE ROLE OF ALTERED SAME METABOLISM IN ALCOHOL-INDUCED ER STRESS AND LIVER INJURY

S24 * S-ADENOSYLMETHIONINE IN THE PATHOGENESIS AND TREATMENT OF ALCOHOLIC LIVER DISEASE * S24.1 * THE ROLE OF ALTERED SAME METABOLISM IN ALCOHOL-INDUCED ER STRESS AND LIVER INJURY

S-Adenosylmethionine regulates connexins sub-types expressed by hepatocytes

Intercellular communication via GAP Junctions plays an important role in tissue homeostasis, apoptosis, carcinogenesis, cell proliferation and differentiation. Hepatocyte connexins (Cx) 26 and 32 levels are decreased during the de-differentiation process of primary hepatocytes in culture, a situation that is also characterized by a decrease in S-Adenosylmethionine (SAMe) levels. In this current study, we show that SAMe supplementation in cultured hepatocytes every 12 h, leads to an up-regulation of Cx26 and 32 mRNA and protein levels and blocks culture-induced Cx43 expression, although it failed to increase Cx26 and 32 membrane localization and GAP junction intracellular communication. SAMe reduced nuclear β-catenin accumulation, which is known to stimulate the TCF/LEF-dependent gene transcription of Cx43. Moreover SAMe-induced reduction in Cx43 and β-catenin was prevented by the proteasome inhibitor MG132, and was not mediated by GSK3 activity. SAMe, and its metabolite 5′-methylthioadenosine (MTA) increased Cx26 mRNA in a process partially mediated by Adenosine A 2A receptors but independent of PKA. Finally livers from MAT1A knockout mice, characterized by low hepatic SAMe levels, express higher Cx43 and lower Cx26 and 32 protein levels than control mice. These results suggest that SAMe maintains a characteristic expression pattern of the different Cxs in hepatocytes by differentially regulating their levels.

S-adenosylmethionine in the chemoprevention and treatment of hepatocellular carcinoma in a rat model

Hepatocellular carcinoma (HCC) remains a common cancer worldwide that lacks effective chemoprevention or treatment. Chronic liver disease often leads to impaired hepatic S-adenosylmethionine (SAMe) biosynthesis, and mice with SAMe deficiency develop HCC spontaneously. SAMe is antiapoptotic in normal hepatocytes but proapoptotic in cancerous hepatocytes. The present study investigated SAMe's effectiveness in prevention and treatment of HCC. Two weeks after injecting 2.5 million H4IIE cells into the liver parenchyma of ACI rats, they typically form a 1-cm tumor. When SAMe (150 mg/kg/day) was delivered through continuous intravenous infusion, hepatic SAMe levels reached 0.7 mM (over 10-fold) 24 hours later. This regimen, started 1 day after injecting H4IIE cells and continued for 10 days, was able to reduce tumor establishment and growth. However, if intravenous SAMe was started after HCC had already developed, it was ineffective in reducing tumor growth for 24 days. Although plasma SAMe levels remained elevated, hepatic SAMe levels were minimally increased (30% higher). Chronic SAMe administration led to induction of hepatic methyltransferases, which prevented SAMe accumulation. To see if SAMe's preventive effect on tumor establishment involves angiogenesis, the effect of SAMe on angiogenesis genes was studied. SAMe treatment of H4IIE cells altered the expression of several genes with the net effect of inhibiting angiogenesis. These changes were confirmed at the protein level and functionally in human umbilical vein endothelial cells. SAMe is effective in preventing HCC establishment but ineffective in treating established HCC because of induction of hepatic methyltransferases, which prevents SAMe level to reach high enough to kill liver cancer cells. SAMe's chemopreventive effect may be related to its proapoptotic action and its ability to inhibit angiogenesis.

Changes in S-adenosylmethionine and GSH homeostasis during endotoxemia in mice

Endotoxemia participates in the pathogenesis of many liver injuries. Lipopolysaccharide (LPS) was shown to inactivate hepatic methionine adenosyltransferase (MAT), the enzyme responsible for S-adenosylmethionine (SAMe) biosynthesis. SAMe treatment was shown to prevent the LPS-induced increase in tumor necrosis factor-α, which may be one of its beneficial effects. SAMe is also an important precursor of glutathione (GSH) and GSH was shown to ameliorate LPS-induced hepatotoxicity. The aims of this work were to examine changes in SAMe and GSH homeostasis during endotoxemia and the effect of SAMe. Mice received SAMe or vehicle pretreatment followed by LPS and were killed up to18 h afterward. Unexpectedly, we found hepatic SAMe level increased 67% following LPS treatment while S-adenosylhomocysteine level fell by 26%, suggesting an increase in SAMe biosynthesis and/or block in transmethylation. The mRNA and protein levels of MAT1A and MAT2A were increased following LPS. However, despite increased MAT1A expression, MAT activity remained inhibited 18 h after LPS. The major methyltransferase that catabolizes hepatic SAMe is glycine N-methyltransferase, whose expression fell by 65% following LPS. Hepatic GSH level fell more than 50% following LPS, coinciding with a comparable fall in the mRNA and protein levels of glutamate-cysteine ligase (GCL) catalytic (GCLC) and modifier subunits (GCLM). SAMe pretreatment prevented the fall in GCLC and attenuated the fall in GCLM expression and GSH level. SAMe pretreatment prevented the LPS-induced increase in plasma alanine transaminases levels but not the LPS-induced increase in hepatic mRNA levels of proinflammatory cytokines. It further enhanced LPS-induced increase in interleukin-10 mRNA level. Taken together, the hepatic response to LPS is to upregulate MAT expression and inhibit SAMe utilization. GSH is markedly depleted largely due to lower expression of GCL. Interestingly, SAMe treatment prevented the fall in GCL and helped to preserve the GSH store and prevent liver injury.

The Transition from Fatty Liver to NASH Associates with SAMe Depletion in db/db Mice Fed a Methionine Choline-Deficient Diet

Nonalcoholic fatty liver disease (NAFLD) is highly prevalent in the Western population. By mechanisms that are not completely understood, this disease may progress to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma (HCC). db/db mice spontaneously develop hepatic steatosis, which progresses to NASH when these mice are fed a methionine choline-deficient (MCD) diet. The goal of our studies was to identify lipid and methionine metabolism pathways affected by MCD feeding to determine potential causal events leading to the development of NASH from benign steatosis. db/db mice fed the MCD diet for 2 weeks exhibited signs of incipient NASH development such as upregulated cytokines and chemokines. At this time point, MCD diet feeding caused S-adenosylmethionine (SAMe) depletion in db/db mice, while wild-type mice on the same diet retained hepatic SAMe levels. SAMe depletion exerts pleiotropic effects upon liver homeostasis and is commonly associated with a variety of liver insults such as thioacetamide, CCL4, and alcohol treatment; thus, SAMe depletion may serve as the second hit in NASH development. It is possible that differences in hepatic lipid and/or methionine metabolism between wild-type and db/db mice underlay the differential maintenance of SAMe levels during methionine and choline restriction. Indeed, db/db mice exhibited inhibited lipid oxidation pathways, which may be a priming factor for NASH development, and db/db mice fed the MCD diet had differential methionine adenosyltransferase (MAT) expression. The occurrence of SAMe depletion at this early, benign stage of NASH development in db/db mice with fatty liver suggests that SAMe supplementation may be (A) targeted to individuals susceptible to NASH (i.e., NAFLD patients) and (B) preventative of NASH before substantial liver injury has occurred.

Role of S-adenosyl-L-methionine in liver health and injury

S-adenosylmethionine (SAMe) has rapidly moved from being a methyl donor to a key metabolite that regulates hepatocyte growth, death, and differentiation. Biosynthesis of SAMe occurs in all mammalian cells as the first step in methionine catabolism in a reaction catalyzed by methionine adenosyltransferase (MAT). Decreased hepatic SAMe biosynthesis is a consequence of all forms of chronic liver injury. In an animal model of chronic liver SAMe deficiency, the liver is predisposed to further injury and develops spontaneous steatohepatitis and hepatocellular carcinoma. However, impaired SAMe metabolism, which occurs in patients with mutations of glycine N-methyltransferase (GNMT), can also lead to liver injury. This suggest that hepatic SAMe level needs to be maintained within a certain range, and deficiency or excess can both lead to abnormality. SAMe treatment in experimental animal models of liver injury shows hepatoprotective properties. Meta-analyses also show it is effective in patients with cholestatic liver diseases. Recent data show that exogenous SAMe can regulate hepatocyte growth and death, independent of its role as a methyl donor. This raises the question of its mechanism of action when used pharmacologically. Indeed, many of its actions can be recapitulated by methylthioadenosine (MTA), a by-product of SAMe that is not a methyl donor. A better understanding of why liver injury occurs when SAMe homeostasis is perturbed and mechanisms of action of pharmacologic doses of SAMe are essential in defining which patients will benefit from its use.

S-adenosylmethionine prevents chronic alcohol-induced mitochondrial dysfunction in the rat liver

An early event that occurs in response to alcohol consumption is mitochondrial dysfunction, which is evident in changes to the mitochondrial proteome, respiration defects, and mitochondrial DNA (mtDNA) damage. S-adenosylmethionine (SAM) has emerged as a potential therapeutic for treating alcoholic liver disease through mechanisms that appear to involve decreases in oxidative stress and proinflammatory cytokine production as well as the alleviation of steatosis. Because mitochondria are a source of reactive oxygen/nitrogen species and a target for oxidative damage, we tested the hypothesis that SAM treatment during alcohol exposure preserves organelle function. Mitochondria were isolated from livers of rats fed control and ethanol diets with and without SAM for 5 wk. Alcohol feeding caused a significant decrease in state 3 respiration and the respiratory control ratio, whereas SAM administration prevented these alcohol-mediated defects and preserved hepatic SAM levels. SAM treatment prevented alcohol-associated increases in mitochondrial superoxide production, mtDNA damage, and inducible nitric oxide synthase induction, without a significant lessening of steatosis. Accompanying these indexes of oxidant damage, SAM prevented alcohol-mediated losses in cytochrome c oxidase subunits as shown using blue native PAGE proteomics and immunoblot analysis, which resulted in partial preservation of complex IV activity. SAM treatment attenuated the upregulation of the mitochondrial stress chaperone prohibitin. Although SAM supplementation did not alleviate steatosis by itself, SAM prevented several key alcohol-mediated defects to the mitochondria genome and proteome that contribute to the bioenergetic defect in the liver after alcohol consumption. These findings reveal new molecular targets through which SAM may work to alleviate one critical component of alcohol-induced liver injury: mitochondria dysfunction.

Impaired liver regeneration in mice lacking methionine adenosyltransferase 1A.

Methionine adenosyltransferase (MAT) is an essential enzyme because it catalyzes the formation of S-adenosylmethionine (SAMe), the principal biological methyl donor. Of the two genes that encode MAT, MAT1A is mainly expressed in adult liver and MAT2A is expressed in all extrahepatic tissues. Mice lacking MAT1A have reduced hepatic SAMe content and spontaneously develop hepatocellular carcinoma. The current study examined the influence of chronic hepatic SAMe deficiency on liver regeneration. Despite having higher baseline hepatic staining for proliferating cell nuclear antigen, MAT1A knockout mice had impaired liver regeneration after partial hepatectomy (PH) as determined by bromodeoxyuridine incorporation. This can be explained by an inability to up-regulate cyclin D1 after PH in the knockout mice. Upstream signaling pathways involved in cyclin D1 activation include nuclear factor kappaB (NFkappaB), the c-Jun-N-terminal kinase (JNK), extracellular signal-regulated kinases (ERKs), and signal transducer and activator of transcription-3 (STAT-3). At baseline, JNK and ERK are more activated in the knockouts whereas NFkappaB and STAT-3 are similar to wild-type mice. Following PH, early activation of these pathways occurred, but although they remained increased in wild-type mice, c-jun and ERK phosphorylation fell progressively in the knockouts. Hepatic SAMe levels fell progressively following PH in wild-type mice but remained unchanged in the knockouts. In culture, MAT1A knockout hepatocytes have higher baseline DNA synthesis but failed to respond to the mitogenic effect of hepatocyte growth factor. Taken together, our findings define a critical role for SAMe in ERK signaling and cyclin D1 regulation during regeneration and suggest chronic hepatic SAMe depletion results in loss of responsiveness to mitogenic signals.

Role of S-adenosylmethionine in two experimental models of pancreatitis.

Severe necrotizing pancreatitis occurs in young female mice fed a choline-deficient and ethionine-supplemented (CDE) diet. Although the mechanism of the pancreatitis is unknown, one consequence of this diet is depletion of hepatic S-adenosylmethionine (SAM). SAM formation is catalyzed by methionine adenosyltransferases (MATs), which are encoded by liver-specific (MAT1A) and non-liver-specific (MAT2A) genes. In this work, we examined changes in pancreatic SAM homeostasis in mice receiving the CDE diet and the effect of SAM treatment. We found that both MAT forms are expressed in normal pancreas and pancreatic acini. After 48 h of the CDE diet, SAM levels decreased 50% and MAT1A-encoded protein disappeared via post-translational mechanisms, whereas MAT2A-encoded protein increased via pretranslational mechanisms. CDE-fed mice exhibited extensive necrosis, edema, and acute pancreatic inflammatory infiltration, which were prevented by SAM treatment. However, old female mice consuming the CDE diet that do not develop pancreatitis showed a similar fall in pancreatic SAM level. SAM was also protective in cerulein-induced pancreatitis in the rat, but the protection was limited. Although the pancreatic SAM level fell by more than 80% in the MAT1A knockout mice, no pancreatitis developed. This study thus provides several novel findings. First, the so-called liver-specific MAT1A is highly expressed in the normal pancreas and pancreatic acini. Second, the CDE diet causes dramatic changes in the expression of MAT isozymes by different mechanisms. Third, in contrast to the situation in the liver, where absence of MAT1A and decreased hepatic SAM level can lead to spontaneous tissue injury, in the pancreas the roles of SAM and MAT1A appear more complex and remain to be defined.

Folate deficiency, methionine metabolism, and alcoholic liver disease

Methionine metabolism is regulated by folate, and both folate deficiency and abnormal hepatic methionine metabolism are recognized features of alcoholic liver disease (ALD). Previously, histological features of ALD were induced in castrated male micropigs fed diets containing ethanol at 40% of kilocalories for 12 months, whereas in male micropigs fed the same diets for 12 months abnormal methionine metabolism and hepatocellular apoptosis developed. Folate deficiency may promote the development of ALD by accentuating abnormal methionine metabolism. Intact male micropigs received eucaloric diets that were folate sufficient, folate deficient, or each containing 40% of kilocalories as ethanol for 14 weeks. Folate deficiency alone reduced hepatic folates by one half, and ethanol feeding alone reduced methionine synthase, S-adenosylmethionine (SAM), and glutathione (GSH) levels and elevated plasma malondialdehyde (MDA) levels. The combined regimen elevated plasma homocysteine, hepatic S-adenosylhomocysteine (SAH), urinary 8-hydroxy-2-deoxyguanosine (oxy 8dG), an index of DNA oxidation, and serum aspartate aminotransferase (AST) levels. Terminal hepatic histopathologic characteristics included typical features of steatonecrosis and focal inflammation in pigs fed the combined diet, with no changes in the other groups. Hepatic SAM levels correlated with those of GSH, whereas urinary oxy 8dG and plasma MDA levels correlated with the SAM:SAH ratio and to hepatic GSH. The results demonstrate the linkage of abnormal methionine metabolism to products of DNA and lipid oxidation and to liver injury. The finding of steatonecrosis and focal inflammation only in the combined diet group supports the suggestion that folate deficiency promotes and folate sufficiency protects against the early onset of methionine cycle–mediated ALD.

Effect of propargylglycine on synthesis of glutathione in mice

Effects of propargylglycine (PPG) treatment on the hepatic glutathione (GSH) synthesis were examined in adult male ICR mice. Administration of PPG (200 μmole/kg, ip) to mice resulted in a complete inhibition of the hepatic cystathionine γ-lyase (CγL) activity measured in cytosol fraction for 40 hr after the treatment. A single injection of PPG rapidly reduced the hepatic GSH levels, which appeared to be sustained at least for 40 hr. The GSH concentration in plasma was significantly decreased for 20 hr, but recovered to the control level in 40 hr. Renal GSH levels did not appear to be changed by PPG treatment. The cysteine concentrations in liver, kidney and plasma were also decreased by PPG. The effect of PPG pretreatment was examined in mice challenged with methionine (1 mmole/kg, po), the sulfur donor in the transsulfuration pathway. Methionine administration elevated S-adenosylmethionine (SAM) and GSH concentrations in liver significantly when measured 3 hr following the treatment. In PPG pretreated mice the hepatic SAM level was increased, however, elevation of GSH by methionine was inhibited completely suggesting that the supply of cysteine from the methionine cycle for GSH synthesis in liver was blocked by PPG. The results show that generation of cysteine in the transsulfuration pathway has a critical role in the hepatic synthesis of GSH, and PPG can be used as an effective tool for study of sulfur-containing amino acid metabolism in liver.

Increasing levels of dietary homocystine with carotid endarterectomy produced proportionate increases in plasma homocysteine and intimal hyperplasia

Purpose: The role that homocysteine may play in post-carotid endarterectomy (CEA) restenosis due to intimal hyperplasia is not well understood. This study was designed to investigate the effects of different levels of dietary homocystine on: (1) plasma homocysteine; (2) post-CEA intimal hyperplasia; and (3) levels of the methyl donor S-adenosylmethionine (SAM) and its counterpart S-adenosylhomocysteine (SAH) in the homocysteine pathway. Methods: Male rats were fed specialized diets for 2 weeks pre- and post-CEA. Groups included control (0 homocystine added, n=9), 1.5 (1.5 g/kg homocystine added, n=10), 3.0 (3.0 g/kg homocystine added, n=9), and 4.5 (4.5 g/kg homocystine added, n=11). The rats underwent a surgical carotid endarterectomy. Endpoints included; plasma homocysteine, intimal hyperplasia, replicative index using with α-SM actin and BrdU, hepatic SAM levels, SAH levels, and the hepatic activities of methylenetetrahydrofolate reductase (MTHFR) and cystathionine β-synthase (CBS). Results: Increasing dietary homocystine produced a proportionate increase in plasma homocysteine and an increase in intimal hyperplasia. Regression analysis of plasma homocysteine levels and intimal hyperplasia showed a significant correlation ( r=0.71, P=0.003). Plasma homocysteine levels above 15 μM were associated with significant increases in intimal hyperplasia above 6.5% ( P=0.04). Elevation of plasma homocysteine levels to moderate levels (5–25 μM) resulted in significant post-CEA intimal hyperplasia. Cellular analysis of the area of intimal hyperplasia in all diet groups showed comparable amounts of cells positive for α-SM actin. However, with increasing levels of dietary homocystine and plasma homocysteine there was an increase in replicative index ( P<0.001) as determined by BrdU staining. Increasing dietary homocystine increased plasma homocysteine and was followed by increases in the replicative index thus producing increased intimal hyperplasia and lumenal stenosis. In hepatic measurements the 1.5 and 3.0 g/kg homocystine diets caused: increased liver activity of MTHFR ( P=0.03) and decreased hepatic levels of SAM, SAH and SAM/SAH ratios compared to controls. Homocystine treatment did not cause significant alterations in CBS levels ( P=0.992). These studies also showed no correlation of the MTHFR and CBS enzymes with plasma homocysteine levels or intimal hyperplasia. However, hepatic levels of SAM showed significant negative correlations with plasma homocysteine ( r=−0.58; P=0.006) and with BrdU percentages of cellular proliferation ( r=−0.69; P=0.06). Conclusion: The degree of post-CEA intimal hyperplasia in a rat model is directly related to the plasma level of homocysteine. The hyperplastic effects of homocysteine may be mediated in part by a physiological insufficiency of methyl donors as shown by decreases in SAM. Thus, increasing levels of plasma homocysteine enhanced and accelerated the smooth muscle cell response after CEA which led to increased intimal hyperplasia and lumenal stenosis.

Co-administration of equimolar doses of betaine may alleviate the hepatotoxic risk associated with niacin therapy

High-dose niacin has versatile and substantial efficacy for the treatment of hyperlipidemias, but its utility is compromised by various side effects, the most serious of which is liver damage. It is proposed that this hepatotoxicity reflects the high demand for methyl groups imposed by niacin catabolism, leading to a reduction in hepatic levels of S-adenosylmethionine (SAM). Depletion of the hepatic SAM pool has likewise been shown to mediate, at least in part, the hepatotoxic effects of ethanol, methotrexate, and niacinamide. If niacin does indeed decrease SAM, a likely consequence would be a counterproductive elevation of plasma homocysteine. Conceivably, methyl group deficiency, by altering membrane properties of skeletal muscle, also contributes to niacin-induced insulin resistance. Concurrent betaine supplementation – preferably administered as a complex with equimolar amounts of niacin – may represent the most cost-effective way to prevent niacin-mediated depletion of SAM and thus avoid hepatotoxicity (and possibly other adverse effects) while controlling homocysteine. Betaine also merits evaluation as an adjuvant to methotrexate and niacinamide therapies.

Betaine effects on hepatic methionine metabolism elicited by short-term ethanol feeding

Previous studies in this laboratory have shown that feeding of ethanol to rats produces prompt inhibition of methionine synthetase (MS) as well as a subsequent increase in activity of betaine homocysteine methyltransferase (BHMT). Further studies have shown that supplemental dietary betaine enhanced methionine metabolism and S-adenosylmethionine (SAM) generation in control and ethanol-fed rats. Because MS and BHMT are both involved in the formation of SAM, this study was conducted to determine early effects of ethanol on hepatic SAM levels and the influence of betaine supplementation on parameters of methionine metabolism during the early periods of MS inhibition and enhanced BHMT activity. Results showed that ethanol feeding produced a significant loss in SAM in the first week with a return to normal SAM levels in the second week. Betaine feeding enhanced hepatic betaine pools in control as well as ethanol-fed animals. This feeding attenuated the early loss of SAM in ethanol-fed animals, produced an early increase in BHMT activity, and generated increased levels of SAM in both control and ethanol-fed groups. Furthermore, betaine lowered significantly the accumulation of hepatic triglyceride produced by ethanol after 2 weeks of ingestion.

Hepatic Content of S-Adenosylmethionine, S-Adenosylhomocysteine and Glutathione in Rats Receiving Treatments Modulating Methyl Donor Availability

Because of evidence linking methyl group deficiency and increased tumor formation in experimental animals, we explored other possible methods of producing a methyl group deficiency. Rats fed a low methionine diet lacking choline (MCD) were injected intraperitoneally daily for 3 wk with large doses of nicotinamide. Hepatic levels of lipids were elevated, S-adenosylmethionine (SAM) levels and the SAM:S-adenosylhomocysteine (SAH) ratio were decreased, and SAH level was not consistently changed. In livers of rats fed the MCD diet without folate (MCFD), lipids were also elevated and SAM reduced as compared to MCD-fed rats. In rats fed the MCD diet plus a methionine (Met) supplement (MCD + Met), hepatic SAM levels and the SAM:SAH ratio were higher and lipid levels lower than in MCD-fed rats, indicating that the MCD diet is marginally deficient in methyl donor groups. The injection of nicotinamide or the removal of folate from the MCD diet increased the severity of methyl donor deficiency, as shown by lower hepatic SAM levels and higher hepatic lipid levels. Hepatic glutathione levels were similar in MCD- and MCFD-fed rats and were lower than in rats fed the methionine-supplemented MCD diet or injected with nicotinamide.