S-Adenosylmethionine (AdoMet) is the major biological methyl donor. AdoMet's methyl group arises both from the diet (eg, methionine, choline, and betaine) and from de novo synthesis by the process of methylneogenesis. At least 50 AdoMet-dependent methylation reactions have been identified in mammals, and genomic analyses suggest that the final number will be much higher. Such methylation reactions play major roles in biosynthesis, regulation, and detoxification. Creatine synthesis is thought to account for the use of >70% of AdoMet-derived methyl groups in humans. This is not consistent with recent studies in mice, in which the phosphatidylethanolamine methyltransferase gene was deleted (PEMT-/-). Loss of this hepatic enzyme resulted in a 50% decrease in plasma homocysteine, which suggests that it accounts for a major component of whole-body AdoMet utilization. A reexamination of human creatine metabolism showed that dietary creatine can account for as much as 50% of daily creatine requirements in nonvegetarians and, therefore, that estimates of creatine synthesis need to be reduced. We suggest that creatine synthesis is responsible for a smaller proportion of AdoMet-derived methyl groups than has been suggested and that phosphatidylcholine synthesis via phosphatidylethanolamine methyltransferase is a major consumer of these methyl groups.

S-adenosylmethionine, formed by the adenylation of methionine via S-adenosylmethionine synthase, is the methyl donor in virtually all known biological methylations. These methylation reactions produce a methylated substrate and S-adenosylhomocysteine, which is subsequently metabolized to homocysteine. The methylation of guanidinoacetate to form creatine consumes more methyl groups than all other methylation reactions combined. Therefore, we examined the effects of increased or decreased methyl demand by these physiological substrates on plasma homocysteine by feeding rats guanidinoacetate- or creatine-supplemented diets for 2 wk. Plasma homocysteine was significantly increased (~50%) in rats maintained on guanidinoacetate-supplemented diets, whereas rats maintained on creatine-supplemented diets exhibited a significantly lower (~25%) plasma homocysteine level. Plasma creatine and muscle total creatine were significantly increased in rats fed the creatine-supplemented or guanidinoacetate-supplemented diets. The activity of kidney L-arginine:glycine amidinotransferase, the enzyme catalyzing the synthesis of guanidinoacetate, was significantly decreased in both supplementation groups. To examine the role of the liver in mediating these changes in plasma homocysteine, isolated rat hepatocytes were incubated with methionine in the presence and absence of guanidinoacetate and creatine, and homocysteine export was measured. Homocysteine export was significantly increased in the presence of guanidinoacetate. Creatine, however, was without effect. These results suggest that homocysteine metabolism is sensitive to methylation demand imposed by physiological substrates.

Mild hyperhomocysteinemia is an independent risk factor for cardiovascular disease. Homocysteine, a nonprotein amino acid, is formed from S-adenosylhomocysteine and partially secreted into plasma. A potential source for homocysteine is methylation of the lipid phosphatidylethanolamine to phosphatidylcholine by phosphatidylethanolamine N-methyltransferase in the liver. We show that mice that lack phosphatidylethanolamine N-methyltransferase have plasma levels of homocysteine that are ϳ50% of those in wild-type mice. Hepatocytes isolated from methyltransferase-deficient mice secrete ϳ50% less homocysteine. Rat hepatoma cells transfected with phosphatidylethanolamine N-methyltransferase secrete more homocysteine than wild-type cells. Thus, phosphatidylethanolamine N-methyltransferase is an important source of plasma homocysteine and a potential therapeutic target for hyperhomocysteinemia.Mild hyperhomocysteinemia is an independent risk factor for cardiovascular (1, 2) and atherosclerotic disease (3). Total plasma homocysteine (Hcy) 1 values of ϳ10 M for men and ϳ8 M for women are in the normal range. However, even a small increase (ϳ5 M) in total plasma Hcy is associated with a 60% increased risk of coronary artery disease for men and 80% for women (3). Moreover, a number of studies have demonstrated that smoking, excessive drinking of alcohol, obesity, type II diabetes, and an unhealthy diet contribute to mild hyperhomocysteinemia (1, 2). In addition, elevated plasma Hcy has recently been linked to Alzheimer's disease and cognitive impairment in the elderly (4, 5).Hcy is a non-protein amino acid derived from the catabolism of S-adenosylhomocysteine (AdoHcy), an immediate product of trans-methylation reactions that utilize S-adenosylmethionine (AdoMet) (6). Hcy has three possible fates: 1) methylation to methionine with N-5-methyltetrahydrofolate or betaine as the methyl donor, 2) conversion to cysteine via the trans-sulfuration pathway, and 3) release into extracellular fluids (e.g. plasma and urine). AdoMet-dependent methyltransferases catalyze many critical reactions including methylation of RNA, DNA, proteins, and small molecules such as guanidinoacetate and glycine (6). The potential of methyltransferases to regulate plasma Hcy is not well defined.Phosphatidylethanolamine N-methyltransferase (PEMT) is a liver-specific enzyme that generates AdoHcy during the conversion of one membrane lipid, phosphatidylethanolamine, into another membrane lipid, phosphatidylcholine (PC) (7). PEMT accounts for the formation of ϳ30% PC made in liver (8, 9). With this large capacity for PC synthesis and the generation of three AdoHcy molecules for each PC molecule synthesized, we hypothesized that the PEMT reaction might contribute significantly to Hcy in plasma. We have utilized the Pemt Ϫ/Ϫ mouse, hepatocytes derived from these mice, and overexpression of PEMT in McArdle RH7777 (rat hepatoma) cells to test this hypothesis. The results show that PEMT expression enhances plasma Hcy levels and the secretion of Hcy from hepatocyte...

Epidemiological studies have provided strong evidence that an elevated plasma homocysteine concentration is an important independent risk factor for cardiovascular disease. We have shown, in the rat, that the kidney is a major site for the removal and subsequent metabolism of plasma homocysteine [Bostom, Brosnan, Hall, Nadeau and Selhub (1995) Atherosclerosis 116, 59-62]. To characterize the role of the kidney in homocysteine metabolism further, we measured the disappearance of homocysteine in isolated renal cortical tubules of the rat. Renal tubules metabolized homocysteine primarily through the transulphuration pathway, producing cystathionine and cysteine (78% of homocysteine disappearance). Methionine production accounted for less than 2% of the disappearance of homocysteine. Cystathionine, and subsequently cysteine, production rates, as well as the rate of disappearance of homocysteine, were sensitive to the level of serine in the incubation medium, as increased serine concentrations permitted higher rates of cystathionine and cysteine production. On the basis of enrichment profiles of cystathionine beta-synthase and cystathionine gamma-lyase, in comparison with marker enzymes of known location, we concluded that cystathionine beta-synthase was enriched in the outer cortex, specifically in cells of the proximal convoluted tubule. Cystathionine gamma-lyase exhibited higher enrichment patterns in the inner cortex and outer medulla, with strong evidence of an enrichment in cells of the proximal straight tubule. These studies indicate that factors that influence the transulphuration of homocysteine may influence the renal clearance of this amino acid.

Biological methylation reactions and homocysteine (Hcy) metabolism are intimately linked. In previous work, we have shown that phosphatidylethanolamine N-methyltransferase, an enzyme that methylates phosphatidylethanolamine to form phosphatidylcholine, plays a significant role in the regulation of plasma Hcy levels through an effect on methylation demand (Noga, A. A., Stead, L. M., Zhao, Y., Brosnan, M. E., Brosnan, J. T., and Vance, D. E. (2003) J. Biol. Chem. 278, 5952-5955). We have further investigated methylation demand and Hcy metabolism in liver-specific CTP:phosphocholine cytidylyltransferase-␣ (CT␣) knockout mice, since flux through the phosphatidylethanolamine N-methyltransferase pathway is increased 2-fold to meet hepatic demand for phosphatidylcholine. Our data show that plasma Hcy is elevated by 20 -40% in mice lacking hepatic CT␣. CT␣-deficient hepatocytes secrete 40% more Hcy into the medium than do control hepatocytes. Liver activity of betaine:homocysteine methyltransferase and methionine adenosyltransferase are elevated in the knockout mice as a mechanism for maintaining normal hepatic S-adenosylmethionine and S-adenosylhomocysteine levels. These data suggest that phospholipid methylation in the liver is a major consumer of AdoMet and a significant source of plasma Hcy.Elevations in plasma homocysteine (Hcy), 1 a nonprotein sulfur-containing amino acid, is an independent risk factor for cardiovascular (1, 2) and atherosclerotic diseases (3). In humans, total plasma Hcy concentration normally ranges from 8 to 12 M. However, a small elevation (ϳ5 M) in plasma Hcy increases the risk of coronary artery disease by as much as 60% in men and 80% in women (3). Hyperhomocysteinema has also been correlated with smoking, obesity, diabetes, hypertension, and impaired B vitamin status (1, 2). Furthermore, altered Hcy metabolism has been observed in Alzheimer's disease (4) and in the elderly with cognitive impairment (5).Homocysteine is formed during the metabolism of methionine. Methionine is activated by methionine adenosyltransferase to form S-adenosylmethionine (AdoMet), an important biological methyl donor (6). Numerous methyltransferases catalyze the transfer of the methyl group from AdoMet to a methyl acceptor, producing a methylated product and S-adenosylhomocysteine (AdoHcy), which is subsequently hydrolyzed to form adenosine and Hcy. Homocysteine has several metabolic fates; it can be remethylated to methionine using either betaine, catalyzed by betaine:Hcy methyltransferase (BHMT), or N 5 -methyltetrahydrofolate, catalyzed by methionine synthase (MAT), both of which are methyl donors. The catabolism of Hcy is accomplished by the transsulfuration pathway, which consists of two enzymes, cystathionine ␤-synthase and cystathionine ␥-lyase. Finally, Hcy can be secreted from cells into the circulation.It is clear that biological methylation and Hcy metabolism are closely related. However, the potential of specific methyltransferases to regulate plasma Hcy levels has been understudied. We have previously...

Elevated plasma homocysteine is a risk factor for cardiovascular disease and Alzheimer's disease. To understand the factors that determine the plasma homocysteine level it is necessary to appreciate the processes that produce homocysteine and those that remove it. Homocysteine is produced as a result of methylation reactions. Of the many methyltransferases, two are, normally, of the greatest quantitative importance. These are guanidinoacetate methyltransferase (that produces creatine) and phosphatidylethanolamine N-methyltransferase (that produces phosphatidylcholine). In addition, methylation of DOPA in patients with Parkinson's disease leads to increased homocysteine production. Homocysteine is removed either by its irreversible conversion to cysteine (transsulfuration) or by remethylation to methionine. There are two separate remethylation reactions, catalyzed by betaine:homocysteine methyltransferase and methionine synthase, respectively. The reactions that remove homocysteine are very sensitive to B vitamin status as both the transsulfuration enzymes contain pyridoxal phosphate, while methionine synthase contains cobalamin and receives its methyl group from the folic acid one-carbon pool. There are also important genetic influences on homocysteine metabolism.

Recent evidence suggests that an increased plasma concentration of the sulphur amino acid homocysteine is a risk factor for the development of vascular disease. The tissue(s) responsible for homocysteine production and export to the plasma are not well known. However, given the central role of the liver in amino acid metabolism, we developed a rat primary hepatocyte model in which homocysteine (and cysteine) production and export were examined. The dependence of homocysteine export from incubated hepatocytes on methionine concentration fitted well to a rectangular hyperbola, with half-maximal homocysteine export achieved at methionine concentrations of approx. 0.44 mM. Hepatocytes incubated with 1 mM methionine and 1 mM serine (a substrate for the transulphuration pathway of homocysteine removal) produced and exported significantly less homocysteine (25-40%) compared with cells incubated with 1 mM methionine alone. The effects of dietary protein on homocysteine metabolism were also examined. Rats fed a 60% protein diet had a significantly increased total plasma homocysteine level compared with rats fed a 20% protein diet. In vitro effects of dietary protein were examined using hepatocytes isolated from animals maintained on these diets. When incubated with 1 mM methionine, hepatocytes from rats fed the high protein diet exported significantly more homocysteine compared with hepatocytes from rats fed the normal protein diet. Inclusion of serine significantly lowered homocysteine export in the normal protein group, but the effect was more marked in the high protein group. In vivo effects of serine were also examined. Rats fed a high protein diet enriched with serine had significantly lower total plasma homocysteine (25-30%) compared with controls. These data indicate a significant role for the liver in the regulation of plasma homocysteine levels.

An elevated plasma level of homocysteine is a risk factor for the development of cardiovascular disease. The purpose of this study was to investigate the effect of glucagon on homocysteine metabolism in the rat. Male Sprague-Dawley rats were treated with 4 mg/kg/day (3 injections per day) glucagon for 2 days while control rats received vehicle injections. Glucagon treatment resulted in a 30% decrease in total plasma homocysteine and increased hepatic activities of glycine N-methyltransferase, cystathionine ␤-synthase, and cystathionine ␥-lyase. Enzyme activities of the remethylation pathway were unaffected. The 90% elevation in activity of cystathionine ␤-synthase was accompanied by a 2-fold increase in its mRNA level. Hepatocytes prepared from glucagon-injected rats exported less homocysteine, when incubated with methionine, than did hepatocytes of saline-treated rats. Flux through cystathionine ␤-synthase was increased 5-fold in hepatocytes isolated from glucagon-treated rats as determined by production of 14 CO 2 and ␣-[1-14 C]ketobutyrate from L-[1-14 C]methionine. Methionine transport was elevated 2-fold in hepatocytes isolated from glucagon-treated rats resulting in increased hepatic methionine levels. Hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine, allosteric activators of cystathionine ␤-synthase, were also increased following glucagon treatment. These results indicate that glucagon can regulate plasma homocysteine through its effects on the hepatic transsulfuration pathway.An elevated plasma concentration of homocysteine, a sulfurcontaining amino acid derived from methionine, has been recognized as an independent risk factor for the development of vascular disease (1). Methionine is adenylated by methionine adenosyltransferase to form S-adenosylmethionine, an important biological methyl donor. Numerous methyltransferases catalyze the transfer of a methyl group from S-adenosylmethionine to a methyl acceptor, producing a methylated product and S-adenosylhomocysteine, which is subsequently hydrolyzed to form adenosine and homocysteine. Homocysteine has several possible fates: 1) remethylation to form methionine via either the cobalamin-dependent methionine synthase (using N 5 -methyltetrahydrofolate as a methyl donor) or betaine:homocysteine methyltransferase (using betaine as a methyl donor); 2) catabolism by the transsulfuration pathway, ultimately forming cysteine; 3) export to the extracellular space. Two vitamin B 6 -dependent enzymes comprise the transsulfuration pathway: cystathionine ␤-synthase, which condenses homocysteine with serine to form cystathionine, and cystathionine ␥-lyase, which cleaves cystathionine to cysteine, NH 4 ϩ , and ␣-ketobutyrate.Altered flux through the remethylation or transsulfuration pathways as a result of genetic mutations or impaired vitamin status has been shown to affect plasma homocysteine levels (2, 3). In recent years it has also become apparent that certain hormones can affect homocysteine metabolism. It has been shown that hypothyroid ...