Glycine N-methyltransferase (GNMT) is the main enzyme responsible for catabolism of excess hepatic S-adenosylmethionine (SAMe). GNMT is absent in hepatocellular carcinoma (HCC), messenger RNA (mRNA) levels are significantly lower in livers of patients at risk of developing HCC, and GNMT has been proposed to be a tumor-susceptibility gene for liver cancer. The identification of several children with liver disease as having mutations of the GNMT gene further suggests that this enzyme plays an important role in liver function. In the current study we studied development of liver pathologies including HCC in GNMTknockout (GNMT-KO) mice. GNMT-KO mice have elevated serum aminotransferase, methionine, and SAMe levels and develop liver steatosis, fibrosis, and HCC. We found that activation of the Ras and Janus kinase ( T he first steps in mammalian methionine metabolism are conversion to S-adenosylmethionine (SAMe) and transfer of the methyl group of SAMe to a large variety of substrates (including DNA, RNA, histones, and small molecules such as glycine, guanidinoacetate, and phosphatidylethanolamine) with the formation of S-adenosylhomocysteine (SAH), an inhibitor of many SAMe-dependent methyltransferases. 1 Although there are a large number of SAMe-dependent methyltransferases, 2 methylation of glycine by glycine Nmethyltransferase (GNMT) to form sarcosine (N-methylglycine) is one of the reactions that contribute most to total transmethylation flux. 3 The importance of GNMT is to remove excess SAMe and maintain a constant hepatic SAMe/SAH ratio to avoid aberrant methylation. 2 Consistent with this function, the activation of GNMT in rats by the administration of retinoic acid causes a reduction in plasma methionine and homocysteine levels, as well as in liver DNA methylation. 4,5 In GNMT-knockout (GNMT-KO) mice, liver SAMe content is elevated 35-fold, and the SAMe/SAH ratio increases about 100-fold, 6 and individuals with GNMT mutations, which leads to inactive forms of the enzyme, have elevated plasma levels of methionine and SAMe but a normal concentration of homocysteine. 7,8 GNMT is expressed in the liver, pancreas, and prostate 9 and is absent in hepatocellular carcinoma (HCC) 10 and down-regulated in the livers of patients at risk of Abbreviations: GNMT, HCC, hepatocellular carcinoma; H3K27me3, trimethylated Received September 10, 2007; accepted November 26, 2007. Supported by NIH grants AA12677, AA13847, and AT-1576 (to S.C.L. and J.M.M.); DK15289 (to C.W.), PN IϩD SAF 2005-00855, HEPADIP-EULSHM-CT-205, and ETORTEK 2005 (to J.M.M. and M.L.M.-C.); Program Ramón y Cajal (to M.L.M.-C.); and Fundación "La Caixa" (to M.L.M.-C., R.M., and A.M.A.).

Cultured monkey kidney cells are shown to possess a cell-surface receptor that binds 5-methyltetrahydro [3',5',7,9-3H4] Folates are a class of pteridine compounds that are essential for normal growth and maturation. Reduced folic acid coenzymes are involved in one-carbon transfer reactions such as those necessary for the biosynthesis of methionine, serine, deoxythymidylic acid, and purines. Although these folatemediated reactions have been studied extensively and many of the key enzymes that catalyze these reactions have been purified and characterized (1), little is known about the overall mechanism ofcellular folate homeostasis. The normal plasma folate concentration in humans is 10-20 nM. However, tissue concentrations are 3 orders of magnitude higher (2-30 nmol/g of wet weight). Even when the plasma concentration of folate is experimentally increased 20-to 40-fold above normal, the steady-state tissue concentration remains constant (2). Therefore, most likely folate enters cells by a high-affinity uptake process that can be regulated.To better understand folate metabolism, many investigators have utilized tissue culture systems. The generally accepted model is that folate uptake is mediated by a membrane carrier that either facilitates the diffusion of folate across the membrane or actively transports the molecule into the cell (3). Another possibility is that folate is internalized by high-affinity receptors on the cell surface by a process that is similar to the receptor-mediated endocytosis of macromolecules. In the latter model, as in the case of vitamin MATERIALS AND METHODSBuffers and Tissue Culture Medium. Dulbecco's phosphate-buffered saline (PBS) was obtained from Hazelton Laboratories (Denver, PA). Medium 199 (M199) with Earle's salts and folic acid was purchased from GIBCO and supplemented with glutamine (GIBCO). RPMI 1640 medium without folic acid was formulated in our laboratory. Organic components were purchased from Sigma; the inorganic salts were obtained from Fisher Scientific. The RPMI medium was supplemented with adenine sulfate (10 mg/liter), thymine (0.3 mg/liter), xanthine (0.3 mg/liter), and hypoxanthine (0.3 mg/liter) (Sigma), and it was buffered with Hepes (25 mM, Sigma) and sodium bicarbonate (2 g/liter). All tissue culture medium contained fetal bovine serum (Hazelton Laboratories). To prepare folate-deficient medium, the serum was treated with activated charcoal (Norit A, Fisher Scientific) as detailed elsewhere (8) 5983The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Three human cases having mutations in the glycine N-methyltransferase (GNMT) gene have been reported. This enzyme transfers a methyl group from S-adenosylmethionine (SAM) to glycine to form S-adenosylhomocysteine (SAH) and N-methylglycine (sarcosine) and is believed to be involved in the regulation of methylation. All three cases have mild liver disease but they seem otherwise unaffected. To study this further, gnmt deficient mice were generated for the first time. This resulted in the complete absence of GNMT protein and its activity in livers of homozygous mice. Compared to WT animals the absence of GNMT resulted in up to a 7-fold increase of free methionine and up to a 35-fold increase of SAM. The amount of SAH was significantly decreased (3 fold) in the homozygotes compared to WT. The ratio of SAM/SAH increased from 3 in WT to 300 in livers of homozygous transgenic mice. This suggests a possible significant change in methylation in the liver and other organs where GNMT is expressed.

Plasma S-adenosylhomocysteine appears to be a much more sensitive indicator of the difference between patients with cardiovascular disease and control subjects than is homocysteine. Both plasma total homocysteine and S-adenosylhomocysteine are significantly correlated with plasma creatinine in patients.

This paper reports clinical and metabolic studies of two Italian siblings with a novel form of persistent isolated hypermethioninaemia, i.e. abnormally elevated plasma methionine that lasted beyond the first months of life and is not due to cystathionine beta-synthase deficiency, tyrosinaemia I or liver disease. Abnormal elevations of their plasma S-adenosylmethionine (AdoMet) concentrations proved they do not have deficient activity of methionine adenosyltransferase I/III. A variety of studies provided evidence that the elevations of methionine and AdoMet are not caused by defects in the methionine transamination pathway, deficient activity of methionine adenosyltransferase II, a mutation in methylenetetrahydrofolate reductase rendering this activity resistant to inhibition by AdoMet, or deficient activity of guanidinoacetate methyltransferase. Plasma sarcosine (N-methylglycine) is elevated, together with elevated plasma AdoMet in normal subjects following oral methionine loads and in association with increased plasma levels of both methionine and AdoMet in cystathionine beta-synthase-deficient individuals. However, plasma sarcosine is not elevated in these siblings. The latter result provides evidence they are deficient in activity of glycine N-methyltransferase (GNMT). The only clinical abnormalities in these siblings are mild hepatomegaly and chronic elevation of serum transaminases not attributable to conventional causes of liver disease. A possible causative connection between GNMT deficiency and these hepatitis-like manifestations is discussed. Further studies are required to evaluate whether dietary methionine restriction will be useful in this situation.