Glycine N-methyltransferase Research Articles

Impaired liver regeneration in mice lacking glycine N‐methyltransferase†

Hepatic S-adenosylmethionine (SAMe) is maintained constant by the action of methionine adenosyltransferase I/III (MATI/III), which converts methionine into SAMe and glycine N-methyltransferase (GNMT), which eliminates excess SAMe to avoid aberrant methylation reactions. During liver regeneration after partial hepatectomy (PH) MATI/III activity is inhibited leading to a decrease in SAMe. This injury-related reduction in SAMe promotes hepatocyte proliferation because SAMe inhibits hepatocyte DNA synthesis. In MATI/III-deficient mice, hepatic SAMe is reduced, resulting in uncontrolled hepatocyte growth and impaired liver regeneration. These observations suggest that a reduction in SAMe is crucial for successful liver regeneration. In support of this hypothesis we report that liver regeneration is impaired in GNMT knockout (GNMT-KO) mice. Liver SAMe is 50-fold higher in GNMT-KO mice than in control animals and is maintained constant following PH. Mortality after PH was higher in GNMT-KO mice than in control animals. In GNMT-KO mice, nuclear factor kappaB (NFkappaB), signal transducer and activator of transcription-3 (STAT3), inducible nitric oxide synthase (iNOS), cyclin D1, cyclin A, and poly (ADP-ribose) polymerase were activated at baseline. PH in GNMT-KO mice was followed by the inactivation of STAT3 phosphorylation and iNOS expression. NFkappaB, cyclin D1 and cyclin A were not further activated after PH. The LKB1/AMP-activated protein kinase/endothelial nitric oxide synthase cascade was inhibited, and cytoplasmic HuR translocation was blocked despite preserved induction of DNA synthesis in GNMT-KO after PH. Furthermore, a previously unexpected relationship between AMPK phosphorylation and NFkappaB activation was uncovered. These results indicate that multiple signaling pathways are impaired during the liver regenerative response in GNMT-KO mice, suggesting that GNMT plays a critical role during liver regeneration, promoting hepatocyte viability and normal proliferation.

Phenotypic differences between two Gnmt−/− mouse models for hepatocellular carcinoma†

We would like to express some technical concerns about the results presented in the article by Mato and coworkers in the April 2008 issue of HEPATOLOGY.1 We have identified the gene for glycine N-methyltransferase (GNMT) as a liver cancer susceptibility gene, generated Gnmt−/− mice, and reported on their tendencies toward chronic hepatitis, glycogen storage disease, and liver cancer.2-6 Although the Gnmt−/− mouse models described by Dr. Mato's team and our group share similar strain (129 substrain/B6) backgrounds, they have at least three significant differences in terms of phenotype: (1) Dr. Mato's group reported that their male Gnmt−/− mice developed hepatocellular carcinoma (HCC) at 8 months old, whereas our Gnmt−/− mice did not develop HCC until a minimum of 14 months of age (up to 24 months); (2) we showed that seven of seven (100%) female Gnmt−/− mice develop HCC, whereas Dr. Mato did not mention whether their female Gnmt−/− mice had developed HCC; and (3) Dr. Mato's team described global DNA hypermethylation in the liver tissues of their Gnmt−/− mice using capillary electrophoresis, whereas we used both [3H]methyl group labeling and a commercially available enzyme immunoassay to identify global DNA hypomethylation in liver tissues from both male and female Gnmt−/− mice.1, 6 Published reports indicate that global DNA hypomethylation and promoter gene cytosine-guanosine dinucleotide hypermethylation are both present in human HCC.7 However, with the exception of Dr. Mato's report, no one has stated that gene knockout mice can develop HCC as early as 8 months of age. One possible explanation for the differences between these two Gnmt−/− models is the completeness of the Gnmt gene section being knocked out. The Gnmt gene has six exons; we knocked out exons 1-4 and part of exon 5,4 whereas Dr. Mato and his collaborator, Dr. Wagner, only knocked out exon 1.8 Results from a nucleotide sequence analysis indicate that the disrupted allele generated in Dr. Wagner's model contained three potential promoters in intron 1 (Fig. 1). In addition, four potential in-frame ATG codons were located downstream from the potential promoters (three in intron 1 and one in exon 2). Targeted modification of mouse Gnmt gene locus. The disrupted allele generated in Dr. Wagner's model contains three potential promoters in intron 1, and four potential start codons in the downstream promoter region and exon 2. Filled arrows point to start codons. Open triangles indicate three potential promoter sites in intron 1. The full-length mouse Gnmt contains 293 amino acids (aa); an incomplete knockout strategy may generate an artificial truncated form containing 220 amino acids. This type of disrupted gene may generate a truncated form of GNMT that lacks the N-terminal 73 amino acids. Previous reports show (1) Gnmt uses its N-terminal 20 amino acids to form a tetramer in the cytosol and (2) the presence of a phosphorylation site in serine-9 of the Gnmt protein.9, 10 We feel there is a need to clarify whether Dr. Mato's Gnmt−/− mice generated such a truncated form of Gnmt, which may cause it to act as a dominant-negative protein, thereby implying its involvement in tumorigenesis. If this is the case, then the mouse model they generated is not a complete Gnmt−/− mouse model. In summary, we believe we are the first group to demonstrate that GNMT is a liver cancer susceptibility gene. Although both teams showed HCC development in Gnmt−/− mice, without further data, we suspect that Dr. Mato's model is an incomplete Gnmt knockout model. Yi-Ming Arthur Chen* , Yi-Jen Liao , Shih-Ping Liu , Ting-Fen Tsai§, * Department of Microbiology, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Molecular Medicine Program, Institute of Public Health, School of Medicine, National Yang-Ming University, Taipei, Taiwan, Center for Neuropsychiatry, China Medical University and Hospital, Taichung, Taiwan, § Faculty of Life Sciences and Institute of Genome Sciences, National Yang-Ming University, Taipei, Taiwan.

Abnormal methyl group metabolism and epigenetic regulation in the Zucker (type 2) diabetic fatty rat

Methyl group metabolism and DNA methylation have been shown to be altered in a streptozotocin‐induced model of type 1 diabetes. The Zucker diabetic fatty (ZDF) rat exhibits similar perturbations of methyl group metabolism, but DNA methylation status and the expression of epigenetic regulatory proteins have not been characterized in this model of type 2 diabetes. Because epigenetic regulation could be linked to secondary complications associated with type 2 diabetes, we chose to examine methyl group metabolism, including DNA methylation, in the liver and pancreas, as well as the heart and kidney. At 12 wk of age, 6 ZDF and 6 lean rats were sacrificed for blood and tissue analysis. As expected, ZDF rats were diabetic (2X increase in glucose and insulin) and exhibited marked hypohomocysteinemia. Glycine N‐methyltransferase (GNMT), a regulator of transmethylation, was divergently regulated in different tissues. In ZDF rats, hepatic GNMT activity and abundance were increased, whereas pancreatic activity was decreased. No change was noted in renal GNMT activity and abundance, or cardiac GNMT abundance, despite a lack of activity. In contrast to type 1 diabetes, hepatic DNA was hypermethylated in ZDF rats. Mechanistically, it appears that expression of epigenetic regulatory proteins (i.e. DNA methyltransferases and methyl CpG binding proteins) was abnormal in diabetic animals as well. (Support: Amer. Heart Assoc.)Grant Funding SourceAmerican Heart Association

Characterization of homocysteine and methyl group metabolism in female non‐obese diabetic (NOD) mice

Methyl groups are critical for numerous biological reactions and proper regulation of methyl group and homocysteine (hcy) metabolism is essential for disease prevention. The hepatic protein glycine N‐methyltransferase (GNMT) is a key regulator of supply and utilization of methyl groups from the primary donor, S‐adenosylmethionine. Chemically‐induced type 1 diabetes has been shown to alter hcy and methyl group metabolism, resulting in a methyl deficient state, including DNA hypomethylation. The objective of this study was to characterize hcy and methyl group metabolism using a genetic model of diabetes, the non‐obese diabetic (NOD) mouse, which spontaneously develops autoimmune‐mediated type 1 diabetes. NOD mice and their controls (ICR), were sacrificed at 12, 18, and 24 wk of age for collection of blood and liver tissue. Plasma hcy concentrations in NOD mice were only 63% of the control values in 18 wk old ICR animals. NOD mice exhibited a 1.4‐fold increase in GNMT activity compared to ICR mice at 18 wk. Neither metabolic parameter was altered in 12 wk old animals. The NOD mouse, a genetic model for type 1 diabetes, exhibits similar perturbations in methyl group and hcy metabolism as the chemically‐induced rat model. This validation will be important in future studies directed at understanding the relation between diabetes, perturbation of methyl groups, and specific pathologies (Support: NIH HD057315‐02).

Folate‐independent remethylation of homocysteine is increased by dietary egg white protein

Proper maintenance and regulation of methyl group and homocysteine (hcy) metabolism is essential for optimization of health. Methyl deficiency and/or elevations in plasma hcy concentrations have been associated with numerous pathologies, such as vascular disease. It is important to identify dietary components that positively impact these metabolic pathways; thus, we examined dietary protein sources with varying sulfur amino acid content. Rats were fed either a casein or egg white protein (20%) diet for 2 wk after which half the rats in each group were fed folate‐deficient diets for 4 wk. Compared to casein, egg white protein resulted in: (1) elevated hepatic betaine‐hcy S‐methyltransferase activity, responsible for folate‐independent hcy remethylation; (2) increased hepatic activity of glycine N‐methyltransferase, a key methyl group regulatory protein; and (3) reduced plasma glutathione concentrations. Moreover, folate deficiency was without affect on plasma hcy levels in the egg white group. In a parallel study, soy protein did not differ from casein with respect to altering methyl group and hcy metabolism. In conclusion, dietary egg white protein appears to promote hcy remethylation at the expense of catabolism via transsulfuration. These findings may be attributed to the greater met‐cys content of egg white protein compared to casein and soy (Support: Amer. Egg Board, Egg Nutr. Center, IA Egg Council).

Proteomics reveal a concerted upregulation of methionine metabolic pathway enzymes, and downregulation of carbonic anhydrase-III, in betaine supplemented ethanol-fed rats

We employed a proteomic profiling strategy to examine the effects of ethanol and betaine diet supplementation on major liver protein level changes. Male Wistar rats were fed control, ethanol or betaine supplemented diets for 4weeks. Livers were removed and liver cytosolic proteins resolved by one-dimensional and two-dimensional separation techniques. Significant upregulation of betaine homocysteine methyltransferase-1, methionine adenosyl transferase-1, and glycine N-methyltransferase were the most visually prominent protein changes observed in livers of rats fed the betaine supplemented ethanol diet. We hypothesise that this concerted upregulation of these methionine metabolic pathway enzymes is the protective mechanism by which betaine restores a normal metabolic ratio of liver S-adenosylmethionine to S-adenosylhomocysteine. Ethanol also induced significant downregulation of carbonic anhydrase-III protein levels which was not restored by betaine supplementation. Carbonic anhydrase-III can function to resist oxidative stress, and we therefore hypothesise that carbonic anhydrase-III protein levels compromised by ethanol consumption, contribute to ethanol-induced redox stress.

Glycine N-methyltransferase affects the metabolism of aflatoxin B 1 and blocks its carcinogenic effect

Previously, we reported that glycine N-methyltransferase (GNMT) knockout mice develop chronic hepatitis and hepatocellular carcinoma (HCC) spontaneously. For this study we used a phosphoenolpyruvate carboxykinase promoter to establish a GNMT transgenic (TG) mouse model. Animals were intraperitoneally inoculated with aflatoxin B 1 (AFB 1) and monitored for 11 months, during which neither male nor female GNMT-TG mice developed HCC. In contrast, 4 of 6 (67%) male wild-type mice developed HCC. Immunofluorescent antibody test showed that GNMT was translocated into nuclei after AFB 1 treatment. Competitive enzyme immunoassays indicated that after AFB 1 treatment, the AFB 1–DNA adducts formed in stable clones expressing GNMT reduced 51.4% compared to the vector control clones. Experiments using recombinant adenoviruses carrying GNMT cDNA (Ad-GNMT) further demonstrated that the GNMT-related inhibition of AFB 1–DNA adducts formation is dose-dependent. HPLC analysis of the metabolites of AFB 1 in the cultural supernatants of cells exposed to AFB 1 showed that the AFM 1 level in the GNMT group was significantly higher than the control group, indicating the presence of GNMT can enhance the detoxification pathway of AFB 1. Cytotoxicity assay showed that the GNMT group had higher survival rate than the control group after they were treated with AFB 1. Automated docking experiments showed that AFB 1 binds to the S-adenosylmethionine binding domain of GNMT. Affinity sensor assay demonstrated that the dissociation constant for GNMT–AFB 1 interaction is 44.9 μM. Therefore, GNMT is a tumor suppressor for HCC and it exerts protective effects in hepatocytes via direct interaction with AFB 1, resulting in reduced AFB 1–DNA adducts formation and cell death.

Evaluations of the trans-sulfuration pathway in multiple liver toxicity studies

Drug-induced liver injury has been associated with the generation of reactive metabolites, which are primarily detoxified via glutathione conjugation. In this study, it was hypothesized that molecules involved in the synthesis of glutathione would be diminished to replenish the glutathione depleted through conjugation reactions. Since S-adenosylmethionine (SAMe) is the primary source of the sulfur atom in glutathione, UPLC/MS and NMR were used to evaluate metabolites involved with the transulfuration pathway in urine samples collected during studies of eight liver toxic compounds in Sprague-Dawley rats. Urinary levels of creatine were increased on day 1 or day 2 in 8 high dose liver toxicity studies. Taurine concentration in urine was increased in only 3 of 8 liver toxicity studies while SAMe was found to be reduced in 4 of 5 liver toxicity studies. To further validate the results from the metabonomic studies, microarray data from rat liver samples following treatment with acetaminophen was obtained from the Gene Expression Omnibus (GEO) database. Some genes involved in the trans-sulfuration pathway, including guanidinoacetate N-methyltransferase, glycine N-methyltransferase, betaine-homocysteine methyltransferase and cysteine dioxygenase were found to be significantly decreased while methionine adenosyl transferase II, alpha increased at 24 h post-dosing, which is consistent with the SAMe and creatine findings. The metabolic and transcriptomic results show that the trans-sulfuration pathway from SAMe to glutathione was disturbed due to the administration of heptatotoxicants.

Characterization of a glycine N‐methyltransferase gene knockout mouse model for hepatocellular carcinoma: Implications of the gender disparity in liver cancer susceptibility

Hepatocellular carcinoma (HCC) is the fifth common cancer in the world and it mainly occurs in men. Glycine N-methyltransferase (GNMT) participates in one-carbon metabolism and affects DNA methylation by regulating the ratio of S-adenosylmethionine to S-adenosylhomocystine. Previously, we described that the expression of GNMT was diminished in human HCC. Here, we showed that 50% (3/6) male and 100% (7/7) female Gnmt-/- mice developed HCC, and their mean ages of HCC development were 17 and 16.5 months, respectively. In addition, 42.9% (3/7) of female Gnmt-/- mice had hemangioma. Wnt reporter assay demonstrated that Gnmt is a negative regulator for canonical Wnt signaling pathway. Beta-catenin, cyclin D1 and c-Myc, genes related to Wnt pathway, were upregulated in the liver tissues from both 11 weeks and HCC stage of Gnmt-/- mice. Furthermore, global DNA hypomethylation and aberrant expression of DNA methyltransferases 1 and 3b were found in the early and late stages of HCC development. Hierarchical cluster analysis of 6,023 transcripts from microarray data found that gene expression patterns of HCC tumors from male and female Gnmt-/- mice were distinctively different. Real-time PCR confirmed that Gadd45a, Pak1, Mapk3 and Dsup3 genes of mitogen-activated protein kinase (MAPK) pathway were activated in Gnmt-/- mice, especially in the female mice. Therefore, GNMT is a tumor suppressor gene for liver cancer, and it is associated with gender disparity in liver cancer susceptibility.

Type I Diabetes Leads to Tissue-Specific DNA Hypomethylation in Male Rats

Numerous perturbations of methyl group and homocysteine metabolism have been documented as an outcome of diabetes. It has also been observed that there is a transition from hypo- to hyperhomocysteinemia in diabetes, often concurrent with the development of nephropathy. The objective of this study was to characterize the temporal changes in methyl group and homocysteine metabolism in the liver and kidney and to determine the impact these alterations have on DNA methylation in type 1 diabetic rats. Male Sprague-Dawley rats were injected with streptozotocin (60 mg/kg body weight) to induce diabetes and samples were collected at 2, 4, and 8 wk. At 8 wk, hepatic and renal betaine-homocysteine S-methyltransferase activities were greater in diabetic rats, whereas methionine synthase activity was lower in diabetic rat liver and kidney did not differ. Cystathionine β-synthase abundance was greater in the liver but less in the kidney of diabetic rats. Both hepatic and renal glycine N-methyltransferase (GNMT) activity and abundance were greater in diabetic rats; however, changes in renal activity and/or abundance were present only at 2 and 4 wk, whereas hepatic GNMT was induced at all time points. Most importantly, we have shown that genomic DNA was hypomethylated in the liver, but not the kidney, in diabetic rats. These results suggest that diabetes-induced perturbations of methyl group and homocysteine metabolism lead to functional methyl deficiency, resulting in the hypomethylation of DNA in a tissue-specific fashion.

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.

S-adenosylmethionine and proliferation: new pathways, new targets

SAMe (S-adenosylmethionine) is the main methyl donor group in the cell. MAT (methionine adenosyltransferase) is the unique enzyme responsible for the synthesis of SAMe from methionine and ATP, and SAMe is the common point between the three principal metabolic pathways: polyamines, transmethylation and transsulfuration that converge into the methionine cycle. SAMe is now also considered a key regulator of metabolism, proliferation, differentiation, apoptosis and cell death. Recent results show a new signalling pathway implicated in the proliferation of the hepatocyte, where AMPK (AMP-activated protein kinase) and HuR, modulated by SAMe, take place in HGF (hepatocyte growth factor)-mediated cell growth. Abnormalities in methionine metabolism occur in several animal models of alcoholic liver injury, and it is also altered in patients with liver disease. Both high and low levels of SAMe predispose to liver injury. In this regard, knockout mouse models have been developed for the enzymes responsible for SAMe synthesis and catabolism, MAT1A and GNMT (glycine N-methyltransferase) respectively. These knockout mice develop steatosis and HCC (hepatocellular carcinoma), and both models closely replicate the pathologies of human disease, which makes them extremely useful to elucidate the mechanism underlying liver disease. These new findings open a wide range of possibilities to discover novel targets for clinical applications.

Glycine N‐methyltransferase is a favorable prognostic marker for human cholangiocarcinoma

Glycine N-methyltransferase (GNMT) is a susceptibility gene for human hepatocellular carcinoma (HCC). We previously reported that GNMT expression is diminished in HCC. Here we report our examination of GNMT expression patterns in cholangiocarcinoma and the relationship between its expression and prognosis. We analyzed GNMT expression in tumor tissues from 33 cholangiocarcinoma patients (19 male) using immunohistochemistry (IHC) procedures with a GNMT monoclonal antibody (mAb 4-17). GNMT expression intensity and percentages were scored on a scale of 0 to 6. The association between GNMT expression and survival was analyzed using the Kaplan-Meier method, and prognostic factors were evaluated with a multivariate Cox proportional hazards regression model. High GNMT expression was found in epithelial cells of normal bile ducts. Six of 33 (18.2%) cholangiocarcinoma tissues had no GNMT expression. A statistically significant difference was noted in GNMT expression between male and female patients (68.4% vs 100%, P < 0.05). Compared to patients with GNMT expression scores > 3, the death hazard ratio for patients with GNMT scores <or= 3 was 3.68 (95% confidence interval = 1.17-11.59, P < 0.05). GNMT expression is a favorable prognosis predictor for cholangiocarcinoma.

Acetylation of N-terminal valine of glycine N-methyltransferase affects enzyme inhibition by folate

Native liver glycine N-methyltransferase (GNMT) is N-acetylated while the recombinant enzyme is not. We show here that acetylation of the N-terminal valine affects several kinetic parameters of the enzyme. Glycine N-methyltransferase is a regulatory enzyme mediating the availability of methyl groups by virtue of being inhibited by folate. N-acetylation does not affect the overall structure of the protein and does not affect basal enzyme activity of GNMT. Binding of both the mono- and pentaglutamate forms of 5-methyltetrahydrofolate is the same for the acetylated and non-acetylated forms of the enzyme, however the pentaglutamate form is bound more tightly than the monoglutamate form in both cases. Although binding of the folates is similar for the acetylated and non-acetylated forms of the enzyme, inhibition of enzyme activity differs significantly. The native, N-acetylated form of the enzyme shows 50% inhibition at 1.3 µM concentration of the pentaglutamate while the recombinant non-acetylated form shows 50% inhibition at 590 µM. In addition, the binding of folate results in cooperativity of the substrate S-adenosylmethionine (AdoMet), with a Hill coefficient of 1.5 for 5-methyltetrahydrofolate pentaglutamate.

Tissue‐specific regulation of glycine N‐methyltransferase (GNMT) activity and abundance in the prediabetic ZDF (type 2) rat: evidence for two regulated GNMT isoforms

Perturbations of methyl group metabolism have been demonstrated in diabetic rat models. Altered expression and activity of key enzymes were evident at 5 and 11 wk of age in the Zucker diabetic fatty (ZDF) rat liver. We have shown that hepatic GNMT is induced in type 1 diabetic rats, concomitant with DNA hypomethylation. The objective of this study was to determine how these alterations, particularly in GNMT, in the ZDF rat might be related to diabetic complications, such as cardiovascular disease. Blood and tissues were collected from ZDF (fa/fa) and lean controls (+/?) at 8 wk of age. Blood glucose was elevated 100% and plasma homocysteine was decreased 51% in ZDF rats compared to controls. Using a GNMT peptide antibody, two immunoreactive proteins (32 and 36 kDa) were clearly detected. Interestingly, both the lighter (L) and heavier (H) forms of GNMT were observed in hepatic and pancreatic samples, whereas only H‐GNMT was present in renal and cardiac tissue. Measurable GNMT enzymatic activity was detected in all tissues except heart. There were no differences between the ZDF rats and controls in the kidney, but both pancreatic activity and abundance of L‐GNMT were lower in the diabetic animals, whereas H‐, but not L‐GNMT, was greater in the diabetic rat liver. These results suggest there is tissue‐specific regulation of both L‐ and H‐GNMT during the progression of type 2 diabetes in the prediabetic ZDF rat. (Am Heart Assoc)

Methyl group metabolism in maternal and fetal hepatic tissues is altered by diabetes and retinoic acid treatment

Adequate methyl group supply is vital for numerous biological reactions, especially during development. A lack of methyl groups, B‐vitamins, and changes in relevant enzymes, can result in a host of pathologies, including neural tube defects. If inappropriately activated, hepatic glycine N‐methyltransferase (GNMT) a regulatory enzyme in folate and methyl group metabolism, leads to methyl deficiency. We have shown that both a diabetic state and retinoic acid (RA) treatment result in activation of GNMT. The goal of this pilot study was to determine if induction of GNMT during pregnancy alters methyl group metabolism in the fetus. Non‐diabetic and diabetic (STZ) pregnant rats treated with and without RA, were sacrificed on d 18 of gestation for collection of blood and liver tissue. Two hepatic immunoreactive proteins were clearly detected (32 and ~36 kD) in liver tissue. The lighter form (L‐GNMT) was more responsive in maternal liver, whereas the heavier protein (H‐GNMT) dominated in fetal liver. Rats receiving both RA and STZ exhibited elevated maternal hepatic GNMT activity, L‐GNMT abundance, and DNA hypomethylation, and reduced circulating homocysteine levels. Fetal liver expressed only H‐GNMT which was slightly responsive to treatment; however, L‐GNMT expression was evident in the combined treatment group. Fetal hepatic tissue did not exhibit changes in DNA methylation, due to the lack of L‐GNMT expression.

Loss of the glycine N‐methyltransferase gene leads to steatosis and hepatocellular carcinoma in mice†

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 GNMT-knockout (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 (JAK)/signal transducer and activator of transcription (STAT) pathways was increased in liver tumors from GNMT-KO mice coincidently with the suppression of the Ras inhibitors Ras-association domain family/tumor suppressor (RASSF) 1 and 4 and the JAK/STAT inhibitors suppressor of cytokine signaling (SOCS) 1-3 and cytokine-inducible SH2-protein. Finally, we found that methylation of RASSF1 and SOCS2 promoters and the binding of trimethylated lysine 27 in histone 3 to these 2 genes was increased in HCC from GNMT-KO mice. These data demonstrate that loss of GNMT induces aberrant methylation of DNA and histones, resulting in epigenetic modulation of critical carcinogenic pathways in mice.

Using Grote−Hynes Theory To Quantify Dynamical Effects on the Reaction Rate of Enzymatic Processes. The Case of Methyltransferases

Dynamical effects have recently received much attention in the context of the theoretical investigation of enzymatic catalysis. In this paper we use a combination of Grote-Hynes theory with quantum mechanical/molecular mechanical modeling that is a powerful tool to understand and quantify these dynamical effects in a particular enzyme, the glycine N-methyltransferase (GNMT). Comparison of the results obtained for this enzyme with another methyltransferase (catechol O-methyltransferase, COMT) allows us to understand the different nature of the coupling of the environment to the reaction coordinate as a function of the electrostatic interaction established by the reactive subsystem. The transmission coefficients obtained using Grote-Hynes theory are in excellent agreement with molecular dynamics estimations and show that the coupling is higher in GNMT than in COMT. The larger friction observed in GNMT is explained on the basis of the interaction established by the substrate in the active site. The larger value of the friction leads to a smaller value of the reaction frequency and thus also to a larger disagreement with the estimation of the transmission coefficient based on the frozen environment approach.

Glycine N-methyltransferase−/− mice develop chronic hepatitis and glycogen storage disease in the liver

Glycine N-methyltransferase (GNMT) affects genetic stability by regulating DNA methylation and interacting with environmental carcinogens. To establish a Gnmt knockout mouse model, 2 lambda phage clones containing a mouse Gnmt genome were isolated. At 11 weeks of age, the Gnmt-/- mice had hepatomegaly, hypermethioninemia, and significantly higher levels of both serum alanine aminotransferase and hepatic S-adenosylmethionine. Such phenotypes mimic patients with congenital GNMT deficiencies. A real-time polymerase chain reaction analysis of 10 genes in the one-carbon metabolism pathway revealed that 5,10-methylenetetrahydrofolate reductase, S-adenosylhomocysteine hydrolase (Ahcy), and formiminotransferase cyclodeaminase (Ftcd) were significantly down-regulated in Gnmt-/- mice. This report demonstrates that GNMT regulates the expression of both Ftcd and Ahcy genes. Results from pathological examinations indicated that 57.1% (8 of 14) of the Gnmt-/- mice had glycogen storage disease (GSD) in their livers. Focal necrosis was observed in male Gnmt-/- livers, whereas degenerative changes were found in the intermediate zones of female Gnmt-/- livers. In addition, hypoglycemia, increased serum cholesterol, and significantly lower numbers of white blood cells, neutrophils, and monocytes were observed in the Gnmt-/- mice. A real-time polymerase chain reaction analysis of genes involved in the gluconeogenesis pathways revealed that the following genes were significantly down-regulated in Gnmt-/- mice: fructose 1,6-bisphosphatase, phosphoenolpyruvate carboxykinase, and glucose-6-phosphate transporter. Because Gnmt-/- mice phenotypes mimic those of patients with GNMT deficiencies and share several characteristics with GSD Ib patients, we suggest that they are useful for studies of the pathogenesis of congenital GNMT deficiencies and the role of GNMT in GSD and liver tumorigenesis.

Destabilization of human glycine N‐methyltransferase by H176N mutation

In the presence of moderate (2-4 M) urea concentrations the tetrameric enzyme, glycine N-methyltransferase (GNMT), dissociates into compact monomers. Higher concentrations of urea (7-8 M) promote complete denaturation of the enzyme. We report here that the H176N mutation in this enzyme, found in humans with hypermethioninaemia, significantly decreases stability of the tetramer, although H176 is located far from the intersubunit contact areas. Dissociation of the tetramer to compact monomers and unfolding of compact monomers of the mutant protein were detected by circular dichroism, quenching of fluorescence emission, size-exclusion chromatography, and enzyme activity. The values of apparent free energy of dissociation of tetramer and of unfolding of compact monomers for the H176N mutant (27.7 and 4.2 kcal/mol, respectively) are lower than those of wild-type protein (37.5 and 6.2 kcal/mol). A 2.7 A resolution structure of the mutant protein revealed no significant difference in the conformation of the protein near the mutated residue.