Exogenous AdoMet Research Articles

Double-Blind, Placebo-Controlled, Randomized Clinical Trial of the Effect of the Dietary Supplement S-Adenosyl-L-Methionine (AdoMet) on Plasma Homocysteine (Hcy) Levels in Healthy Human Subjects.

BACKGROUND: S-adenosyl-L-methionine (AdoMet or SAM-e®) is a commonly used nutritional supplement available in the United States since 1999. AdoMet is metabolized to homocysteine (Hcy), a potential cardiovascular risk factor. A few open-label, single-arm studies have reported on the effect of exogenous AdoMet on the levels of Hcy in humans; however, this has not been tested in a double-blind, randomized clinical trial. As a nutritional supplement, AdoMet is subject only to limited regulation by the FDA, despite being used to treat clinical diseases such as depression and osteoarthritis. AdoMet is the methyl donor for small molecule, DNA, RNA, and protein methylation reactions; therefore, further understanding the biology of the AdoMet/Hcy system is important. We hypothesized that exogenous AdoMet would increase plasma Hcy levels.METHODS: In a double-blind, placebo-controlled, randomized clinical trial, 93 healthy human subjects were screened and 52 were treated with placebo (26) or 800 mg per day AdoMet (26) pills for 4 weeks. Pre- and post-treatment Hcy levels were measured. The primary endpoint was change in Hcy level. Secondary endpoints included an interim Hcy level, high sensitivity C-reactive protein (hsCRP) levels, lipid profile, and transaminases. Exclusion criteria included pregnancy and concurrent use of medications associated with changes in Hcy.RESULTS: Of 52 subjects enrolled, 45 were evaluable at the end of treatment. Subject characteristics and dropout rates were similar between placebo and control groups. Adverse events were minor and were not different between placebo and AdoMet. The primary endpoint, change in Hcy, was not significantly different between the groups (mean (umol/L), baseline: 7.43 (placebo), 8.25 (AdoMet), P=0.358; 4 week: 7.66 (placebo), 8.06 (AdoMet), P = 0.683; Baseline − 4 week: 0.23 (placebo), −0.19 (AdoMet), P = 0.427). No statistically significant difference in change in Hcy or hsCRP at 2 or 4 weeks was noted. This was true for both absolute differences as well as relative percent changes. A small decrease in ALT was observed at 2 weeks in the AdoMet group compared to the placebo group (P = 0.027). AdoMet is used in the treatment of liver diseases. There was a small, but statistically significant (P = 0.028) decrease in total cholesterol in the AdoMet group as compared to the placebo group. Interestingly, a subject with the highest baseline Hcy level had a decline in Hcy on AdoMet. Study limitations include no evaluation of AdoMet serum levels or measurement of the effect of AdoMet on DNA methylation patterns.CONCLUSIONS: AdoMet seems well tolerated and in a dose of 800 mg/day for 4 weeks does not appear to significantly affect Hcy levels in the blood. Future clinical trials of AdoMet should monitor Hcy levels with extended use of AdoMet to confirm its safety with long term use.Clinicaltrials.gov ID: NCT00284011.

Exogenous AdoMet and its analogue sinefungin differentially influence DNA cleavage by R.EcoP15I—Usefulness in SAGE

While it has been demonstrated that AdoMet is required for DNA cleavage by Type III restriction enzymes, here we show that in the presence of exogenous AdoMet, the head-to-head oriented recognition sites are cleaved only on a supercoiled DNA. On a linear DNA, exogenous AdoMet strongly drives methylation while inhibiting cleavage reaction. Strikingly, AdoMet analogue sinefungin results in cleavage at all recognition sites irrespective of the topology of DNA. The cleavage reaction in the presence of sinefungin is ATP dependent. The site of cleavage is comparable with that in the presence of AdoMet. The use of EcoP15I restriction in presence of sinefungin as an improved tool for serial analysis of gene expression is discussed.

Involvement of S-adenosylmethionine in G1 cell-cycle regulation in Saccharomyces cerevisiae.

S-adenosyl-l-methionine (AdoMet) is a molecule central to general metabolism, serving as a principal methyl donor for methylation of various cellular constituents. The alteration in the availability of AdoMet has profound effect on cell growth. A mutant allele of Saccharomyces cerevisiae gene SAH1 encoding S-adenosyl-l-homocysteine (AdoHcy) hydrolase, was isolated as a mutation that suppressed the Ca(2+)-sensitive phenotypes of the zds1Delta strain, such as the Ca(2+)-induced, Swe1p- and Cln2p-mediated G(2) cell-cycle arrest, and polarized bud growth. The mutation (sah1-1) led the cells to accumulate AdoMet besides AdoHcy, the substrate of Sah1p. The cells treated with exogenous AdoMet and AdoHcy had markedly decreased levels of SWE1 and CLN2 mRNA, providing the basis for the suppression of the Ca(2+) sensitivity by the sah1-1 mutation. Exogenous AdoMet transiently led the cells to G(1) cell-cycle delay whereas AdoHcy caused growth inhibition irrelevant to the cell cycle. The effect of AdoMet in inducing the cell-cycle delay was exerted in a manner independent of Met4p, an overall transcriptional activator for MET genes. Our observation provides an insight into the role played by AdoMet in cell cycle regulation.

S-adenosylmethionine (AdoMet) modulates endotoxin stimulated interleukin-10 production in monocytes.

IL-10 is produced by a large variety of cells including monocytes, macrophages, B and T lymphocytes, as well as natural killer cells and is an important suppressor for both immunoproliferative and inflammatory responses. IL-10 exerts antifibrotic effects in the liver, and decreased monocyte synthesis of IL-10 is well documented in alcoholic cirrhosis. Intracellular deficiency of S-adenosylmethionine (AdoMet) is a hallmark of toxin-induced liver injury. Although the administration of exogenous AdoMet attenuates this injury, the mechanisms of its actions are not fully established. This study was performed to investigate the effect of exogenous AdoMet on IL-10 production in LPS-stimulated RAW 264.7 cells, a murine macrophage cell line. Our results demonstrated that exogenous AdoMet administration enhanced both protein production and gene expression of IL-10 in RAW 264.7 cells. Ethionine, an inhibitor for methionine adenosyltransferases, inhibited LPS-stimulated IL-10 both at the protein and mRNA levels. Exogenous AdoMet increased the intracellular cAMP concentration as early as 3 h and continued for 24 h after AdoMet treatment; however, the inhibitors for both adenylyl cyclase and PKA did not significantly affect IL-10 production. On the basis of these results, we conclude that AdoMet administration may exert its anti-inflammatory and hepatoprotective effects, at least in part, by enhancing LPS-stimulated IL-10 production.

S-Adenosylmethionine, cytokines, and alcoholic liver disease

Hepatic deficiency of S-adenosylmethionine (AdoMet) is a critical acquired metabolic abnormality in alcoholic liver disease (ALD) and in many experimental models of hepatotoxicity. Subnormal AdoMet, elevated serum tumor necrosis factor (TNF), and endotoxemia (LPS) are hallmarks of ALD and experimental liver injury. AdoMet deficiency is attributed to its subnormal synthesis, but mechanisms for increased TNF are not known. AdoMet deficiency may affect the critical balance of proinflammatory (e.g., TNF) and antiinflammatory [e.g., interleukin (IL)-10] cytokines. Rats maintained on a choline-deficient diet with limited amounts of methionine (MCD diet) developed AdoMet deficiency. When challenged with LPS, rats fed MCD diet had significantly increased serum TNF levels and worse liver injury compared with findings for controls. Exogenous AdoMet attenuated liver injury and serum TNF levels. Results of in vitro studies with the use of RAW 264.7 cells demonstrated that exogenous AdoMet supplementation lowered LPS-induced TNF formation in a dose-dependent manner, and AdoMet deficiency enhanced TNF secretion and TNF gene expression. AdoMet also dose-dependently decreased LPS-stimulated TNF production from monocytes obtained from patients with alcoholic hepatitis. Finally, AdoMet supplementation stimulated production of the antiinflammatory cytokine IL-10. Interleukin-10 plays a critical role in the modulation of TNF production, and IL-10 may inhibit hepatic fibrosis. This article will review (1) the role of AdoMet in ALD/liver injury, (2) the role of TNF/proinflammatory cytokines in ALD, (3) potential roles of AdoMet in TNF/proinflammatory cytokine regulation in ALD, and (4) conclusions and future directions.

S-Adenosylmethionine attenuates the lipopolysaccharide-induced expression of the gene for tumour necrosis factor α

Intracellular deficiency of S-adenosylmethionine (AdoMet) and elevated serum concentrations of tumour necrosis factor alpha (TNF) are hallmarks of toxin-induced liver injury. In these models, the administration of either exogenous AdoMet or antibody/soluble receptor for TNF attenuates the injury. We have demonstrated previously that the administration of exogenous AdoMet to AdoMet-deficient rats attenuated lipopolysaccharide (LPS)-induced liver injury and serum TNF concentrations. Here we report that AdoMet lowered the amount of TNF secreted by LPS-stimulated murine macrophage cells (RAW 264.7) in a dose-dependent manner. The inhibition of TNF release was correlated with changes in the steady-state TNF mRNA concentrations. Changes in TNF mRNA were not due to its altered stability and might have been due to an attenuation of the transcription rate of the TNF gene. The inhibition of TNF release in RAW cells was not mediated by GSH because treatment with AdoMet did not increase intracellular GSH. In addition, N-acetylcysteine, whereas it did increase GSH concentration, had no effect on LPS-stimulated TNF release in these cells. Exogenous AdoMet also attenuated LPS-induced serum TNF levels in normal rats sensitized with lead. Thus AdoMet administration might exert its hepatoprotective effects at least in part by its inhibitory effect on expression of the gene for TNF.

Kinetics ofS-Adenosylmethionine Cellular Transport and Protein Methylation inTrypanosoma brucei bruceiandTrypanosoma brucei rhodesiense

African trypanosomes of theTrypanosoma bruceigroup are agents of disease in man and animals. They present unique biochemical characteristics such as the need for preformed purines and have extensive salvage mechanisms for nucleoside recovery. In this regard we have shown that trypanosomes have a dedicated transporter forS-adenosylmethionine (AdoMet), a key metabolite in transmethylation reactions and polyamine synthesis. In this study we compared the apparent kinetics of AdoMet transport, cytosolic AdoMet pool formation, and utilization of AdoMet in protein methylation reactions using two isolates:Trypanosoma brucei brucei,a veterinary parasite, andTrypanosoma brucei rhodesiense,a human pathogen that is highly refractory and has greatly reduced susceptibility to standard trypanocidal agents active againstT. b. brucei.The apparentKmvalues for [methyl-3H]AdoMet transport, derived by Hanes–Woolf analysis, forT. b. bruceiwas 4.2 and 10 mM forT. b. rhodesiense,and theVmaxvalues were 124 and 400 μmol/liter/min, respectively. Both strains formed substantial cytosolic pools of AdoMet, 1600 nmol/109T. b. bruceiand 3500 nmol/109T. b. rhodesienseafter 10 min incubation with 25 mM exogenous AdoMet. Data obtained from washed trichloroacetic acid precipitates of cells incubated with [methyl-3H]AdoMet indicated that the rate of protein methylation inT. b. bruceiwas fourfold greater than inT. b. rhodesiense.These results demonstrate that the unique rapid uptake and utilization of AdoMet by African trypanosomes is an important consideration in the design and development of new agents of potential use in chemotherapy.

S-adenosylmethionine deficiency and TNF-alpha in lipopolysaccharide-induced hepatic injury.

S-adenosylmethionine (Adomet) is a substrate for de novo synthesis of choline. Adomet deficiency occurs in certain types of liver injury, and the injury is attenuated by exogenous Adomet. Tumor necrosis factor-alpha (TNF-alpha) is also a mediator of these models of hepatotoxicity. We investigated the role of Adomet in lipopolysaccharide (LPS)-induced liver injury in rats made deficient in both Adomet and choline. Rats were maintained on either a methionine-restricted and choline-deficient (MCD) diet or a diet containing sufficient amounts of all nutrients [methionine and choline sufficient (MCS)] and then administered either LPS or saline. MCS-LPS rats had normal liver histology and no change in serum transaminases compared with the MCS-saline control group. MCD-saline rats had hepatosteatosis but no necrosis, and a five- to sevenfold increase in transaminases vs. the MCS-saline group. MCD-LPS rats additionally had hepatonecrosis and a 30- to 50-fold increase in transaminases. Exogenous Adomet administration to MCD-LPS rats corrected the hepatic deficiency of Adomet but not of choline, prevented necrosis but not steatosis, and attenuated transaminases. Serum TNF-alpha was sixfold higher in MCD rats even without LPS challenge and 300-fold higher with LPS challenge. Exogenous Adomet attenuated increased serum TNF-alpha in MCD-LPS rats.

Novel evidence for an ecto-phospholipid methyltransferase in isolated rat hepatocytes.

Phospholipids of isolated rat hepatocytes were labelled by preincubation with either 2 microM -methyl-14C-S-adenosylmethionine (AdoMet) or 2 microM [methyl-14C]methionine. Subsequent addition of phospholipase C to the suspension removed 95% of the radioactivity from phospholipids methylated by [methyl-14C]AdoMet within a few minutes, but was without effect on phospholipids methylated by [methyl-14C]methionine radioactivity from the latter could, nevertheless, be removed by phospholipase C after permeabilization of the cells with digitonin. The results clearly show that the methyl group of exogenous AdoMet, contrary to that of methionine, is transferred on to phospholipids located on the external face of the plasma membrane. Accordingly, pretreatment of isolated hepatocytes with trypsin prevented the methylation of phospholipids from exogenous AdoMet by 60-80%, whereas it was almost without effect when exogenous methionine was the methyl donor. Our data corroborate previous work [Bontemps and Van den Berghe (1997) Biochem. J. 327, 383-389], which indicated that AdoMet methylates hepatocyte phospholipids without penetrating the cells.

Metabolism of exogenous S-adenosylmethionine in isolated rat hepatocyte suspensions: methylation of plasma-membrane phospholipids without intracellular uptake.

Administration of S-adenosylmethionine (AdoMet), the main biological methyl donor, has been shown to exert favourable effects on liver disorders in man and animal models. The mechanism of action of AdoMet has, however, remained elusive, mainly owing to controversies with respect to its capacity to enter intact liver cells. Incubation of isolated rat hepatocytes with 2 or 50 microM -methyl-14C-AdoMet showed that it was utilized predominantly to methylate cellular phospholipids, forming mainly phosphatidylcholine, although less than 0.2% of labelled AdoMet was found inside the cells. The concentration of neither AdoMet nor S-adenosylhomocysteine (AdoHcy), its demethylation product, was significantly elevated inside the cells. A slight elevation of intracellular AdoMet was only recorded on incubation with concentrations of AdoMet above 200 microM. AdoHcy, which does not penetrate cells, inhibited phospholipid methylation from [methyl-14C]AdoMet but not from [methyl-14C]Met. Elevation of intracellular AdoHcy by adenosine dialdehyde, an inhibitor of AdoHcy hydrolase, inhibited phospholipid methylation from [methyl-14C]Met, but virtually not at all from [methyl-14C]AdoMet. Taken together, these data indicate that exogenous AdoMet does not penetrate hepatocytes significantly but is utilized for phospholipid methylation on the outer surface of the plasma membrane.

Effects of carboxylmethylation and polyamine synthesis inhibitors on methylation of Trypanosoma brucei cellular proteins and lipids.

The fate of methionine in eukaryotic cells is divided between protein synthesis and the branched pathway encompassing polyamine synthesis, methylation of proteins and lipids, and transsulphuration reactions. Aside from protein synthesis, the first step to all other uses of methionine is conversion to S-adenosylmethionine. Blockade of polyamine synthesis in African trypanosomes by the ornithine decarboxylase inhibitor DL-alpha-difluoromethylornithine (Ornidyl, DFMO) the AdoMet decarboxylase inhibitor 5'-[[(Z)-4-amino-2-butenyl]-methylamino]-5'-deoxyadenosine or the protein methylase inhibitor sinefungin induces dramatic increases in intracellular AdoMet. In a previous study, distribution and pool sizes of [35S] or [U-14C]methionine were followed in bloodform trypanosomes as incorporation into the total TCA precipitable fractions. In the present study, the effects of pretreatment with DFMO (1 mM), MDL 73811 (1 microM) and sinefugin (2 nM) on [35S] and [U-14C]methionine incorporation were studied in blood forms. DFMO or MDL 73811 pretreatment increased protein methylation 1.5-fold through incorporation of [U14C]methionine, while sinefungin caused a 40% reduction of incorporation. The increases in incorporation of [U-14C]methionine due to DFMO and MDL 73811 were reduced 40% to 70% by including cold AdoMet (1 mM) in the incubation medium, an indication of AdoMet transport by bloodform trypanosomes and the utilization of [U-14C]methionine as AdoMet. Exogenous AdoMet had no effect on [35S]methionine incorporation. The agents studied are curative for African trypanosomiasis infections, either clinically (DFMO) or in model infections (MDL 73811, sinefungin) and thus highlight interference with AdoMet metabolism and methylation reactions as biochemical consequences of these agents.

Inhibition of S-Adenosyl-L-Methionine (AdoMet) Decarboxylase by the Decarboxylated AdoMet Analog 5'-[(Z)-4-Amino-2-Butenyl]Methylamino-5'-Deoxyadenosine (MDL 73811) Decreases the Capacities of Trypanosoma cruzi to Infect and Multiply within a Mammalian Host Cell

A decarboxylated S-adenosyl-L-methionine (AdoMet) analog, 5'-([(Z)-4-amino-2-butenyl]methylamino)-5'-deoxyadenosine (MDL 73811), that specifically and irreversibly inhibits AdoMet decarboxylase (DC) was used to investigate the role of AdoMetDC in the regulation of host cell invasion and intracellular replication by Trypanosoma cruzi. The presence of MDL 73811 in cocultures of T. cruzi and rat heart myoblasts (RHM) significantly inhibited host cell infection in a dose-dependent manner. This effect, evidenced by reductions in the proportion of infected RHM and the number of parasites per 100 RHM, was due to MDL 73811 action on T. cruzi as it was reproduced when the parasites, but not the RHM, were pretreated with the inhibitor. Significant inhibition of infectivity required a 10-min treatment with MDL 73811 although greater effects ensued with additional incubation time. We could not detect signs of recovered infectivity up to 6 hr after the removal of nonincorporated MDL 73811, suggesting that either AdoMetDC turnover in T. cruzi is a relatively slow process or the amounts of MDL 73811 taken up could not be blocked or eliminated during this time period. MDL 73811-mediated inhibition of infectivity was not bypassed by the addition of exogenous spermidine or spermine, suggesting that the mechanism of action does not involve reduced production of these polyamines due to AdoMetDC inhibition. Intracellular accumulation of AdoMet appeared to be a more plausible explanation in view of the fact that exogenous AdoMet significantly inhibited infectivity. When added to infected RHM cultures, MDL 73811 or AdoMet inhibited also intracellular T. cruzi growth.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification, crystallization, and preliminary X-ray diffraction analysis of an M.HhaI-AdoMet complex.

The type-II DNA-(cytosine-5)-methyltransferase M.HhaI was overexpressed in Escherichia coli and purified to apparent homogeneity. The purification scheme exploits a unique high salt back-extraction step to solubilize M.HhaI selectively, followed by FPLC chromatography. The yield of purified protein was 0.75-1.0 mg per gram of bacterial paste. M.HhaI could be isolated in two forms: bound with its cofactor S-adenosylmethionine (AdoMet) or devoid of the cofactor. The AdoMet-bound form was capable of methylating DNA in vitro in the absence of exogenous AdoMet. From kinetic studies of the purified enzyme, values for KmAdoMet (60 nM), KiAdoHye (0.4 nM), and Kcat (0.22 s-1) were determined. The purified enzyme bound with its cofactor was crystallized by the hanging drop vapor diffusion technique. Crystals were of monoclinic space group P2(1) and had unit-cell dimensions of a = 55.3 A, b = 72.7 A, c = 91.0 A, and beta = 102.5 degrees, with two molecules of M.HhaI in each of the two asymmetric units. The crystals diffract beyond 2.5 A and are suitable for structure determination.

Modulation of polyamine-biosynthetic activity by S-adenosylmethionine depletion.

The methionine-analogue inhibitor of S-adenosylmethionine (AdoMet) synthetase, L-2-amino-4-methoxy-cis-but-3-enoic acid (L-cisAMB), was used to study the early effects of AdoMet depletion on polyamine biosynthesis. In the presence of decreased methionine (30 microM) in the medium, treatment of cultured L1210 cells with 1 mM-L-cisAMB resulted in a near-total (95%) depletion of cellular AdoMet pools by 4 h. This was accompanied by a 3-fold increase in ornithine decarboxylase (ODC) activity, a 2.5-fold increase in AdoMet decarboxylase (AdoMetDC) activity and a 20% decrease in spermidine and spermine pools. The increase in enzyme activities seemed to be partially due to prolongation of enzyme activity half-life, since that of ODC was extended from 30 to 50 min and that of AdoMetDC from 65 to 310 min. By temporal sequence characterization (0-4 h), the onset of elevations of enzyme activity (0.5-1 h) seemed to be causally related to an earlier (0-0.5 h) decline in AdoMet pools, as opposed to the 20% decrease in spermidine and spermine pools, which occurred much later (2-4 h); the latter are known to regulate decarboxylase activities negatively. Drug-induced elevations in ODC and, to a lesser extent, AdoMetDC activities were reversed by later treatment with exogenous AdoMet. However, because the latter also increased spermine pools (which could not be prevented with various enzyme inhibitors), the reversal of elevations in enzyme activities could not be directly linked to AdoMet. Although not definitive, the data raise the interesting possibility that, in addition to being negatively regulated by polyamines, ODC and AdoMetDC activities may also be subject to negative control by cellular AdoMet (or an AdoMet metabolite). The net effect of either or both of these influences would be to conserve polyamine-biosynthetic activity in the face of declining AdoMet supplies.