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Nutrient Interactions and Toxicity
Research Communication

Betaine Lowers Elevated S-Adenosylhomocysteine Levels in Hepatocytes from Ethanol-Fed Rats¹

Anthony J. Barak^*,,2, Harriet C. Beckenhauer^*, Mark E. Mailliard^*,, Kusum K. Kharbanda^*, and Dean J. Tuma^*,

^* VA Alcohol Research Center, Department of Veterans Affairs Medical Center, Omaha, NE 68105 and Department of Internal Medicine, University of Nebraska Medical Center, Omaha, NE 68198

²To whom correspondence should be addressed. E-mail: ajbarak{at}yahoo.com.

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Previous studies showed that chronic ethanol administration inhibits methionine synthase activity, resulting in impaired homocysteine remethylation to form methionine. This defect in homocysteine remethylation was shown to increase plasma homocysteine and to interfere with the production of hepatic S-adenosylmethionine (SAM) in ethanol-fed rats. These changes were shown to be reversed by the administration of betaine, an alternative methylating agent. This study was undertaken to determine additional effects of ethanol on methionine metabolism and their functional consequences. The influences of methionine loading and betaine supplementation were also evaluated. Adult Wistar rats were fed ethanol or a control Lieber-DeCarli liquid diet for 4 wk, and metabolites of the methionine cycle were measured in vitro in isolated hepatocytes under basal and methionine-supplemented conditions. S-Adenosylhomocysteine (SAH) concentrations were elevated in hepatocytes isolated from ethanol-fed rats compared with controls and in hepatocytes from both groups when supplemented with methionine. The addition of betaine to the methionine-supplemented incubation media reduced the elevated SAH levels. The decrease in the intracellular SAH:SAM ratio due to ethanol consumption inhibited the activity of the liver-specific SAM-dependent methyltransferase, phosphatidylethanolamine methyltransferase. Our data indicate that betaine, by remethylating homocysteine and removing SAH, overcomes the detrimental effects of ethanol consumption on methionine metabolism and may be effective in correcting methylation defects and treating liver diseases.

KEY WORDS: • hepatocytes • ethanol • S-adenosylhomocysteine • betaine • phosphatidylethanolamine methyltransferase

Previous studies have shown that chronic ethanol administration alters methionine metabolism in the liver (1,2). Inhibition of methionine synthase (MS)² activity appears to be a major defect elicited by ethanol consumption, resulting in impaired remethylation of homocysteine to form methionine (2,3). This defect ultimately leads to a decrease in hepatic levels of the universal methylating agent, S-adenosylmethionine (SAM) (2,4,5) and impaired methylation reactions (6). In addition, increased homocysteine, a potentially toxic agent, is generated and released from the liver as a consequence of impaired MS activity (7). Further studies showed that betaine, which can also provide methyl groups for the remethylation of homocysteine via betaine-homocysteine-methyltransferase (BHMT), prevented the ethanol-induced changes in methionine metabolism by restoring hepatic SAM levels (5,8) and by preventing the increased release of homocysteine by the liver (7). Furthermore, betaine was also shown to prevent and reverse ethanol-induced hepatic steatosis (5,8).

The purpose of the present study was to continue our work examining the ethanol-induced changes of hepatic methionine metabolism and the ability of betaine to reverse and/or counteract these alterations. Here, we investigated the effects of ethanol on hepatocyte S-adenosylhomocysteine (SAH) levels and the influence of betaine on these effects. Examination of SAH levels is important because SAH is the metabolic precursor of homocysteine and is formed as a product of methyl transfer reactions involving SAM (9). In addition, SAH has been shown to be a potential inhibitor of methyltransferases; therefore, it is an important regulator of methylation reactions in the cell (10,11).

Because dietary methionine was shown to elevate plasma homocysteine levels (12) and methionine loading in fasting patients is used to stress homocysteine pathways to reveal disturbances in methionine metabolism (13), the effect of methionine on hepatocyte levels of SAM and SAH in control and ethanol-fed rats was also studied. In addition, the effect of betaine under these conditions was investigated.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Diet formulation.

Nutritionally adequate liquid diets were formulated according to the method of Lieber and DeCarli (14) and purchased from Dyets (Bethlehem, PA). The ethanol-containing diet consisted of 18% of total energy as protein, 35% as fat, 11% as carbohydrate and 36% as ethanol. All control rats were pair-fed the same diet as the ethanol-fed rats except that ethanol was replaced isoenergetically with carbohydrate. Both ethanol-fed and control rats ingested identical amounts of all nutrients except carbohydrates.

Ethanol feeding procedure.

Male Wistar rats (Charles River Laboratories, Wilmington, MA) weighing 150–175 g were initially fed the Purina 5001 diet (Ralston Purina, St. Louis, MO) until they reached body weights of 200–220 g; they were then divided into two groups. The rats were housed individually and acclimated to the control diet for 3 d. These rats were then weight-matched and paired so that one rat received the liquid diet containing ethanol as 36% of total energy and the second was pair-fed the isoenergetic control diet. As with all of our previous studies with betaine, the rats were pair-fed for 4 wk. During the 24 h before isolation of hepatocytes, the liquid diets were fed in three portions: 25% at 0900 h, 50% at 1600 h and the final 25% at 0700 h. This meal-feeding regimen was used to minimize variations in feeding patterns between the ethanol-fed rats and their pair-fed controls before isolation of hepatocytes. The care and the use of the rats as well as all procedures were approved by the Institutional Animal Care and Use Committee at the Omaha Veterans Affairs Medical Center and were in accordance with the guidelines of the Institutional Care and Use Committee of the National Institute on Drug Abuse and the NIH (15).

Isolation of hepatocytes.

Hepatocytes were obtained from the livers of control and ethanol-fed rats by a modified collagenase-perfusion technique previously described by our laboratory (16). Viabilities of the different cell populations were determined by trypan blue exclusion, and only cell preparations attaining a viability of >85% were used. Cell suspensions (1 x 10⁹ cells/L) were then incubated in Kreb’s-Hensleit buffer containing 12.5 mmol/L HEPES and 2.5 mmol/L Ca⁺⁺ under an atmosphere of 95% O₂/5% CO₂. Before any treatment, cells were equilibrated for 30 min at 37°C. After this period, the cells were incubated for 4 h in the absence and presence of L-methionine (0.5 mmol/L), betaine (1.0 mmol/L) or methionine (0.5 mmol/L) plus betaine (1.0 mmol/L) at 37°C.

Measurement of SAM and SAH.

After incubation, hepatocyte suspensions were immediately centrifuged (150 x g for 5 min) to pellet the cells. The cell pellets were then treated with 0.5 mol/L HClO₄, and the resulting supernatants were filtered through a Millipore membrane (0.22 µm; Millipore, Bedford, MA). The filtered acid extracts were subjected directly to HPLC for the determination of hepatocellular SAM and SAH levels, using the method described by Fu et al. (17).

Preparation of the hepatocyte microsomal fraction.

Hepatocytes, isolated from rats fed the Purina diet, were sonicated in ice-cold 10 mmol/L Tris HCl (pH, 7.4) containing 0.25 mol/L sucrose. The cell lysate was centrifuged at 16,000 x g for 20 min at 4°C and the supernatant was centrifuged again at 105,000 x g for 60 min at 4°C. The pellet of this last centrifugation (microsomal fraction) was resuspended in an appropriate volume of 0.25 mol/L sucrose, and protein content determined by the BCA (bicinchoninic acid) protein assay (Pierce, Rockford, IL) using bovine serum albumin (BSA) as the standard.

Phosphatidylethanolamine methyltransferase (PEMT) assay.

The PEMT activity in the microsomal fraction was determined by measuring the incorporation of [³H]methyl group from S-adenosyl-L-(methyl-³H) with endogenous microsomal phosphatidylethanolamine (PE) as the substrate (18). The concentration of SAM was kept constant (100 µmol/L). The concentration of SAH was varied to generate SAM:SAH ratios from 2.5 to 25, which correspond to the ratios in the hepatocytes of control or ethanol-fed rats under basal conditions and after methionine load and betaine administration. Briefly, the assay mixture (in a total volume of 500 µL) contained 10 mmol/L HEPES, pH 7.3, 0.25 mmol/L dithiothreitol, 5 mmol/L MgCl₂, 100 µmol/L S-adenosyl-L-methionine, 74 kBq S-adenosyl-L-(methyl-³H)-methionine and 500 µg microsomal protein. SAH was added in varying amounts to the assay mixtures such that the ratio of SAM:SAH in the mixture was 2.5, 5, 7.5, 10 or 25. The reaction was initiated by the addition of a mixture of unlabeled and labeled S-adenosyl-L-(methyl-³H)-methionine, followed by a 10-min incubation at 37°C and was terminated by pipetting 100 µL of assay mixture in duplicate into 2 mL of chloroform/methanol/2 mol/L HCl (6:3:1) for lipid extraction. The aqueous phase was aspirated after centrifugation at 1800 x g for 5 min and the chloroform phase washed three times with 1 mL of 0.5 mol/L KCl in 50% methanol. After the last wash, the entire chloroform phase was pipetted into a counting vial, dried under a stream of nitrogen, dissolved into 8 mL of scintillation liquid and counted. The PEMT activity was expressed as pmol methylated PE products/(min · mg microsomal protein).

Statistics.

Results are expressed as means ± SEM. The data were analyzed by two-way ANOVA. Planned comparisons of treatment means were evaluated by direct contrasts of means using the statistical computer program Systat 9.0 (Richmond, CA). Differences between means were considered significant when P < 0.05. A one-tailed test was used for postulated unidirectional effects. Univariate repeated-measures ANOVA was used for determining the effects of different ratios on PEMT activity.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
After 4 wk of dietary treatment, SAM levels in freshly isolated hepatocytes from the ethanol-fed rats tended to be lower than in the pair-fed controls [F(1,4) = 1.06, P = 0.36, Table 1]. Incubation of hepatocytes in the presence of betaine [F(1,28) = 23.13, P < 0.0001) or methionine [F(1,28) = 131.22, P < 0.0001) markedly increased the levels of SAM in both groups. The effects of betaine or methionine on SAM levels were similar in both hepatocyte types. The addition of betaine to the methionine-containing incubation media had no further influence on SAM levels in cells from either the ethanol-fed or control rats [significant interaction between methionine and betaine F(1,28) = 16.78, P < 0.0005 (Table 1)].

View larger version (30K): [in this window] [in a new window]   FIGURE 1 Effect of the ratio of S-adenosylmethionine (SAM) to S-adenosylhomocysteine (SAH) on rat liver phosphatidylethanolamine methyltransferase (PEMT) activity. Data are expressed as pmol methylated phosphatidylethanolamine (PE) products formed/(min · mg microsomal protein) and are means ± SEM, n = 6. *Different from the SAM:SAH ratio of 5.0, P < 0.05; #Different from the SAM:SAH ratio of 2.5, P < 0.01.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The results of this study identified another consequence of the ethanol-induced alterations of methionine metabolism. We observed a marked increase in SAH levels in hepatocytes from ethanol-fed rats, and these SAH levels were further elevated when the hepatocytes were incubated in the presence of methionine. In agreement with these results, others have also reported increases in SAH levels in livers of ethanol-fed rats (22,23). Elevated levels of SAH could have detrimental effects in the cell because this metabolite, rather than SAM, is an important indicator and regulator of cellular methylation status (10,11). The potential pathogenicity of intracellular SAH lies in its high affinity binding to the catalytic region of most SAM-dependent methyltransferases, enabling it to act as a potent product inhibitor, with K_i in the submicromolar to low micromolar range. In fact, the K_i for SAH is often less than the K_m for SAM for many of the methyltransferases (24). Consistent with this, we demonstrated in this study that decreasing the SAM:SAH ratio by increasing the SAH levels under conditions of constant levels of SAM significantly decreased the activity of microsomal PEMT. Recent reports (25,26) suggested an important role for the phosphatidylcholine generated via the PEMT pathway in the formation of VLDL and transport of triglycerides from the liver. One of the earliest manifestations of ethanol-induced liver injury is the accumulation of triglycerides in the liver. Our present study supports the hypothesis that the decrease in the activity of PEMT due to the decreased intracellular SAM:SAH ratio in hepatocytes of ethanol-fed rats (leading to a defect in secretion of VLDL) may be responsible in part for the hepatic steatosis seen in these rats. The decrease in the SAM:SAH ratio may also be responsible for the impaired DNA methylation reported in rats fed intragastrically with ethanol and a high fat diet (21) by affecting the SAM-dependent DNA methyltransferases.

From the above, it is clear that removal of hepatic SAH is essential to correct the SAM:SAH ratio and the resultant defect in the transport of triglycerides from hepatocytes. The reaction that converts SAH to homocysteine and adenosine is reversible and catalyzed by SAH hydrolase. The thermodynamics of the reaction favor SAH synthesis over homocysteine synthesis; however, in vivo, the reaction proceeds toward hydrolysis only if the products are removed (9). The efficient removal of homocysteine also allows for the efficient removal of SAH. The most important finding of this study is that betaine, by remethylating homocysteine, is very effective in reducing the ethanol-induced elevation of SAH levels in isolated hepatocytes. Our previous study showed that betaine administration ameliorated ethanol-induced steatosis (8). On the basis of the results of this study, it is likely that betaine reduces the elevated SAH levels and corrects the defect in PEMT activity. Thus, in addition to promoting the generation of hepatic SAM, lowering homocysteine release from the liver and ameliorating steatosis (5,7,8), betaine is also effective in attenuating the elevation of SAH levels in the liver, increasing the SAM:SAH ratio and ultimately correcting the methylation defect elicited by chronic ethanol consumption. It is the decrease in the SAM:SAH ratio that appeared to be responsible for the reduced phosphatidylcholine synthesis and resultant steatosis and may also have been responsible for the altered signaling and genomic hypomethylation reported after ethanol consumption (21).

Despite the decrease in SAH levels when hepatocytes were incubated in the presence of both methionine and betaine, SAM levels did not increase correspondingly. This apparent paradoxical effect occurs because reduced SAH levels would result in decreased inhibition of hepatic methyltransferases, thus allowing increased utilization of SAM for methylation reactions. In addition, when hepatocytes from ethanol-fed rats were incubated in the presence of methionine, betaine reduced the increase in SAH levels but did not restore them to the low levels of controls (no methionine load). A likely explanation for these results is that under the stress of a methionine load in hepatocytes from ethanol-fed rats, betaine-elicited remethylation of homocysteine via BHMT is the only effective pathway operable because the alternate pathway for remethylation via MS is impaired by ethanol.

Overall, our data indicate that betaine, by virtue of its ability to remethylate homocysteine, lowers SAH levels, which in turn corrects defective cellular methylation and the resultant sequela of events of steatosis, DNA hypomethylation and altered signaling. Thus, betaine could be very effective in treating liver diseases as well as other diseases associated with elevated homocysteine and defective methylation.

	ACKNOWLEDGMENTS

 
The authors thank Gerri Siford for her excellent technical assistance and Roger D. Reidelberger for his guidance and assistance in the data analysis.

	FOOTNOTES

 
¹ Supported by the Department of Veterans Affairs Alcohol Research Center, Omaha, NE.

³ Abbreviations used: BHMT, betaine-homocysteine-methyltransferase; BSA, bovine serum albumin; MS, methionine synthase; PE, phosphatidylethanolamine; PEMT, phosphatidylethanolamine methyltransferase; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine.

Manuscript received 8 April 2003. Initial review completed 4 May 2003. Revision accepted 16 June 2003.

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TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 

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