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Research Communication

Retinoid Compounds Activate and Induce Hepatic Glycine N-Methyltransferase in Rats^{1 ,2}

Department of Food Science and Human Nutrition, Iowa State University, Ames, IA 50011

³To whom correspondence should be addressed. E-mail: kschalin{at}iastate.edu

	ABSTRACT

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Glycine N-methyltransferase (GNMT) functions to regulate S-adenosylmethionine (SAM) levels and the ratio of SAM/S-adenosylhomocysteine (SAH). SAM is a universal methyl group donor and the up-regulation of GNMT may lead to wastage of methyl groups required for transmethylation reactions. Previously, we demonstrated that dietary treatment of rats with 13-cis-retinoic acid (CRA) decreased the hepatic concentration of SAM and the SAH ratio. Here, we examined the ability of CRA, as well as all-trans-retinoic acid (ATRA), to regulate hepatic GNMT as a potential basis for our earlier observations. Rats were fed either a control (10% casein + 0.3% L-methionine) diet or a control diet supplemented with L-methionine (10 g/kg diet). Rats from each group were orally given ATRA, CRA (both at 30 µmol/kg body), or vehicle daily for 7 d. For control rats, administration of both CRA and ATRA elevated the hepatic GNMT activity 49% and 34%, respectively, compared with the control group. Similar results were exhibited by rats fed the methionine-supplemented diet. Moreover, the retinoid-induced elevations in enzyme activity were reflected in the abundance of GNMT protein. To our knowledge, this is the first report of a nutritional compound that induces GNMT activity at the transcriptional and/or translational level.

KEY WORDS: • retinoic acid • S-adenosylmethionine • transmethylation • glycine N-methyltransferase • rats

	INTRODUCTION

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Methyl group and folate-dependent one-carbon metabolism (Fig. 1 ) are interrelated pathways that play an important role in health and disease. Methyl groups are important substrates for a number of S-adenosylmethionine (SAM)⁴ -dependent transmethylation reactions (1 ,2) and, thus, an adequate supply, derived from the diet and/or the one-carbon pool, is essential. A lack of dietary methyl groups can result in hepatocarcinogenesis (3 ,4) , a consequence that may be related to reduced methylation of DNA by SAM. Likewise, suboptimal folate status and subsequent down-regulation of SAM-dependent transmethylation have been implicated as a potential mechanism in neoplastic development (5) .

View larger version (13K): [in this window] [in a new window]   Figure 1. Interrelationship between methyl group and folate metabolism. Hepatic SAM, a methyl group donor in a number of transmethylation reactions, is produced from methionine, via the diet and from the remethylation of homocysteine in a vitamin B-12-dependent reaction that uses the folate-dependent one-carbon pool as a methyl group source (i.e., 5-methyl-tetrahydrofolate, 5-methyl-THF). SAH, the product of SAM-dependent transmethylation reactions, is a potent inhibitor of most methyltransferases and, thus, the ratio of SAM/SAH is an index of transmethylation potential. SAM is also an allosteric inhibitor of 5,10-methylene-THF reductase, the enzyme that catalyzes the irreversible reduction of 5,10-methylene-THF to 5-methyl-THF. In turn, 5-methyl-THF is an inhibitor of GNMT, a key protein involved in the regulation of transmethylation by controlling the ratio of SAM/SAH. Both of these inhibitory relationships are indicated by the dashed arrows.

Previous work in our laboratory (11 ,12) has demonstrated that administration of the retinoid 13-cis-retinoic acid (CRA) results in conditions indicative of enhanced methionine catabolism. This conclusion is based on our observations that CRA-treated rats exhibited a significant reduction in both the hepatic concentration of SAM and the ratio of SAM/SAH. In an attempt to understand the role of retinoid compounds to alter methionine metabolism and to induce methyl group loss, the work presented here focused on activation of GNMT as a potential mechanism. Moreover, we have extended our work by also evaluating the ability of all-trans-retinoic acid (ATRA), an active form of vitamin A, to perturb methyl group metabolism.

	MATERIALS AND METHODS

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Chemicals and reagents.

Reagents were obtained from the following: S-adenosyl-L-[methyl-³H]methionine, New England Nuclear (Boston, MA); phenylmethylsulfonylflouride, Calbiochem (La Jolla, CA); goat anti-rabbit immunoglobulin G horseradish peroxidase, Southern Biotechnology (Birmingham, AL); ECL Western blotting detection reagents, Amersham Pharmacia (Piscataway, NJ); and S-adenosyl-L-methionine, Sigma Chemical (St. Louis, MO). GNMT antibody was kindly provided by Dr. Conrad Wagner, Vanderbilt University. ATRA and CRA were provided courtesy of Hoffman-LaRoche (Nutley, NJ). All other chemicals were of analytical grade.

Animals and diets.

All animal experiments were approved by and conducted in accordance with Iowa State University Laboratory Animal Resources Guidelines. Male Sprague-Dawley (Harlan Sprague-Dawley, Indianapolis, IN) rats were housed in suspended wire-mesh cages in a room with a 12-h light:dark cycle and given free access to food and water. The composition of the control diet was the same as previously described (11) . The methionine-supplemented (MS) diet contained additional L-methionine (10 g/kg diet) at the expense of glucose monohydrate.

After an 11-d acclimation period during which rats were adapted to both the control diet and the oral administration of corn oil, they were divided into six treatment groups consisting of five rats per group. Rats were fed one of the two diets (control or MS) and were orally given vehicle (corn oil, 1 µL/g body), vehicle containing CRA, or vehicle containing ATRA daily. Both retinoids were administered at a level of 30 µmol/kg of body. After the 7-d treatment period, rats were anesthetized and liver samples were rapidly removed for analysis.

Measurement of GNMT activity.

The enzymatic activity of GNMT was assayed as described by Cook and Wagner (13) with minor modifications. Portions of liver were homogenized in three volumes of ice-cold phosphate buffered (10 mmol/L, pH 7.0) sucrose (0.25 mol/L) containing 1 mmol/L EDTA, 1 mmol/L sodium azide, and 0.1 mmol/L phenylmethylsulfonylflouride. After centrifugation at 20,000 x g for 30 min, the resulting supernatant was removed and 2-mercaptoethanol was added to a final concentration of 10 mmol/L. The assay mixture consisted of 0.1 mol/L Tris buffer (pH 9.0), 5 mmol/L dithiothreitol, 1 mmol/L glycine, and 1 mmol/L S-adenosyl-L-[methyl-³H]methionine (4.77 x 10⁶ Bq/mmol). The reaction was initiated upon addition of 250 µg of sample protein. The assay was linear with respect to time and protein concentration. For the determination of total soluble protein in the tissue extract, a commercial kit (Coomassie Plus, Pierce, Rockford, IL) based on the method of Bradford (14) was used with bovine serum albumin as a standard.

Measurement of GNMT protein.

For the determination of the abundance of GNMT protein, immunoblotting with chemiluminescence detection was used. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was performed using a 10%–20% gradient gel and 75 µg sample protein per lane. After separation, the proteins were electrophoretically transferred to nitrocellulose and the membrane was incubated at room temperature in a blocking solution containing nonfat dry milk (50 g/L) in Tween 20 + Tris-buffered saline (TTBS) buffer consisting of 20 mmol/L Tris, (pH 7.5) and 500 µl/L Tween 20. Affinity-purified polyclonal GNMT antibody in bovine serum albumin (10 g/L)-TTBS (1:1000) was added and the blot was incubated at 4°C overnight. The blot was incubated for 1 h at room temperature with goat anti-rabbit horseradish peroxidase in TTBS (1:5000), followed by a 1-min incubation in Western blot chemiluminescent detection reagents before multiple exposures to autoradiography film. Densitometric analysis was performed using the National Institutes of Health Image software.

Statistical analysis.

The means of each treatment group were subjected to a two-way ANOVA. When the ANOVA was significant (P < 0.05), the means were compared using the Fisher least significant difference procedure (15) .

	RESULTS

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Retinoid treatment did not alter rat growth rates.

Body weights were measured during both the acclimation phase (data not shown) and retinoid treatment period of the study (Fig. 2 ). All rat groups exhibited similar growth patterns regardless of treatment, and no significant differences were detected in either the initial (81 ± 2 g) or final (181 ± 3 g) body weights. As indicated in Figure 2 , no significant differences were observed in the cumulative weight gain across the treatment groups, indicating that neither the MS diet nor the retinoids were overtly toxic to the animals. We have shown previously that administration of CRA can induce hepatic steatosis in rats fed a similar diet (12) . This was also found to be the case in the present study for both CRA and ATRA (data not shown).

View larger version (23K): [in this window] [in a new window]   Figure 2. Administration of CRA and ATRA to rats for 7 d did not alter their growth rates. Data are means ± SEM, n = 5 and were compared across treatment groups at each time point. After an adaptation period, an equal number of rats in both the control and MS (10 g/kg diet) groups were treated with vehicle (corn oil), CRA (30 µmol/kg body) or ATRA (30 µmol/kg body) daily. For all rats (n = 30), mean (± SEM) body weights at treatment d = 0 and d = 7 were 146 ± 3 g and 181 ± 3 g, respectively. NS, not statistically significant, P > 0.05.

Administration of CRA and ATRA consistently elevated the enzymatic activity of hepatic GNMT (Fig. 3 ). For rats fed the control diet, CRA administration increased GNMT activity 49%. ATRA also increased enzyme activity (34%); however, this difference was not significant (P = 0.081). In contrast, both CRA and ATRA significantly induced GNMT activity 41% and 45%, respectively, in rats fed the MS diet. Adding L-methionine alone (10g/kg diet) was without significant effect. Previous studies have demonstrated that induction of GNMT activity by dietary methionine requires a level closer to 20 g/kg diet (16) .

View larger version (38K): [in this window] [in a new window]   Figure 3. Administration of CRA and ATRA to rats increased the hepatic activity of GNMT in both control and MS (10 g/kg diet) rats. Data are means ± SEM, n = 5. Bars without a common letter are significantly different (P < 0.05). All assays were performed in triplicate.

Using immunoblotting with a GNMT antibody, we determined whether the retinoid-induced increase in GNMT activity was due to changes in the production of the enzyme (Fig. 4 ). The bar graph reflects the mean result from all of the experimental animals, whereas the immunoblot shown above is a representative example with the relative fold induction shown for each lane. Both CRA and ATRA markedly induced GNMT protein abundance in control rats and in those receiving the MS diet. The induction of GNMT protein by retinoids across treatment groups ranged from 8- to 16-fold. Similar to the enzyme activity data, the MS diet alone did not significantly alter GNMT protein levels based on mean values. However, it did seem to have some effect as shown in the representative blot (3.1-fold induction). There was no difference in actin protein abundance across the treatment groups. Interestingly, in both the bar graph and representative immunoblot, the effect of the MS diet and retinoids on GNMT protein induction appeared to be additive. As suggested by others (16) , we have directly confirmed that the addition of graded levels of L-methionine to the diet and subsequent induction of GNMT enzyme activity is reflected in an increase in GNMT protein abundance (Rowling and Schalinske, unpublished data).

View larger version (47K): [in this window] [in a new window]   Figure 4. Administration of CRA and ATRA to rats increased the hepatic abundance of GNMT in both control and MS (10 g/kg diet) rats. Data are means ± SEM, n = 5. Bars without a common letter are significantly different (P < 0.05). Above the bar graph is a representative immunoblot with the relative fold induction provided below each lane.

	DISCUSSION

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In addition, we have extended our earlier observations with CRA (11 ,12) by demonstrating that ATRA can alter methionine metabolism and GNMT activity in a similar manner. This has important nutritional implications, because ATRA represents the active form of vitamin A for a number of its functions. The retinoid doses used in this study (30 µmol/kg) are well above the recommended clinical dosage for humans (2–6 µmol/kg) as well as the level of vitamin A provided to rats in the AIN-93G vitamin mix (0.5 µmol/kg). However, we have found in preliminary dose-response studies that retinoid doses as low as 1 µmol/kg are sufficient to significantly induce GNMT (Rowling and Schalinske, unpublished data).

A number of previous studies have reported compounds or conditions that result in an increase in GNMT activity, including ethanol administration (17) and folate deficiency (18) . Although folate deficiency increased GNMT activity as much as 2.6-fold, this increase was not reflected in the abundance of GNMT protein. Wagner and co-workers (7 ,8) have demonstrated that GNMT can be negatively and positively regulated by 5-methyl-THF and phosphorylation, respectively, posttranslationally. Although we have found that retinoids induce GNMT protein abundance and retinoids are known to function at the transcriptional level, it remains a possibility that posttranslational control, such as depletion in 5-methyl-THF, may play a role in this regulation as well. In support of this possibility, it has been shown that vitamin A status can influence the one-carbon pool and the distribution of folate coenzymes, including a depletion of 5-methyl-THF (19 ,20) .

An intriguing possibility exists that the ability of retinoids to activate GNMT is related to the induction of a gluconeogenic condition. Xue and Snoswell (21) reported that GNMT activity was elevated 65-fold in alloxan-diabetic sheep. Likewise, the report on phosphoregulation of GNMT (8) mentioned that enzymatic activity was increased twofold in diabetic rats. In support of these findings, it has been shown in rats that GNMT can be found predominantly in the gluconeogenic tissues liver and kidney, as well as the pancreas (22) . In turn, it has been demonstrated that ATRA regulates phosphoenolpyruvate carboxykinase gene expression (23) , the rate-limiting enzyme in gluconeogenesis. Moreover, it has been shown in vitro that cyclic adenosine 5'-monophosphate-dependent kinases activate GNMT activity via phosphorylation (8) ; elevations in cyclic adenosine 5'-monophosphate play a role in the stimulation of phosphoenolpyruvate carboxykinase and during diabetes (24) .

Certainly a number of potential mechanisms, both transcriptional/translational and posttranslational, may play a role in the retinoid-induced activation of GNMT. Likewise, the possibility that other factors may mediate the ability of retinoids to alter GNMT needs to be considered. Our laboratory is actively pursuing these areas of research as well as determining the consequences of retinoid-induced alterations in transmethylation and possibly folate-dependent one-carbon metabolism. The results of this ongoing research will be important in future dietary recommendations and in the evaluation of retinoid-derived compounds for clinical use.

	ACKNOWLEDGMENTS

 
We thank Conrad Wagner, Vanderbilt University, for the generous supply of GNMT antibodies, and Robert D. Steele, Pennsylvania State University, in whose laboratory this work began.

	FOOTNOTES

 
¹ A preliminary report of this manuscript was presented at Experimental Biology 2001, Orlando, FL [Rowling, M. J. & Schalinske, K. L. (2001) Glycine N-methyltransferase is up-regulated by all-trans- and 13-cis-retinoic acid in rats. FASEB J. 15: A602 (abs.)].

² Supported in part by Iowa Agriculture and Home Economics Experiment Station (Journal Paper No. J-19189); the Iowa State University Office of Biotechnology; the Center for Designing Foods to Improve Nutrition (U. S. Department of Agriculture Special Projects Grant 99-03168); U. S. Department of Agriculture NRI 01-35200-09854 (to K.L.S.); and the American Institute for Cancer Research 00B078REV (to K.L.S.).

⁴ Abbreviations used: ATRA, all-trans-retinoic acid; CRA, 13-cis-retinoic acid; GNMT, glycine N-methyltransferase; MS, methionine-supplemented; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; THF, tetrahydrofolate; TTBS, Tween 20 + Tris-buffered saline.

Manuscript received January 29, 2001. Revision accepted April 18, 2001.

	REFERENCES

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