Division of Nutritional Sciences, Cornell University, Ithaca, New York 14853
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ABSTRACT |
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To determine the role of nonsulfur vs. sulfur
amino acids in regulation of cysteine metabolism, rats were fed a basal
diet or diets supplemented with a mixture of nonsulfur amino acids (AA), sulfur amino acids (SAA), or both for 3 wk. Hepatic
cysteine-sulfinate decarboxylase (CSDC), cysteine dioxygenase (CDO),
and -glutamylcysteine synthetase (GCS) activity, concentration, and
mRNA abundance were measured. Supplementation with AA alone had no
effect on any of these measures. Supplementation of the basal diet with
SAA, with or without AA, resulted in a higher CDO concentration
(32-45 times basal), a lower CSDC mRNA level (49-64% of
basal), and a lower GCS-heavy subunit mRNA level (70-76%). The
presence of excess SAA and AA together resulted in an additional type
of regulation: a lower specific activity of all three enzymes was
observed in rats fed diets with an excess of AA and SAA. Both SAA and
AA played a role in regulation of these three enzymes of cysteine
metabolism, but SAA had the dominant effects, and effects of AA were
not observed in the absence of SAA.
cysteine-sulfinate decarboxylase; cysteine dioxygenase; -glutamylcysteine synthetase; sulfate; taurine
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INTRODUCTION |
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CYSTEINE is utilized in the synthesis of protein and
for the synthesis of several nonprotein compounds, including taurine, reduced inorganic sulfur, sulfate, and glutathione (GSH), which are
essential for a wide variety of critical functions in the body. The
activities of key regulatory enzymes of cysteine metabolism [cysteine-sulfinate decarboxylase (CSDC), EC 4.1.1.29; cysteine dioxygenase (CDO), EC 1.13.11.20; and -glutamylcysteine synthetase (GCS), EC 6.3.2.2] have been observed to change in liver of rats
fed different levels of dietary protein (2-7). Although cysteine
is metabolized to some extent by many tissues, the liver clearly plays
the dominant role in cysteine metabolism response to sulfate, taurine,
and GSH in intact rats (11, 12), and only the hepatic enzymes of
cysteine metabolism are known to respond to changes in the levels of
dietary protein (6). Thus the liver plays a major role in sulfur amino
acid metabolism, and changes in CSDC, CDO, and GCS activities in
response to diet can affect the utilization of cysteine for synthesis
of essential metabolites.
It is generally assumed that the effects of dietary protein on CSDC, CDO, and GCS are due to the sulfur amino acid content of the protein. In support of this hypothesis, both CDO and GCS are strongly regulated in response to changes in dietary intake of either protein or sulfur amino acids alone (2-7). However, in studies done previously in our laboratory, a lower CSDC activity was observed in isolated hepatocytes in response to increased dietary protein levels (2, 4-7) but not to increased methionine levels (3, 4, 6, 7). When rats were fed diets with very high levels of sulfur amino acids (>2% of the diet), a lower CSDC activity was observed, but methionine was less effective in lowering CSDC activity than an equisulfur amount of protein (6, 7). Similarly, addition of methionine to primary cultures of rat hepatocytes resulted in upregulation of CDO and downregulation of GCS but had no effect on CSDC activity (24). The results from these studies collectively suggest that the hepatic signal for regulation of CSDC is related to the protein content of the diet and not solely to changes in the level of sulfur amino acids.
Jerkins et al. (16) saw a lower CSDC activity in response to an increased dietary protein level that corresponded to changes in CSDC concentration and CSDC mRNA level. In contrast to results from our laboratory, Jerkins and Steele (17, 18) reported a marked decrease in CSDC activity and CSDC protein levels in response to supplementation of a basal diet (10% casein) with methionine to yield a total dietary sulfur amino acid content of 11-16 g/kg of diet vs. 6 g/kg in the basal diet. Despite their observation of a greater response to sulfur amino acids alone compared with observations in our laboratory, Jerkins and co-workers (15, 17, 18) did observe a greater decrease in CSDC activity in response to protein than to an equisulfur amount of methionine, which is in agreement with our findings.
The purpose of this study was to determine the role of excess nonsulfur amino acids, excess sulfur amino acids, or a combination of the two in the regulation of CSDC activity. Although changes in CSDC activity were the major focus of this study, changes in CDO and GCS were also evaluated to more clearly define the dietary component(s) responsible for regulation of these enzymes. By using diets made with purified amino acids, we were able to more clearly elucidate the role of sulfur vs. nonsulfur amino acids in the regulation of CSDC, CDO, and GCS and of cysteine metabolism in vivo.
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MATERIALS AND METHODS |
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Animals and Dietary Treatments
Semipurified diets were prepared as shown in Tables 1 and 2 to contain various levels of sulfur amino acids, nonsulfur amino acids, or a combination of the two. Diets were based on the AIN-76A formulation (1) and the amino acid mixtures of Rogers and Harper (25). Modifications were made to prepare diets that contained 100 g casein/kg diet and either no amino acid supplement (basal; B), 300 g of a sulfur amino acid-free amino acid mixture per kilogram diet (basal + amino acids; B+AA), 9.6 g of L-methionine/kg diet (basal + methionine; B+M), 300 g of the sulfur amino acid-free amino acid mixture plus 9.6 g L-methionine/kg diet (basal + amino acids + methionine; B+AA+M), or 300 g of the sulfur amino acid-free amino acid mixture plus 7.8 g L-cystine/kg diet (basal + amino acids + cystine; B+AA+C). Additions to the basal diet were made at the expense of sucrose. The molar amounts of sulfur amino acids (methionine plus half-cystine) were equivalent for the B+M, B+AA+M, and B+AA+C diets.
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Male Sprague-Dawley rats that weighed ~140 g were purchased from Harlan Sprague Dawley (Indianapolis, IN). Rats were housed individually in stainless steel mesh cages in a room maintained at 20°C and 60-70% humidity with light from 2000 to 0800. Animals were fed a nonpurified diet (RHM 1000, Agway, Syracuse, NY) for 3 days before being assigned to a specific experimental diet. A total of 32 animals were grouped into seven blocks by body weight, and rats within each block were randomly assigned to receive one of the five diets. Thus six or seven animals were assigned to each of the five experimental diets, and the weight distribution of rats was similar for all treatment groups. Rats were fed the experimental diets for 21-23 days and had free access to diet and water for the duration of the experiment.
Beginning on day 18 of the dietary
treatment, one-half of the rats from each group were placed in
individual metabolic cages for collection of 24-h urine samples. Thymol
was used as a preservative for the urine. At the end of the collection
period, urine volume was measured and brought to 25 ml. Urine samples
were then frozen and stored at 20°C. The same procedure was
performed for the remaining rats on day
20 of dietary treatment.
Beginning on day 21 of dietary treatment, 10 or 12 rats per day were killed (CO2 anesthesia + decapitation) on each of 3 days. Rats were killed by assigned block, beginning with the block with the greatest initial body weights. Within assigned blocks, rats were killed in random order. Two to three rats per dietary group were killed on each of the 3 days. The care and use of animals were approved by the Cornell University Institutional Animal Care and Use Committee.
Liver was removed, rinsed with ice-cold saline, blotted, and weighed.
Approximately 100-150 mg of liver from each animal were homogenized in denaturation solution (ToTALLY RNA kit, Ambion, Austin,
TX) and then stored at 70°C for later measurement of CDO,
GCS-heavy subunit (GCS-HS), and CSDC mRNA as we will describe. The
liver was then minced and homogenized in appropriate ice-cold buffers;
homogenate was immediately used for enzyme assays or to obtain the
soluble fraction, which was stored at
70°C for subsequent
determination of the concentrations of CSDC, CDO, and GCS-HS (Western
blot analysis). Aliquots of homogenate or 20,000 g supernatant were frozen for later
analysis of hepatic protein, taurine, and GSH.
Enzyme Assays
CSDC and CDO activities (4, 5) and GCS activity (4) were measured in liver homogenate with methods developed in our laboratory, as described previously.Analysis of Protein, Taurine, GSH, Sulfate, and Creatinine
Urine taurine, sulfate, and creatinine levels were determined as previously described by Bella and Stipanuk (6). Hepatic protein, taurine, and total GSH levels were determined as described by Bella et al. (7).Western, Northern, and Dot-Blot Methods
Sources of antibodies. Rabbit anti-CSDC serum was a gift from Dr. Owen Griffith (Medical College of Wisconsin, Milwaukee, WI). The purified IgG fraction from rabbit anti-CDO serum was a gift from Dr. Yu Hosokawa (National Institute of Health and Nutrition, Tokyo, Japan). Rabbit anti-GCS-HS serum was a gift from Dr. Henry Jay Forman (University of Southern California, Los Angeles, CA). The preparation of these antibodies against rat liver CSDC (31), rat liver CDO (32), and a peptide sequence of GCS-HS (27) has been reported.
Sources of cDNA. An EcoR I-cut cDNA for CDO (14) was a gift of Dr. Yu Hosokawa and Nobuyo Tsuboyama (National Institute of Health and Nutrition, Tokyo, Japan). Probes for CSDC and GCS-HS were prepared as described by Bella et al. (4). DECAprobe template-actin-mouse and DECAprobe template-18S-mouse (Ambion) were used as internal standards for Northern and dot-blot analyses and were labeled with [32P]dCTP by use of the Prime-It RmT random primer labeling kit (Stratagene, La Jolla, CA).
Western blot analysis. Western blot analyses (20, 30) were conducted as previously described by Bella et al. (4). Briefly, total liver supernatant protein was separated by one-dimensional SDS-PAGE, and the proteins were electroblotted onto Immobilon-P membranes (Millipore, Medford, MA). Immunoreactive protein was detected by chemiluminescence with exposure to Kodak X-OMAT XRP film. The film image was scanned using a desktop scanner (Hewlett-Packard Scanjet 3c, Hewlett-Packard, Camas, WA). Two-dimensional quantitative densitometric analysis of the regions of interest was performed using Molecular Analyst software (Bio-Rad Laboratories, Hercules, CA). Single bands corresponding to 54 kDa for CSDC, 23.5 kDa for CDO, and 74 kDa for GCS-HS were detected as noted except in results for CDO; apparent molecular masses were consistent with previously published values (17, 26, 32). Graded amounts of rat liver supernatant were run on all gels and used to generate standard curves for conversion of relative absorbance units to relative protein concentrations.
Isolation of total RNA, Northern blot, and dot-blot hybridization analyses. Northern and dot-blot analyses were performed as previously described by Bella et al. (4). Briefly, total RNA was isolated from liver by use of the ToTALLY RNA kit (Ambion), based on the method of Chomczynski and Sacchi (10), and Northern blot analysis was conducted using 32P-labeled probes as described by Brown (8). Results were quantified using the Bio-Rad GS 363 Phosphorescence Imaging System (Bio-Rad Laboratories, Melville, NY) and the Molecular Analyst program (Bio-Rad Laboratories, Hercules, CA). Graded amounts of liver total RNA were loaded on each gel or membrane to generate standard curves for conversion of relative absorbance units to relative mRNA concentrations.
Statistics
Results were analyzed by ANOVA with factorial analysis for the main effects of nonsulfur amino acids and methionine (Minitab 81.1, State College, PA). Tukey's or Tukey-Kramer's ![]() |
RESULTS |
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Food Intake, Growth, and Hepatic Protein, Taurine, and GSH Concentrations
As shown in Table 3, daily weight gain and final body weights of rats fed both the B and B+AA diets were significantly lower (P < 0.05) than those of rats fed the sulfur amino acid-supplemented diets (B+M, B+AA+M, B+AA+C). These lower rates of weight gain were anticipated because the total sulfur amino acid content of the B and B+AA diets was 0.3% and the total protein content of the B diet was 10%, which are below the National Research Council's recommendation of 0.6% for sulfur amino acids and 12% for protein (23). Despite the low levels of sulfur amino acids and protein, the B diet did support a reasonable rate of weight gain, and intake was similar to that of rats fed the sulfur amino acid-supplemented diets. The daily intake of rats fed the B+AA diet was significantly lower (P < 0.05) than the intake of rats fed the other four diets. The B+AA diet contained 100 g of casein plus 300 g of a sulfur amino acid-free amino acid mixture per kilogram diet; the imbalanced amino acid mixture may have contributed to the lower intake of the diet.
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The relative liver weight (g liver/100 g body weight) was similar for rats fed the B, B+AA, B+AA+M, and B+AA+C diets but was slightly elevated in rats fed the B+M diet. The hepatic protein concentration (mg protein/g liver) was similar for rats fed all five experimental diets, and enzyme concentrations and activities, as well as metabolite concentrations, have been reported per unit of liver protein.
Hepatic taurine and GSH concentrations were lower in rats fed sulfur
amino acid-deficient diets (B and B+AA) than in those fed sulfur amino
acid-supplemented diets (Table 3). The hepatic taurine concentration
was lower and the hepatic GSH concentration was higher in rats fed the
diet supplemented with methionine alone (B+M) than in rats fed the
diets supplemented with both sulfur and nonsulfur amino acids (B+AA+M
and B+AA+C). Analysis of hepatic taurine concentration for treatment
effects indicated a significant effect of sulfur amino acid level
(P < 0.001) but no effect of supplemental nonsulfur amino acids (Table
4). Analysis of hepatic GSH concentration
for treatment effects indicated significant effects of both sulfur
amino acids (P < 0.001) and
nonsulfur amino acids (P = 0.001) and a significant interaction
(P < 0.001) of the two. The observed
patterns of change in hepatic taurine and GSH levels are in agreement
with those reported previously (4, 5).
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CSDC, CDO, and GCS Activities
For all three hepatic enzymes, activity was similar (P < 0.05) in rats fed the B vs. B+AA diets, as shown in Fig. 1. When the basal diet was supplemented with both the excess amino acid mixture and methionine (B+AA+M) or cystine (B+AA+C), CSDC activity was lower (P < 0.05) than that observed with methionine supplementation alone (B+M) (48 or 57% vs. 78%, respectively, of the activity observed in rats fed the B diet). In addition, there was a strong interaction (P = 0.003) between nonsulfur amino acids and methionine (Table 5). This observation of methionine alone being less effective than sulfur plus nonsulfur amino acids in lowering CSDC activity is consistent with previous observations of methionine being less effective than protein at equisulfur levels (2-7).
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CDO activity was significantly higher (P < 0.001) and GCS activity was significantly lower (P < 0.001) with addition of sulfur amino acids to the basal diet. The levels of CDO activity in liver of rats fed the B+M, B+AA+M, and B+AA+C diets were 178-, 138-, and 115-fold, respectively, the activity observed in rats fed the B diet. GCS activities in the liver of rats fed the B+M, B+AA+M, and B+AA+C diets were 52, 27, and 32%, respectively, of the activity in rats fed the B diet.
CSDC, CDO, and GCS-HS Protein Concentrations
As shown in Fig. 2, no difference in the relative protein level for any of the three hepatic enzymes was observed between rats fed the B and B+AA diets. Relative CSDC protein levels in rats fed the B+M, B+AA+M, and B+AA+C diets were 60, 50, and 47%, respectively, of the level observed in rats fed the B diet. Relative hepatic GCS-HS protein levels in the B+M, B+AA+M, and B+AA+C groups were 61, 62, and 54%, respectively, of the level observed in rats fed the B diet.
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The relative CDO levels in the liver of rats fed the B+M, B+AA+M, and B+AA+C diets were 34, 45, and 32 times, respectively, the concentration of CDO observed in rats fed the B diet. In addition, the CDO band detected in samples from rats fed the B+M, B+AA+M, and B+AA+C diets resolved into two distinct bands, with the additional or lower band having an estimated molecular mass of 23.5 kDa compared with the upper or usual band with an estimated molecular mass of 25.5 kDa. The appearance of two bands in liver of rats fed excess protein or methionine has been observed previously (4). The quantitative results for relative CDO protein reported in Fig. 2 for rats fed the three sulfur amino acid-supplemented diets include both bands.
CSDC, CDO, and GCS-HS mRNA Levels
As shown in Fig. 3, no difference in the mRNA level for any of the three hepatic enzymes was observed between rats fed the B and B+AA diets. CSDC mRNA levels in rats fed the B+M, B+AA+M, and B+AA+C diets were 64, 49, and 63%, respectively, of the level observed in rats fed the B diet. As with CSDC activity, differences in CSDC mRNA levels were a result of sulfur amino acid (P < 0.001) but not of nonsulfur amino acid (P = 0.53) supplementation (Table 5). However, no significant interaction of sulfur amino acids and nonsulfur amino acids was observed for CSDC mRNA levels as was observed for CSDC activity.
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No significant effect of dietary treatment on hepatic CDO mRNA levels was observed. CDO mRNA levels tended to be slightly higher in liver of rats fed the B+AA+M and B+AA+C diets than in rats fed the other three diets, but the magnitude of the apparent differences in CDO mRNA concentration was small compared with those observed in CDO activity and concentration.
The levels of GCS-HS mRNA in rats fed the B+M, B+AA+M, and B+AA+C diets were 70, 76, and 71%, respectively, of the level observed in rats fed the B diet. The GCS-HS mRNA levels were significantly affected by dietary methionine level (P = 0.003) but not by nonsulfur amino acid level, and they generally responded to dietary changes with a pattern similar to that observed for GCS-HS concentration.
Urinary Taurine and Sulfate Excretion
Consistent with the increased intake of sulfur amino acids, the absolute taurine excretion by rats fed the B+M, B+AA+M, and B+AA+C diets was 75, 13, and 32 times, respectively, the level excreted by rats fed the B diet (Table 6). Urinary taurine as a percentage of total sulfur excretion was also greater in rats fed the sulfur amino acid-supplemented diets than in rats fed the B or B+AA diets; this is probably the result of increased cysteine-sulfinate-dependent vs. cysteine-sulfinate-independent catabolism of cysteine in rats fed sulfur amino acid-supplemented diets (2). In addition to the effect of sulfur amino acids (P < 0.001), the addition of nonsulfur amino acids consistently and significantly (P < 0.001) lowered both the absolute amount of taurine excreted and the proportion of total sulfur excretion as taurine vs. sulfate (B vs. B+AA; B+M vs. B+AA+M) (Table 7). Cystine appeared to be slightly more effective than methionine in increasing taurine excretion by rats fed the amino acid-supplemented diets (B+AA+C vs. B+AA+M). In general, the patterns of urinary taurine and sulfate excretion observed in this study are similar to the patterns in previous studies in which rats were fed diets supplemented with protein or methionine (6).
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DISCUSSION |
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Regulation of Cysteine Metabolic Enzymes in Response to Sulfur vs. Nonsulfur Amino Acids
By using purified amino acids instead of protein, we further elucidated the effects of dietary nonsulfur amino acids vs. methionine and their interaction in the regulation of CSDC, CDO, and GCS in vivo. We were also able to compare the effects of methionine vs. cystine in the presence of dietary nonsulfur amino acids on these same parameters. The degrees of change in enzyme activity, enzyme concentration, mRNA concentration, and specific activity (enzyme activity divided by the relative enzyme concentration) observed for CSDC, CDO, and GCS-HS in this study are summarized in Table 8.
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Regulation of CSDC. Consistent with previous reports (4, 16), we found that downregulation of CSDC in response to changes in dietary amino acid content occurred at the level of CSDC mRNA concentration. CSDC (protein) concentration and CSDC mRNA level were highly correlated with each other (r2 = 0.94) and with enzyme activity (r2 = 0.92 and r2 = 0.92, respectively). By supplementing the basal diet with either a mixture of nonsulfur amino acids, a sulfur amino acid, or both nonsulfur and sulfur amino acids, we also were able to separate the effects of the sulfur and nonsulfur amino acid components of dietary protein. The CSDC mRNA levels were significantly affected by the dietary sulfur amino acid level (P < 0.001) and not at all affected by the level of nonsulfur amino acids alone (Table 5). It is not known whether the decrease in the CSDC mRNA concentration is a result of a decrease in the rate of transcription of the CSDC gene or of a decrease in CSDC mRNA transcript stability (an increased rate of degradation).
In contrast to changes in the CSDC mRNA level in response to sulfur amino acid intake, the response of CSDC activity to excess sulfur amino acid showed a strong interaction with the level of nonsulfur amino acids in the diet (P = 0.003), suggesting the possibility that additional regulation of CSDC occurs at the posttranslational level. In agreement with previous studies in which a greater decrease in CSDC activity was observed in response to protein than to sulfur amino acids alone (2-4, 6, 7), we observed a greater decrease in CSDC activity in response to an excess of a complete mixture of amino acids (sulfur amino acid plus nonsulfur amino acids that simulated dietary protein) than to an excess of sulfur amino acid alone. A relative calculation of CSDC specific activity (CSDC activity divided by the relative CSDC protein concentration) revealed a slightly higher specific activity in liver of rats fed the B+M diet compared with rats fed the other two diets (B+AA+M and B+AA+C) in which the excess sulfur amino acids were "balanced" by excess nonsulfur amino acids. Changes in CSDC specific activity in response to methionine alone were not observed in a previous study, but changes may not have been apparent because of a relatively weak response of CSDC to either methionine or protein in that study (4). Although not large, this apparent posttranslational regulation of CSDC seems to account for the stronger effect of protein than of sulfur amino acids alone in bringing about downregulation of CSDC activity. The possibility of posttranslational regulation of CSDC activity deserves further study.Regulation of CDO. As previously reported (4), markedly higher steady-state CDO activity and CDO protein concentration were seen in liver of rats fed sulfur amino acid-supplemented diets with or without additional nonsulfur amino acids, whereas no differences were observed in CDO mRNA levels in liver of rats fed these diets. These changes mainly occurred in response to sulfur amino acid supplementation, because supplementation with nonsulfur amino acids alone had no effect on either CDO activity or CDO protein level.
Although not always statistically significant, we have consistently found in several different studies (2-7) higher CDO activity in response to methionine supplementation vs. protein supplementation. We recently reported a lower CDO specific activity in liver of rats fed a protein-supplemented diet compared with rats fed a methionine-supplemented diet with an equivalent level of sulfur amino acid (4). In this study, a borderline significant (P = 0.063) overall effect of nonsulfur amino acids on CDO activity was observed: hepatic CDO activity, but not CDO protein concentration, was lower in rats fed the B+AA+M or B+AA+C diet than in rats fed the B+M diet. As a result, CDO specific activity (relative CDO activity/relative CDO protein concentration) was ~40% lower in liver of rats fed diets supplemented with sulfur plus nonsulfur amino acids compared with those fed diets supplemented with methionine alone. These findings suggest that the presence of nonsulfur amino acids may restrict the magnitude of the increase of CDO activity in response to sulfur amino acid supplementation. This modulation of activity seems to occur via a decrease in the activity state of CDO, but the mechanism by which this occurs is not yet known. It is possible that the apparent changes in the activity state of CDO may be related to changes in the relative proportions of the 23.5- and 25.5-kDa CDO species observed on Western blot analysis, but these two forms of CDO have not yet been adequately separated to test this hypothesis.Regulation of GCS. Regulation of GCS in response to excess sulfur vs. nonsulfur amino acids appears to occur at two levels: GCS-HS mRNA concentration and GCS-HS specific activity. Both GCS-HS protein and GCS-HS mRNA concentrations were lower in rats supplemented with sulfur amino acid, regardless of whether they were also supplemented with additional nonsulfur amino acids. Changes in the GCS-HS mRNA level occurred in response to sulfur amino acid supplementation (P = 0.003) but not in response to supplementation with nonsulfur amino acids (Table 5). These results suggest that changes in GCS activity in response to sulfur amino acid supplementation are a result of decreases in the GCS-HS mRNA level due to a decrease either in the rate of transcription of the GCS-HS gene or in GCS-HS mRNA transcript stability.
In addition to regulation of the steady-state GCS-HS mRNA level, our data suggest that GCS activity is regulated posttranslationally. The specific activity of hepatic GCS-HS decreased to a greater extent in animals fed diets supplemented with both sulfur and nonsulfur amino acids than in those fed the diet supplemented with either methionine alone or the nonsulfur amino acid mixture alone. The presence of a complete mixture of amino acids or of a sulfur amino acid plus some component(s) of the nonsulfur amino acid mixture apparently is required to bring about a decrease in GCS-HS specific activity. Although analysis of hepatic GCS activity for treatment effects showed only borderline significance for nonsulfur amino acids (P = 0.052), the greater decrease in GCS-HS specific activity with an excess of both sulfur and nonsulfur amino acids vs. an excess of methionine alone is probably biologically significant. A similar decrease in the specific activity of hepatic GCS-HS was observed in rats fed protein-supplemented compared with methionine-supplemented diets in a previous study (4). Because an excess of both sulfur amino acids and nonsulfur amino acids seems to be necessary for lowering of GCS specific activity, we would expect to be able to detect a significant interaction between the effects of sulfur and nonsulfur amino acids on GCS activity. Lack of detection of a significant interaction between sulfur and nonsulfur amino acid supplementation in the analysis of GCS activity in this study is probably due to the coexistence of a large effect of sulfur amino acid alone on GCS activity (via a decrease in GCS-HS concentration), resulting in insufficient statistical power to detect this smaller effect on GCS specific activity that is dependent on the interaction of methionine with nonsulfur amino acids.Common Roles of Both Nonsulfur and Sulfur Amino Acids in the Regulation of CSDC, CDO, and GCS
Neither CSDC, CDO, nor GCS activity changed in response to supplemental nonsulfur amino acids alone, which clearly underscores the important role of dietary sulfur amino acids in the regulation of these three enzymes. Changes in the abundance of all three enzymes occurred in response to sulfur amino acid supplementation. The changes in CDO protein and in CSDC and GCS-HS mRNA and protein levels all occurred in response to sulfur amino acid level with no apparent interaction with nonsulfur amino acid level.Nevertheless, nonsulfur amino acids did seem to play a role in modulating the response of each of the three enzymes to sulfur amino acid supplementation. Although the main regulatory effects of sulfur amino acids did not require and were not affected by the presence of excess nonsulfur amino acids, the specific activity of all three hepatic enzymes was lower in rats fed the diet containing both excess methionine and nonsulfur amino acids than in those fed the diet containing only excess methionine.
Role of Methionine vs. Cyst(e)ine in Regulation of CSDC, CDO, and GCS-HS
Overall, the effect of methionine vs. cystine supplementation was similar for all three enzymes, regardless of their mechanism of regulation. Although methionine and cystine were compared in this study only in the presence of excess nonsulfur amino acids, similar effects of methionine and cystine on hepatic CDO and CSDC activities and on the capacity for GSH synthesis were observed in a previous study in which two levels of cystine and methionine were compared in the absence of excess nonsulfur amino acids (3). The similar response of all three enzymes to either methionine or cystine suggests that cysteine or a subsequent metabolite of cysteine probably plays a key role as a cellular signal for the adaptive regulation of hepatic CSDC, CDO, and GCS. Liver of animals consuming higher amounts of sulfur amino acid would clearly be exposed to high sulfur amino acid concentrations in the portal blood, and liver is the major site for transsulfuration of methionine to form cysteine. Thus an increase in either dietary methionine or cystine would be expected to markedly increase the exposure of hepatocytes to cysteine.The effect of changes in dietary methionine or cystine on the levels of hepatic CSDC and GCS-HS mRNAs is also particularly interesting. Expression of the asparagine synthetase gene (13) can be modulated by changes in amino acid concentrations. Marten et al. (21) demonstrated changes in expression of several genes in rat hepatoma cells in response to limitation of either a single amino acid or of several amino acids for 24 h. Because dietary amino acids go directly to the liver, the liver is the major catabolic organ for most amino acids and plays a major role in the fate of amino acids in the body. It is possible that amino acid concentrations or secondary signals affected by changes in amino acid availability, such as the concentration of amino acid metabolites, could serve as cellular effectors for regulation of hepatic gene expression. This possibility seems worthy of further study, because expression of several genes in Escherichia coli is regulated by the cellular concentration of leucine via a leucine-responsive regulatory protein (9). Furthermore, the DNA-binding activities of several liver-specific transcription factors have been shown to be altered in liver of rats fed protein-restricted diets (22).
Dietary Sulfur and Nonsulfur Amino Acids Influence Cysteine Metabolism in Vivo
The factors that influence hepatic taurine and GSH levels have been reviewed extensively elsewhere (5). As seen in other studies, differences in hepatic GSH concentrations in this study reflect the level of cysteine availability, with some apparent modulation by GCS activity. The increases in cysteine availability and in hepatic CDO activity in rats fed the three diets supplemented with sulfur amino acids presumably were associated with greater conversion of cysteine to cysteine-sulfinate and, hence, to taurine. Increased taurine synthesis was reflected by increases in both hepatic taurine level and urinary taurine excretion.Among the three sulfur amino acid-supplemented groups, higher activities of both CDO and CSDC were observed in rats fed the diet supplemented with methionine alone (B+M) than in rats fed the diet supplemented with both methionine and nonsulfur amino acids (B+AA+M). These higher enzyme activities may have resulted in a greater partitioning of cysteine to taurine vs. sulfate (taurine: taurine+sulfate ratio of 47 in the B+M group vs. 11 in the B+AA+M group) and in the much greater rate of taurine excretion (29.9 µmol/mg creatinine in the B+M group vs. 5.3 µmol/mg creatinine in the B+AA+M group) in rats fed the B+M diet than in those fed the B+AA+M diet. These findings are in agreement with those reported previously (6).
Hepatic taurine concentration is less likely to reflect the rate of taurine synthesis in vivo than is urinary taurine excretion. The hepatic taurine concentration in rats fed the B+M diet tended to be lower than that in rats fed the B+AA+M or B+AA+C diet, which is in contrast to observations for urinary taurine. Besides substrate availability and CDO and CSDC activities, the level of hepatic taurine efflux, the plasma taurine concentration, and the regulation of taurine reabsorption in the epithelium of the renal proximal tubules can also affect hepatic and urinary taurine levels and may explain observed differences. Little is known about regulation of these processes except that renal taurine concentration seems to be the signal for changes in renal taurine transporter activity (19, 29).
An independent effect of excess nonsulfur amino acids on urinary
taurine excretion is indicated by the significantly lower taurine
excretion in rats fed the B+AA diet than in rats fed the B diet. This
difference occurred despite similar hepatic taurine concentrations,
cysteine availability, and hepatic CDO and CSDC activities in rats fed
these two diets. This, along with the lower rate of taurine excretion
of rats fed the B+AA+M vs. the B+M diet, suggests the possibility that
the partitioning of cysteine-sulfinate between sulfate and taurine may
be affected by the presence of excess nonsulfur amino acids. There may
be an increase in flux of cysteine-sulfinate to pyruvate and sulfate
via transamination to -sulfinylpyruvate owing to a high rate of
amino acid catabolism/gluconeogenesis or an elevated keto acid
cosubstrate (
-ketoglutarate) concentration for transamination. In
addition, the rate of renal reabsorption of taurine may be different in
rats fed diets with excess levels of nonsulfur amino acids than in rats
fed diets supplemented with both sulfur and nonsulfur amino acids.
The regulation of both CDO and CSDC enzyme activities by changes in diet clearly affects the rates of taurine and sulfate synthesis in vivo, but regulation of CDO activity seems to play the dominant role. In a previous study (4), CDO activity was increased ~10-fold when dietary protein was increased from 10 to 20% casein, whereas no change was observed for CSDC activity over this range of dietary protein. Even greater degrees of change in CDO activity (>100-fold increases) were observed with sulfur amino acid supplementation in this study, whereas only a 23-59% decrease in CSDC activity was observed. Little regulation of CSDC activity, which could alter the partitioning of hepatic cysteine-sulfinate between taurine vs. sulfate, is observed unless the levels of dietary protein (or complete amino acid mixture) are clearly in excess. Even at the lowest observed levels of CSDC activity in this study, increases in CDO and cysteine availability were accompanied by large increases in rates of taurine synthesis and excretion (i.e., the B+AA+M group). Therefore, changes in CDO activity most likely play a greater role in regulation of taurine synthesis in animals consuming levels of protein that are near the requirement, and even in animals consuming very high levels of protein, than do changes in CSDC activity.
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ACKNOWLEDGEMENTS |
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We gratefully acknowledge the guidance and advice of Dr. Patrick Stover and the technical assistance of Larry Hirschberger. We also thank Dr. Jay Forman for the anti-GCS-HS serum, Dr. Owen Griffith for the anti-CSDC serum, Dr. Yu Hosokawa for the anti-CDO IgG, and Dr. Yu Hosokawa and Nobuyo Tsuboyama for the EcoR I cut cDNA for CDO.
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FOOTNOTES |
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This research was supported in part by National Research Initiative Competitive Grants Program/US Department of Agriculture (USDA) Grant 92-37200-7583, USDA/Cooperative State Research, Education, and Extension Service Grant 94-34324-0987, and by the President's Council of Cornell Women. D. L. Bella was supported by a National Institute of Diabetes and Digestive and Kidney Diseases training grant (T32-DK-07158).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: M. H. Stipanuk, 225 Savage Hall, Cornell Univ., Ithaca, NY 14853-6301 (E-mail: mhs6{at}cornell.edu).
Received 10 December 1998; accepted in final form 25 March 1999.
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REFERENCES |
---|
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---|
1.
American Institute of Nutrition.
Report of the American Institute of Nutrition ad hoc committee on standards for nutritional studies.
J. Nutr.
107:
1340-1348,
1977[Medline].
2.
Bagley, P. J.,
and
M. H. Stipanuk.
The activities of rat hepatic cysteine dioxygenase and cysteinesulfinate decarboxylase are regulated in a reciprocal manner in response to dietary casein level.
J. Nutr.
124:
2410-2421,
1994.
3.
Bagley, P. J.,
and
M. H. Stipanuk.
Rats fed a low protein diet supplemented with sulfur amino acids have increased cysteine dioxygenase activity and increased taurine production in hepatocytes.
J. Nutr.
125:
933-940,
1995[Medline].
4.
Bella, D. L.,
L. L. Hirschberger,
Y. Hosokawa,
and
M. H. Stipanuk.
Mechanisms involved in the regulation of key enzymes of cysteine metabolism in rat liver in vivo.
Am. J. Physiol.
276 (Endocrinol. Metab. 39):
E326-E335,
1999
5.
Bella, D. L.,
Y. H. Kwon,
and
M. H. Stipanuk.
Variations in dietary protein but not in dietary fat plus cellulose or carbohydrate levels affect cysteine metabolism in rat isolated hepatocytes.
J. Nutr.
126:
2179-2187,
1996[Medline].
6.
Bella, D. L.,
and
M. H. Stipanuk.
Effects of protein, methionine, or chloride on acid-base balance and on cysteine catabolism.
Am. J. Physiol.
269 (Endocrinol. Metab. 32):
E910-E917,
1995
7.
Bella, D. L.,
and
M. H. Stipanuk.
High levels of dietary protein or methionine have different effects on cysteine metabolism in rat hepatocytes.
In: Taurine 2: Basic and Clinical Aspects, edited by R. Huxtable,
J. Azuma,
M. Nakagawa,
K. Kuriyama,
and A. Baba. New York: Plenum, 1996, p. 73-84.
8.
Brown, C.
Analysis of RNA by Northern and slot blot hybridization.
In: Current Protocols in Molecular Biology, edited by F. M. Ausubel,
R. Brent,
R. E. Kingston,
D. D. Moore,
J. G. Seidman,
J. A. Smith,
and K. Strunl. New York: Wiley-Interscience, 1993, p. 4.9.1-4.9.14.
9.
Calvo, J. M.,
and
R. G. Matthews.
The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli.
Microbiol. Rev.
58:
466-490,
1994[Abstract].
10.
Chomczynski, P.,
and
N. Sacchi.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal. Biochem.
162:
156-159,
1987[Medline].
11.
Coloso, R. M.,
L. L. Hirschberger,
and
M. H. Stipanuk.
Uptake and metabolism of L-2-oxo-[35S]thiazolidine-4-carboxylate by rat cells is slower than that of L-[35S]cysteine or L-[35S]methionine.
J. Nutr.
121:
1341-1348,
1991[Medline].
12.
Garcia, R. A. G.,
and
M. H. Stipanuk.
The splanchnic organs, liver and kidney have unique roles in the metabolism of sulfur amino acids and their metabolites in rats.
J. Nutr.
122:
1693-1701,
1992[Medline].
13.
Guerrini, L.,
S. S. Gong,
K. Mangasarian,
and
C. Basilico.
Cis and trans-acting elements involved in amino acid regulation of asparagine synthetase gene expression.
Mol. Cell. Biol.
13:
3202-3212,
1993[Abstract].
14.
Hosokawa, Y.,
A. Matsumoto,
J. Oka,
H. Itakura,
and
K. Yamaguchi.
Isolation and characterization of a complementary DNA for rat liver cysteine dioxygenase.
Biochem. Biophys. Res. Commun.
168:
473-478,
1990[Medline].
15.
Jerkins, A. A.,
L. E. Bobroff,
and
R. D. Steele.
Hepatic cysteine sulfinic acid decarboxylase activity in rats fed various levels of dietary casein.
J. Nutr.
119:
1593-1597,
1989[Medline].
16.
Jerkins, A. A.,
D. D. Jones,
and
E. A. Kholhepp.
Cysteine sulfinic acid decarboxylase mRNA abundance decreases in rats fed a high-protein diet.
J. Nutr.
128:
1890-1895,
1998
17.
Jerkins, A. A.,
and
R. D. Steele.
Dietary sulfur amino acid modulation of cysteine sulfinic acid decarboxylase.
Am. J. Physiol.
261 (Endocrinol. Metab. 24):
E551-E555,
1991
18.
Jerkins, A. A.,
and
R. D. Steele.
Quantification of cysteine sulfinic acid decarboxylase in male and female rats: effect of adrenalectomy and methionine.
Arch. Biochem. Biophys.
294:
534-538,
1992[Medline].
19.
Jessen, H.
Taurine and -alanine in an established human kidney cell line derived from the proximal tubule.
Biochim. Biophys. Acta
1194:
44-52,
1994[Medline].
20.
Laemmli, U. K.
Cleavage of structural proteins during its assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[Medline].
21.
Marten, N. W.,
E. J. Burke,
J. M. Hayden,
and
D. S. Straus.
Effect of amino acid limitation on the expression of 19 genes in rat hepatoma cells.
FASEB J.
8:
538-544,
1994
22.
Marten, N. W.,
F. M. Sladek,
and
D. S. Straus.
Effect of dietary protein restriction on liver transcription factors.
Biochem. J.
317:
361-370,
1996[Medline].
23.
National Research Council.
Nutrient Requirements of Laboratory Animals. Washington, DC: National Academy of Sciences, 1978, p. 13-16.
24.
Ohta, J.,
and
M. H. Stipanuk.
Taurine production in rat primary hepatocytes.
In: Taurine 2: Basic and Clinical Aspects, edited by R. Huxtable,
J. Azuma,
M. Nakagawa,
K. Kuriyama,
and A. Baba. New York: Plenum, 1996, p. 69-71.
25.
Rogers, Q. R.,
and
A. E. Harper.
Amino acid diets and maximal growth in the rat.
J. Nutr.
87:
267-273,
1965[Medline].
26.
Seelig, G. F.,
R. P. Simondsen,
and
A. Meister.
Reversible dissociation of -glutamylcysteine synthetase into two subunits.
J. Biol. Chem.
259:
9345-9347,
1984
27.
Shi, M. M.,
A. Kugelman,
T. Iwamoto,
L. Tian,
and
H. J. Forman.
Quinone-induced oxidative stress elevates glutathione and induces -glutamylcysteine synthetase activity in rat lung epithelial L2 cells.
J. Biol. Chem.
269:
26512-26517,
1994
28.
Steel, R. G. D.,
and
J. H. Torrie.
Principles and Procedures of Statistics. New York: McGraw-Hill, 1960, p. 99-160.
29.
Sturman, J. A.,
and
R. W. Chesney.
Taurine in pediatric nutrition.
Ped. Clin. No. Am.
42:
879-897,
1995.
30.
Towbin, H.,
T. Staehelin,
and
J. Gordon.
Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets; procedure and some applications.
Proc. Natl. Acad. Sci. USA
76:
4530-4534,
1979[Abstract].
31.
Weinstein, C. L.,
and
O. W. Griffith.
Multiple forms of rat liver cysteinesulfinate decarboxylase.
J. Biol. Chem.
262:
7254-7263,
1987
32.
Yamaguchi, K.,
Y. Hosokawa,
N. Kohashi,
Y. Kori,
S. Sakakibara,
and
I. Ueda.
Rat liver cysteine dioxygenase (cysteine oxidase): further purification, characterization, and analysis of the activation and inactivation.
J. Biochem.
83:
479-491,
1978[Abstract].