Cysteine regulates expression of cysteine dioxygenase and
-glutamylcysteine synthetase in cultured rat
hepatocytes
Young Hye
Kwon and
Martha H.
Stipanuk
Division of Nutritional Sciences, Cornell University, Ithaca, New
York 14853
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ABSTRACT |
Rat
hepatocytes cultured for 3 days in basal medium expressed low levels of
cysteine dioxygenase (CDO) and high levels of
-glutamylcysteine
synthetase (GCS). When the medium was supplemented with 2 mmol/l
methionine or cysteine, CDO activity and CDO protein increased by
>10-fold and CDO mRNA increased by 1.5- or 3.2-fold. In contrast, GCS
activity decreased to 51 or 29% of basal, GCS heavy subunit (GCS-HS)
protein decreased to 89 or 58% of basal, and GCS mRNA decreased to 79 or 37% of basal for methionine or cysteine supplementation,
respectively. Supplementation with cysteine consistently yielded
responses of greater magnitude than did supplementation with an
equimolar amount of methionine. Addition of propargylglycine to inhibit
cystathionine
-lyase activity and, hence, cysteine formation from
methionine prevented the effects of methionine, but not those of
cysteine, on CDO and GCS expression. Addition of buthionine sulfoximine
to inhibit GCS, and thus block glutathione synthesis from cysteine, did
not alter the ability of methionine or cysteine to increase CDO. GSH
concentration was not correlated with changes in either CDO or GCS-HS
expression. The effectiveness of cysteine was equivalent to or greater
than that of its precursors (S-adenosylmethionine,
cystathionine, homocysteine) or metabolites (taurine, sulfate). Taken
together, these results suggest that cysteine itself is an important
cellular signal for upregulation of CDO and downregulation of GCS.
glutathione; hepatocytes
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INTRODUCTION |
OTHER THAN
INCORPORATION INTO PROTEIN, the major fates of cysteine in the
body are incorporation into the tripeptide glutathione (GSH) or
catabolism via cysteinesulfinate-dependent pathways (Fig. 1). Because methionine sulfur is almost
entirely converted to cysteine sulfur via the transsulfuration pathway
before its sulfur is oxidized and excreted, intake of a certain molar
amount of methionine results in synthesis of a nearly equimolar amount
of cysteine (21, 22). Although substantial amounts of
cysteine are incorporated into both protein and GSH, cysteine is
ultimately catabolized to taurine or sulfate. We have previously
reported results of several rat studies in which both hepatic cysteine dioxygenase (CDO, EC 1.13.11.20) and hepatic
-glutamylcysteine synthetase (GCS, EC 6.3.2.2) activities responded to changes in the
protein or dietary sulfur amino acid levels. These two enzymes
responded in opposite directions, with CDO activity increasing and GCS
activity decreasing in response to an increase in the protein or sulfur
amino acid level (2-5). Increases in CDO activity were associated with increases in the urinary
taurine-to-taurine+sulfate ratio (5). Higher
levels of CDO activity were also accompanied by greater rates of both
taurine and sulfate production as well as by an increase in the
proportion of cysteine metabolized to taurine vs. sulfate by isolated
hepatocytes incubated with 0.2 mmol/l L-cysteine (2,
3, 5). Likewise, the lower levels of GCS activity in isolated
hepatocytes were accompanied by lower rates of GSH synthesis (2,
5).

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Fig. 1.
Major pathways of sulfur amino acid metabolism. Steps catalyzed by
cysteine dioxygenase (CDO) and -glutamylcysteine synthetase (GCS)
and steps blocked by DL-propargylglycine (PPG) and
DL-buthionine-[S,R]-sulfoximine (BSO) are indicated.
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Increases in CDO activity in response to an increase in the supply of
sulfur amino acids could be critical for prevention of potential damage
to the cell by rapid removal of excess cysteine. Elevated levels of
cysteine have been shown to be both cytotoxic and neurotoxic, and
increased levels of homocysteine, a precursor of cysteine in the
methionine transsulfuration pathway, have been associated with
increased risk for cardiovascular disease and with the occurrence of
neural tube defects (22). The lower GCS activity present
in animals fed high levels of sulfur amino acids acts to somewhat limit
the rate of GSH synthesis and favors the catabolism of cysteine to
taurine or sulfate. On the other hand, the higher GCS activities and
lower CDO activities that are present in animals fed diets low in
sulfur amino acids appear to ensure that cysteine is efficiently used
for GSH synthesis, rather than being catabolized to taurine and
sulfate, when sulfur amino acid supply is limited.
Studies of the molecular regulation of CDO and GCS activities in rat
liver by dietary sulfur amino acid intake indicated that increases in
CDO activity were accomplished predominantly by increases in CDO
protein concentration, with no effect on CDO mRNA levels and a much
smaller degree of change in activity state (3, 4). Changes
in GCS activity in response to dietary supplementation with methionine
were largely accounted for by changes in the concentration of mRNA
coding for GCS-heavy (or catalytic) subunit (GCS-HS) (3, 4). The precise mechanisms and signals involved in the sulfur amino acid-induced regulation of CDO and GCS are not known, although the regulation of CDO clearly appears to be posttranscriptional (3, 4).
Recently, we reported the development of a primary hepatocyte model for
studies of regulation of CDO and GCS by sulfur amino acids
(16). Supplementation of the culture medium with either methionine or cysteine resulted in higher CDO activity and lower GCS
activity in cultured rat hepatocytes. These studies demonstrated that
hepatocytes in culture could be used as a model for further studies of
the regulation of hepatic CDO and GCS at the cellular level.
Thus rat hepatocytes in primary culture were used for studies designed
to further elucidate the particular sulfur amino acid or metabolite
that was effective at the cellular level in inducing regulatory changes
in hepatic CDO and GCS activities and to determine whether the pattern
of changes in enzyme activity, enzyme protein concentration, and enzyme
mRNA concentrations in cultured hepatocytes were similar to those
observed in liver of intact animals. To determine the particular sulfur
amino or metabolite that acts as a signal for regulatory changes in CDO
and GCS at the cellular level, we cultured hepatocytes with methionine,
cysteine, or one of their metabolites. We also used
DL-propargylglycine (PPG), an inhibitor of cystathionine
-lyase (EC 4.4.1.1), to block transsulfuration of methionine to
cysteine (17) and
DL-buthionine-[S,R]-sulfoximine (BSO), an inhibitor of
GCS, to block GSH synthesis (15) to further assess the
roles of cysteine and GSH, respectively, as mediators of the effect of
sulfur amino acid availability on CDO and GCS activities.
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MATERIALS AND METHODS |
Materials.
Williams' medium E (WE medium), murine natural epidermal growth factor
(EGF), bovine insulin, and antibiotic-antimycotic mixture that
contained the sodium salt of penicillin G, streptomycin sulfate, and
amphotericin B were purchased from GIBCO-BRL (Grand Island, NY). Calf
skin collagen type I, collagenase, dexamethasone,
Na2SeO3, bathocuproine disulfonate (BCS), BSO,
PPG, L-cysteine, L-methionine, S-adenosyl-L-methionine,
L-cystathionine, L-homocysteine, and taurine
were all purchased from Sigma (St. Louis, MO). Tissue culture dishes
(60 × 15 mm) were purchased from Becton-Dickinson (Franklin
Lakes, NJ). [32P]dCTP (3,000 Ci/mmol) was purchased from
Du Pont-New England Nuclear (Boston, MA). All other reagents were of
analytical grade and were obtained from commercial sources.
Antibodies.
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 antibody was raised to CDO purified from rat liver
(13). Rabbit anti-GCS-HS serum was a gift from Dr. Henry
Jay Forman (University of Alabama at Birmingham, Birmingham, AL). The
preparations of these antibodies against rat liver CDO (13) and a peptide sequence of rat kidney GCS-HS
(18) have been reported. Specificity of these antibodies
has been described previously (4).
Primary cultures of rat hepatocytes.
Male Sprague-Dawley rats (Harlan Sprague Dawley, Indianapolis, IN) were
housed in stainless steel mesh cages in a room maintained at 20°C and
60-70% humidity with light from 2000 to 0800. Rats were given ad
libitum access to water and a nonpurified diet (Prolab RMH 1000, Agway,
Syracuse, NY). The care and use of animals was approved by the Cornell
University Institutional Animal Care and Use Committee. Rats weighed
~250-300 g when they were used to obtain hepatocytes.
Hepatocytes were isolated aseptically by collagenase perfusion as
described by Berry et al. (6). The initial viability of
isolated hepatocytes was more than 85% as determined by 0.2% (wt/vol)
Trypan blue exclusion. The freshly isolated hepatocytes were
resuspended in WE medium to give a cell number of 1.5 × 107 cells per ml; the suspended cells then were diluted
with basal WE medium to yield a final cell concentration of 7.5 × 105 cells per ml. The basal WE medium provided 0.49 mmol/l
total L-cysteine, 0.08 mmol/l L-methionine,
and a negligible amount of GSH (0.16 µmol/l) and was prepared to
contain 1 µg/ml insulin, 50 ng/ml EGF, 50 nmol/l dexamethasone, 3 nmol/l Na2SeO3, 100 units/ml penicillin G, 100 µg/ml streptomycin sulfate, and 0.25 µg/ml amphotericin B. Culture
dishes were coated with collagen as described previously (16). Five milliliters of the diluted cell suspension
(0.18 ml/cm2) were plated on each 60-mm-diameter
collagen-coated dish. Cells were allowed to attach in basal medium over
a 4-h period. At 4 h, the basal medium was replaced with fresh
basal medium or with medium that was supplemented with 2 mmol/l
methionine, 2 mmol/l cysteine, or 2 mmol/l of a sulfur amino acid
metabolite as indicated in RESULTS. Incubations with thiols
(i.e., cysteine or homocysteine) also contained 0.05 mmol/l of BCS,
which was added to prevent formation of disulfides (9).
The experimental medium was replaced every 24 h (i.e., at 28 and
52 h). To examine the effect of inhibitors, either 1 mmol/l PPG or
100 µmol/l BSO were added to the designated culture medium when
medium was changed at 28 and 52 h. Cells were harvested after
72 h of treatment (i.e., total of 76 h in culture).
At the end of the culture period, monolayer cultures were washed three
times with 2.5 ml of ice-cold PBS. For measurement of enzyme activities
and Western blot analysis, washed cells were collected by scraping.
Harvested cells were suspended in 50 mmol/l MES, pH 6.0, to give a
final cell concentration of ~6.5 × 106 cells per
ml, and the suspension was sonicated for three 15-s periods using a
High Intensity Ultrasonic Processor (Sonics and Materials, Danbury, CT)
to disrupt the cells. A portion of each cell homogenate was centrifuged
at 20,000 g for 30 min at 4°C to obtain the supernatant
fraction. Protein concentrations in the cell homogenates and
supernatant fractions were measured by the bicinchoninic acid method of
Smith et al. (19). Total GSH concentration in the
homogenate was measured by the method of Fariss and Reed
(10) as modified by Stipanuk et al. (23). For
mRNA analysis (Northern and dot blots), 0.75 ml of denaturation solution (ToTALLY RNA kit, Ambion, Austin, TX) was added to the washed
cells in the culture dish, and the cells were then collected by scraping.
Measurement of enzyme activities.
By use of the cell homogenate, which contained ~8-10 mg
protein/ml, CDO activity was measured according to the method of Bagley and Stipanuk (1), except that the volume of the reaction
mixture was reduced to 0.2 ml and the amount of
[35S]cysteine was increased to 10-15 µCi/0.2 ml of
reaction mixture. For assay of GCS activity, the cell homogenate was
further diluted with 0.5 or 1 volume of
N-(2-hydroxyethyl)piperazine-N'-3-propanesulfonic acid (EPPS) buffer, pH 8.5, to give a final concentration of 50 mmol/l
EPPS buffer. GCS activity was assayed as described previously (4), except that the volume of the reaction mixture was
reduced to 0.5 ml and EPPS buffer was used (instead of HEPES) to
maintain the incubation mixture at pH 8.1. Under these assay
conditions, the GSH present in the cell homogenate had no effect on
measured GCS activity (Y. H. Kwon and M. H. Stipanuk,
unpublished results).
Western blot analysis.
Separation of proteins was carried out by one-dimensional SDS-PAGE
(14). Aliquots of supernatants that contained
~20-150 µg of protein were mixed with equal volumes of
SDS-reducing buffer and then were loaded onto polyacrylamide gels (10 and 15% wt/vol polyacrylamide for GCS-HS and CDO, respectively, with
4% stacking gels). Medium-range molecular weight markers (Promega,
Madison, WI) and Rainbow molecular weight markers (Sigma) were used for estimation of protein molecular weights.
Protein blotting was performed using the procedure for tank transfer
described by Hoefer Scientific (San Francisco, CA) to an Immobilon-P
polyvinylidene difluoride transfer membrane (Millipore, Medford, MA).
Membranes were incubated with the IgG fraction of rabbit polyclonal
anti-rat CDO for 3 h or with rabbit polyclonal anti-rat GCS-HS for
2 h at room temperature. Immunoreactive protein was detected by
chemiluminescence with the use of goat anti-rabbit IgG (Pierce,
Rockford, IL) conjugated to horseradish peroxidase (HRP) and the
Supersignal CL-HRP substrate system (Pierce) with exposure to Kodak
X-OMAT SRP film. Bands were scanned with a Hewlett Packard Scanjet 3C
(Hewlett Packard, Camas, WA), and two-dimensional quantitative
densitometric analysis of the scanned bands was done using the
Molecular Analyst program (Bio-Rad Laboratories, Hercules, CA).
Relative protein amounts were quantitated using standard curves (pixel
density/mm2 vs. µg total protein loaded) run on each gel;
standard curves were generated by loading incremental amounts
(20-50 µg for CDO, 15-45 µg for GCS-HS) of total
supernatant protein from hepatocytes cultured in
methionine-supplemented medium on each gel. The relative amount of
protein was then divided by the actual amount of total protein loaded
for each sample.
Extraction of cellular RNA and mRNA analysis.
Total RNA was isolated from cultured hepatocytes using a ToTALLY RNA
kit (Ambion) based on the method of Chomczynski and Sacchi (8). Northern blot analysis was done as described by Brown (7) with electrophoresis on a 1% (wt/vol)
agarose-formaldehyde gel and blotting onto a Magna Graph nylon membrane
(Micron Separations, Westboro, MA). Membranes were prehybridized using
herring sperm DNA and then hybridized with one of the
32P-labeled cDNA probes (2 × 106 cpm/ml)
for rat CDO, rat GCS-HS, or mouse glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. Probes for CDO and GCS-HS mRNAs were prepared and labeled as described previously (4). GAPDH
mRNA was synthesized using GAPDH mouse DECAprobe template (Ambion). After hybridization, membranes were washed twice in 2× saline-sodium phosphate-EDTA buffer (SSPE) with 0.2% (wt/vol) SDS for 30 min at room
temperature and twice in 0.1× SSPE at 60°C for 15 min and then
autoradiographed using Kodak X-OMAT film. Probes were stripped from the
membrane by boiling the membrane in 0.1× SSPE with 1% (wt/vol) SDS
between hybridization with the three probes.
For quantification of relative levels of mRNA, aliquots of total RNA
(5-15 µg/dot) were applied to a Magna Graph nylon membrane using
a microsample filtration manifold (Minifold, Schleicher and Schuell,
Keene, NH). The membranes were hybridized with the [32P]cDNA for CDO, GCS-HS, or GAPDH mRNA. Results were
quantified using a Bio-Rad GS-363 Phosphorescence Imaging System
(Bio-Rad, Melville, NY) and Molecular Analyst program (Bio-Rad). A
standard curve was generated on each membrane by loading incremental
amounts (1.25-15 µg) of pooled total RNA from hepatocytes
cultured in methionine-supplemented medium. Relative amounts of mRNA
for each enzyme mRNA and for GAPDH were calculated using a standard
curve of pixel density per square millimeter vs. the amount of total RNA loaded. Relative enzyme mRNA amount was calculated from the standard curve and was corrected for loading by dividing the relative enzyme mRNA amount by the relative GAPDH mRNA amount.
Statistics.
Data were analyzed either by the paired t-test or by
analysis of variance (Minitab 10.5., Minitab, State College, PA) and Tukey's
-procedure (20). Differences were accepted at
P
0.05. Data for CDO activity and relative amounts
of CDO protein were transformed to log10 before statistical
analysis. Correlation coefficients were calculated using Microsoft
Excel 5.0 (Microsoft, Cambridge, MA).
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RESULTS |
Effects of methionine, cysteine and their metabolites on the
activities of CDO and GCS.
As shown in Table 1, addition of 2 mmol/l
methionine, homocysteine, or cysteine to the basal medium was effective
in both increasing CDO activity and decreasing GCS activity. Neither
S-adenosylmethionine nor cystathionine was effective in
significantly increasing CDO activity, although both compounds can be
converted to homocysteine and then cysteine.
S-Adenosylmethionine and cystathionine did significantly
decrease GCS activity, but the decrease was less than that obtained
with cysteine, homocysteine, or methionine. Taurine and sulfate, which
are catabolites of cysteine, were not effective as regulators of either
CDO or GCS activity. The changes in CDO and GCS activities across all
treatments were reciprocal, with the correlation coefficient for CDO
activity vs. GCS activity being r =
0.87.
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Table 1.
Effect of addition of methionine, cysteine, or their metabolites to
medium on the activities of CDO and GCS in cultured rat hepatocytes
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Effects of PPG on the ability of methionine and cysteine to affect
expression of CDO and GCS.
As shown in Fig. 2, the addition of PPG
significantly decreased the GSH concentration in cells cultured in
either basal or methionine-supplemented medium but not in cells
cultured in cysteine-supplemented medium. Despite a higher initial GSH
concentration in methionine-supplemented cells, the GSH content of
cells cultured in either basal or methionine-supplemented medium
reached a similar low level of ~10-15 nmol/mg protein
(~1.5-2.5 µmol/g wet weight of hepatocytes) after 2 days of
treatment with PPG. The marked decrease in the GSH content of cells
cultured in basal medium, which provided much more cysteine than
methionine [0.49 mmol/l cyst(e)ine and 0.1 mmol/l methionine], or
in medium supplemented with excess methionine (+ 2.0 mmol/l)
demonstrates the quantitatively important contribution of methionine as
a precursor of cysteine for GSH synthesis. The level of GSH in cells
cultured in cysteine-supplemented medium was not significantly
decreased by PPG, as would be expected when excess preformed cysteine
is available.

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Fig. 2.
Effect of sulfur amino acids and/or PPG on cellular
glutathione (GSH) concentration in cultured hepatocytes. Rat
hepatocytes were cultured in basal medium or medium supplemented with 2 mmol/l L-methionine (+ Met) or 2 mmol/l
L-cysteine (+ Cys) for 72 h. The effect of PPG was
assessed by addition of 1 mmol/l PPG to the culture medium for the last
48 h. Results are expressed as means ± SE for experiments
with hepatocytes isolated from 3 different rats. Values not followed by
a common superscript letter are significantly different
(P < 0.05) by ANOVA and Tukey's -procedure.
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As shown in Fig. 3A, CDO
activity was 14 times higher in cells cultured in medium supplemented
with methionine and 23 times higher in cells cultured in medium
supplemented with cysteine than in cells cultured in basal medium. The
addition of PPG to methionine-supplemented medium significantly reduced
the activity of CDO to the basal level. In contrast, the activity of
CDO in cells in cysteine-supplemented medium remained high, showing no apparent effect of PPG. No effect of PPG on CDO activity was observed in hepatocytes cultured in basal medium in which CDO activity was
already very low.

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Fig. 3.
Effect of sulfur amino acids and/or PPG on the activities
of CDO (A) and GCS (B) in cultured hepatocytes.
Rat hepatocytes were cultured in basal medium or medium supplemented
with 2 mmol/l L-methionine (+ Met) or 2 mmol/l
L-cysteine (+ Cys) for 72 h. The effect of PPG was
assessed by addition of 1 mmol/l PPG to the culture medium for the last
48 h. Results are expressed as means ± SE for experiments
with hepatocytes isolated from 3 different rats. Values not followed by
a common superscript letter are significantly different
(P < 0.05) by ANOVA and Tukey's -procedure.
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GCS activity is cells cultured in medium supplemented with methionine
was 54% of the basal level, and that in cells cultured in medium
supplemented with cysteine was 26% of the basal level (Fig.
3B). Addition of PPG to the methionine-supplemented medium blocked the normally observed decrease in GCS activity in response to
methionine supplementation. However, addition of PPG had no significant
effect on GCS activity in cells cultured in the basal or
cysteine-supplemented medium. Neither CDO nor GCS activity was
significantly correlated with cellular GSH concentration
(r = 0.48 for CDO and
0.61 for GCS).
The relative amounts of CDO and GCS-HS protein, which were quantified
by Western blot analysis of samples from individual experiments, are
shown in Table 2. The band detected by
antibody for CDO corresponded to a molecular mass of ~23.5 kDa. This
band corresponds to the lower of the two apparent forms of CDO (23.5 and 25.5 kDa) observed by Bella et al. (4, 5) for rat
liver CDO. No other CDO antibody-reactive bands were observed. The
level of CDO protein in cells cultured in basal medium was too low to be quantified in some experiments. Nevertheless, this basal level clearly was significantly increased in hepatocytes cultured in medium
supplemented with methionine or cysteine to levels that were at least
10 or 15 times basal, respectively. Addition of PPG to the
methionine-supplemented medium prevented the increase in CDO protein
level, but addition of PPG did not affect the level of CDO protein in
cells cultured in cysteine-supplemented medium. These effects on CDO
protein are consistent with observed changes in the activity of CDO in
response to sulfur amino acids and PPG.
On Western analysis, the band detected by antibody for GCS-HS had a
molecular mass of ~74 kDa, which corresponds to the reported molecular mass of rat kidney GCS-HS (18) and to that
reported by Bella et al. (4) for rat liver GCS-HS. No
other GCS-HS antibody-reactive bands were observed. Addition of
methionine or cysteine to the medium resulted in levels of GCS-HS
protein that were 10 or 37% less, respectively, than those in cells
cultured in basal medium; the difference was significant between cells
cultured in basal vs. cysteine-supplemented, but not
methionine-supplemented, medium (Table 2). The more marked effect of
cysteine than of methionine in decreasing GCS-HS protein level is
consistent with the greater effect of cysteine than of methionine in
increasing CDO protein. PPG treatment did not have a significant effect
on GCS-HS concentration in cells cultured in methionine-supplemented
medium, which is not unexpected because methionine supplementation
alone did not cause a significant decrease in the GCS-HS protein level
in this study. As observed for GCS activity, the treatment of cells
cultured in cysteine-supplemented medium with PPG did not significantly affect the concentration of GCS-HS, which remained low.
The relative amounts of CDO and GCS-HS mRNAs, which were determined on
individual samples by quantitative dot blot analysis with correction
for relative level of GAPDH mRNA in the sample, are reported in Fig.
4. Northern blots for CDO and GCS-HS
mRNAs, which were run on pooled samples for each treatment group, are shown below the bar graphs. The cDNA probe for CDO mRNA hybridized with
a single band of ~1.7 kb, which is consistent with the reported size
of rat liver CDO mRNA (13). The GCS-HS probe hybridized with a single band ~3.7 kb, which is consistent with the reported size of rat kidney GCS-HS mRNA (27).

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Fig. 4.
Effect of sulfur amino acids and/or PPG on the relative
levels of CDO (A) and GCS heavy subunit (GCS-HS;
B) mRNAs in cultured hepatocytes. Rat hepatocytes were
cultured in basal medium or medium supplemented with 2 mmol/l
L-methionine (+ Met) or 2 mmol/l L-cysteine (+ Cys) for 72 h. The effect of PPG was assessed by addition of 1 mmol/l PPG to the culture medium for the last 48 h. The amounts of
CDO and GCS-HS mRNA were adjusted for the amount of
glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each aliquot
of RNA loaded onto the gel. Results are expressed as means ± SE
for experiments with hepatocytes isolated from 3 different rats. Values
not followed by a common superscript letter are significantly different
(P < 0.05) by ANOVA and Tukey's -procedure.
Northern blot analysis of CDO and GCS-HS mRNAs, which was done on
pooled samples from each treatment group (n = 3;
samples pooled on the basis of equal amounts of total RNA), is shown
below the bar graphs.
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The CDO mRNA level was significantly higher in cells cultured with
either supplemental methionine (1.5-fold higher) or cysteine (4-fold
higher) than in cells cultured in basal medium (Fig. 4A). Supplementation with cysteine resulted in significantly higher levels
of CDO mRNA than did supplementation with methionine. Treatment of
cells with PPG did not significantly reduce the concentration of CDO
mRNA in cells cultured in either basal or cysteine- or methionine-supplemented medium.
The concentration of GCS-HS mRNA in cells cultured in methionine- or
cysteine-supplemented medium was 88 or 41%, respectively, of that in
cells cultured in basal medium (Fig. 4B). Supplemental methionine did not have a statistically significant effect on the
GCS-HS mRNA level, whereas supplemental cysteine resulted in a
significantly lower GCS-HS mRNA concentration. Addition of PPG to the
medium tended to increase the GCS-HS mRNA concentration in cells
cultured in either basal or sulfur amino acid-supplemented medium, but
the effect of PPG was significant only for methionine-supplemented cells.
Effects of buthionine sulfoximine on the ability of methionine and
cysteine to affect expression of CDO and GCS.
Although cellular GSH concentrations were not closely correlated with
CDO or GCS activity in the studies with PPG, the possible role of GSH
concentration in regulation of CDO activity was studied further using
buthionine sulfoximine (BSO) to block GSH synthesis. As shown in Fig.
5, the GSH concentration tended to be
higher in hepatocytes cultured in medium supplemented with sulfur amino acids and without BSO. Addition of BSO decreased the concentration of
GSH by >90% in cells cultured in either basal or sulfur amino acid-supplemented medium. GSH concentration was low and similar in all
cells cultured with BSO (~2-4 nmol/mg protein, or
~0.3-0.7 µmol/g wet wt of hepatocytes), regardless of whether
or not the medium was supplemented with sulfur amino acid.

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Fig. 5.
Effect of sulfur amino acids and/or BSO on cellular GSH
concentration in cultured hepatocytes. Rat hepatocytes were cultured in
basal medium or medium supplemented with 2 mmol/l
L-methionine (+ Met) or 2 mmol/l L-cysteine (+ Cys) for 72 h. The effect of BSO was assessed by addition of 100 µmol/l BSO to the culture medium for the last 48 h. Results are
expressed as means ± SE for experiments with hepatocytes isolated
from 3 different rats. Values not followed by a common superscript
letter are significantly different (P < 0.05) by ANOVA
and Tukey's -procedure.
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Although the cellular GSH level was significantly depleted by addition
of BSO to the medium, the activity of CDO was not affected (Fig.
6A). The lack of effect on CDO
activity clearly indicates that the response of CDO activity to sulfur
amino acids is not mediated by the changes in GSH concentration. The
correlation coefficient for CDO activity vs. GSH concentration was
0.19.

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Fig. 6.
Effect of sulfur amino acids and/or BSO on the activities
of CDO (A) and GCS (B) in cultured hepatocytes.
Rat hepatocytes were cultured in basal medium or medium supplemented
with 2 mmol/l L-methionine (+ Met) or 2 mmol/l
L-cysteine (+ Cys) for 72 h. The effect of BSO was
assessed by addition of 100 µmol/l BSO to the culture medium for the
last 48 h. Results are expressed as means ± SE for
experiments with hepatocytes isolated from 3 different rats. Values not
followed by a common superscript letter are significantly different
(P < 0.05) by ANOVA and Tukey's -procedure.
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The addition of BSO to basal medium decreased GCS activity by
~75%, whereas its addition resulted in an apparent 50% decrease in
GCS activity in cells cultured in sulfur amino acid-supplemented media
(Fig. 6B). The decrease was statistically significant only for cells cultured in basal medium, which had the highest level of GCS
activity in the absence of BSO. Nevertheless, GCS activity was low and
similar in cells grown in media with BSO, regardless of whether or not
the medium was supplemented with methionine or cysteine. Because BSO
was added to inhibit GCS activity and thus to deplete the cellular GSH
concentration, low GCS activity was expected.
Results for quantification of CDO and GCS-HS protein by Western blot
analysis of samples from individual experiments are shown in Table
3. Treatment with BSO did not
significantly affect the level of CDO protein in cells cultured in
basal or sulfur amino acid-supplemented medium, which is consistent
with the observed lack of response of CDO activity to BSO. As seen in
the other studies, supplementation of the culture medium with either
methionine or cysteine significantly increased CDO protein level, with
a greater increase in response to cysteine than to methionine.
The level of GCS-HS protein in cells cultured in the presence of
supplemental cysteine was 42% lower than the level in cells cultured
in basal medium. The apparent 13% decrease in GCS-HS protein in
response to supplemental methionine was not statistically significant.
BSO treatment tended to increase the level of GCS-HS protein compared
with values for cells cultured without BSO, but these apparent
increases were not significant.
Results from dot blot analysis of CDO and GCS-HS mRNAs in individual
samples are reported in Fig. 7 as the
relative amounts of CDO and GCS-HS mRNAs. Northern blots for CDO and
GCS-HS mRNAs, which were run using pooled samples from each treatment
group, are shown below the bar graphs. Culture of hepatocytes with
additional methionine or cysteine significantly increased CDO mRNA to
2.4 or 3.3 times the basal level, respectively (Fig. 7A).
The 48-h exposure to BSO tended to decrease CDO mRNA, but these
apparent decreases were not significant for cells cultured in any of
the media.

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Fig. 7.
Effect of sulfur amino acids and/or BSO on the relative
levels of CDO (A) and GCS-HS mRNAs (B) in
cultured hepatocytes. Rat hepatocytes were cultured in basal medium or
medium supplemented with 2 mmol/l L-methionine (+ Met) or 2 mmol/l L-cysteine (+ Cys) for 72 h. The effect of BSO
was assessed by addition of 100 µmol/l BSO to the culture medium for
the last 48 h. The amounts of CDO and GCS-HS mRNAs were adjusted
for the amount of GAPDH mRNA in each aliquot of RNA loaded onto the
gel. Results are expressed as means ± SE for experiments with
hepatocytes isolated from 3 different rats. Values not followed by a
common superscript letter are significantly different
(P < 0.05) by ANOVA and Tukey's -procedure.
Northern blot analysis of CDO and GCS-HS mRNAs, which was done on
pooled samples from each treatment group (n = 3;
samples pooled on the basis of equal amounts of total RNA), is shown
below the bar graphs.
|
|
As shown in Fig. 7B, GCS-HS mRNA was significantly lower in
hepatocytes cultured in medium with supplemental methionine or cysteine
than in cells cultured in basal medium. The exposure of cultured
hepatocytes to BSO significantly increased GCS-HS mRNA concentration in
cells cultured in both basal and sulfur amino acid-supplemented media,
and this increase in GCS-HS mRNA abundance presumably was in response
to the GSH depletion produced by BSO.
 |
DISCUSSION |
Reciprocal regulation of CDO and GCS.
In all three of the studies reported in this paper, CDO activity was
greater and GCS activity was less in hepatocytes cultured in medium to
which either methionine or cysteine had been added. CDO activity
increased up to 24 times the basal level with cysteine supplementation
of the medium and up to 15 times the basal level with methionine
supplementation of the medium. In contrast, GCS activity in hepatocytes
cultured with methionine decreased to as little as 33% of the
level found in cells cultured in basal medium, and GCS activity in
cells cultured with cysteine decreased to as little as 24% of the
basal level. Thus the capacity for GSH synthesis decreased and the
capacity for cysteine catabolism increased in response to an increased
availability of sulfur amino acid.
Although we used 2 mmol/l of supplemental amino acid or metabolite in
this study, we would expect to see significant effects of lower
concentrations of cysteine or methionine on CDO and GCS activities. In
our initial studies with hepatocytes in culture, we noted a
dose-response relationship for both CDO and GCS activities between 0.1 and 0.5 mmol/l cysteine or methionine and a plateau in responses over
the range of 0.5-5 mmol/l (16). Changes in sulfur
amino acid concentration in the medium were not monitored over the 24-h
period after each replacement of medium with fresh medium, but a copper
chelator was included in thiol-supplemented medium to minimize thiol oxidation.
This pattern of response of CDO and GCS activities in hepatocytes to
sulfur amino acids in the culture medium is consistent with the pattern
reported previously for these hepatic enzymes in rats fed diets that
contained methionine, cysteine, or protein in excess of the requirement
level (3, 4). For example, supplementation of a basal diet
that contained 100 g of casein/kg with 10 g of
L-methionine/kg resulted in an increase of CDO activity to
35 times the basal level and in a decrease of GCS activity to 47% of
the basal level (4). However, although the pattern of
response was very similar, it should be noted that the CDO activity
observed in cultured hepatocytes was markedly lower than the values
reported for liver from intact rats or for freshly isolated hepatocytes
(3, 4, 5). CDO activity appears to be lost with
dedifferentiation of hepatocytes in culture and is also low in liver
cell lines (16). A second notable difference between
studies with intact rats and these experiments with cultured rat
hepatocytes is that addition of cysteine to the culture medium consistently yielded a greater increase in CDO activity and a smaller
decrease in GCS activity than did an equimolar amount of methionine,
whereas methionine was more effective than cysteine when the sulfur
amino acids were added to a low protein diet (2, 3).
Differences in the rates of intestinal absorption or the rates of
hepatic uptake of methionine and cyst(e)ine in intact rats, differences
in the rate or efficiency of conversion of methionine to cysteine in
intact rats vs. cultured hepatocytes, or differences in the rates of
removal of methionine and cysteine by various pathways could explain
this apparent difference in response of liver cells to methionine vs. cysteine.
Changes in enzyme mRNA and protein concentrations in response to
sulfur amino acid supplementation.
The relative changes in CDO and GCS activities, protein concentrations,
and mRNA concentrations are summarized in Table
4. Increases in CDO activity in response
to sulfur amino acids were associated with similar degrees of changes
in CDO protein concentration and with much smaller changes in CDO mRNA
abundance (r = 0.97 for CDO activity vs. CDO protein
and r = 0.89 for CDO protein vs. CDO mRNA). In previous
studies with rats fed diets supplemented with methionine or cysteine or
with high levels of protein, increases in both CDO activity and CDO
protein, but no changes in CDO mRNA abundance, were observed. In
cultured hepatocytes the increases in CDO activity could be explained
by an increase in the mRNA level (2.5-5.0 times basal) and
increases in CDO protein or activity that were 4-6 times as much
as those predicted from the increases in CDO mRNA. The meaning of the
differences in CDO mRNA levels is uncertain, as the amount of CDO mRNA
found in freshly isolated hepatocytes decreases markedly with
maintenance of these cells in primary culture over 24 h and
remains low through 76 h in culture (Y. H. Kwon, L. L. Hirschberger, and M. H. Stipanuk, unpublished observations).
Although cultured hepatocytes do not exactly model hepatocytes in situ,
the major regulatory change, which was an increase in CDO activity
associated with a similar increase in CDO protein, was consistent
between the animal and cell culture studies. Thus these studies in cell
culture also indicate that regulation of CDO activity in response to
sulfur amino acids is accomplished predominantly via changes in CDO
concentration. The possible role of changes in CDO mRNA abundance and
the significance of the two isoforms of CDO need further study, as does
the mechanism by which the concentration of CDO is increased.
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Table 4.
Relative effects of methionine and cysteine on CDO and GCS activities,
concentrations, and mRNA levels in cultured rat hepatocytes
|
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Decreases in GCS activity in response to supplementation of the culture
medium with sulfur amino acids were associated with similar trends for
GCS-HS mRNA abundance and GCS-HS protein concentration (r = 0.96 for GCS activity vs. GCS-HS protein and
r = 0.95 for GCS-HS protein vs. GCS-HS mRNA). However,
the decreases in GCS-HS protein concentration tended to be less than
the decreases in GCS activity or GCS-HS mRNA concentration, suggesting
that regulation of GCS involves multiple mechanisms. Both the pattern
and the magnitude of the changes in GCS activity, GCS-HS protein
concentration, and GCS-HS mRNA in response to sulfur amino acid
supplementation were similar to those observed in liver of rats fed
diets supplemented with sulfur amino acid plus a mixture of nonsulfur
amino acids or diets high in casein (3, 4). However, when
diets were supplemented with sulfur amino acid alone, the magnitudes of
the decreases in GCS-HS protein concentration and GCS activity were similar, suggesting that the decrease in activity state in vivo depends
on the presence of nonsulfur amino acids as well as sulfur amino acids
(3, 4). Thus again, although the overall changes in
response to sulfur amino acids appear to be similar in liver in vivo
and in hepatocytes in culture, they are not identical, and this
indicates the importance of also doing studies in vivo.
Expression of both subunits of GCS is known to be transcriptionally
regulated, and GCS activity also can be modulated by posttranslational mechanisms (24, 25). The initiating signal for GCS
induction in response to an increase in cysteine is not clear. It seems likely that cysteine has a regulatory effect on GCS-HS expression that
is independent of changes in GSH concentration, because cellular GSH
concentrations were not closely associated with GCS-HS mRNA levels in
these studies with cultured hepatocytes. Additionally, the apparent
decrease in activity state of GCS-HS (GCS activity/GCS-HS protein
concentration) observed in cells cultured in medium supplemented with
sulfur amino acids does not appear to be explained by feedback inhibition of GCS by GSH. The assay conditions used to measure GCS
activity result in GSH concentrations that are too low to have a
measurable effect on activity, and cellular GSH concentrations were not
closely correlated with GCS activity state. Note, for example, that
cells cultured in basal medium had markedly different ratios of GCS
activity to GCS-HS protein ratios, as well as different GCS-HS mRNA
levels, than did cells cultured in cysteine-supplemented media, despite
similar GSH concentrations.
In these studies with cultured rat hepatocytes, we observed induction
of CDO at the level of both mRNA and protein, indicating that
regulation of CDO in response to cysteine may be complex. In studies
with intact rats, no changes in CDO mRNA levels were observed,
indicating that regulation of CDO expression in vivo is clearly
posttranscriptional (3, 4). Yamaguchi et al. (26) showed that injection of cysteine into rats increased
the activity of hepatic CDO without a lag phase, suggesting the rapid activation of a previously existing endogenous CDO by cysteine or the
stabilization of CDO by its substrate. The half-life of CDO is on the
order of 2 to 3 h and has been reported to be increased to 6 h in the presence of excess cysteine, favoring the stabilization hypothesis (11, 12). However, the induction of CDO
activity by cysteine was partially inhibited by cycloheximide
(26), suggesting an involvement of protein synthesis as
well. Although CDO protein concentration and activity were closely
correlated in these studies with cultured hepatoyctes, it should be
noted that preliminary results from our laboratory indicate that the
activity of the CDO isoform with the lower apparent molecular weight,
which is the dominant isoform in cultured hepatocytes, is much less
than that of the other isoform, which is most abundant in liver in vivo
(L. L. Hirschberger and M. H. Stipanuk, unpublished
observations). This observation raises the hypothesis that some type of
posttranslational modification may be involved in regulation of CDO
activity. Clearly the details of the molecular mechanisms involved in
upregulation of CDO remain to be elucidated.
Cellular mediator of response to sulfur amino acids.
When methionine and cysteine and their metabolites were tested for
effectiveness in upregulating CDO and downregulating GCS, methionine,
homocysteine (an intermediate in the transsulfuration pathway), and
cysteine were found to be effective in regulating the activities of
both enzymes. S-Adenosylmethionine and cystathionine, intermediates in the transsulfuration pathway, were effective in
downregulating GCS activity but not in upregulating CDO. The lesser
effect of S-adenosylmethionine and cystathionine may be related to lower rates of cellular uptake of these compounds compared with methionine, cysteine, and homocysteine (21). Taurine
and sulfate, which are metabolites of cysteine, had no effect on either GCS or CDO activity. Because cysteine was as effective as any of its
precursors, it seems very likely that either cysteine or a closely
related compound must play an essential role in the hepatic response to
sulfur amino acids.
A key role of cysteine is also supported by the observation that, in
these studies with cultured hepatocytes, the magnitudes of the effects
of sulfur amino acid supplementation in increasing CDO activity, CDO
concentration, and CDO mRNA concentration or in decreasing GCS
activity, GCS-HS concentration, and GCS-HS mRNA concentration were
consistently greater when cysteine was added than when methionine was added.
To clarify the role of cysteine, we used PPG to block the
transsulfuration of methionine to cysteine at the level of
cystathionine. Inhibition of cystathionine
-lyase (cystathionase)
blocks the last step of the methionine transsulfuration pathway in
which cystathionine is cleaved to release cysteine,
-ketobutyrate, and ammonia. By blocking transsulfuration at the level of
cystathionine, addition of PPG should limit cysteine formation from
methionine sulfur and serine and also result in higher concentrations
of cystathionine and perhaps other transsulfuration intermediates. The
marked decrease in GSH concentration that resulted from treatment of
cells cultured in either basal or methionine-supplemented medium with
PPG indicated the effectiveness of PPG in inhibiting transsulfuration; GSH synthesis depends upon cysteine availability as well as on GCS
activity. Addition of PPG to culture medium supplemented with methionine blocked the increase in CDO activity and the decrease in GCS
activity, but PPG had no effect on CDO or GCS activity in hepatocytes
cultured in medium supplemented with cysteine. Thus methionine was
ineffective in regulating these two enzymes when its conversion to
cysteine was blocked, clearly indicating that cysteine, rather than
methionine or an intermediate in the transsulfuration pathway, is
essential for bringing about sulfur amino acid-induced changes in CDO
and GCS activities in hepatocytes.
Because GSH concentration is closely associated with cysteine
availability (supplied as cysteine or formed from methionine via
transsulfuration), additional studies were done to test the possibility
that GSH, rather than cysteine, may be the mediator of the effect of
sulfur amino acids on CDO and GCS activities. The effectiveness of BSO
in inhibiting GCS, the enzyme that catalyzes the first step in GSH
synthesis, is clear from the low GCS activities (but not GCS-HS protein
concentrations) and low GSH concentrations in hepatocytes cultured with
BSO. The effects of BSO on GCS activity and GSH concentration were
observed regardless of the sulfur amino acid level in the medium.
The consistent lack of response of CDO activity, CDO protein, and CDO
mRNA to BSO despite the marked decreases in the level of GSH that were
induced by BSO indicate that CDO is not regulated by the cellular
content of GSH. GSH concentrations were not correlated with CDO
activity in either the study with PPG or the study with BSO, also
indicating that GSH is not the mediator of the effect of cysteine on
CDO activity.
Although GCS activity was low in cells cultured with BSO due to its
inhibition of GCS, cells cultured with BSO had higher concentrations of
GCS-HS mRNA and GCS-HS protein. This suggests that the low GSH
concentration, or oxidative stress related to it, caused upregulation
of GCS-HS expression. On the other hand, variations in GSH
concentration in cells cultured with or without PPG and with or without
sulfur amino acid were not correlated with GCS activity, suggesting
that a signal other than cellular GSH concentration and/or oxidative
stress is also involved in regulation of GCS activity. It is possible
that cysteine itself, in addition to GSH, is involved in the
downregulation of GCS in response to sulfur amino acid supplementation.
These studies provide strong evidence that cysteine itself, rather than
a precursor or metabolite of cysteine, acts as an initial signal for
regulation of CDO and GCS activities in hepatocytes. Clearly, further
studies of the mechanisms by which cysteine downregulates GCS activity
and upregulates CDO activity need to be conducted, and cultured rat
hepatocytes appear to be a suitable model system for use in some of
those studies.
 |
ACKNOWLEDGEMENTS |
We gratefully acknowledge the assistance of Larry L. Hirschberger.
We also thank Dr. Jay Forman for the anti-GCS-HS serum, Dr. Yu Hosokawa
for the anti-CDO IgG, and Drs. Yu Hosokawa and Nobuyo Tsuboyama for the
EcoR I-cut cDNA for CDO.
 |
FOOTNOTES |
This research was supported in part by United States Department of
Agriculture/Cooperative State Research, Education, and Extension
Service Grants 94-34324-0987 and 99-34324-8120.
Address for reprint requests and other correspondence: M. H. Stipanuk, 225 Savage Hall, Div. of Nutritional Sciences, Cornell University, Ithaca, NY 14853-6301 (E-mail:
mhs6{at}cornell.edu).
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. Section 1734 solely to indicate this fact.
Received 5 October 2000; accepted in final form 26 January 2001.
 |
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