Regulation of hepatic glutathione synthesis: current
concepts and controversies
SHELLY C. LU1
USC Liver Disease Research Center, the Division of Gastrointestinal and Liver Diseases, Department
of Medicine, University of Southern California School of Medicine, Los Angeles, California 90033,
USA
ABSTRACT
Glutathione (GSH) is an important
intracellular peptide with multiple functions ranging
from antioxidant defense to modulation of cell
proliferation. GSH is synthesized in the cytosol of all
mammalian cells in a tightly regulated manner. The
major determinants of GSH synthesis are the availability of cysteine, the sulfur amino acid precursor,
and the activity of the rate-limiting enzyme, g-glutamylcysteine synthetase (GCS). In the liver, major
factors that determine the availability of cysteine are
diet, membrane transport activities of the three
sulfur amino acids cysteine, cystine and methionine,
and the conversion of methionine to cysteine via the
trans-sulfuration pathway. Many conditions alter
GSH level via changes in GCS activity and GCS gene
expression. These include oxidative stress, activators
of Phase II detoxifying enzymes, antioxidants, drugresistant tumor cell lines, hormones, cell proliferation, and diabetes mellitus. Since the molecular
cloning of GCS, much has been learned about the
regulation of this enzyme. Both transcriptional and
post-transcriptional mechanisms modulate the activity of this critical cellular enzyme.—Lu, S. C. Regulation of hepatic glutathione synthesis: current concepts and controversies. FASEB J. 13, 1169 –1183
(1999)
Key Words: g-glutamylcysteine synthetase z cysteine availability z detoxification z antioxidant
CURRENT CONCEPTS: STRUCTURE AND
FUNCTIONS OF GSH
Glutathione is a ubiquitous tripeptide, g-glutamylcysteinyl glycine, found in most plants, microorganisms, and all mammalian tissues. Glutathione
exists in the thiol-reduced (GSH)2 and disulfideoxidized (GSSG) forms (1). Eukaryotic cells have
three major reservoirs of GSH. Almost 90% of cellular GSH are in the cytosol, 10% in the mitochondria,
and a small percentage in the endoplasmic reticulum (2– 4). In the endoplasmic reticulum, where
GSH is implicated in protein disulfide bond formation, the GSH to GSSG ratio is 3:1 (2). In the
0892-6638/99/0013-1169/$02.25 © FASEB
cytoplasm and mitochondria, ratios exceed 10:1 (3,
4). Cytosolic GSH in the rat liver turns over rapidly
with a half-life of 2–3 h. The g-glutamyl linkage
promotes intracellular stability and the sulfhydryl
group is required for GSH’s functions (Fig. 1). The
peptide bond linking the amino-terminal glutamate
and the cysteine residue of GSH is through the
g-carboxyl group of glutamate rather than the conventional a-carboxyl group. This unusual arrangement resists degradation by intracellular peptidases
and is subject to hydrolysis by only one known
enzyme, g-glutamyltranspeptidase (GGT), which is
on the external surfaces of certain cell types (1, 4).
Furthermore, the carboxyl-terminal glycine moiety
of GSH protects the molecule against cleavage by
intracellular g-glutamylcyclotransferase (4). As a
consequence, GSH resists intracellular degradation
and is only metabolized extracellularly.
GSH serves several vital functions, including 1)
detoxifying electrophiles; 2) maintaining the essential thiol status of proteins by preventing oxidation of
-SH groups or by reducing disulfide bonds induced
by oxidant stress; 3) scavenging free radicals; 4)
providing a reservoir for cysteine; and 5) modulating
critical cellular processes such as DNA synthesis,
microtubular-related processes, and immune function (1, 4 – 6). Some of the functions of GSH are
discussed briefly, as this review focuses on the synthesis of GSH.
1
Correspondence: HMR Rm. 415, Department of Medicine, USC School of Medicine, 2011 Zonal Ave., Los Angeles,
CA, 90033, USA. E-mail: [email protected]
2
Abbreviations: ARE, antioxidant response element; AP-1,
activator protein-1; AP-2, activator protein-2; BSO, buthionine
sulfoximine; CMK, Ca21/calmodulin kinase II; DBcAMP,
dibutyryl cyclic AMP; DEM, diethyl maleate; EpRE, electrophile response element; GCS, g-glutamylcysteine synthetase;
GCS-HS, GCS heavy subunit; GCS-LS, GCS light subunit;
GGT, g-glutamyltranspeptidase; GSH, reduced glutathione;
GSSG, oxidized glutathione; IL-1, interleukin-1; IL-1b, interleukin-1 beta; MRE, metal response element; MRP, human
multidrug resistance protein; NF-kB, nuclear factor kappa B;
NO, nitric oxide; TGF-b1, transforming growth factor b1;
TNF, tumor necrosis factor.
1169
(6). Normally, cellular GSSG is kept extremely low
(,1% of total GSH pool) so that protein mixed
disulfide formation may be limited. The thiol-disulfide equilibrium within the cell is known to regulate
a diverse number of metabolic processes including
enzyme activity, transport activity, and gene expression via alteration of redox-sensitive trans-activating
factors (1, 6, 7).
Antioxidant function of GSH
Figure 1. The structure of glutathione, g-glutamylcysteinyl
glycine. The amino-terminal glutamate and cysteine are
linked by the g-carboxyl group of glutamate.
Detoxifying functions of GSH
Detoxification of xenobiotics or their metabolites is
one of the major functions of GSH. These compounds
are electrophiles and form conjugates with GSH either
spontaneously or enzymatically in reactions catalyzed
by GSH S-transferase (1, 4). The conjugates formed are
usually excreted from the cell and, in the case of
hepatocytes, into bile. The metabolism of GSH conjugates begins with cleavage of the g-glutamyl moiety by
GGT, leaving a cysteinyl-glycine conjugate. The cysteinyl-glycine bond is cleaved by dipeptidase, resulting in
a cysteinyl conjugate. This is followed by N-acetylation
of the cysteine conjugate, forming a mercapturic acid
(Fig. 2). The metabolism of GSH conjugates to mercapturic acid begins either in the biliary tree, intestine,
or kidney, but the formation of the N-acetylcysteine
conjugate usually occurs in the kidney (1). In addition
to exogenous compounds, many endogenously formed
compounds also follow similar metabolic pathways.
Some examples include estradiol-17-b, leukotrienes,
and prostaglandins (4). Although the majority of the
conjugation reactions to GSH result in detoxification
of the compound, occasionally the product itself is
highly reactive (1). One such example is the GSH
conjugate of dibromoethane (1). GSH conjugation
irreversibly consumes intracellular GSH.
As a consequence of aerobic metabolism, all aerobic
organisms are subject to a certain level of physiological oxidative stress. The intermediates that are
formed, such as superoxide (O22z) and hydrogen
peroxide, can lead to the further production of toxic
oxygen radicals that can cause lipid peroxidation
and cell injury. The endogenously produced hydrogen peroxide is reduced by GSH in the presence of
selenium-dependent GSH peroxidase (Fig. 3). As a
result, GSH is oxidized to GSSG, which in turn is
reduced back to GSH by GSSG reductase at the
expense of NADPH, forming a redox cycle. Either
GSH peroxidase or GSH S-transferase can reduce
organic peroxides. Hydrogen peroxide can also be
reduced by catalase, which is present only in the
peroxisome. In the mitochondria, GSH is particularly important because there is no catalase. Recent
studies have shown that mitochondrial GSH is critical in defending against both physiologically and
pathologically generated oxidative stress (1, 7, 8). A
selective reduction in the mitochondrial GSH pool
has been reported in rats fed alcohol and may play
an important pathogenetic role in the development
of the liver disease (see below) (7, 8).
Severe oxidative stress may overcome the ability of
the cell to reduce GSSG to GSH, leading to accumulation of GSSG within the cytosol. To protect the cell
Maintenance of essential thiol status
As the dominant nonprotein thiol in mammalian
cells, GSH is essential in maintaining the intracellular redox balance and the essential thiol status of
proteins (1, 4). To achieve this, GSH undergoes
thiol-disulfide exchange in a reaction catalyzed by
thiol-transferase, as follows:
protein-SSG 1 GSH 3 protein-SH 1 GSSG
Since this reaction is a reversible reaction, the equilibrium is determined by the redox state of the cell,
which depends on the concentrations of GSH and
GSSG (proportional to the log of [GSH]2/[GSSG])
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Vol. 13
July 1999
Figure 2. The mercapturic pathway. X, compounds with an
electrophilic center can form GSH conjugate in a reaction
catalyzed by GSH S-transferase. The g-glutamyl moiety is then
cleaved by g-glutamyltranspeptidase, liberating the cysteinylglycine conjugate. This is further broken down by dipeptidase, resulting in the formation of the cysteinyl conjugate.
This is followed by N-acetylation of the cysteine conjugate
catalyzed by N-acetylase, resulting in the formation of a
mercapturic acid.
The FASEB Journal
LU
rated into protein, depending on the need of the
cell, and some is degraded into sulfate and taurine.
For most cells, this mechanism provides a continuous source of cysteine. Thus, the g-glutamyl cycle
allows the efficient utilization of GSH as cysteine
storage.
SYNTHESIS OF HEPATIC GSH
Figure 3. Antioxidant function of GSH. Hydrogen peroxide,
which is generated as a result of aerobic metabolism, can be
metabolized by GSH peroxidase in the cytosol and by catalase
in the peroxisome. To maintain the cellular redox equilibrium, GSSG is reduced back to GSH by GSSG reductase at the
expense of NADPH, thereby forming a redox cycle. Organic
peroxides can be reduced by either GSH peroxidase or GSH
S-transferase. Under severe oxidative stress, the ability of the
cell to reduce GSSG to GSH may be overcome, leading to
accumulation of GSSG within the cytosol. To avoid a shift in
the redox equilibrium, GSSG can either be actively transported out of the cell or react with a protein sulfhydryl (PSH)
to form a mixed disulfide (PSSG).
The liver has one of the highest organ contents of
GSH and is unique in two aspects of GSH biosynthesis. First, the hepatocyte has the unique ability to
convert methionine to cysteine through the transsulfuration pathway (see below); second, the rate of
GSH biosynthesis in the hepatocyte is balanced by its
rate of export into plasma, bile, and mitochondria
via distinct GSH transport systems (7, 9). The mitochondrial GSH transporter maintains the GSH pool
in the mitochondria, which cannot biosynthesize
GSH (7, 8). This transporter is selectively impaired
in rats fed alcohol and accounts for the fall in
mitochondrial GSH in this liver disease model (7, 8).
Pathways that consume GSH, such as formation of
GSH conjugates and mixed disulfides, account for
very little of the hepatocyte GSH utilization under
normal physiological conditions. The importance of
hepatic GSH to the interorgan GSH homeostasis is
from a shift in the redox equilibrium, GSSG can be
actively exported out of the cell or react with a
protein sulfhydryl group, leading to the formation of
a mixed disulfide. Thus, severe oxidative stress depletes cellular GSH (1, 4, 7).
GSH as cysteine storage and the g-glutamyl cycle
One of the most important functions of GSH is to
store cysteine because cysteine is extremely unstable
extracellularly and rapidly auto-oxidizes to cystine, in
a process producing potentially toxic oxygen free
radicals (4, 7). The g-glutamyl cycle (Fig. 4), first
described by Meister in the early 1970s (see ref 4),
allows GSH to serve as a continuous source of
cysteine. Here, GSH is released from the cell by
carrier-mediated transporter(s) (7) and the ectoenzyme GGT then transfers the g-glutamyl moiety of
GSH to an amino acid (the best acceptor being
cystine), forming g-glutamyl amino acid and cysteinylglycine. The g-glutamyl amino acid can then be
transported back into the cell to complete the cycle.
Once inside the cell, the g-glutamyl amino acid can
be further metabolized to release the amino acid and
5-oxoproline, which can be converted to glutamate
and used for resynthesis of GSH. Cysteinylglycine is
broken down by dipeptidase to generate cysteine
and glycine. Cysteine is readily taken up by most if
not all cells. Once inside the cell, the majority of
cysteine is incorporated into GSH; some is incorpoHEPATIC GLUTATHIONE SYNTHESIS
Figure 4. GSH as cysteine storage and the g-glutamyl cycle.
One of the most important functions of the g-glutamyl cycle
is to provide a continuous source of cysteine. Cysteine is taken
up readily by most cells; once it enters the cell, most of it is
incorporated into GSH. Cysteine is also incorporated into
newly synthesized proteins and some is broken down into
sulfate and taurine. GSH is exported from the cell [by
carrier-mediated transporter(s)] and the ecto-enzyme GGT
then transfers the g-glutamyl moiety of GSH to an amino acid
(the best acceptor being cystine), forming g-glutamyl amino
acid and cysteinylglycine (cys-gly). The g-glutamyl amino acid
can then be transported back into the cell to complete the
cycle. Once inside the cell, the g-glutamyl amino acid can be
further metabolized to release the amino acid and 5-oxoproline, which can be converted to glutamate. Cysteinylglycine is
broken down by dipeptidase (DP) to generate cysteine and
glycine, which are then transported back into the cell to be
reincorporated into GSH.
1171
underscored by the fact that plasma GSH and cysteine levels are largely determined by the sinusoidal
efflux of hepatic GSH (9). Therefore, a better understanding of the regulation of hepatic GSH synthesis is central to understanding interorgan GSH
homeostasis under normal and pathological conditions.
GSH is synthesized from precursor amino acids in
the cytosol of virtually all cells (1, 4, 7). The synthesis
of GSH from its constituent amino acids, L-glutamate, L-cysteine, and L-glycine, involves two ATPrequiring enzymatic steps:
[1] L-glutamate 1 L-cysteine 1 ATP 3 g-glutamylL-cysteine 1 ADP 1 Pi
[2] g-glutamyl-L-cysteine 1 L-glycine 1 ATP 3
GSH 1 ADP 1 Pi
The first step of GSH biosynthesis is rate-limiting
and catalyzed by g-glutamylcysteine synthetase
(GCS), which exhibits an absolute requirement for
either Mg21 or Mn21. GCS is composed of a heavy
(GCS-HS, Mr ; 73,000) and a light (GCS-LS, Mr ;
30,000) subunit, which are encoded by different
genes in both rat and human (10 –13). The enzyme
may be dissociated under nondenaturing conditions
by treatment with dithiothreitol (14). The heavy
subunit obtained after this treatment exhibits all the
catalytic activity of the isolated enzyme as well as
feedback inhibition by GSH (14). Although the
heavy subunit is active catalytically, it has a higher Km
value for glutamate (18.2 vs. 1.4 mM) and a lower Ki
value for GSH (1.8 vs. 8.2 mM) compared with the
holoenzyme (15). Thus, the light subunit plays an
important regulatory role for the overall function of
the enzyme and allows the holoenzyme to be catalytically more efficient and less subject to inhibition by
GSH than the heavy subunit alone. The low affinity
of the heavy subunit for glutamate and the high
feedback inhibition exerted by GSH (Ki value for
GSH is about one-third of the normal physiological
liver GSH level) suggest that the heavy subunit alone
is not likely to be active physiologically. This remains
to be proved.
GCS is specific for the glutamyl moiety, and Lys-38
of the heavy subunit may be involved in binding to
glutamate (16). GCS in both rat and human is
regulated physiologically by:
1) Feedback competitive inhibition by GSH (17,
18). Inhibition by GSH is nonallosteric and involves
binding of GSH to the glutamate site and another
site on the enzyme. This latter binding appears to
involve interaction with the thiol moiety of GSH but
not with a methyl group (17).
2) The availability of its precursor, L-cysteine (1, 4,
7). The apparent Km values of GCS for glutamate and
cysteine are 1.8 and 0.1– 0.3 mM, respectively, in
both rat and human (18, 19). The intracellular
glutamate concentration is several folds higher than
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Vol. 13
July 1999
the Km value of GCS for glutamate, but the intracellular cysteine concentration approximates the apparent Km value of GCS for cysteine (7, 19). Therefore,
the availability of intracellular cysteine and the activity of GCS most significantly influence the rate of
GSH synthesis.
The second step in the synthesis of GSH is catalyzed by GSH synthetase. This enzyme has not been
studied as extensively as GCS. GSH synthetase purified from rat kidney has an Mr of ;118,000 daltons
and is composed of two apparently identical subunits
(20). Studies of the mapping of the substrate binding sites of the enzyme with methyl-substituted and
other analogs of the substrates indicate that the
regions of the active site that bind glycine and the
cysteinyl moiety of g-glutamylcysteine are highly specific. The L-g-glutamyl moiety, on the other hand,
can be replaced by a variety of analogs, indicating
that this binding site is not specific (20). This
enzyme has been cloned recently from the rat kidney
(21). The isolated rat kidney enzyme is known to
contain a small amount (2%) of carbohydrate and
two asparagine residues (residues 124 and 171) in
the amino acid sequence, fitting the requirement for
N-linked protein glycosylation, and one serine residue, agreeing with the general pattern for an Olinked N-acetylglucosamine addition site (20, 21).
However, the significance of glycosylation and the
overall regulation of this enzyme remain poorly
understood. Early studies in hog and pigeon liver
suggested that ADP may play a regulatory role (22,
23). GSH synthetase is not subject to feedback inhibition by GSH. One recent study in the yeast Saccharomyces cerevisiae showed that this enzyme is dispensable for growth under both normal and oxidative
stress conditions due to an accumulation of g-glutamylcysteine, which was able to protect against
oxidative stress (24). Overexpression of GSH synthetase failed to increase GSH level whereas overexpression of GCS increased the GSH level, consistent
with the fact that GCS is the rate-limiting enzyme of
GSH synthesis (24). However, GSH synthetase deficiency in humans can result in dramatic metabolic
consequences because the accumulated g-glutamylcysteine is converted to 5-oxoproline, which can
cause severe metabolic acidosis (21).
Factors that determine the availability of cysteine
One of the major determinants of the rate of GSH
synthesis is the availability of cysteine. Cysteine is
normally derived from the diet and protein breakdown, and in the liver from methionine via the
transsulfuration pathway (7, 25). Cysteine differs
from other amino acids because its sulfhydryl form,
cysteine, is predominant inside the cell whereas its
disulfide form, cystine, is predominant outside the
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LU
cell. Cysteine readily autoxidizes to cystine in the
extracellular fluid; once it enters the cell, cystine is
rapidly reduced to cysteine (25). Therefore, the key
factors that regulate the hepatocellular level of cysteine other than diet include membrane transport of
cysteine, cystine, and methionine as well as the
activity of the transsulfuration pathway (25–27). Although glutamate and glycine are also precursors of
GSH, there is no evidence to suggest that their
transport influences GSH synthesis since they are
synthesized via several metabolic pathways within
hepatocytes (25).
Dietary influence
Studies from Tateishi and co-workers have shown
that hepatic GSH level is closely related to nutritional conditions, especially the cysteine content of
the diet (28, 29). Rat liver normally contains 7– 8
mmol GSH/g tissue, mostly in the reduced state.
Starvation for 48 h results in a significant fall in liver
GSH content to between two-thirds and one-half of
the normal levels. On refeeding, the GSH level
returns to normal within a few hours (28). Fasting
did not affect the activities of GCS and GSH synthetase. The effects of fasting and refeeding on
hepatic GSH level were not affected by pretreatment
with actinomycin D or cycloheximide, suggesting
that the amount of enzymes involved in GSH synthesis was unaffected (28). This shows the strong dependency of hepatic GSH level on food intake.
Cysteine transport
Cysteine is transported almost entirely as a neutral
amino acid, and in rat hepatocytes is transported
mainly by the ASC system (26, 30, 31). This system is
Na1 dependent and especially reactive with neutral
amino acids with short to intermediate length side
chains such as serine and alanine. This system has
high stereospecificity (it does not take up D-cysteine), is pH sensitive (a change from 7.4 to 6.5
resulted in suppression of L-cysteine transport by
30%), and is not sensitive to adaptive regulation or
insulin and glucagon stimulation under conditions
producing these effects for System A (26, 31). The
ASC system mediates both inward and outward flows
of its substrate amino acids and is subject to transstimulation (32): a System ASC amino acid at one
side of the membrane stimulates the transport of
another System ASC amino acid present at the other
side of the membrane. Therefore, the intracellular
cysteine concentration depends on the intracellular
and extracellular levels of not only cysteine, but also
other System ASC amino acids. Higher levels of
extracellular cysteine will raise its intracellular level,
but higher levels of other System ASC amino acids
HEPATIC GLUTATHIONE SYNTHESIS
will inhibit competitively the influx of cysteine (cisinhibition) and stimulate the efflux of cysteine
(trans-stimulation).
Cystine transport
The transport of cystine is distinct from that of
cysteine (30, 33, 34). Cystine has four ionizable
groups and is present mostly as the tetrapolar ion at
neutral pH. However, because the pKs of the two
amino groups of cystine are ; 7.9 and 8.8, a considerable part of cystine (; 20% of total cystine at pH
7.4) is present as the tripolar ion at physiological pH
(25). The system that transports this anionic form of
cystine is System Xc2. Glutamate also exists in anionic form at physiological pH and is the only other
significant substrate for System Xc2. This system is
Na1 independent and mediates one-to-one exchange of cystine for glutamate (34). The physiologically significant flow via System Xc2 is an entry of
cystine accompanied by an exit of glutamate, because cells are usually rich in glutamate, which is also
transported by systems other than Xc2 and synthesized from its precursor, glutamine, but not in cystine (25). The driving force of this exchange appears
to be a steep concentration gradient of glutamate
(outside the fibroblast-1.6 mM, inside-24 mM) (25).
Probably this high intracellular glutamate concentration functions to stimulate the influx of cystine to
maintain an adequate balance between cysteine and
glutamate inside the cells.
Cystine transport in hepatocytes is also mediated
by System Xc2. Under normal physiological conditions, cystine is taken up poorly by hepatocytes (27,
30, 35). However, on culturing rat hepatocytes, the
activity of System Xc2 emerges after a 12 h lag in
response to insulin and dexamethasone. The increase in System Xc2 activity is dependent on de novo
synthesis of both RNA and protein (27). This may be
an adaptive response of cultured cells since there is
no detectable cysteine in the culture medium (almost completely autoxidized). The activity of System
Xc2 can also be induced by treatment with electrophilic agents and O2 (25, 35). In both fibroblasts and
isolated hepatocytes, treatment with electrophilic
agents depletes intracellular GSH levels, followed by
enhanced cystine uptake via System Xc2 and restoration of cell GSH levels. When cystine uptake was
inhibited by glutamate or homocysteate, restoration
of cell GSH level was prevented (35). This suggests
that System Xc2 is involved in the cell defense
mechanism against an electrophilic attack by facilitating increased synthesis of GSH, which in turn may
serve for detoxification of the electrophiles. The
exact mechanisms of this induction is unclear at
present, and it is not known whether it occurs in
intact liver. Although normal hepatocytes do not
1173
transport cystine, GSH released from the cells can
undergo thiol-disulfide exchange with cystine, liberating cysteine, which is then available to the hepatocytes (36). This phenomenon has also been observed
in HepG2 cells (37). In fact, this was the sole
mechanism for providing cysteine for GSH synthesis
in HepG2 cells, which are not able to convert methionine to cysteine (see below) (37).
Methionine transport
Methionine is transported mainly by System L in
hepatocytes, as the increase in the intracellular GSH
level by methionine was almost completely inhibited
by the presence of 2-aminobicyclo-(2, 2, 1)heptane2-carboxylic acid (BCH, a model substrate of System
L), but was scarcely affected by a-methylamino isobutyrate (MeAIB, a model substrate of System A) (26,
38). System L is Na1 independent and not responsive to either adaptive control or hormonal stimulation. System L also exhibits trans-stimulation. Studies
from Kilberg and co-workers (26) of the kinetics of
substrate uptake by System L revealed two components to this system. Component I has high affinity,
low capacity (estimated Km values ,200 mM) whereas
component II has low affinity and high capacity
(estimated Km values between 2 and 5 mM) (26). In
rat hepatocytes, methionine is transported biphasically by both components of system L (39). In terms
of the relative rates of uptake of these three sulfur
amino acids by rat hepatocytes cultured for 24 h, the
rate of uptake of cysteine is about 3-fold higher than
that of methionine and 13-fold higher than that of
cystine (38).
Transsulfuration pathway
The ability for the liver cell to convert methionine to
cysteine is important since the liver is the major site
of methionine catabolism and the major storage
organ for GSH. That cysteine can be synthesized
from methionine was first demonstrated by Tarver
and co-workers in 1939 (40). This pathway is termed
the transsulfuration or the cystathionine pathway
(Fig. 5). This pathway is active and unique to the
liver cell, as it is absent or insignificant in other
GSH-synthesizing systems either in normal or in
transformed tissues (41). The activity of this pathway
in the liver is also markedly impaired or absent in the
fetus and newborn infant, cirrhotic patients, and
patients with homocystinemia (42, 43). Liver cancer
cells such as HepG2 and HuH-7 cells have a block in
the transsulfuration pathway proximal to the formation of homocysteine. These cells are unable to form
GSH from methionine but are able to from homocysteine (37). The mechanism of this block is unknown.
1174
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July 1999
Figure 5. Hepatic methionine metabolism and GSH synthesis.
The transsulfuration pathway converts methionine to cysteine, which is then converted to GSH via the GSH synthetic
pathway. Methionine can also be resynthesized from homocysteine. ①, methionine adenosyltransferase; ②, transmethylation reactions; ③, S-adenosylhomocysteine hydrolase; ④,
cystathione b-synthase; ⑤, g-cystathionase; ⑥, g-glutamylcysteine synthetase; ⑦, GSH synthetase; ⑧, methionine synthase;
⑨, betaine-homocysteine methyltransferase.
Characterization of the transsulfuration pathway in
the liver has been achieved mainly through the efforts
of Reed and co-workers using isolated hepatocytes (44,
45). Methionine is sequentially converted to cysteine
via several enzymatic steps. The first step requires ATP
and involves the activation of methionine to S-adenosylmethionine in a reaction catalyzed by methionine
adenosyltransferase. In contrast to the two GSH-synthesizing enzymes, which have a low Km for ATP (;0.1
mM), the Km of hepatic methionine adenosyltransferase for ATP is high (;2 mM) (46). Therefore,
hypoxic depletion of ATP is more likely to affect GSH
synthesis from methionine than cysteine. Subsequent
demethylation and the removal of the adenosyl moiety
yields homocysteine. Homocysteine condenses with
serine to form cystathionine in a reaction catalyzed by
cystathionine synthase. Cleavage of cystathionine, catalyzed by cystathionase, then releases free cysteine. In
this pathway methionine and homocysteine are readily
interconvertible, but the subsequent step, the formation of cystathionine, is irreversible (Fig. 5). Proparglyglycine is a potent irreversible inhibitor of cystathionase; its use has been instrumental in demonstrating
the importance of this pathway in the biosynthesis of
GSH (44).
The main regulatory controls of the transsulfuration pathway appear to center on keeping a fine
control on the methionine level. The liver methionine level is relatively insensitive to changes in
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TABLE 1. Conditions known to alter GCS activity and GCS subunit expression
Treatments/conditions
Oxidative stressa
Drug-resistant tumor cell lines
Cytokines (TNF, IL-1b)
Heat shock
Antioxidants
Heavy metals
GSH-conjugating agentsb
Copper deficiency
Hormones:c insulin, glucocorticoids
Growth and cell cycle related
Nitric oxide
Esai hyperbilirubinemic rats
GSH level
Miscellaneous: high glucose
TGFb1
cycloheximide
Effects on GCS-HS
Effects on GCS-LS
References
1 Transcription
mRNA stabilization
1 Transcription
1 Transcription
1 mRNA level
1 mRNA level
1 mRNA level
1 Transcription
mRNA stabilization
1 mRNA level
1 Transcription
1 Transcription
Depends on the
cell
1 mRNA level
Controversial
2 Transcription
2 Transcription
mRNA stabilization
1 Transcription
mRNA stabilization
Controversial
?
?
?
?
1 Transcription
mRNA stabilization
?
No effect
No effect
1 mRNA level in aortic
smooth muscle cell
?
?
?
?
?
60–65, 72, 76, 81–83,
87–93
53–59, 80, 84, 94
64, 73, 74
70
69, 71
60
65, 71, 72
75
19, 65, 66
65–68, 85
95, 96
78
65, 73, 92
73
86
79
a
4-Hydroxy-2-nonenal increased transcription and mRNA stabilization of both GCS subunits (93).
DEM increased GCS subunit mRNA levels by both increased transcription and mRNA stabilization (72).
Hormone addition in cultured hepatocytes increased transcription of GCS-HS but hormone deficiency in vivo resulted in decreased
GCS-HS mRNA level. ? 5 unknown, not examined.
b
c
dietary protein intake (47). The control appears to
be exerted at the level of homocysteine: when methionine is needed, homocysteine is remethylated by
methionine synthase or betaine-homocysteine methyltransferase; when methionine is in excess, catabolism of homocysteine via the cystathionine synthase
reaction is accelerated (48).
Hepatic conversion of methionine to S-adenosylmethionine represents the major catabolic pathway
for methionine, accounting for nearly half of the
daily intake of methionine (43). Patients with liver
cirrhosis often exhibit hypermethioninemia and
have impaired methionine clearance (49, 50) because the activity of hepatic methionine adenosyltransferase is significantly impaired (43). This impairment has been postulated to be one of the
mechanisms of a decreased hepatic GSH level (43).
In support of this, administration of S-adenosylmethionine to patients with liver cirrhosis resulted in
increased hepatic GSH level (51). Preliminary results
showing an improved 2-year survival in alcoholic liver
disease patients treated with S-adenosylmethionine
have also been presented (52). However, the protective mechanisms of S-adenosylmethionine remain ill
defined.
Regulation of GCS
The activity of GCS is the other major determinant of
the rate of GSH synthesis. GCS is a heterodimer
made up of a catalytic (heavy, 73 kDa) and a regulatory (light, 30 kDa) subunit. Changes in GCS
activity can result from regulation at multiple levels
HEPATIC GLUTATHIONE SYNTHESIS
affecting only the heavy or both the heavy and light
subunits. Transcriptional and post-transcriptional
regulation of both subunits have been described.
Post-transcriptional regulation includes both mRNA
stabilization/destabilization and post-translational
modification. Most of the studies examined transcriptional regulation of GCS. Table 1 lists conditions known to alter GCS activity and GCS expression
(19, 53–96). Almost all of the reports published
before 1997 focused on the regulation of the heavy
subunit. These conditions fall into several major
categories, including oxidative stress, drug-resistant
tumor cells, hormones, and growth related. For
simplicity, Table 1 lists cytokines and GSH-conjugating agents as separate categories, but these may also
induce oxidative stress. Inducers of Phase II detoxifying enzymes such as b-naphthoflavone can also
induce oxidative stress. Most of these conditions
[with the exception of drug-resistant tumor cell
lines, heat shock, heavy metals, glucose, cycloheximide, and transforming growth factor b1 (TGFb1)]
altered GCS expression in liver cells.
Oxidative stress
Agents ranging from methyl mercury, quinones,
hydrogen peroxide, menadione, cytokines such as
tumor necrosis factor (TNF), buthionine sulfoximine (BSO), tert-butylhydroquinone, diethyl maleate
(DEM), okadaic acid, 4-hydroxy-2-nonenal, and ionizing radiation, which cause oxidative stress in a
variety of cells and organs, increased GCS activity
and GCS-HS transcription (60 – 65, 72, 76, 81– 83,
1175
87–93). In addition to increased gene transcription,
DEM and 4-hydroxy-2-nonenal treatments have also
been found to stabilize the GCS-HS mRNA (72, 93).
Recently, increased transcription of GCS-LS has also
been found in response to DEM, BSO, tert-butylhydroquinone, b-naphthoflavone, and 4-hydroxy-2nonenal (63, 65, 82, 83, 93, 97). 4-Hydroxy-2-nonenal also stabilized the GCS-LS mRNA (93). Thus,
many causes of oxidative stress have similar effects on
the expression of both GCS subunits.
Recent efforts have focused on elucidating the
molecular mechanism(s) of oxidative stress induced
increase in GCS expression. The 59-flanking regions
of both human GCS subunits have been cloned and
sequenced (63, 83, 97–99). Putative nuclear factor
kappa B (NF-kB), Sp-1, activator protein-1 (AP-1),
activator protein-2 (AP-2), metal response (MRE),
and antioxidant response (ARE)/electrophile-responsive elements (EpRE) have been identified in
the promoter of the heavy subunit (63, 98). The
promoter of the light subunit also contains AP-1,
AP-2, MRE and ARE/EpRE elements (83, 97). The
region from 2817 to 145 nucleotide sequence of
the human GCS-HS contains AP-1 or AP-1 likeresponsive elements that are critical in mediating the
effects of cigarette smoke condensate solution, menadione, hydrogen peroxide, TNF, and ionizing radiation on GCS-HS promoter activity (64, 76, 77, 87,
88). In menadione and hydrogen peroxide-mediated
increase in GCS-HS gene transcription, the most
important AP-1 element was found to be located at
2269 to 2263 (87). The same element was also
found to be responsible for induction of GCS-HS
expression in BSO-resistant cell lines (91). Recent
studies from Sekhar et al. and Morales et al. have
further confirmed the importance of AP-1 activity in
mediating the effect of oxidative stress on GCS-HS
transcription (81, 88). On the other hand, Mulcahy
et al. described a critical distal ARE element (ARE4),
located ;3.1kb upstream of the transcriptional start
site, which mediated constitutive and b-naphthoflavone inducible expression in HepG2 cells (63).
b-Naphthoflavone is known to activate Phase II enzymes via AREs (63). AREs are also referred to as
electrophile-responsive elements (EpRE), which is
the more appropriate term since these elements are
clearly induced by pro-oxidants (99). The ARE4
element of the human GCS-HS has a consensus ARE
sequence (59-GTGACTCAGCG-39) and an embedded PMA-responsive element (TRE, underlined),
which can bind AP-1 binding proteins (99). Thus,
AP-1 binding proteins may still be involved in activating ARE4. This possibility was recently investigated by Wild and colleagues (99). They demonstrated that the AP-1 binding sequence of the
AREA4-mediated constitutive expression of the
GCS-HS promoter, but not b-naphthoflavone-in1176
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July 1999
duced expression, and that other unknown protein
complexes are involved (99). An adjacent ARE
(ARE3), located 34 bases downstream from ARE4,
also was not required for b-naphthoflavone’s effect
on GCS-HS gene expression (99).
EpRE and AP-1 have also been found to be important in mediating the effect of b-naphthoflavone on
GCS-LS expression (97). There is a consensus EpRE
site located around 300 base pairs upstream from the
translational start site and a consensus AP-1 binding
site located 33 base pairs upstream of the EpRE.
Both are capable of supporting b-naphthoflavone
inducibility, but the EpRE is more potent. Basal
expression is influenced by the AP-1 site whereas
EpRE is not required for maximal basal expression
(97). Thus, both of the GCS subunits share in
common the presence of functional AP-1 and EpRE,
which likely mediate the effect these various prooxidants have on GCS subunit gene expression. The
nature of the trans-activating protein(s) that bind the
ARE or EpRE sites remain to be defined.
Although the effect of oxidative stress and inducers of Phase II enzymes on the GCS subunits expression appear to be similar, different signaling pathways are involved at least in the case of 4-hydroxy-2nonenal (93). This agent increased the transcription
and stabilized the mRNA of both subunits, but de
novo protein synthesis was required only for the light
subunit (93). The molecular mechanisms underlying
these differences remain to be elucidated.
Drug-resistant tumor cells
Resistance to commonly used alkylating agents or
platinum chemotherapeutic drugs is often multifactorial, involving altered drug transport, biotransformation, and enhanced detoxification capacity (53–
59, 80, 84). GSH has been found to be elevated in a
number of drug-resistant tumor cell lines and in
tumor cells isolated from patients whose tumors are
clinically resistant to drug therapy (53). The increase
in cell GSH is a major contributing factor to drug
resistance by binding to or reacting with drugs,
interacting with reactive oxygen moieties, preventing
damage to proteins or DNA, or by participating in
DNA repair processes. Although increased GSH in
drug-resistant tumor cells has been documented for
over 20 years, the mechanism for the increase was
elucidated more recently. Increased GCS activity,
steady-state GCS-HS mRNA level, and GCS-HS gene
transcription have been found (53–58, 80, 84).
Whether or not GCS-LS is also altered is controversial. MRP (human multidrug resistance protein), a
member of the superfamily of ATP binding cassette
membrane transporters, is capable of conferring
resistance to multiple classes of chemotherapeutic
agents (57, 58). Several studies have shown coordi-
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nated overexpression of GCS-HS and MRP in drugresistant tumor cell lines, human colorectal tumors,
and human lung cancer specimens after platinum
drug exposure (57, 58). In contrast, no correlation
was found between MRP and GCS-LS expression
(57). Godwin et al. found cisplatin-resistant ovarian
tumor cell lines exhibited increased levels of GCS-HS
and GCS-LS proteins (56). However, Yao et al. found
no difference in the mRNA level of GCS-LS among
different ovarian cancer cell lines with varying cisplatin resistance (84). The reason for the discrepancy is
unclear. In the latter work, AP-1 binding was highly
associated with cisplatin resistance and increased
GCS-HS expression, whereas AP-2 and NF-kB binding activities were not (84). Using a cisplatin-resistant
lung cancer cell line SBC-3, Tomonari et al. described the 59-flanking sequence from 2192 to 191
(especially 2108 to 228 and 134 to 191 bp) to be
involved in cisplatin-induced transcriptional up-regulation of GCS-HS (80). These regions contain two
AP-2 sites, an Sp1 site and a GCF binding site, but no
AP-1 site (80). Different mechanisms and cis-acting
elements may be involved in mediating transcriptional changes of GCS-HS in different cells.
Hormonal and cell cycle regulation of GCS
Transcriptional regulation
We and others have
shown that GCS activity and GCS-HS expression are
under hormonal and growth-related regulation (19,
65– 68, 85). Specifically, in primary cultures of rat
hepatocytes the activity of GCS and the GCS-HS
mRNA level can be induced by insulin or hydrocortisone treatment or by lowering the initial plating
cell density (19, 65– 67). The mechanism was an
increase in the transcription of GCS-HS without any
change in the stability of the GCS-HS mRNA (66).
The effects of the hormones in cultured cells were
confirmed using in vivo models such as insulindeficient diabetic or adrenalectomized rats. Both
exhibited lower hepatic GSH levels and GCS activity,
which were prevented with hormone replacement
(19). However, glucocorticoid administration to
sham-operated animals did not result in increased
hepatic GSH or GCS activity (19). Thus, it appears
these hormones are required for maintenance of
normal hepatic GCS activity. Abnormality in GCS
activity was observed only when there was hormonal
deficiency, not excess. In diabetic rats, the steadystate hepatic GCS-HS mRNA level was decreased
compared with controls, but normal if treated with
insulin (66). Thus, in these animal models, a lower
hepatic GSH and antioxidant defense may play a
pathogenetic role in the associated complications. It
should be stressed that these hormones may not
exert similar effect in other cell types. In fact, a
recent study showed that dexamethasone actually
HEPATIC GLUTATHIONE SYNTHESIS
had the opposite effect, namely, lowering of cell
GSH and GCS activity, in a type II alveolar epithelial
cell line (100). Thus, results obtained from one cell
type may not be applicable to another and results
obtained from cell lines may not be comparable to
those obtained from primary cultures or in vivo.
In the case of cell density, lowering the initial
plating density effectively shifts adult rat hepatocytes
from the Go to the G1 phase of the cell cycle,
analogous to liver regeneration after partial hepatectomy or after cell death (101). Consistent with
findings in cultured rat hepatocytes, a doubling of
the hepatic GSH level, GCS activity, and the steadystate mRNA levels of GCS-HS were noted 12 h after
two-thirds partial hepatectomy (68). The increase in
GSH under low cell density and after partial hepatectomy was due to both an increase in GCS activity
and cysteine availability (67, 68). The increase in
GSH occurred before the increase in DNA synthesis
during liver regeneration (68). Some speculations
can be made about the significance of the increase in
hepatic GSH and cysteine levels prior to DNA synthesis. Previous studies involving lymphocytes and
fibroblasts have shown that an increased GSH level
was associated with an early proliferative response
and was essential for the cell to enter the S phase
(102–105) However, other studies did not find a
correlation between GSH levels and cell cycle (106,
107). The requirement for increased GSH or thiols
prior to DNA synthesis may be related to the fact that
proliferating cells require increased amounts of pentoses and thiols. DNA synthesis depends absolutely
on the formation of pentoses and on their conversion into deoxyribose by ribonucleotide reductase
(108). The activity of this rate-limiting enzyme in
DNA synthesis requires reduced glutaredoxin or
thioredoxin, which are maintained by GSH with
concomitant oxidation to GSSG via glutathione reductase or oxidation of NADPH via thioredoxin
reductase, respectively. Alternatively, an increase in
the cellular GSH content may change the thiol-redox
status of the cell that activates genes essential for G1
to S transition. Future studies examining the molecular mechanism(s) of the cell-cycle effect on GCS-HS
gene expression as well as the significance of the
increase in hepatic GSH during liver regeneration
will address these possibilities.
In contrast to oxidative stress, which increased the
transcription of both heavy and light subunits of
GCS, hormones and plating hepatocytes at low cell
density had no influence on the expression of
GCS-LS (65). Consistent with findings in cultured
cells, GCS-LS mRNA level also remained unchanged
after partial hepatectomy (68). Thus, at least in
hepatocytes, there may be more GCS-LS available
than GCS-HS, so that a change in the GCS-HS alone
changed the GCS activity. That the two subunits are
1177
under different regulatory controls have been suggested by others. Gipp et al. found no correlation
between the steady-state mRNA levels of the two GCS
subunits in different tissues (12). Huang et al. hinted
that liver might synthesize more light subunit than
other tissues relative to the amount of heavy subunit,
although no data were provided (11). Using HepG2
cells transfected with antisense directed at GCS-LS,
we observed a 80% reduction in the amount of
GCS-LS mRNA, accompanied by a significant reduction in the protein level, but no change in the cell
GSH level (unpublished observation). This supports
the notion that there is more GCS-LS in hepatocytes.
Kijima et al. used hammerhead ribozymes designed
against the two GCS subunits and showed that in
Min-6 mouse islet cells transfected with these ribozymes, this strategy resulted in a decrease of the
GCS-HS and GCS-LS expression to 27% and 86% of
control, respectively (109). Despite a minimal
change in the GCS-LS expression, cell GSH was
depleted even more in the cells treated with ribozyme against GCS-LS. The authors speculated that
the low GSH level induced the expression of GCS-LS
mRNA level and reversed the decrease in GCS-LS
expression by the ribozyme. They concluded that
both subunits contributed equally to the enzyme
activity. However, whether the ribozyme against
GCS-LS also affected the expression of GCS-HS is
unknown and the effect of the ribozymes on the GCS
subunit protein levels were not examined. Finally,
there is likely to be cell-specific differences in the
differential regulation of the two GCS subunits.
Posttranslational regulation of GCS
GCS is also
regulated post-translationally. We and others reported that GSH synthesis and GCS activity were
acutely inhibited by hormone-mediated activation of
various signal transduction pathways (110, 111).
These hormones are secreted under stressful conditions, many of which have associated lower hepatic
GSH levels (7, 112, 113). The fall in hepatic GSH
level can be explained by both an increase in sinusoidal GSH efflux and an inhibition of GSH synthesis
(110, 114). This may represent the hepatic stress
response by increasing the systemic delivery of GSH
and cysteine to where they are needed and channeling cysteine away from GSH synthesis to synthesis of
stress proteins, many of which are rich in cysteine
(115). Since the liver GSH level is normally very high
(5–10 mM), a slight fall in the GSH level would not
jeopardize its defense capacity. To explain the inhibition of GCS activity, we showed that GCS-HS is
phosphorylated directly by activation of protein kinase A, protein kinase C or Ca21-calmodulin kinase
II (CMK) (116). The degree of phosphorylation
correlated with loss of GCS activity (20% under
physiological Mg21 concentration) and no additional inhibition was observed when GCS was phosphorylated in the presence of all three kinases. In
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addition, 2-dimensional phosphopeptide mapping
studies showed that the same five phosphopeptides
were phosphorylated by these kinases, suggesting
that these kinases were acting on the same site(s).
Phosphorylation of GCS-HS was also demonstrated
in cultured hepatocytes after treatment with dibutyryl cAMP (DBcAMP) or phenylephrine using
specific antibodies that immunoprecipitated the
phosphorylated GCS-HS. There was basal GCS phosphorylation, which increased after treatment with
DBcAMP or phenylephrine, suggesting that GCS
may be under a basal inhibitory tone. The demonstration of phosphorylation of GCS by using cultured
hepatocytes suggests that phosphorylation-dephosphorylation may be an important physiological regulator of GCS. The inhibition of GCS activity after
phosphorylation of purified GCS was 20% at 1 mM,
a physiologically relevant Mg21 concentration, which
is similar to what we observed when cultured rat
hepatocytes or perfused rat livers were treated with
DBcAMP, glucagon, or phenylephrine (110, 116).
Although the degree of inhibition in GCS activity is
small, the effect on GSH synthesis and hepatic GSH
turnover may be significant. The turnover of normal
hepatocyte GSH is estimated at 20%/h or 12 nmol/
106 cells/h for a repleted cell, which has 60 nmol
GSH/106 cells (36). This is the amount of GSH that
is lost per hour, which is normally balanced by
synthesis of GSH. An adult rat liver effluxes 12 mmol
of GSH per hour (assuming 108 cells/g and 10
g5liver weight). Thus, even a 20% inhibition in GCS
activity would cause a significant reduction of GSH
synthesis by the liver (2.4 mmol per hour). Since
many pathological and toxic conditions result in
sustained increase in cytosolic free Ca21, which can
lead to activation of CMK and phosphorylation of
GCS, inhibition of GCS may theoretically further
contribute to toxicity under these conditions. The
report by Lauterburg and Mitchell that toxic doses of
acetaminophen administered in vivo suppressed hepatic GSH synthesis in rats raises this possibility
(117).
Post-translational regulation of the light subunit
has not been well studied. In our work on phosphorylation of the heavy subunit, no phosphorylated light
subunit was immunoprecipitated when using antiGCS-HS antibodies at the end of the reaction (116).
Thus, the light subunit is probably not phosphorylated.
Other conditions that alter GCS expression
In addition to oxidative stress, drug-resistance, hormones, and plating density, several other conditions
are also known to influence the GCS activity and the
steady-state GCS-HS mRNA level. These include
treatments with several antioxidants such as buty-
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lated hydroxyanisole, GSH-conjugating agents such
as DEM and phorone, heavy metals, heat shock,
copper deficiency, cycloheximide, TGFb1, high glucose, and nitric oxide (NO) (60, 70 –75, 79, 86, 95,
96). Several of these treatments such as DEM, phorone and heavy metals can also induce oxidative
stress. All of these treatments, with the exception of
TGFb1 and high glucose concentration, increased
steady-state GCS-HS mRNA level. The mechanism
for the increase in most of these treatments was
increased transcription. In the case of cycloheximide, the increase in GCS-HS mRNA level occurred
by stabilization of the mRNA (79). Finally, cellular
GSH level itself may also modulate GCS expression
(see Areas of Controversy).
In addition to hormone (insulin and glucocorticoids) deficiency (19, 66), two other treatments that
decreased GCS-HS transcription are TGFb1 and high
glucose concentration (28 mM) (73, 86). Arsalne et
al. showed that TGFb1 decreased GCS-HS transcription in a lung epithelial cell line (86). They speculated whether overexpression of TGFb1 in the alveolar epithelial cells may contribute to low levels of
GSH in the alveolar epithelial lining fluid of patients
with fibrotic lung disorders (86). The molecular
mechanism remains to be elucidated. Regarding
high glucose concentration, Urata et al. showed that
GCS-HS mRNA level and transcriptional rates in
mouse endothelial cells were increased by treatment
with cytokines such as TNF and IL-1b (73). The basal
GCS-HS mRNA level was decreased and the effect of
the cytokines disappeared when cells were grown in
media containing high glucose concentration (28
mM). This raises the question of whether the decreased GCS activity and GCS-HS mRNA level observed in diabetic rat livers is due to lack of insulin or
hyperglycemia or both. In our work, cultured hepatocytes were grown in medium containing 17.5 mM
glucose 6 insulin (19, 66). Thus, it is likely that
insulin was involved in increasing GCS-HS gene
transcription in cultured hepatocytes. However, glucose can influence gene expression by itself and the
effect of insulin may or may not require glucose
(118, 119). More work is needed to elucidate the
exact mechanism of how insulin and glucose influence GCS-HS gene expression.
The effect of NO on GCS expression is dependent
on the cell type (95, 96). In rat hepatocytes, NO
exerted no influence on the basal GSH level or GCS
expression (95). However, when these cells were
treated with interleukin-1 (IL-1), which stimulated the
expression of inducible nitric oxide synthase, the GSH
level was depleted and GCS expression decreased if
NO synthesis was blocked (95). This would suggest that
in response to oxidative stress induced by IL-1, rat
hepatocytes up-regulate GCS expression by a mechanism that depends on NO synthesis. In contrast, treatHEPATIC GLUTATHIONE SYNTHESIS
ment of rat aortic vascular smooth muscle cells with
NO at physiological concentrations resulted in increased cell GSH levels and expression of both GCS
subunits (96). This illustrates the cell type-specific
complex interactions between NO and GSH.
Finally, we showed recently that the GCS-HS
mRNA level was increased in mutant Eisai Hyperbilirubinemic rats (78). This along with increased
cysteine availability contributed to the high GSH
levels found in liver and many other organs (78).
The mechanism for the increased GCS-HS mRNA is
unknown and it is speculative whether retained GSH
conjugates may have contributed to the increase.
AREAS OF CONTROVERSY
Some of the controversial areas have been discussed,
but three others deserve special mention: the role of
GSH in apoptosis; the role of NF-kB in modulation
of the GCS-HS gene expression, and the role of cell
GSH itself in the modulation of GCS expression.
GSH in apoptosis
Apoptotic cell death is one of the most intensely
studied research areas. Whether GSH and/or the
intracellular redox state modulate apoptosis is controversial. Numerous studies have shown that apoptotic cells accumulate oxidized proteins and lipids
and are presumably exposed to some degree of
oxidative stress; antioxidants, including GSH, have
been shown to protect against or delay apoptosis
triggered by many different stimuli (120 –124). However, apoptosis has been shown to occur under very
low oxygen environments indicating that reactive
oxygen species are unlikely to be essential mediators
under these conditions (121). Another study showed
that the protective effect of thiol agents may be
related to down-regulation of Fas expression on T
lymphocytes rather than their antioxidative properties (123). What is clear is that there is accelerated
GSH efflux from the cell stimulated to undergo
apoptosis. This was shown by several groups in
several cell types (mostly lymphocytes) with different
proapoptotic stimuli (120, 121, 124). GSH loss occurred prior to chromatin fragmentation or development of oxidative stress, required protease activation, and was blocked by agents known to inhibit
GSH transport (121). Thus, increased export of GSH
represents an oxygen-independent mechanism for a
fall in the cellular redox state. However, apoptotic
degradation of nuclear DNA still occurred with the
same kinetics even when GSH efflux was blocked
(121). Similar results were obtained by Ghibelli et al.
(120). GSH loss occurred concomitant with the
onset of apoptosis, and modulation of intracellular
1179
GSH did not influence the overall extent of apoptosis. These findings seem to dispute the importance of
the redox state in modulating apoptosis. What is also
unclear is the significance of the GSH efflux during
apoptosis and whether this occurs in other cell types.
One speculation is that depletion of cell GSH will
facilitate apoptosis to occur, provide antioxidants
extracellularly, and possibly stimulate phagocytic
cells to engulf the apoptotic cell (121).
Urata et al. (73). It is unclear whether the discrepancy is related to the cell type studied, the method
used to increase GSH, or other unknown factors.
Whether the light subunit is also affected by an
increase in cell GSH is unknown. These unresolved
issues deserve further investigation.
Role of NF-kB
Since the importance of GSH was recognized and
the enzymes of GSH synthesis characterized almost
half a century ago, we have come a long way in
understanding how the synthesis of GSH is regulated. Advances in molecular biology have led to an
explosion of knowledge in understanding how the
rate-limiting enzyme GCS is regulated at the molecular level. Areas of future investigation should include gaining a better understanding of the role of
GSH in cell proliferation, apoptosis, and GCS expression, the pathogenetic importance of impaired
GSH synthesis in certain disease states such as diabetes mellitus, cell type-specific differential regulation
of the two GCS subunits, post-translational regulation of GCS, and characterization of the signal
transduction pathways and trans-activating factors
that regulate GCS subunit gene expression. Results
from these studies should improve the treatment
and prevention of complications that may result
from altered GSH synthesis.
Another area of controversy is the role of NF-kB in
modulation of the GCS-HS gene expression. There is
a NF-kB consensus element ;1kb upstream of the
transcriptional start site of the human GCS-HS (63).
Oxidative stress is known to induce NF-kB activity,
and Urata et al. showed that blocking activation of
NF-kB by antisense strategies prevented the cytokineinduced increase in GCS-HS transcription in mouse
endothelial cells (73). We also showed blocking the
activation of NF-kB prevented the increase in the
GCS-HS mRNA level induced by BSO and tert-butylhydroquinone in rat hepatocytes (65). Iwanaga et al.
also implicated the NF-kB consensus element, but
not AP1 or ARE, in mediating the effect of ionizing
radiation on GCS-HS expression in a human glioblastoma cell line (90). However, the NF-kB consensus element was shown not to be involved in mediating the effect of TNF, okadaic acid, and ionizing
radiation on GCS-HS expression in HepG2 cells (64,
81, 88). The explanations for these discrepant results
are unclear, and whether there is species and/or cell
type differences in mediating the effect of these
agents at the GCS-HS promoter level remains to be
examined.
SUMMARY AND FUTURE DIRECTIONS
This work was supported by National Institutes of Health
grant DK45334.
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