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Mol. Cells, Vol. 2, pp. 39-42
Induction of y-Gultamyl Transpeptidase by a Thyroid Hormone in
Primary Cultures of Rat Hepatocytes and in Rat liver
Kang Yeol Yu, Byoung Yul Soh, Sang-Bae Hant, Jin-Ho Kim, Mun-Kuk Park2,
Gook Hyun Chung and Tai-Boong Ubm 1*
Department of Biology, 'Department of Food Science and Technology, and 2Department of
Biological Education, Chonbuk National Univen"ty, Chonju 560-756, Korea
(Received on February 1, 1992)
We have studied the induction of gamma-g1utamyl trans peptidase (GGT) by a thyroid
hormone, triiodothyronine ([3) in primary cultures of rat hepatocytes. When added to
cultures for 5-6 days, T3 increased GGT activity to levels 2.5- to 3-times those of control
cultures. In rat liver, T3 also causes a 1.8-fold increase in GGT activity when hyperthyroidism in rats was induced. Considering highly related structures between the nuclear receptors for retinoic acid, thyroid and steroid hormones, induction mechanism of GGT by
T3 may be similar to those by retinoic acid and steroid hormones, which can also induce
GGT activity.
Gamma-g1utamyl transpeptidase (GGT) «5-g1utamyl)-peptide:amino acid 5-g1utamyl transferase, EC
2.3.2.2), a plasma membrane associated enzyme, is a
key enzyme in the regulation of the level of glutathione via the gamma-glutamyl cycle. It catalyzes transfer of the gamma-glutamyl moiety of glutathione to
a variety of acceptors such as amino acids and dipeptides (transpeptidation) and to water (hydrolysis).
The highest GGT activity is found in the kidney,
but GGT of liver and liver-derived cells has attracted
considerable attention, because in human liver GGT
activities increase highly during chronic alcoholism
(Rosalki and Rau, 1972) or in patients with hepatocellular carcinoma (Gerber and Thung, 1980). Also, in
primary culture of rat hepatocytes, the GGT activity
is influenced by treatment with various xenobiotics
such as dexamethasone (Cotariu et al., 1988) and ste-
their receptors, in order to meet cellular needs (Umesono et al., 1988). If this hypothesis is correct, thyroid
hormone also may act as a positive regulator for the
transcription of GGT gene, because the structure of
the thyroid hormone receptor is closely related to
those of the retinoic acid and steroid hormone receptors.
We now report that thyroid hormone induces the
activity of GGT in primary culture of rat hepatocytes
and in vivo experiments and propose a possible mechanism of GGT induction in the presence of thyroid
hormone.
Materials and Methods
Animals and materials
Male Sprague-Dawley rats weighing 150-200 g were
roid hormones (Billon et aI., 1980) and retinoic acid
used as sources of liver cells in all experiments. Triio-
([sao and Batist, 1988).
It has been known that steroid and thyroid hormones and retinoic acids coordinate complex events involved in development, differentiation, and physiological response to diverse stimuli. Furthermore, recent
studies of steroid receptors have led to the identification of a superfamily of ligand-inducible regulatory
proteins including receptors for thyroid hormones and
retinoic acid (Ham and Parker, 1989).
These receptors are homologous to those for the
steroid hormones and these appear to function by similar mechanisms. This family of receptors regulates
the transcription of certain genes by binding as a hormone-receptor complex to specific DNA sequences termed response elements (RE). Thus, retinoic acid and
steroid hormones may also induce GGT gene expression by binding to a common RE as complexes with
dothyronine ([3) and dexamethasone were purchased
from Sigma Chemical Co. (St. Louis, MO).
* To whom correspondence should be addressed
Hepatocyte isolation and culture
Hepatocytes were isolated by a collagenase perfusion procedure (Whiting and Edwards, 1979). Cultures were established by adding about 5 X 106 freshly
isolated hepatocytes to collagen-coated Falcon 3001
tissue culture plates in Williams' medium E (Williams
et aI., 1977) with 5% fetal bovine serum. Cells were
incubated at 37 °C in a humidified 5% COJ 95% air
incubator, and allowed for attachment for 12 h. Hepatocyte integrity and viability were routinely checked
by microscopical examination and by lactate dehydrogenase release in the medium as compared to the initial dispersion. The time of seeding was designated
The abbreviations used are: GGT, gamma-giutamyl transpeptidase; RE, reponse element; T), 3,5,3-triiodo-L-thyronine.
© 1992 The Korean Society of Molecular Biology
40
Mol. Cells
Thyroid Honnone Induction of y-Glutamyl Transpeptidase
time O. After 24 h the medium was replaced with fresh
medium containing 10 nM T3 or 1 ~ dexamethasone. Cultures were maintained for 5 days with daily
medium exchanges.
Treatment of animals for in vivo induction
Chronic hyperthyroidism was induced by a slight
modification of the method of Narayan et al. (1984).
It was induced by intraperitoneal injection of 35 /lg
of T3 per 100 g of body weight per day for 6 days.
Groups of five animals were fed standard chow diet
(Miwon). Six h after the final injection rats were sacrifised under CO 2 anesthesia, and the livers were removed and weighed. Pieces (2 g) of the left median lobes
of the liver were then homogenized in 4 rnl of 0.1
M Tris-HCl buffer (PH 7.6 at 4 "c) containing 0.25
M sucrose and 10 mM MgCb. Following centrifugation at 1,000 X g for removal of cell debris, a membrane preparation was made by centrifuging the supernatant fraction at 105,000 X g for 1 h. The pellet
was resuspended in buffer containing 0.1 M Tris-HCl
and 10 mM MgCb.
Assays of GGT and protein
At the indicated times plates were rinsed twice with
0.15 M NaCl, and the attached monolayers were scraped off, resuspended in saline and sedimented by centrifugation for 3 min at 100 X g. Cells were homogenized in 50 mM Tris-HCl/250 mM sucrose buffer
(pH 7.4) in a glass-glass homogenizer. GGT was assayed in homogenates at 37 "c by the method of Tate
and Meister (1974) using L-gamma glutamyl-p-nitroanilide and glycylglycine as substrates. One unit of enzyme activity is that yielding I nmole product per
minute at 37 °C. Protein was measured in cell homogenates by the method of Lowry (1951). Statistical
analysis for the significance of differences between
groups was done by means of Student's t-test. Differences were considered statistically significant when P<
0.05.
Results
Effect of T3 on hepatocyte GGT
Figure 1 shows changes in GGT activity with time
in culture when hepatocytes were maintained in defined Williams' medium. Under control culture the
activity of GGT increased slowly during the first 5
days. Addition of T3 (final concentration of 10 nM)
to mono1ayers increassed activity significantly after 3
days (P<O.OOl) and after 5-6 days with T3, GGT activities were about 2.5-3.0 times those in control cultures.
Mode of GGT induction by dexamethasone (1 ~),
a well-known GGT inducer, was found to exhibit
time-dependence similar to that for induction by T3
(Fig. 1).
Microscopic observations showed that control and
T 3-treated cells were same in their morphological properties during cultures (data not shown). Also, we did
not detect any releae of lactate dehydrogenase in the
culture medium from either control or T 3-treated cells
600
.§ 500
e
p.,
bll
§ 400
E
~
:~
~
~
8
300
200
100
OL---~----~----~----~--~
1
2
3
5
Time, days
Figure 1. Induction of GGT activity by T3 ce) and dexamethasone CO). T3 (10 nM) and dexamethasone (1 f.LM) were
added to the medium after 24 h in culture; controls ()).
Monolayers were incubated for the periods shown in the
figure after which hepatocytes were harvested for GGT and
protein assays. The means and standard errors of 5 or 6
plates are shown.
(data not shown), indicating that no significant cell
damage occurred at the concentration of 10 nM T3.
The time-dependent increase in GGT in the presence of T3 appeared to represent enzyme induction since the increase was completely prevented by addition
of the protein synthesis inhibitor cycloheximide (100
~) to 3-day cultures for a 24-h test period and reduced about 50% by adding the RNA synthesis inhibitor,
actinomycin D (0.5 ~) for a similar test period. Moreover, incubation of hepatocyte cells in the presence
of cycloheximide (100 ~) or actinomycin D (0.5 ~)
together with 10 nM T3 for 3 days prevented the hormonal effect.
Induction of GGT in hepatocyte primary cultures
also depended on the concentration of T 3. As shown
in Figure 2, lower concentrations of T3 up to 10 nM
caused an increase in GGT activity, which was maximal at 10-50 nM T 3. At the higher T3 concentrations,
above 200 nM, the activity rather declined.
In vivo GGT induction by T3
Hepatic membrane GGT activity in T 3-treated rats
was significantly higher than activity in the controls
after 5-day exposure (P<O.Ol), although the inducing
effect was small compared to that in primary hepatocyte cell culture (Table 1).
Discussion
In the present report, T3 as well as dexamethasone
specifically promoted an induction of GGT with time-
Kang Yeol Yu et al.
Vol. 2 (1992)
41
::::::::::::;::::~i~i;?r:
500
=
e
Plasma Membrane
Nucleus Membrane
T
.~
~T)-specific Receptor
~
eo
\
E
::l
--E
6
.;;
'p
u
co
20
IThyroid I
Promoter
Hormone
RE
f-
0
0
T)-Rec e ptor Complex
as a Transcriptional
Factor/
GGT gene
I
Transcription
100
0
0
GGT mRNA
10
20
30
40
50 200
T3, nM
Figure 2. The dependence of induction of GGT activity on
the concentration of T3 in the culture medium. Primary cell
cultures were uniformaly exposed to the various concentration of T3 for 4 days prior to measuring their GGT specific
activities. Values represent the means and standard errors
of 5 or 6 plates.
Table 1. Induction of GGT activity in rat liver after treatment of T3 for 5 days·
Treatment
GGT activity
(mU/mg proteint
84± 19
157 ± 27'
See the text for detailed procedure.
Means ± S.D. from five animals.
' Significantly greater than control (P<O.OI).
a
h
and dose-dependent mode in cultured rat hepatocytes.
Also, induction of GGT in the liver in vivo was caused
by the treatment of T 3.
A structurally diverse group of xenobiotics and steroids have been shown to elevate GGT activity in
hepatocytes in primary culture. Also, retinoic acid increased the level of the GGT mRNA in transformed
cell lines. This effect apparently involves time-dependent induction of the enzyme, requiring continuing
RNA and protein synthesis. These findings suggest
that GGT induction by T3 may also be modulated
via a similar mechanism.
Consistent with this view, thyroid hormone such
as T 3, retinoic acid, and steroid hormones and their
derivatives which induce GGT have been reported to
be inducers for growth and differentiation of the cell.
The metabolic turnover of glutathione is closely geared
to the activity of GGT. Also, GGT functions in cellular transport of amino acids, detoxication and cellular
+
GGT
Figure 3. A hypothetical model for GGT induction by TJ •
Binding of T3 to a T3-specific nuclear receptor induces an
activated T3-nuclear receptor complex. This leads to the
binding to the thyroid hormone response element, which
can also act as a common response element both for the
binding of retinoic acid- and steroid hormone-nuclear receptor complexes.
neutralization by ammonia production (Tate, 1980).
Therefore, the induction of GGT activity may represent a necessary physiological adaptation in the cell
differentiation, growth, and the increase of metabolic
rates by upregulating the synthesis of cellular glutathione.
Recently, it was found that the family of steroid
hormone receptors also includes receptors for thyroid
hormone, a number of vitamines and several other
ligands yet to be identified (Evans, 1988). Because of
the highly related structures among the above receptors, a human retinoic acid receptor expressed from
cloned complementary DNA or the endogenous retinoic acid receptor present in teratocarcinoma cells
could activate gene expression from promoters fused
to a natural or synthetic thyroid hormone respone
element (Umesono et aI., 1988). The implication of
these findings is that the thyroid and steroid hormones (or their derivatives) and retinoic acid, acting through their respective receptors, could control overlapping gene networks involved in the regulation of vertebrate morphogenesis and homeostasis.
We here propose a posssible mechanism of GGT
induction by T3 as follows, based on our results and
others. T 3, like steroid hormones, diffuses freely across
the plasma membrane and binds specifically with
high affinity to receptors in the nucleus of liver. Next,
the Trnuclear receptor complex binds to a thyroid
hormone RE with high affinity. Or the Trreceptor
complex may bind to a steroid and/or retinoic acid
RE to activate GGT expression as a common respo-
42
Thyroid Hormone Induction of y-Glutamyl Transpeptidase
nse element. This binding can then stimulate promoter
activity of GGT gene (Fig. 3). Major concern in this
hypothesis is that a common response element for
binding of T r , steroid hormone-, and retinoic acid-receptor complexes may exist near the promoter site of
the GGT gene. This should be solved by analyzing
the regulation sites of GGT gene.
However, other mechanisms can be proposed as
well. One of these could be the induction of a factor
which in tum directly activates the target enzyme.
That is, a Tr induced intermediate affects the enzyme
conformation without changing the mRNA level.
However, cycloheximide and actinomycin D prevented
the induction, suggesting that protein and mRNA synthesis were necessary for this process. Specific mRNA
assays will be necessary for further studies.
Acknowledgment
This work was supported by a Genetic Engineering
Research grant to T. B. Uhm from Ministry of Education in 1990.
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