1 Laboratorio de
Fisiología, The liver of adult
mammals contains various classes of polyploid hepatocytes produced by a
process that is partially regulated by hormones. However, it is not
well understood how the hormones affect the rate of hepatocyte
proliferation under physiological conditions. Here we have studied the
specific roles of 3,5,3'-triiodothyronine (T3), growth hormone (GH), and
sex steroids on the percentage of diploid nuclei in S phase and on the
population of liver tetraploid (4C) cell nuclei in several rat model
systems. Gonadal steroids had no effect on the S phase but account for
gender differences in the 4C nuclei. Hypophysectomy in adult male rats
produced a moderate decrease in 4C nuclei that was reversed by
treatment with 25 µg
T3 · kg
liver polyploidy; flow cytometry; cell cycle
POLYPLOIDY IS A characteristic feature of mammalian
hepatocytes (3). During normal developmental growth, the rat liver parenchyma undergoes dramatic changes characterized by a progressive polyploidization by which hepatocytes of several ploidy classes emerge
as the result of modified cell-division cycles. In newborn rats, the
parenchyma contains diploid cells that divide intensively. Binucleate
hepatocytes (formed by acytokinetic mitosis) appear after several days
and markedly increase in number after a few weeks. Then mononucleate
tetraploid cells emerge, followed by binucleate cells with two
tetraploid (4C) nuclei and finally by mononucleate octoploid cells.
This succession of hepatocyte cell classes has been established by
early cytophotometric studies (1, 30) and more recently by flow
cytometry studies (16, 33, 34). The entire process of hepatocyte
polyploidization is considered to be a mechanism of evolutionary
adaptation, reflecting an increasing degree of irreversible
hepatocellular differentiation adopted to decrease the high risk of
genomic damage to which the liver is exposed (4).
The adult liver retains a high proliferative capability. It responds to
tissue injuries such as chemical agents or partial hepatectomy by
priming quiescent hepatocytes to enter the cell cycle. The
hepatocellular growth shifts to a nonpolyploidizing growth pattern, and
expansion of the diploid hepatocyte population has been found to take
place (16, 33, 34). This proliferating response makes the liver
susceptible to genomic damage, especially during regeneration, which
may lead to the efficient development of rodent models of
hepatocarcinogenesis that, in many aspects, resemble human hepatocarcinomas.
The molecular events that cause nuclear growth and polyploidization
remain elusive (4, 19). Morphometric studies have revealed that mitotic
activity and the numbers of the hepatocyte of each ploidy class are
altered by hormones. The regulation, as established 30 years ago, is
predominantly carried out by growth hormone (GH), with modulatory
effects of both sex steroids and thyroid hormones (5, 18, 20). However,
no further studies have been published, and the separate effect of each
hormone remains obscure. The influence of some hormones on the process
that leads the hepatocyte to enter the cell cycle has been studied from
the point of view of hormone-induced hepatocarcinogenesis (26, 39, 40).
In this aspect, the risk of hepatocarcinogenesis would increase if the
rate of hepatocyte proliferation were increased by manipulating the
endocrine status and vice versa. However, the physiological effect of
hormones on hepatocyte proliferation has not been explored in depth.
Here we have studied the influence of thyroid hormones, GH, and sex
steroids on hepatocyte proliferation and polyploidization under
physiological conditions to develop rat model systems with preestablished hepatocyte proliferation rates. We used various rat
model systems that had been successfully applied in our laboratory (2,
7, 8, 13, 21) to study the endocrine regulation of a steroid-binding
liver microsomal protein, LAGS (low-affinity glucocorticoid binding
site). The rates of hepatocyte proliferation and polyploidization were
studied by a procedure based on obtaining the liver nuclei by
fine-needle aspiration (FNA), followed by determination of their DNA
content by flow cytometry. This procedure is simple, easily
reproducible, and can be used to carry out these studies in
anesthetized rats. The results obtained allowed us to clarify the
predominant role of thyroid hormones in the regulation of hepatocyte
proliferation and polyploidization.
Human recombinant GH (hGH; Saizen) was kindly donated by Serono
Laboratories (Milan, Italy). Human recombinant insulin-like growth
factor I (IGF-I) was kindly donated by Nordisk Hispania (Madrid,
Spain), 17 Male and female Wistar rats were obtained from Charles River (St. Aubin
les Elbeuf, France). During the treatment, animals were kept under
standardized conditions (controlled environmental conditions, 12:12-h
light-dark cycle and free access to normal chow and water). The rats
were killed by decapitation, and their livers were quickly removed,
weighed, and rapidly frozen at Orchiectomy and ovariectomy were performed under ether anesthesia.
Pseudooperated rats were used as control groups. Treatment with EB was
performed by dissolving the steroid in corn oil. Implants were
constructed of 2.5-cm lengths of Dow-Corning Silastic tubing (0.062 in.
ID × 0.125 in. OD) that were capped at each end by Silastic type
A adhesive. Individual implants containing either 25 µg EB, 500 µg
TP, or vehicle (corn oil) were subcutaneously placed in the dorsal
cervical region using light ether anesthesia.
Hypothyroidism was induced by administering 0.02% MMI to the dams in
their drinking water on the 12th day of pregnancy and keeping the
newborn rats under that treatment until they were killed (10).
GH deficiency was provoked by treatment of neonatal male and female
Wistar rats with monosodium glutamate (MSG), at a dose of 4 mg/g body
wt, dissolved in a minimum volume of sterile saline, on
days
2, 4,
6, 8,
and 10 of life (25). The effectiveness
of treatment was monitored by checking the weight increase every week
until the rats were killed.
Where indicated, hGH was administered in sterile physiological saline
at a dose of 200 µg · rat FNA was used to obtain liver nuclei from fresh or thawed tissue
specimens. A 0.5 × 16 mm (25-gauge) needle on an empty 10-ml disposable syringe fitted on a one-hand-operated handle (Cameco, Täby, Sweden) was used. Suction was applied when the point of the
needle was in the tissue. The needle was moved back and forth in
different directions in the tissue and aspiration was repeated two or
three times until a sufficient amount of sample was obtained. The
aspirates were flushed with citrate buffer (3.4 mM trisodium citrate,
1.5 mM spermine tetrahydrochloride, 0.5 M Tris, and 0.1% Nonidet P-40,
pH 7.6). The suspensions were then clarified by filtering through a
50-µm nylon mesh into tubes and centrifuging at 200 g for 5 min. The pellets were
resuspended in 100 µl of citrate buffer and treated according to the
method of Vindelov and Christensen (41). Liver cells were obtained from
living rats through a small incision made in the right ventral side
under ether anesthesia. A 0.9 × 40 mm (20-gauge) needle on a
10-ml disposable syringe was used to biopsy the liver. These aspirates
were flushed with PBS, mechanically disaggregated by serial pipetting
against the tube wall, collected by centrifugation at 200 g, and stained with propidium iodide
(5 µg/ml) containing RNase A (30 µg/ml).
Cells or cell nuclei were analyzed on an Epics XL-MCL cytometer
(Coulter, Hialeah, FL). Quality control of the flow cytometer was
carried out using Epics DNA check alignment fluorospheres (Coulter).
Peak vs. integrated red fluorescence, gated along the diagonal, was
used to analyze 20,000 cell nuclei or 10,000 hepatocytes. Half-peak
coefficients of variation were always <5%. Generated histograms were
analyzed for cell cycle compartments and background debris subtraction
by using the Multicycle software for cell cycle analysis (Phoenix Flow
Systems, San Diego, CA). The data are expressed as the means of 4C
nuclei ± SE. Where stated, the 4C nuclei are referred to per the
relative liver weight (RLW), using the following equation: 4C/RLW = %4C nuclei/(liver wt × 100/body wt).
Statistical differences were determined by ANOVA, using the
Student-Newman-Keuls test to allow for multiple comparisons.
Statistical significance was reported if
P < 0.05 was achieved.
FNA procedure.
The FNA procedure provided a nuclear suspension with very little
debris, suitable for flow cytometry studies. Representative flow
cytometry histograms are shown in Fig. 1.
At birth, male and female rats showed a similar percentage of both 4C
nuclei and diploid (2C) nuclei in S phase, but both parameters were
significantly higher in adult males than in adult females
(P < 0.0001). The FNA procedure provided a nuclear ploidy distribution similar to the
procedures based in tissue disruption to obtain the nuclear fraction
(9, 14, 36). Table 1 shows a comparison of
the FNA procedure with those based in the two-step collagenase
preparation of whole hepatocytes or hepatocyte nuclei (17, 35). The
latter procedure produced 10% more 4C nuclei than the FNA applied to frozen liver tissue. This is due to the presence of nonparenchymal cells in the fine-needle aspirates and does not represent a limitation on the validity of this study. The discrepancy between 4C nuclei, obtained by any procedure, with the 4C cells (Table 1) is due to the
contribution of binucleate hepatocytes with 2C nuclei that cannot be
separated from the mononucleate cells with 4C nuclei. The octoploid
cell nuclei were undetectable by the FNA procedure in rats under 40 days of age and were ~1% in adult rats. For this reason, they were
not included in the study. The FNA procedure is easily reproducible as
proved by its low inter- and intra-assay coefficient of variation (4.7 and 3.9%, respectively). It is sufficiently sensitive to estimate the
level of 4C nuclei and the number of 2C nuclei in S phase, two
parameters that are altered by endocrine manipulations.
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
1 · day
1,
whereas treatment with 200 µg human recombinant GH
(hGH) · kg
1 · day
1
was ineffective. Rats made hypothyroid by methimazole treatment of dams
and pups until death showed a low S phase and only 5% of 4C nuclei at
70 days of age. T3 significantly
increased the S phase 24 h after administration and restored the adult
normal level of 4C nuclei after 10 days of treatment. hGH did not
affect the 4C nuclei or the S phase in the hypothyroid rats. These
results suggest that the processes of hepatocyte proliferation and
polyploidization of the rat liver are under endocrine control, with
thyroid hormones playing the essential regulatory role.
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
-estradiol 3-benzoate (EB), testosterone propionate (TP),
3,5,3'-triiodothyronine
(T3),
1-methyl-2-mercaptoimidazole (methimazole; MMI) propidium iodide, and
all other products used in this study were purchased from Sigma
Chemical (St. Louis, MO).
70°C. The international
criteria for the use and care of experimental animals in research were observed.
1 · day
1,
divided into two daily subcutaneous injections.
T3 was dissolved in a minimum
volume of 0.01 N NaOH and brought up to the final concentration with
sterile saline. It was administered as a single daily intraperitoneal
injection at a dose of 25 µg/kg body wt. IGF-I was administered as a
single daily subcutaneous injection at a dose of 300 µg/rat. In each
case, control animals received the same volume of the appropriate vehicle.
RESULTS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
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Fig. 1.
Flow cytometry histograms of liver nuclei. Representative histograms of
fine-needle aspirated whole liver nuclei from newborn female rat
(A), adult female rat
(B), newborn male rat
(C), and adult male rat
(D). The peak at 32 fluorescence
units (arbitrary units of DNA content) represents the diploid cell
nuclei (2C) and that at 64 units, the tetraploid cell nuclei (4C). The
octoploid cell nuclei was <1% at best and is not visible in this
histogram. Shaded area represents 2C nuclei in S phase (SPF). Results
shown in
A-D
represent means ± SE of no. of rats
(n) stated in each case.
Table 1.
Values of rat liver 4C nuclei and 2C nuclei in S phase
Age and sex variations of liver 4C nuclei and S phase.
The age variations in the percentage of 2C nuclei in S phase and of 4C
liver nuclei from newborn to adult male and female rats are shown in
Fig. 2. The percentage of 2C nuclei in S
phase was similar in both sexes. It decreased with age from 17% on the day of birth to 1-2% at 40 days
(P < 0.001) and
thereafter.
|
Effect of sex steroids on liver 4C cell nuclei and S phase.
To study whether the gender differences in the percentage of 4C liver
nuclei and 2C nuclei in S phase were attributable to sex hormones, we
tested their effect in the following two model systems: prepuberal, 20 day-old rats and adult rats castrated at 60 days of age. Prepuberal
rats were treated with Silastic capsules containing either EB, TP, or
vehicle for 10 days. TP significantly increased the 4C
nuclei in both males and females, whereas EB was effective only in
males (Table 2). No
significant change in the 2C nuclei in S phase was found after any
treatment.
|
Effect of GH on 4C liver nuclei and S phase.
The starting point for the study of the effect of GH on the ploidy of
the liver nuclei was the observation that male Sprague-Dawley rats
hypophysectomized at 60 days and killed 1 mo later had a significantly
lower percentage of 4C nuclei than the sham-operated controls
(27.9 ± 5.4 vs. 43.8 ± 2.7%, respectively;
P < 0.001). Treatment with 200 µg hGH/day for 10 days failed to significantly modify the percentage of 4C nuclei (34.6 ± 0.8%), whereas
treatment with 25 µg
T3 · kg1 · day
1
significantly increased the 4C nuclei (41.5 ± 2.8%;
P < 0.05).
|
Effect of thyroid hormones on liver 4C nuclei and S phase. To study the effects of thyroid hormones on the processes of hepatocyte proliferation and polyploidization, we used rats that were rendered hypothyroid by administering propylthiouracil (PTU) in the drinking water. PTU treatment started when the rats were 70 days old. After 21 days, these rats showed a percentage of 4C nuclei and S phase similar to the controls (not shown). When the rats were treated with T3, the percentage of 4C nuclei and the number of 2C nuclei in S phase remained unaltered. These results suggested that hypothyroidism provoked in adult rats may not have an effect on these parameters. However, as in the case of hypophysectomized rats, the 70-day-old rats already contain a high percentage of 4C nuclei and a low S phase, and the half-life of hepatocytes is longer than the period that rats were subjected to treatment with PTU. This led us to use another rat model system to study the effect of thyroid hormones on 4C liver nuclei and S phase: MMI-induced hypothyroid rats.
Rats were rendered hypothyroid by MMI treatment to dams and newborn rats until they were killed. Rats withdrawn from MMI treatment showed a significant increase in body growth, S phase, and 4C nuclei after 20 days (P < 0.001), thus showing that the effect of MMI on these parameters was reversible. Hypothyroid animals (Table 4) showed significantly lower levels of 4C liver nuclei than their corresponding age-matched controls (P < 0.001) and, in females, also with the weight-matched controls (P < 0.05).
|
Time-course of T3 effect in vivo on
hepatocyte ploidy and S phase.
To validate the previous results, we obtained liver cells by aspiration
with a 20-gauge needle from the livers of anesthetized rats. This
procedure provided 25,000-50,000 hepatocytes, which were enough to
measure the different ploidy cell classes and the number of 2C cells in
S phase. Figure
3A
shows a typical DNA content histogram for adult male hypothyroid rat
hepatocytes.
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DISCUSSION |
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Three main findings can be highlighted from this study: 1) the age- and hormone-dependent changes undergone by the liver 2C nuclei in S phase, which had not been previously quantified, 2) the essential regulatory role of T3 in the processes of hepatocyte proliferation and polyploidization, which is in contrast with previous studies suggesting that the effect of T3 is indirectly mediated by GH, and 3) the introduction of an in vivo estimate of liver ploidy and S phase that allowed the demonstration of a separate regulation for both the cell multiplication and the polyploidization processes.
The high percentage of 2C cell nuclei in S phase at birth and during the first 4 wk of life reveals a strong stimulation of liver cell proliferation that leads to a predominance of 2C nuclei. After the S phase stabilizes at ~30 days of age, a higher population of 4C nuclei emerges and predominates for the rest of life. These findings suggest that the factors promoting the liver cell multiplication until the rat reaches 30 days of age are replaced by those operating afterward that promote a higher hepatocyte polyploidization.
The ontogeny of 4C cells resembles that of the liver thyroid hormone
receptor TR- (32). It also resembles that of some liver proteins
under multihormone regulation, such as
2-µglobulin (6) or LAGS (7).
These proteins are at a very low level before 30 days of life and then
abruptly increase to reach a plateau at 60-70 days of life, in
coincidence with the process of the anterior pituitary maturity. The
similarity in the ontogenies of 4C nuclei and these proteins suggests
that anterior pituitary hormones, and/or those under its
control, could be responsible for the hepatocyte polyploidization
process. Therefore, the effects of each pituitary hormone and their
subsidiary hormones on the 4C nuclei and S phase were investigated.
The gender differences in the percentage of 4C nuclei observed here have previously been described in rats (37) and also in mice (23, 31). We have also observed that the level of 4C liver nuclei in humans is much lower than in rats, but the liver from men contains a significantly higher percentage of 4C nuclei than that from women (7.6 ± 1.7 vs. 4.5 ± 0.5%, respectively; P < 0.05; unpublished results). These differences may be attributable to sex steroids, which play a role in the process of hepatocyte polyploidization. To study that possibility, we performed a series of experiments with both prepubertal and castrated rats. We found that castration did not have a significant effect on the percentage of 2C cells in S phase, but it did increase the percentage of 4C nuclei in females and decrease it in males. TP treatments proved to have a mild effect as promoters of polyploidization, as did EB in some experiments. Castration at birth did not significantly alter the pattern of 4C nuclei formation (38, and data not shown), and it appears that the sex differences observed in the percentage of 4C nuclei were attributable to an effect exerted through the liver growth. However, this difference persists when the data were normalized taking into account the RLW. These results suggest that the probable effect of sex steroids is that estrogen delays, and testosterone promotes, the polyploidization process. The lack of effect of steroid hormones on the percentage of 2C cells in S phase is in contrast with previous studies (7, 26, 39, 40) that found increases after ethynil estradiol treatment. This is probably due to the high doses used in these studies (7, 26, 39, 40), which may produce different effects than the physiological level of estrogen used in this study.
Because sex hormones seem to play a minor role in the hormone regulation of liver cell proliferation and polyploidization, we studied the effects of GH and T3 in a series of rat model systems. The MMI-induced hypothyroid rats of both genders showed 5% of 4C nuclei at 70 days of age, which is similar to that observed at birth. MMI-withdrawn rats underwent an increase in the 4C nuclei level after 21 days. This proves that the effect of MMI on 4C nuclei is reversible. Because these rats are also deficient in GH, they provided the most useful model to study the hormone regulation of liver polyploidization. Whereas hGH had little effect in hypothyroid females and none in males, T3 was very effective in increasing both 4C nuclei and S phase in all the situations studied.
The endocrine regulation of the growth and polyploidization of liver nuclei has been extensively studied in the past. Carriere stated in a review (5) that the regulation of DNA synthesis and mitosis in liver nuclei involves the concerted action of several hormones and the effects of thyroxine, GH, and testosterone were exerted separately. However, when summarizing the role for pituitary-mediated hormones, it was suggested (5) that the action of thyroid hormone is largely mediated through an effect on the release of anterior pituitary GH. Through flow cytometry, Mendecki and co-workers (27) investigated the effect of T3 in thyroidectomized 6-wk-old male rats on the ploidy of liver nuclei. A complete cessation of 4C nuclei formation was reported in these rats. As expected, treatment of thyroidectomized rats with a single dose of 10 mg T3/kg body wt abruptly increased the percentage of 4C nuclei. Despite the large dose used, Mendecki et al. (27) confirm the effect of T3 in the process of 4C nuclei formation and that the most prominent effect occurred 6 days after the injection. However, Mendecki and colleagues (27) did not examine the question of whether the effects of thyroid hormone were derived from its direct action on liver cells or were mediated indirectly by GH.
The effect of hGH on liver growth is clear in all the studied model systems, which is in agreement with its effect on the 2C nuclei in S phase. These results suggest that GH plays a relevant role in the induction of cell multiplication. GH acts as a growth stimulus for liver cells, thus increasing liver cell proliferation, but it is not essential to liver 4C nuclei formation. The changes in polyploid nuclei formation depend on T3. In fact, when T3 is highly depleted and exogenous GH was supplied (MMI plus hGH rats), the only evident effect was an increase in body and liver weight as well as in the percentage of 2C nuclei in S phase. On the other hand, when GH is highly depleted and T3 was unaffected (MSG control rats), the polyploid growth pattern develops as in normal rats. The normal percentage of 4C nuclei in GH-deficient male rats and its mild effect in other model systems suggest that GH plays modulatory roles of variable intensity, depending on the gender and hormone environment.
Hormone regulation of very complex processes such as those investigated in this study opens many questions about the putative molecules implicated in each step, such as the role played by cyclins, cyclin-dependent kinases (22, 24, 43), and other cell cycle regulatory molecules (12, 28, 42). These molecules are usually studied in tissue cultures, in which physiological situations such as pulsatile secretion of GH are difficult to mimic (11, 29). In the case of T3, the difficulty is exemplified by a lack of effect of T3 alone on DNA synthesis by hepatocytes in vitro, in contrast with its strong effect in vivo (15). To initiate an approach to study the molecular events, we used the MMI-treated hypothyroid rat model system where rats were anesthetized to obtain whole hepatocytes by FNA in vivo. This procedure provided enough hepatocytes to study ploidy and S phase and also to study molecules of interest by immunocytofluorescence and flow cytometry or by PCR-derived procedures. We used FNA here to carry out a time-course experiment to study the T3 induction of 4C nuclei formation in individual rats in vivo. We found that T3 powerfully induces hepatocytes to enter the S phase 24 h after injection, whereas a significant increase in the percentage of tetraploid hepatocytes required 6 days. These results suggest that liver growth and polyploidization are processes that are probably under different regulatory signals. Clearly, through manipulating T3, it is possible to create model systems with a wide range of hepatocyte proliferation and polyploidization rates. These systems would be useful for studies at the molecular level of chemical or hormonal induction of liver carcinogenesis, as well as in creating favorable conditions for transfection of new genes in vivo that require high proliferation rates (15).
In conclusion, the results presented here support the hypothesis that the process of hepatocyte polyploidization of the rat liver is under endocrine control, with thyroid hormones playing the essential regulatory role. GH seems to be an important factor in inducing the liver cell proliferation. Together with sex steroids, GH may also play modulatory roles of variable intensity in the process of liver cell polyploidization, depending on the age and gender of the rats.
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ACKNOWLEDGEMENTS |
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We thank A. Lleó for help in animal management, N. Granados and R. Medina for technical assistance, and Dr. F. Vidal-Vanaclocha, Dr. Sergio Moreno, and S. Cranfield for help in manuscript preparation.
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FOOTNOTES |
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This work was supported by grants from the Dirección General de Universidades e Investigación, Consejeriá de Educación Cultura y Deportes (Comunidad Autónoma de Canarias, 90/126 to B. N. Díaz-Chico), Instituto Canario de Investigación y Desarrollo de la Consejería de Hacienda (BOC 90-1995), and Comisión Interministerial de Ciencia y Tecnología (Spain, SAF95/0581 to B. N. Díaz-Chico).
Part of this work was presented as a poster at the Recent Progress in Hormone Research Meeting (1996, Stevenson, WA) and also at the Endocrine Society Meeting (1997, Minneapolis, MN).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests: B. N. Díaz-Chico, Laboratorio de Fisiología, Centro de Ciencias de la Salud, Universidad de Las Palmas de Gran Canaria, Apdo. 550, E-35080 Las Palmas de Gran Canaria, Canary Islands, Spain.
Received 11 May 1998; accepted in final form 24 September 1998.
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