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INTRODUCTION |
Hepatocytes show unique differences in gene expression patterns in
the liver lobule. Some genes are expressed throughout the liver lobule,
whereas others exhibit gradients between portal and perivenous areas
(1-9). Associations between transcriptional gene switching, such as
albumin and
-fetoprotein genes, and selective gene expression during
development, as seen for cytochrome P450 genes have been interpreted in
the context of cell differentiation mechanisms (10-12). Demonstrations
of specialized functions along with other parameters in hepatocyte
subsets, including those involved in drug disposition in perivenous
areas, led to speculations concerning the presence of less
differentiated hepatic progenitor cells in periportal areas with less
comprehensive gene expression repertoire, followed by the acquisition
of specialized functions in more differentiated hepatocytes in
perivenous areas (12). This feature of hepatic gene expression was
augmented by interspersed instances of markedly restricted gene
expression in the liver lobule. For example, glutamine synthetase and
GLUT-1 are expressed in only a small rim of hepatocytes immediately
adjacent to the terminal hepatic venule (7, 9). The involvement of
transcriptional, as well as post-transcriptional, mechanisms in the
regulation of specific genes, such as glutamine synthetase,
phenobarbitone-inducible cytochrome P450IIB1,2 genes, and GLUT-1 in
perivenous areas, has been proposed to reflect compartmentation of
specialized function in specific hepatocyte subsets (2). The
possibility of other mechanisms, including substrate availability and
regulation by soluble circulatory signals led to complex studies of
vascular mechanisms, such as alterations in portal blood flow in intact
animals (13).
The recent ability to repopulate the liver with genetically marked
hepatocytes offers unique opportunities for studying physiological processes in the liver (14). One transplantation system utilizes F344
rats deficient in dipeptidyl peptidase IV enzyme activity (DPPIV
) as
recipients of DPPIV+ syngeneic hepatocytes (15). In this system,
transplanted hepatocytes can be conveniently localized by histochemical
visualization of DPPIV and dual-label studies can be conducted to
address gene expression or cell proliferation mechanisms (16).
Transplanted hepatocytes are originally located in periportal areas due
to their arrival in the liver lobule along with portal blood and
entrapment in proximal hepatic sinusoids due to the smaller size of the
latter. An important element concerns integration of transplanted
hepatocytes in the liver parenchyma, as shown by the presence of hybrid
plasma membrane structures, such as gap junctions and bile canaliculi
among conjoint transplanted and host hepatocytes (14). Transplanted
hepatocytes can be induced to replicate, especially under conditions
involving loss of host hepatocytes, such that toxins producing
perivenous liver injury that would spare transplanted cells in
periportal areas cause these to proliferate, along with uninjured host
hepatocytes (17). Repeated self-limited liver injury to the host can
induce multiple proliferation cycles in transplanted hepatocytes.
During these cycles of liver injury, transplanted hepatocytes migrate
from periportal toward perivenous areas, in a process resembling
"wound repair."
We considered that examination of position-dependent gene
regulation mechanisms in the repopulated liver would be instructive. The fate of transplanted cells induced to change their position in the
liver lobule could be compared with control animals receiving cells
from the same donor animal but without undergoing any change from their
original periportal location. This would allow analysis of cell
age-related versus position-specific mechanisms in gene regulation. Also, we reasoned that transplantation of fully
differentiated mature hepatocytes into another microenvironment, such
as the liver of recent-born pups, would allow further analysis of
position-specific gene expression. Finally, we asked whether gene
expression could be regulated in similar fashions in hepatocytes
isolated from periportal and perivenous areas of the liver lobule, if
their microenvironment were altered. The latter studies were
facilitated by our recent characterization of hepatocytes fractionated
from periportal or perivenous areas of the same animal followed by analysis in cell culture (18).
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EXPERIMENTAL PROCEDURES |
Animals--
Eight- to 10-week old DPPIV
F344 rats weighing
160-180 g and 3-day-old DPPIV
pups weighing 7-9 g were provided by
the Special Animal Core of the Marion Bessin Liver Research Center.
Normal F344 rats were obtained from the National Institutes of Health (Bethesda, MD) and weighed 250-300 g when used. Animals were utilized in groups of at least three to four animals each under protocols approved by the institutional Animal Care and Use Committee, according to the Guide for the Care and Use of Laboratory Animals (National Institutes of Health publication number 86-23, revised 1985).
To induce perivenous hepatic injury (19), 1.45 ml/kg carbon
tetrachloride (CCl4) was administered intramuscularly after diluting 1:1 (v/v) in mineral oil (Sigma). To induce CYP450 activity in
hepatocyte recipients, 0.5 g/liter phenobarbitone (Sigma) was administered in drinking water for 4 days to adult rats, which amounted
to 10-12.5 mg of phenobarbitone/rat/day as 20-25 ml water was
consumed per 24 h. Hepatocyte donors were treated with
phenobarbitone as above for 7 days. Rat pups received 1 mg of
phenobarbitone intramuscularly for 7 days with continued nursing by
mothers. The animals were housed in the Institute for Animal Studies at Albert Einstein College of Medicine under 14 h light, 10 h
dark cycles and received standard pelleted food and water ad
libitum.
Hepatocyte Isolation and Cell Fractionation--
Cells were
isolated from F344 rats with two-step perfusion using 0.025% (w/v)
collagenase, as described previously (15). The cell viability was
tested by trypan blue dye exclusion and attachment to tissue culture
plastic in RPMI 1640 medium containing 100 units/ml penicillin, 100 µg/ml streptomycin, and 10% heat-inactivated fetal bovine serum
(Life Technologies, Inc.). To fractionate cells, Percoll (Amersham
Pharmacia Biotech, Uppsala, Sweden) was diluted in phosphate-buffered
saline, pH 7.4 (PBS)1
containing 5 mg/ml bovine serum albumin (Sigma), as described (18).
Hepatocytes were suspended in 1 ml of RPMI 1640 medium, and 1-1.5 × 107 cells were placed at the top of Percoll gradients in
15-ml polystyrene tubes, followed by centrifugation under 1000 × g for 30 min at 4 °C. Cells localizing in discrete bands
were designated H4 (at the bottom) and H2 (toward the top). These cells
were obtained by pipetting and washed twice in RPMI 1640 medium.
Characterization of fractionated hepatocytes included flow cytometric
determination of cell ploidy, analysis of
-glutamyl transpeptidase,
glucose-6-phosphatase (Glu-6-P), glycogen, and P450 activities by
histochemical and immunocytochemical methods, as described previously
(18).
Hepatocyte Transplantation--
Animals were anesthetized with
inhaled ether (Fisher). Adult rats received 2 × 107
cells, and pups received 0.5 × 106 cells via spleen,
as described previously (15). After closing the abdomen, animals were
monitored closely until full recovery. Pups were returned to the
mothers immediately after recovery from anesthesia.
Histological Analysis for Transplanted Cell Localization--
To
localize F344 rat hepatocytes in DPPIV
animals, histochemical
staining was performed on frozen tissue sections as reported previously
(15). Integrations of transplanted cells in the liver parenchyma were
demonstrated by colocalization of DPPIV activity and bile canalicular
ATPase activity. Tissues were counterstained with hematoxylin.
To document the position of transplanted cells in tissues, serial wedge
liver biopsies were obtained from individual CCl4-treated adult rats. The position of transplanted cells in the liver of pups was
verified by sampling of animals. To determine the position of
transplanted cells in liver lobules, morphometric analysis was
performed with multiple microphotographs using a standard magnification
(× 40). At a minimum, 50 liver lobules were analyzed per animal with
determination of the number of transplanted cells in various zones of
the liver lobule. In addition, the number of cells in each liver lobule
was determined. The actual distance of transplanted cells from the
center of the nearest portal vein was measured in microphotographs. To
document whether equivalent areas of the liver were analyzed, we
measured portal vein diameters and the distance between portal and
central veins.
Gene Expression in the Intact Liver--
Colocalization studies
were performed to determine gene expression in transplanted cells.
Glu-6-P activity was determined in unfixed cryostat tissue, followed by
acetone-chloroform fixation and DPPIV histochemistry for localizing
transplanted cells, as described previously (15). Tissue glycogen was
determined by first performing DPPIV histochemistry in cryostat
sections fixed with ethyl alcohol-acetic acid followed by incubation in
1% aqueous periodic acid for 5 min and Schiff's reagent for another 5 min (18). After washing repeatedly in 0.5% sodium bisulfite, slides were immersed in running tap water for 10 min. To verify the
specificity of glycogen staining, control tissues were preincubated
with salivary amylase. The intensity of staining for Glu-6-P and
glycogen was arbitrarily graded from 0 (negative) to 4+ (maximally
positive). To demonstrate differences in gene expression, at least 50 cells per condition were graded in each animal.
A polyclonal goat antibody directed against rat CYPIIB1 isoform
(Gentest Inc., Woburn, MA) was used for analyzing
phenobarbitone-induced P450 expression (18). Cryostat tissue sections
or cytospun hepatocytes were fixed in alcohol-acetone (99:1 v/v) and
ethanol alone at 
20 °C for 5 min each. Tissues were air-dried,
rehydrated in PBS, blocked with nonimmune goat serum (diluted 1:20 in
PBS) for 20 min, and incubated with the primary antibody (diluted 1:20 in PBS) for 1 h at room temperature. After washing with PBS three times, antibody binding was localized with peroxidase-conjugated goat
IgG for 30 min at room temperature using the diaminobenzidine substrate (Sigma).
In Vitro Analysis of P450 Activity--
Chemicals and reagents
were from Sigma. Freshly isolated hepatocytes were cultured in 24-well
tissue culture plates coated with rat tail collagen (0.5-1 × 105 cells/cm2). Cells were allowed to attach
for 1-2 h in RPMI 1640 culture medium containing 10% fetal bovine
serum, penicillin, streptomycin, and 10
8 M
dexamethasone. To induce P450 activity, either 2 mM
phenobarbitone or 2 µM 3-methylcholanthrene were added to
the culture medium immediately after cell attachment, with replacements
of these additives on days 2 and 3 of culture. P450 activity was
assayed 1, 2, 3, and 5 days after initiating hepatocyte cultures,
according to an assay described previously with minor modifications
(20). Cells were washed twice with cold PBS followed by the addition of
8 µM 7-ethoxyresorufin and 10 µM dicumarol
with incubation for 1 h at 37 °C. From some wells, culture
medium and cells were harvested separately, whereas only medium was
harvested from other wells for sequential studies. To 0.3-ml aliquots
of culture medium, 0.2 ml of ethanol was added, and samples were
centrifuged for 10 min in a microcentrifuge to solubilize resorufin
products, followed by fluorescence measurements at 530 nm (excitation)
and 590 nm (emission). Purified resorufin was used to generate standard curves in linear range and data normalized by measuring protein in
aliquots with the Bio-Rad assay.
Experimental Design--
Studies using adult rats addressed gene
expression in unprimed transplanted hepatocytes. The strategy was to
transplant hepatocytes isolated from a single donor into 18 DPPIV
F344 rats. The animals were divided into two groups of nine rats each.
One group served as controls with cells in periportal areas, since
intrasplenic injection is known to deposit hepatocytes into these areas
(15). The other animal group was given three doses of CCl4
at 10-day intervals to cause perivenous injury and shift the position
of transplanted hepatocytes from periportal to perivenous areas during the ensuing liver repair (Fig.
1A).

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Fig. 1.
Experimental strategies for in vivo
experiments. A shows the scheme of cell
transplantation experiments in adult rats. Transplanted cells were
allowed to engraft and integrate in the liver parenchyma for 2 months
after which three cycles of CCl4 were administered
separated by 10 days each. Serial biopsies were obtained from several
animals before and after CCl4. Some animals were given
phenobarbitone in drinking water for 4 days prior to sacrifice at 24 months after cell transplantation. B shows the scheme of
cell transplantation into 3-day-old suckling pups. Hepatocytes were
from animals treated with phenobarbitone for 7 days, which induced P450
activity. Pups intended for sacrifice after 1 week were treated with
intramuscular phenobarbitone starting on the day of cell
transplantation until sacrifice. Pups intended for sacrifice at 4 weeks
were treated with intramuscular phenobarbitone for the preceding 7 days.
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A second experiment concerned transplantation of hepatocytes into
suckling DPPIV
rat pups with P450 induction by treating the donor rat
with phenobarbitone for 7 days. These hepatocytes were transplanted
into nine 3-day-old DPPIV
pups (Fig. 1B). The pups were
killed in groups of 2-3 animals each at 2 h, 7 days, and 4 weeks
after cell transplantation. The pups received phenobarbitone daily for
7 days each prior to being killed at 7 days and 4 weeks after
cell transplantation.
Statistics--
Data are expressed as mean ± S.D.
SigmaStat 2.0 software was used for data analysis (Jandel Corp., San
Rafael, CA). The significance of differences was analyzed as
appropriate by Student's t test, Chi-square test,
Mann-Whitney rank sum tests, or analysis of variance.
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RESULTS |
The experiments were first conducted in adult rats. After
transplantation via spleen, hepatocytes engrafted in the liver with integrations in the hepatic parenchyma, which was documented by colocalization of bile canalicular DPPIV and ATPase activities (not shown).
Changing the Position of Transplanted Hepatocytes in the Adult Rat
Liver--
In normal control rats, transplanted hepatocytes were
distributed mainly in periportal areas of the liver. This pattern was generally unchanged throughout the 2-year duration of the studies (Fig.
2). The findings were compatible with
previous studies of liver repopulation in normal mice (21). However, at
24 months, transplanted hepatocytes showed somewhat larger cell
clusters in F344 rats, which indicated the possibility of spontaneous
cell proliferation in this setting, although transplanted cells still remained in periportal locations. In contrast, in response to CCl4-induced liver injury, the position of transplanted
cells changed progressively. In this situation, transplanted cells were localized in periportal areas (zone 1), deeper inside (zone 2), as well
as perivenous areas (zone 3) of the liver lobule.

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Fig. 2.
Switching the position of transplanted
hepatocytes in the adult rat liver. Control rat liver from an
individual animal at 2 months (A), 3 months (B),
and 24 months (C) after hepatocyte transplantation.
Transplanted hepatocytes are visualized by histochemical localization
of DPPIV activity. Note that transplanted cells (arrows)
appear in periportal areas at all these times in control animals. The
remaining panels show transplanted cells in an animal subjected to
CCl4 treatments. D shows a liver biopsy at 2 months after cell transplantation, before CCl4 was given.
Transplanted cells are in periportal locations, similar to control
animals. E shows the liver following two cycles of
CCl4, when the position of transplanted cells began to
shift toward perivenous areas. F shows a large cluster of
transplanted hepatocytes after cells shifted to the perivenous area at
24 months. Also, there is spontaneous proliferation of biliary cells in
F, which is a known feature of the aging F344 rat liver.
p = portal area; c = perivenous
area.
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Detailed morphometric analysis of sequential biopsies in three animals
showed that at the end of all three CCl4 treatments, significantly fewer transplanted hepatocytes were in zone 1 of the
liver lobule, whereas more hepatocytes were in zone 3 (Table I). To assure that various tissue samples
were comparable, we evaluated the dimensions of the liver lobules under
study by using portal vein diameters and the distance between portal
and central veins as reference points. These parameters were not
different in the tissues analyzed. The mean distance of transplanted
cells from the center of portal vein shifted significantly after
CCl4. Also, the overall number of transplanted hepatocytes
increased in the liver, consistent with cell proliferation during
recovery from CCl4-induced liver injury. Analysis of the
number of transplanted cells showed 6 ± 2 cells per liver lobule
before CCl4 and 15 ± 3 cells per liver lobule after
the completion of all three CCl4 treatments,
p < 0.02 (t test). However, some cell
clusters in CCl4 recipients were large and contained
>100-200 cells each. The findings were compatible with the ability to
shift the position of transplanted hepatocytes from periportal areas to
additional locations without altering the overall organization of the
liver lobule.
Liver Gene Regulation in Transplanted Cells Engrafted in the Adult
Liver--
Although Glu-6-P and glycogen are present in hepatocytes
throughout the liver lobule, during previous studies to demonstrate gene expression in transplanted cells, we recognized gradients of
expression in the F344 rat liver. We found that while Glu-6-P showed
greater histochemical activity in periportal areas (3+ to 4+ grade),
compared with perivenous locations (0 to 2+ grade), this situation was
reversed in the case of glycogen (Fig.
3). These findings allowed us to
determine whether gene expression was regulated in transplanted
hepatocytes following shifts in their position. In normal control
animals, as well as in animals before CCl4 treatments,
periportally located transplanted hepatocytes showed greater Glu-6-P
activity (3+ to 4+), similar to adjacent host hepatocytes, compared
with host hepatocytes in perivenous areas (0 to 2+). In contrast, when
the position of transplanted hepatocytes was shifted toward perivenous
areas, Glu-6-P expression began to progressively decline, such that in
hepatocytes immediately adjacent to the terminal hepatic venule,
Glu-6-P expression was markedly attenuated (p < 0.01, t test). Interestingly, in the CCl4-treated
liver where transplanted cells were simultaneously localized elsewhere,
there was much greater Glu-6-P activity in periportal areas, indicating
that the change in gene expression was not simply due to some other
cellular alteration during CCl4-induced proliferation.
Furthermore, changes in the glycogen content of transplanted
hepatocytes mirrored those of Glu-6-P. In transplanted hepatocytes
situated in periportal areas, as well as adjacent host hepatocytes,
less glycogen was visualized (1+ to 2+) compared with hepatocytes in
perivenous areas (3+ to 4+), p < 0.01, t
test. This situation became reversed when transplanted hepatocytes
shifted to perivenous areas.

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Fig. 3.
Gene regulation in the adult liver.
A, glycogen staining showing a gradient in the liver lobule
with greater glycogen content in perivenous hepatocytes. B,
Glu-6-P staining showing a reversed gradient, with greater expression
in periportal areas. C, transplanted hepatocytes
(arrow) in a control animal showing less glycogen content,
similar to adjacent host hepatocytes in periportal areas compared with
host hepatocytes in perivenous areas. D, Glu-6-P staining in
liver from the same control animal showing a reversed pattern, with
greater Glu-6-P expression in transplanted periportal hepatocytes
compared with perivenous hepatocytes. E, glycogen staining
in the liver from an animal subjected to repeated CCl4
treatments showing extensive glycogen expression in perivenous areas.
F, attenuation of Glu-6-P expression in transplanted
hepatocytes localized in perivenous areas with some hepatocytes
immediately adjacent to the terminal hepatic venule showing very little
Glu-6-P activity (short arrows). In contrast, transplanted
hepatocytes situated in adjacent periportal areas show much greater
Glu-6-P activity (long arrows).
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To demonstrate regulation of cytochrome P450 system, we analyzed
changes in response to induction by phenobarbitone. Hepatocyte recipients were given phenobarbitone for 4 days immediately before sacrificing animals at 24 months. Immunolocalization of CYPIIB1 activity, along with DPPIV staining to localize transplanted cells, showed that whereas host and transplanted hepatocytes in perivenous areas were induced by phenobarbitone, neither transplanted nor host
hepatocytes in periportal areas could express CYPIIB1 activity (not
shown). The findings were again compatible with regulation of gene
expression in transplanted hepatocytes by the ambient microenvironment
of the liver lobule.
Gene Regulation in Transplanted Hepatocytes in the Developing Rat
Liver--
These studies were performed in suckling F344 rat pups to
elucidate regulation of gene expression in hepatocytes isolated from
adult rats. The premise was that transplanted hepatocytes would engraft
and function in the developing liver. Immediately after injection of
cells into the spleen, transplanted hepatocytes appeared in portal
areas and hepatic sinusoids (Fig. 4).
Dual staining for bile canalicular DPPIV and ATPase activities showed that transplanted hepatocytes were integrated in the liver parenchyma at 7 days of transplantation. The suckling rat liver showed a gradient
of Glu-6-P, which was similar to the adult rat liver, and supported
abundant expression of Glu-6-P activity in transplanted hepatocytes (3+
to 4+). In contrast, hepatic glycogen stores were less pronounced (1+)
in the suckling rat liver and showed no gradients in the liver lobule
at 7 days of age. This was reflected in transplanted hepatocytes, many
of which showed very little glycogen. However, glycogen staining became
more pronounced in both transplanted and host hepatocytes at the age of
4 weeks (2+ to 3+), consistent with further maturation of the
liver.

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Fig. 4.
Gene regulation in the suckling liver.
A, hepatocytes integrated in the liver of rat pups as shown
here at 7 days after cell transplantation. The liver was first stained
for DPPIV activity (red color, short arrows) and
then for ATPase activity (brown color, long
arrows) to demonstrate networks of hybrid bile canaliculi in liver
plates. B, the gradient of Glu-6-P activity in the suckling
rat liver, which was similar to the adult rat liver. The
arrow is pointing to transplanted hepatocytes at 7 days
after injection into the pup. C, higher magnification view
of the same area as in B showing Glu-6-P expression in
transplanted hepatocytes. D, glycogen staining in the
7-day-old suckling rat liver showing relatively faint staining compared
with the adult liver and an absence of a gradient in the liver lobule.
Transplanted hepatocytes (arrows) show little glycogen
activity. E, at 4 weeks after cell transplantation, when
animals had not yet been weaned, more glycogen was apparent in the
liver. However, at this stage, glycogen staining was more pronounced in
periportal areas compared with perivenous areas, which was in
contradistinction with the adult liver. Transplanted hepatocytes
(arrow) began to demonstrate greater glycogen content as
well. F shows magnified view of transplanted hepatocytes at
4 weeks with glycogen content similar to that of adjacent periportal
hepatocytes of the host.
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The regulation of phenobarbitone-inducible CYPIIB1 activity in the
suckling liver was instructive. When hepatocytes were isolated from a
donor with extensive P450 induction following 7 days of treatment with
phenobarbitone, 80-90% cells showed immunostaining for CYPIIB1
activity. Upon transplantation of these cells into the suckling rat
liver, CYPIIB1 expression was present in transplanted cells at 1 h
of transplantation (Fig. 5). However, all
transplanted cells rapidly lost CYPIIB1 activity in the suckling rat
liver subsequently (p < 0.001, Chi-square) when
animals were studied at 1 week after cell transplantation, despite
phenobarbitone administration to pups for these 7 days. Moreover, P450
activity did not return in transplanted hepatocytes at 4 weeks,
although perivenous cells in the suckling liver were faintly stained
with anti-CYPIIB1 at this time following 7 days of phenobarbitone
treatment. The findings indicated that in response to signals emanating
from the nonpermissive host hepatic microenvironment, P450 activation
was lost in transplanted hepatocytes.

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Fig. 5.
Phenobarbitone-inducible CYPIIB1 activity in
the suckling liver. A, donor rat liver showing
extensive immunoreactivity with an antibody against CYPIIB1. The animal
was treated with phenobarbitone for 7 days prior to isolation of cells.
B, the suckling liver from pups treated with phenobarbitone
for 7 days did not react with the antibody. Control tissues from an
uninduced normal adult rat liver or when the primary antibody was
omitted also showed no reaction product. C, showing
transplanted hepatocytes staining with CYPIIB1 antibody in portal areas
and hepatic sinusoids of a suckling pup within 1 h of intrasplenic
injection. D, at 7 days after cell transplantation, CYPIIB1
immunostaining was detected in neither transplanted hepatocytes
(arrows) nor host liver.
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Regulation of Gene Expression in Hepatocytes Isolated from
Different Positions of the Liver Lobule--
We reasoned that if
external signals were important determinants in hepatic gene
regulation, it should be possible to test whether specific function
could be induced in hepatocytes isolated from permissive and
nonpermissive areas of the liver lobule. For this purpose, we utilized
Percoll gradients to isolate hepatocytes enriched in periportal or
perivenous areas of the same liver. In parallel experiments, cell
fractions were characterized by a variety of parameters, including
differences in their ploidy states and phenotypic analysis to ascertain
the lobular origin (18). The hepatocytes were isolated from a normal
F344 rat. Perivenous hepatocytes exhibited greater abundance of
tetraploid nuclei on flow cytometry, greater glycogen content and P450
activity, and lesser Glu-6-P activity. None of the isolated hepatocytes expressed
-glutamyl transpeptidase, indicating an absence of contamination with biliary cells. When these isolated hepatocytes were
cultured with phenobarbitone and 3-methylcholanthrene, CYP450 activity
using resorufin conversion as a measure was not apparent at 24 h
of cell culture. Subsequently, although basal P450 activity was greater
in cultured perivenous hepatocytes (H2 fraction) (up to 3-fold greater,
p < 0.001), periportal hepatocytes (H4 fraction) rapidly acquired this facility in culture conditions. Indeed, the
relative magnitude of P450 induction in both cell fractions was
comparable after 2, 3, and 5 days in culture, although P450 inducibility declined in cultured hepatocytes with time (Fig. 6). In some experiments,
3-methylcholanthrene-inducible P450 activity was greater in H4 cell
fraction compared with H2 cell fraction (p < 0.001, t test) at 2 days in culture, but then became similar in
both cell fractions.

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Fig. 6.
Induction of P450 expression in periportal
hepatocytes nonpermissive in vivo. Hepatocytes
isolated from the same rat liver were cultured as described under
"Experimental Procedures." Utilization of 7-ethoxyresorufin is
shown in H2 cells (perivenous hepatocyte fraction) and H4 cells
(periportal hepatocyte fraction) following culture with phenobarbitone.
The magnitude of resorufin conversion in the two fractions was similar
with no difference upon statistical analysis (t tests,
p = not significant). None of the cell fractions showed
significant resorufin conversion at 24 h. Results of experiments
with 3-methylcholanthrene induction of cells were essentially similar.
The data were representative of at least six independent experiments
performed.
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DISCUSSION |
These findings establish that signals emanating from the
microenvironment play critical roles in directing liver gene
expression. Our findings are novel in respect with the use of
transplanted hepatocytes as reporters to analyze physiological
mechanisms in the liver. Also, the results demonstrate unequivocally
that hepatocytes can both acquire and lose specialized functions
depending upon the permissiveness or otherwise of the microenvironment.
All three of our approaches, namely shifting the position of reporter
hepatocytes in the adult liver, switching hepatocytes from a permissive
environment to a nonpermissive one, and analysis of hepatocytes from
perivenous and periportal areas in the neutral territory of identical
culture conditions and substrate availability provided mutually
compatible results.
Our studies were greatly facilitated by the recent development of novel
cell transplantation systems and insights into mechanisms of liver
repopulation with transplanted hepatocytes (14). A most critical
feature concerned the ability of transplanted hepatocytes to enter
hepatic cords in the adult liver, which was demonstrated previously
(16), as well as in the neonatal liver, as shown here. While
transplanted hepatocytes can proliferate extensively in the diseased
liver with constant depletion of host hepatocytes (22, 23), in the
setting of self-limited liver injury, such as acute CCl4
toxicity, proliferation of transplanted hepatocytes was tightly
regulated (17). Although it could potentially be argued that the
progeny of transplanted hepatocytes could have shown differences in
gene regulation mechanisms, such was not the case in our experience.
Despite significantly increased sizes of hepatocyte clusters in animals
treated repeatedly with CCl4, gene expression in
transplanted hepatocytes resembled that in adjacent host cells. During
extensive repopulation of the mouse liver, transplanted adult
hepatocytes have also been shown to display gene expression patterns
consistent with their position in the liver lobule (23).
A multitude of studies established previously that periportal and
perivenous hepatocytes differ in their biochemical capacities (18,
24-29). Our findings of differences in the abundance of Glu-6-P and
glycogen content were compatible with models of metabolic zonation in
the liver (30, 31). It was interesting that the periportal preference
of Glu-6-P activity in the suckling rat liver was similar to the adult
liver, whereas glycogen stores were less pronounced and showed no zonal
predilection in very young suckling pups. Metabolic activities
localized preferentially in periportal areas are broadly classified to
be involved in glucose and amino acid utilization and urea, cholesterol
and bile synthesis, etc., whereas glycogen, fatty acid and glutamine
synthesis, and xenobiotic disposal are preferentially localized in
perivenous areas (30, 31). Our findings concerning restriction of P450 activity in perivenous areas of the intact adult liver are compatible with metabolic zonation of gene expression. This peculiar zonal selection of gene expression has been correlated with structural features of cells, e.g. the occurrence of greater
hepatocellular ploidy in perivenous areas of the liver lobule (32).
Similarly, post-translationally regulated distributions of gene
products in cellular compartments and plasma membrane domains have been interpreted as unique position-specific features of some hepatocytes (2). On the other hand, our findings concerning transplantation of
P450-expressing hepatocytes in suckling pups indicated that this
activity was reversible. The findings suggested regulation at a
functional rather than structural level. Moreover, we found that
hepatocytes isolated from periportal areas of the liver were able to
rapidly acquire P450 inducibility in the presence of appropriate substrates. The findings were compatible with the previous
demonstrations of phenobarbitone-inducible CYPIIB1 mRNA expression
in the developing rat liver (33). Studies with in situ
mRNA hybridization showed that CYPIIB1 mRNAs were distributed
across the hepatic lobule in this situation, except for periportal
cells, as in our studies of adult rats with immunostainable CYPIIB1
protein following phenobarbitone induction. The lack of immunostainable
CYPIIB1 in our suckling rats is in agreement with post-transcriptional
regulation if previous mRNA expression data are extrapolated (33).
Nonetheless, our findings should help understand previously
unreconciled results concerning regulation of hepatic alcohol
dehydrogenase activity. In vivo studies showed that alcohol
dehydrogenase localized principally in perivenous areas of the adult
liver (1). In contrast, studies using periportal and perivenous
hepatocytes isolated by enzymatic digestion of the liver showed that
the rates of ethanol metabolism were similar in both cell fractions
(34).
The studies suggest that position-specific gene expression must reflect
the consequence of complex interactions among cellular transcription
factors, signals from adjacent cells, extracellular matrix components,
hormones, intermediary metabolites, etc. Regulation of
position-specific gene expression in the liver by manipulating ambient
oxygen or hormone concentrations has been documented (35, 36). Recent
studies showed changes in the distribution of specific cellular
transcription factors, as well as abundance of cytokines in various
zones of the liver lobule (37, 38). Similarly, gradients of
extracellular matrix components are recognized in the liver and may
have important influences upon hepatic gene expression (39). Cell-cell
interactions have also been shown to regulate hepatic gene expression,
e.g. in cocultures of hepatic stellate cells and
hepatocytes, differentiated hepatocyte functions are retained in a
superior fashion (40). Our findings indicate that hepatocytes do not
possess irrevocable mechanisms to account for position-specific gene
expression patterns and are in agreement more with a major role of
external influences, albeit not necessarily exclusive ones.
The results have implications upon further analysis of liver gene
regulation and characterization of specific liver cell subpopulations. For instance, there is burgeoning interest in searching for hepatic stem cells. Cell lines capable of differentiating into hepatocytes have
been obtained from progenitor liver cells (41). One goal of these
general efforts is to establish mechanisms for bio-artificial liver
devices to support the failing liver, which would benefit from suitable
devices that can reproduce the hepatic microenvironment and offer
greater preservation of desired functions in immobilized cells. Other
investigations are aimed at using progenitor cells for cell and gene
therapy approaches. In undertaking characterization of progenitor liver
cells with analysis of gene regulation, our findings concerning the
effects of microenvironment on cell differentiation should be
especially germane.