Cell volume regulates liver
phosphoenolpyruvate carboxykinase
and fructose-1,6-bisphosphatase genes
Stephan
Kaiser
Department of Medicine, University Hospital of Tübingen, 72076 Tübingen, Germany
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ABSTRACT |
Hypertonic-induced
cell shrinkage increases glucose release in H-4-II-E rat hepatoma
cells. This is paralleled by a concomitant increase in the mRNA levels
of the rate-limiting enzymes of the pathway of gluconeogenesis,
phosphoenolpyruvate carboxykinase (PCK) and fructose-1,6-bisphosphatase (FBP), of seven- and fivefold, respectively. In contrast, hypotonic-induced swelling of the cells results in a transient decrease in PCK and FBP mRNAs to 15% and 39%
of control levels. The antagonistic effects of hyper- and hypotonicity
mimic the counteracting effects of adenosine 3',5'-cyclic monophosphate (cAMP) and insulin on PCK and FBP mRNA levels. The hypertonic-induced increase in mRNA levels is due to an enhanced transcriptional rate, whereas the decrease in mRNAs caused by hypotonicity results from a decrease in transcription as well as mRNA
stability. The inductive effect of hypertonicity does not require
ongoing protein synthesis and acts independently of the cAMP-dependent
protein kinase and protein kinase C pathways. These results suggest
that cell volume changes in liver cells may play an important role in
regulating hepatic glucose metabolism by altered gene expression.
gluconeogenesis; cell volume regulation; liver gene
expression
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INTRODUCTION |
CELL VOLUME HAS LONG BEEN recognized as an important
determinant for cellular function and metabolism in a variety of
prokaryotic as well as eukaryotic cells (8). More recently, it has
become clear that especially in the kidney and the brain certain genes are controlled by hyperosmotic challenge (7). This is not surprising in
view of the importance of a naturally occurring osmotic gradient in the
medulla of the kidney and of the sensitivity of brain function toward
alterations in osmotic pressure. Regarding the liver, a control of
cellular function by cell volume appears less obvious; however, changes
in cell volume have been shown to affect metabolic liver function (15).
When hepatocytes are exposed to anisotonic medium, the cells are able
to counterregulate their volume within minutes. This volume-regulatory
response, however, restores cell volume only partially, thus leaving
the cells in a slightly swollen or shrunken state throughout the
anisotonic exposure. Recent studies have shown that these cell volume
changes occur not only under anisotonic conditions but also under the
influence of hormones such as insulin and adenosine
3',5'-cyclic monophosphate (cAMP) (1) or during cumulative
substrate uptake of certain amino acids such as glutamine (15). Among
other effects, the changes in cell volume lead to subsequent
alterations in hepatic carbohydrate metabolism. Thus cell swelling
inhibits and cell shrinkage stimulates lactate, pyruvate, and glucose
release in the isolated perfused rat liver, pointing to an inhibition
or stimulation of glycogenolysis during cell swelling or shrinkage,
respectively (15). Furthermore, glycogen synthesis has been shown to be
stimulated during hyposmotic cell swelling (24). Although the effects
of cell volume on metabolic flux rates have been studied extensively,
little information is available regarding the effects of cell volume on
the regulation of gene expression in the liver. Recent observation of a
transcriptional regulation of albumin gene expression by osmotic
pressure (26) has indicated a regulatory mechanism also on the
molecular level.
Because previous reports showed that cell volume changes affect glucose
metabolism in the liver and in isolated hepatocytes, the possibility of
an effect of cell volume changes on hepatic gluconeogenesis and the
expression of the genes coding for the two key enzymes
phosphoenolpyruvate carboxykinase
(PCK) and fructose-1,6-bisphosphatase (FBP) (27) was investigated. PCK
and FBP activities are not subject to allosteric regulation but rather
are determined solely by the abundance of enzyme. Therefore, the chief
mechanism by which PCK and FBP activities are controlled is by
changes in gene expression. The expression of the PCK
gene is increased by cAMP, dexamethasone, thyroid hormone, and
retinoic acid (27) and inhibited by insulin (25), the phorbol ester
phorbol 12-myristate 13-acetate (PMA) (9), vanadate (4), okadaic acid
(27), and lithium (5). Expression of the FBP gene is also
increased in the presence of cAMP, whereas it is decreased by insulin
(11, 12). Most importantly, the dominant antagonistic regulation of
both genes is exerted by insulin and cAMP, and these two hormones also
exert counteracting effects on liver cell volume.
The present study shows that cell volume changes modulate both PCK and
FBP gene expression in H-4-II-E rat hepatoma cells. This response is
also found in primary rat and human hepatocytes. Anisotonic-induced
cell volume changes stimulate alterations in the rate of transcription
as well as in mRNA stability and involve a signal transduction pathway
apparently distinct from cAMP-dependent protein kinase and protein
kinase C.
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EXPERIMENTAL PROCEDURES |
Materials.
H-4-II-E cells were from American Type Culture Collection (ATCC;
CRL-1548 and CRL-1560). Tissue culture media, fetal calf serum, and ribonuclease (RNase) T1 were from GIBCO-BRL.
[
-32P]dCTP (sp act
3,000 Ci/mmol),
[
-32P]UTP
(sp act 3,000 Ci/mmol), and
[3H]UTP (sp act 46.8 Ci/mmol) were from Amersham, and GeneScreen and GeneScreen Plus nylon
membranes were from Du Pont-New England Nuclear.
Nucleoside-5'-diphosphate kinase, yeast tRNA, glutamate dehydrogenase, restriction enzymes, and 8-(4-chlorophenylthio)-cAMP (CPT-cAMP) were purchased from Boehringer Mannheim, and the
oligolabeling kit was obtained from Pharmacia Biotechnology. RNasin and
T3 and T7 polymerases were purchased from Promega. AG501-X8 resin and sodium dodecyl sulfate (SDS) were from Bio-Rad, and agarose was from
FMC. Guanidinium thiocyanate and sodium
N-lauroyl sarcosine were from Fluka,
and protein kinase inhibitors were from LC Laboratories. All other chemicals were obtained from either Sigma Chemical or Merck.
The pPCK-10 and pFru-1,6-P2ase
plasmids were obtained from R. Hanson (Case Western Reserve University
School of Medicine, Cleveland, OH) (31) and S. Pilkis (State University
of New York, Stony Brook, NY) (12), respectively. pRLCGAP and pALB
containing the 1.3-kb rat glyceraldehyde-3-phosphate dehydrogenase
(GAPDH) and the albumin mouse cDNA were obtained from ATCC. The
isolated cDNA fragments derived from pPCK-10, pFBPase, and pRLCGAP
subcloned into pBluescript II SK(
) were used to synthesize the
respective sense and anti-sense cRNA probes as described.
Cell culture.
H-4-II-E cells were grown to confluency on 100-mm plates using a 50:50
mixture of Dulbecco's modified Eagle's medium (DMEM) and Ham's F-12
nutrient solutions containing 5% (vol/vol) fetal calf serum, 5 mM
glucose, and 25 mM sodium bicarbonate and adjusted to pH 7.4. The cells
were maintained at 37°C in an atmosphere of 95% air-5%
CO2. Anisotonic conditions were
obtained by varying the sodium chloride concentration by either adding
or omitting 50 mmol/l or as indicated. Cell viability under the various
experimental conditions was assessed by trypan blue exclusion and
lactate dehydrogenase release and was not significantly affected by the
employed anisosmotic conditions for the used time periods.
The effect of dexamethasone was determined using cells that were
maintained in serum-free medium for 24 h before the addition of the
glucocorticoid.
Cell volume of H-4-II-E cells was determined using a calibrated
resistance cytometer (30). Cells were kept in shaking water flasks in
DMEM-F12 under otherwise identical conditions as described above and
were then injected into a capillary where the measured resistance is
directly proportional to the calibrated amount of medium removed from a
single cell, which again is dependent on the diameter of the cell. With
multiple measurements the volume of the cell can be determined. The
obtained data correlated well with previous volume determinations
obtained with isolated perfused rat liver under otherwise similar
experimental conditions (15).
Northern analysis.
Total cellular RNA was isolated by guanidinum
thiocyanate-phenol-chloroform-isoamyl alcohol extraction and ethanol
precipitation as described (21). Two additional precipitations in 75%
and 100% ethanol were carried out, and the RNA was lyophilized for 30 min and then dissolved in diethyl pyrocarbonate-treated water at a
concentration of 2-3 µg/ul and stored at
70°C.
Formaldehyde-agarose gel electrophoresis and Northern blot
hybridization were carried out as described previously (21). Slot blot
experiments were performed using a Schleicher and Schuell Minifold II
manifold. The probes used were the 1.6-kb
Bgl II fragment of the pPCK-10
plasmid, which encodes the cytosolic PCK, and the 0.5-kb
Nde
I/Xba I fragment of the
pFru-1,6-P2ase plasmid coding for
fructose-1,6-bisphosphatase. The blots were hybridized overnight in
50% deionized formamide, 0.25 M
Na2HPO4,
pH 7.2, 0.25 NaCl, 1 mM EDTA, 7% SDS, and 100 µg/ml salmon sperm DNA
containing 2 × 106 cpm of
32P-labeled probe/ml and then
washed at high stringency. All blots were stripped by washing at
80°C with 2% glycerol and were rehybridized with a GAPDH probe.
Levels of GAPDH were unaffected by the various experimental conditions.
Quantification of PCK, FBP, and GAPDH mRNA levels was accomplished by
densitometer scanning of suitably exposed autoradiograms. The levels of
PCK and FBP mRNA were standardized relative to those of GAPDH mRNA.
Nuclear run-on transcription assay.
Nuclei were isolated and transcription run-on experiments were
performed as described (17). This method yielded ~0.3 × 105 nuclei/dish. The transcription
reaction mixture (200 µl) contained 0.21 µM
[
-32P]UTP (3,000 Ci/mmol), 25% glycerol, 75 mM sodium
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (pH 7.5), 100 mM KCl, 5 mM magnesium acetate, 1 mM
MnCl2, 50 µM EDTA, 1 mM ATP, 0.5 mM GTP and CTP, 4 mM dithiothreitol, 0.1 mg/ml heparin, 8.8 mM
phosphocreatine, 40 µg/ml creatine phosphokinase, 200 U/ml
RNasin, 0.1 mg/ml nucleoside-5'-diphosphate kinase, and ~2 × 107 nuclei. After 20 min
of incubation at 25°C, nuclei were digested with RNase-free
deoxyribonuclease I. Yeast tRNA was added, and nuclear RNA was isolated
by using a solution containing 4 M guanidinium thiocyanate, 25 mM
sodium citrate (pH 7.0), 0.5% sodium lauroyl sarcosine, 0.1 M
2-mercaptoethanol, 2 M sodium acetate (pH 4.0), water-saturated phenol,
and chloroform/isoamyl alcohol (24:1). The labeled nuclear RNA was
precipitated with isopropyl alcohol, washed in 70% ethanol, dissolved,
and centrifuged through a Sephadex G-50 spin column. Aliquots
containing 10 µg of the anti-sense RNAs were immobilized on a
GeneScreen Plus membrane using a Schleicher and Schuell Minifold II
apparatus. Hybridizations were carried out as described at 53°C for
3 days. The membranes were then washed and exposed to RNase A (5 µg/ml) and RNase T1 (5 U/ml), followed by proteinase K (50 µg/ml).
The hybridized RNAs were quantitated by densitometry as described
above. Hybridization efficiency (~30%) was determined simultaneously
by quantitating the extent of binding of
3H-labeled sense PCK or FBP RNA.
All data were calculated as specific hybridization relative to that of
GAPDH.
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RESULTS |
Effect of anisotonicity on PCK and FBP mRNA levels.
H-4-II-E rat hepatoma cells were grown to confluency in DMEM-F12 (1:1)
medium and then exposed to anisotonic conditions. Exposure to
hypertonic conditions (405 mosmol/l) for 6 h increased the levels of
PCK and FBP mRNA 6.7- and 5.3-fold, respectively, whereas hypotonic
conditions (205 mosmol/l) for the same time period decreased the mRNAs
of PCK and FBP to 33% and 42% of normotonic (305 mosmol/l) control
levels, respectively (Fig.
1, Table 1). Similar
results were obtained when experiments were carried out with Hep G2 and Hep 3B human hepatoma cells or primary rat and human
hepatocytes (Table 2). Incubation of the
cells with CPT-cAMP (50 µM) or with dexamethasone (1 µM) increased
PCK gene expression (Fig. 1, Table 1), whereas only CPT-cAMP was able
to enhance FBP expression, in line with previous reports (12, 18).
Insulin had a strong repressive effect on both mRNAs, even compared
with basal levels. Hypotonicity was able to partially block the effects
of the cyclic nucleotide or dexamethasone on the induction of the
mRNAs; the decreased tonicity of 205 mosmol/l caused a 35% repression
of the normally observed induction of PCK mRNA by CPT-cAMP and a 46%
repression of the effect on FBP mRNA levels. Conversely, the effect of
hypertonicity (405 mosmol/l) was synergistic with the effects of
CPT-cAMP and dexamethasone. The effect of anisotonicity was osmolarity
dependent (Fig. 2). Interestingly, the
greatest variation of both PCK and FBP mRNA levels occurred around an
osmolarity of 300 mosmol/l. Thus the modulating effect of anisotonicity
appeared to be greatest within the region of physiological osmolarity.

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Fig. 1.
Effect of anisotonicity on
phosphoenolpyruvate carboxykinase
(PCK) mRNA levels in H-4-II-E rat hepatoma cells. Northern blot
analysis was performed from RNA isolated from confluent plates of H35
hepatoma cells grown under normal (305 mosmol/l) or anisotonic (405 and
205 mosmol/l) conditions for 6 h. Anisotonic conditions were obtained
by addition or omission of 50 mM NaCl. RNA was also obtained from cells
in which insulin (100 nM), 8-(4-chlorophenylthio)-cAMP (cAMP) (50 µM), or dexamethasone (1 µM) had been added for the same time
period. Total RNA (20 µg) was analyzed by Northern blotting and
subsequently hybridized with a
32P-labeled probe for PCK. FBP,
fructose-1,6-bisphosphatase.
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Fig. 2.
Osmolarity dependence of PCK and FBP mRNA levels. Osmolarity was varied
by the appropriate addition or omission of sodium chloride.
A: total RNA (20 µg) was analyzed by
Northern blotting and subsequently hybridized with a probe for PCK.
B: relative mRNA levels for PCK and
FBP are expressed as fold difference to standardized value obtained for
the condition of 305 mosmol/l, which was expressed relative to that of
GAPDH and taken as 1. Values represent means ± SD from 3 or 4 different experiments.
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In contrast to the effects of anisotonicity on PCK and FBP mRNA levels,
the mRNAs of GAPDH, tyrosine aminotransferase, liver-type glutaminase,
and glutamine synthetase remained unchanged. Albumin mRNA levels were
decreased after hypotonic exposure for more than 8 h by about one-half,
in line with previous observations (26). Hypotonic exposure led to
slight increases in
-actin and tubulin mRNA levels; however, these
effects were not significant. Thus the effect of anisotonicity on PCK
and FBP mRNA levels appeared to be specific, and hybridization with
GAPDH cDNA was used for normalization of the mRNA on the blots.
Cell volume affects PCK and FBP mRNA levels.
Treatment of the cells with 100 mM of the nonmetabolizable sugars
raffinose or sucrose led to an induction of PCK and FBP mRNAs of a
magnitude similar to that under exposure with increased sodium chloride
(+50 mM) concentration. Furthermore, treatment with glycerol (100 mM)
or urea (100 mM) at concentrations yielding hyperosmotic conditions
(405 mosmol/l) but with no effect on cell volume did not change PCK and
FBP mRNA levels. When normal osmotic pressure of hypotonic medium (205 mosmol/l) was restored by adding raffinose (100 mM) or sucrose (100 mM)
instead of sodium ions, PCK and FBP mRNA levels remained unchanged
(Table 1). These observations ruled out the possibility that the
anisotonic-induced changes in PCK and FBP mRNAs were due to changes in
ionic strength or osmolarity. However, the observations also strongly
suggested that cell volume was the actual determinant of the observed
effects. Furthermore, when the cell volume of H-4-II-E
cells under the various experimental conditions was measured using a
calibrated resistance cytometer, the cell volume determinations
correlated well with the corresponding tonicity, hormone, or amino acid
(Table 3). Also, these data
are in line with previous estimations made with isolated rat liver
perfusions under otherwise similar experimental conditions (15).
Kinetics of the alterations of PCK and FBP mRNAs.
All cells were grown to confluency in medium containing 5 mM glucose
and 155 mM NaCl and then maintained at either 105 mM NaCl (= 205 mosmol/l) or 205 mM NaCl (= 405 mosmol/l) for various times. The cell
swelling induced by hypertonicity resulted in a biphasic response of
PCK mRNA levels with an initial twofold increase, which was followed by
a slight decrease and a second increase that reached a maximum
induction of 6.7-fold at 6 h and a subsequent decrease plateauing at a
fivefold induction. The mRNA of FBP increased constantly to reach a
plateau of a fivefold induction at 6-12 h (Fig.
3). In contrast, the level of GAPDH mRNA
remained unchanged when the cells were transferred to hypertonic medium. When the cells were transferred to hypotonic conditions, PCK
and FBP mRNAs showed a rapid and coordinate decline to a minimum of
16% and 39%, respectively, of levels observed under control conditions. However, the decrease was transient and both
mRNAs reached levels comparable to normotonic levels again by
12-24 h (Fig. 4). This response thus
paralleled to a great extent the insulin-induced repression of PCK mRNA
levels in the same cell line (19).

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Fig. 3.
Time dependence of the induction of PCK and FBP mRNA levels after
initiation of hypertonic conditions (405 mosmol/l). Cells were
maintained under hypertonic conditions for indicated periods of time.
Northern analysis was performed as described in Fig. 1 legend. The fold
induction is expressed as the ratio of the PCK or FBP mRNA level to the
GAPDH level with the 305 mosmol/l (= 155 mM NaCl) value set at an
arbitrary unit of 1. Values represent means ± SD from at least 3 different experiments.
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Fig. 4.
Time dependence of repression of PCK and FBP mRNA levels after onset of
hypotonic conditions (205 mosmol/l). Cells were maintained under
hypotonic conditions for indicated periods of time.
A: representative Northern blot using
a probe for PCK. B: PCK and FBP mRNA
levels expressed relative to the level of GAPDH mRNA of a normotonic
medium of 305 mosmol/l. Values are means ± SD from at least 3 different experiments.
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Effect of cell volume on transcription rates.
To identify the possible mechanism responsible for the altered levels
of PCK and FBP mRNAs under anisotonic conditions, transcription run-on
assays were performed using nuclei isolated at different times after
onset of hypertonic (405 mosmol/l) or hypotonic (205 mosmol/l)
conditions. The relative transcription rates of the PCK and FBP genes
increased five- and fourfold, respectively, by 5 h after initiation of
hypertonic conditions (Fig. 5). In contrast, hypotonic exposure resulted in a significant decrease in the
transcription rate of the PCK gene to 11% and of the FBP gene to 28%
of control levels by 6 h. The transcription of GAPDH was unaffected.
The initial increase in transcription rates correlated with the
increase in the respective mRNAs. Therefore, enhancement of
transcription could account for the initial induction of the two mRNAs.

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Fig. 5.
Nuclear run-on transcription assay using isolated nuclei from
H-4-II-E hepatoma cells. Nuclei from H35 cells obtained under
normotonic (305 mosmol/l) and hypertonic (405 mosmol/l) conditions
were incubated with
[ -32P]CTP.
[ -32P]CTP-labeled
nuclear RNA was hybridized with a membrane containing 15 µg of
PCK and GAPDH anti-sense RNAs. The blank lane contained the plasmid
vector with no insert (pBlue). As a further control, experiments
carried out in presence of -amanitin (2 µg/ml) showed no
hybridization signal.
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Effect of cell volume on mRNA half-life.
H-4-II-E hepatoma cells were maintained under normotonic or anisotonic
conditions for 6 h, after which the RNA synthesis inhibitor actinomycin
D was added and the decrease in PCK and FBP mRNAs relative to that of
GAPDH was determined. In all cases, the observed decrease occurred with
first-order kinetics. The calculated half-lives for PCK mRNA under
normotonic and hypertonic conditions were 4.4 and 3.7 h, respectively;
the half-life under hypotonic conditions was 2.3 h (Fig.
6A). The
corresponding half-lives for FBP mRNA were 5.4 and 4.8 h under
normotonic and hypertonic conditions, respectively, with a decrease in
half-life under hypotonic conditions to ~3.4 h (Fig.
6B). Thus destabilization could
contribute to the decrease of PCK and FBP mRNAs under hypotonic
conditions, while a slight but not significant destabilizing effect of
hypertonic exposure on both mRNAs was observable. Such destabilization
has already been shown to play a significant role in the repressive effect of PMA on renal PCK mRNA. However, the decline of both mRNAs
under hypotonic exposure was due to both decreased transcription and
mRNA stability.

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Fig. 6.
Effect of anisotonicity on mRNA half-life. Disappearance of PCK
(A) and FBP mRNAs
(B) was observed under normal (305 mosmol/l), hypertonic (405 mosmol/l), and hypotonic (205 mosmol/l)
conditions in the presence of actinomycin D (5 µg/ml). Cells were
grown for 6 h under normal or anisotonic conditions, after which
actinomycin D was added. Cells were harvested at the indicated time
points after actinomycin D addition. Values represent means ± SD
from 3 different experiments and are expressed relative to the mRNA
level before addition of actinomycin D.
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Recovery from hypertonicity.
When cells were maintained under hypertonic conditions for 24 h and
then returned to normotonic conditions, the levels of PCK and FBP mRNAs
returned to normal within 4-6 h. The decrease occurred with an
apparent half-life of 30-40 min for both mRNAs (Fig.
7). This calculated half-life correlates
well with the half-life of ~30 min obtained for liver PCK previously
(16). However, this process occurred more rapidly than the apparent
half-life of PCK and FBP mRNA measured in the presence of actinomycin
D. Thus selective inactivation might also contribute to the rapid disappearance of the two mRNAs during recovery. Alternatively, the
estimation of mRNA half-lives with actinomycin D might be erroneously
high, since this drug has been shown to stabilize mRNA by itself.

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Fig. 7.
Time course of decrease of PCK and FBP mRNA levels after recovery from
hypertonic exposure. Cells were maintained under hypertonic conditions
(405 mosmol/l) for 24 h and then medium was adjusted to 305 mosmol/l.
Values are means ± SD from 3 different experiments and are
expressed relative to the level before recovery, which was taken as
100.
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Cell volume affects PCK and FBP gene expression independently of
cAMP-dependent protein kinase and protein kinase C.
The observation that both cell shrinkage and cAMP induced increases in
PCK and FBP mRNA levels suggested that the effect of cell shrinkage may
be mediated by cAMP. However, the response of both mRNAs to the two
effectors was synergistic (Fig. 1). Furthermore, the cAMP-dependent
protein kinase inhibitor HA-1004 (50 µM) completely blocked the
inductive effect of cAMP but had only a minor effect on the adaptation
caused by cell shrinkage (Table 4). These
data thus largely rule out an involvement of the cAMP-dependent signal transduction pathway in mediating the inductive effect of cell volume
decrease on PCK and FBP gene expression.
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Table 4.
Effect of various protein kinase activators and inhibitors on the
effect of anisotonicity on levels of PCK and FBP mRNAs
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As both cell swelling and the phorbol ester PMA exhibit a repressive
effect on PCK and FBP mRNA levels, the question arose as to whether the
repressive effect of cell swelling was transduced by a pathway
involving protein kinase C. However, downregulation of protein kinase C
by exposure of H-4-II-E cells to PMA for 24 h did not abolish the
repressive effect of hypotonicity. Furthermore, the protein kinase C
inhibitors chelerythrine (100 nM), staurosporine (25 nM),
bisindolylmaleimide (100 nM), and Gö-6976 (25 nM) were able to
block the PMA effect but not the hypotonicity-induced repression of the
two mRNAs (Table 4).
Pretreatment of H-4-II-E cells with the protein synthesis inhibitor
cycloheximide (CHX) (200 µg/ml) did not abolish the increase in the
levels of PCK or FBP mRNA after hypertonic exposure (405 mosmol/l) (PCK
mRNA, 6.72 ± 0.81 vs. 6.43 ± 0.76 with CHX; FBP mRNA, 5.35 ± 0.94 vs. 5.21 ± 1.23 with CHX). Also, the repressive effect
of hypotonic conditions (205 mosmol/l) on the two mRNAs was unaffected
by pretreatment with CHX (PCK mRNA, 0.33 ± 0.04 vs. 0.32 ± 0.06 with CHX; FBP mRNA, 0.42 ± 0.07 vs. 0.39 ± 0.12 with CHX). Thus
ongoing protein synthesis did not appear to be required for the effects
of anisotonicity on PCK or FBP gene expression in H-4-II-E cells.
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DISCUSSION |
Hepatic carbohydrate metabolism, more specifically gluconeogenesis, is
known to be modulated by liver cell volume changes (1, 15, 24).
However, the precise molecular mechanisms remain to be identified. In
an attempt to provide insight into the putative cellular mechanisms
involved, the effect of anisotonic-induced cell volume changes on gene
expression of the rate-controlling enzymes of the gluconeogenic pathway
was studied in H-4-II-E rat hepatoma cells. The present study shows
that anisotonic exposure of H-4-II-E rat hepatoma cells resulted in
profound changes in PCK and FBP mRNA levels. Hypertonic cell swelling
increased whereas hypotonic cell shrinkage decreased both mRNAs. In
addition, anisotonicity also affected PCK and FBP mRNAs in Hep G2 and
Hep 3B human hepatoma cells and in primary rat and human hepatocytes.
Moreover, hypotonic exposure was able to at least partially counteract
the increased mRNA levels caused by cAMP or dexamethasone in H-4-II-E
hepatoma cells. The repressive effect of hypotonicity by itself was
similar in magnitude to that of insulin or phorbol esters such as PMA. However, the counteracting effect in the presence of cAMP or
dexamethasone was less pronounced. The inductive effect of
hypertonicity was comparable to that of both cAMP and
dexamethasone and when administered simultaneously was synergistic.
The effect of hypertonicity on the mRNAs of the two genes could be
mimicked by exposure to raffinose or sucrose at concentrations yielding
the same osmolarity. When the osmolarity of hypotonic, sodium-chloride
depleted medium was restored to normal by addition of a corresponding
amount of raffinose, the levels of both mRNAs remained unchanged.
Furthermore, increasing medium osmolarity by addition of urea or
glycerol had no effect on PCK and FBP mRNAs. Thus the above-described
effects appeared to be due to changes in liver cell volume.
Furthermore, the inductive effect of cAMP on PCK and FBP gene
expression was in line with the corresponding cell volume
decrease exerted by this hormone (15) and the shrinkage-induced induction of the two genes. Conversely, the repressive effects of
both insulin and hypotonicity on PCK and FBP gene expression correspond
to the cell swelling properties of insulin (1).
The effects of anisotonicity on PCK and FBP mRNAs in hepatoma cells
were osmolarity dependent. Even though changes of ±40 mosmol/l were
already effective, stronger effects appeared with greater osmolarity
changes. It is noteworthy that cell volume changes caused by osmolarity
changes of ±40 mosmol/l are similar in extent to that caused by
several hormones as well as amino acids that are taken up by cumulative
substrate transport (15). At these changes in cell volume, effects on
hepatic glucose release (15) and glycogen metabolism (24) are notable.
The changes in the levels of PCK and FBP mRNAs caused by anisotonicity
were detected after 1 h of cell incubation. This rapid response to
anisotonicity suggests that at least the initial phase was not caused
by modification of mRNA stability. Nuclear run-on data demonstrated
that an increased rate of transcription could account for the initial
increases of both mRNAs on hypertonic exposure. Conversely, hypotonic
conditions decreased the rate of transcription of the two genes.
Surprisingly, the apparent half-lives of PCK and FBP mRNAs were even
slightly, although not significantly, decreased by growth in hypertonic
medium, whereas hypotonic exposure reduced the mRNA half-lives by
~50%. Thus increased PCK and FBP mRNA levels apparently resulted
solely from increased transcription. In contrast, the decrease of both
mRNAs during hypotonic exposure was due to both a decrease in the rate
of transcription and a decrease in mRNA stability.
Changes in PCK and FBP gene expression could be related to
modifications in hepatic glucose metabolism. In this regard,
hepatocytes treated with anisotonic medium show corresponding changes
in gluconeogenesis from different precursors (unpublished data), in
that hypertonic exposure increases and hypotonicity decreases glucose
production. The effect of hypotonicity thus closely mimicked the effect
of insulin, suggesting that insulin-induced cell swelling might
contribute to the well-known repressive effect of insulin on PCK and
FBP gene expression. Furthermore, this concept would be in line with data showing that cell volume affects hepatic glycogen metabolism (24).
A major question arising from this study concerns the mechanism by
which anisotonicity-induced cell volume changes exert their effects on
PCK and FBP gene transcription. Jun has been shown to stimulate
transcription from the PCK promoter in hepatoma cells, an effect which
can be reversed by cotransfection of an expression vector containing
the gene for c-Fos (14). In this context it is of interest to note that
hypotonic conditions increase the mRNA of c-Fos, whereas c-Jun mRNA is
increased under hypertonic exposure in H-4-II-E hepatoma cells (20).
However, de novo protein synthesis is not required for the effects of
anisotonicity on PCK gene expression, thus making the possibility that
at least part of the cell volume-induced changes in mRNA levels may be mediated by changes in the levels of Fos and Jun rather unlikely. It
appears more likely that preexisting transcription factors bind to the
PCK promoter due to an altered phosphorylation state induced by
anisotonicity.
Protein kinase C has been shown to inhibit transcription from the PCK
promoter and exert its effects at the CRE-1 element and to interfere
with the binding and/or stimulation of transcription from the
PCK promoter normally associated with the cAMP regulatory element
binding protein (27). Likewise, okadaic acid (27), vanadate (4), and lithium (5) both decrease the basal rate of PCK gene
transcription and block the positive effect of cAMP on transcription by
interaction with the CRE-1 element. It remains a possibility that
hypotonic-induced cell volume increase mediates its negative effect on
PCK transcription via the same promoter sequence.
The signal transduction mechanism by which the anisotonicity-induced
cell volume changes exert their effect on cellular processes in
hepatocytes is not clear at the moment, whereas osmosensing pathways in
yeast are better understood (6). It is known that cell swelling leads
to a transient increase in intracellular calcium concentration (23), a
hyperpolarization of the cell membrane (10), a decrease in
intracellular pH (13), an increase in potassium conductance (29), and
stimulation of inositol 1,4,5-trisphosphate formation (2). In addition,
participation of the cytoskeleton in cell volume regulation by
hepatocytes has been suggested. However, there are no data at present
relating these mechanisms to the effects of cell volume on hepatic
glucose metabolism and PCK and FBP gene expression. The repressive
effect of hypotonicity on PCK and FBP mRNAs was not inhibited by
protein kinase C inhibitors or prevented by downregulation of protein
kinase C. Furthermore, the inductive effect of hypertonicity could not
be completely blocked by the cAMP-dependent protein kinase inhibitor
HA-1004 at concentrations sufficient to abolish cAMP-mediated
induction. Preliminary experiments showed no difference in
cAMP-dependent protein kinase activity between cells kept in normotonic
(305 mosmol/l) and hypertonic medium (405 mosmol/l). Thus the effects of anisotonicity on PCK and FBP gene expression do not appear to be
mediated by either of these two protein kinases. It is conceivable, however, that changes in cell volume and cell shape perturb the cytoskeletal network, which in turn alters the configuration of signal
transduction systems across the plasma membrane. For example, this
could lead to an agonist-independent activation of certain signal
transduction pathways. Thus it has been demonstrated that osmotic
changes may specifically alter the phosphorylation state of histonelike
proteins (28). From the above results it is not possible to estimate
whether such a mechanism could be operational in the signal
transduction between cell volume and PCK and FBP gene expression. An
alternative explanation could be postulated based on the observation
that the transcriptional activity of certain genes is dependent on the
association of chromatin with the nuclear matrix. Changes in cell
volume and the resulting perturbation of cytoskeletal structures
possibly alter the integrity of the nuclear matrix, which in turn
modifies its interaction with chromatin, resulting in changes in gene
activity. Interestingly, a recent study showed an inductive effect of
H-4-II-E cells grown at high density on PCK mRNA levels (3). It appears
possible that this observation is related to the effects observed in
the present study and would strengthen the concept that the expression
of the PCK gene is coupled to the liver cell shape.
In conclusion, the present study provides evidence in support for a
role of cell volume in regulating PCK and FBP gene expression in the
liver, regardless of whether volume changes are induced by
anisotonicity, nonmetabolizable sugars, or hormones. The mechanism involves changes in transcription rates as well as mRNA stability of
the two genes, is apparently independent of cAMP-dependent protein
kinase and protein kinase C pathways, and does not require ongoing
protein synthesis. The H-4-II-E rat hepatoma cells should provide a
system that will make feasible the further characterization of this
potentially novel type of signal transduction mechanism by which liver
cells react to changes in cell volume and tranduce this to alter the
expression of specific genes.
 |
ACKNOWLEDGEMENTS |
Fruitful discussions and critical reading of the manuscript by Dr.
J. J. Hwang are gratefully acknowledged.
 |
FOOTNOTES |
This work was supported in part by the Deutsche Forschungsgemeinschaft.
Address reprint requests to Medizinische Universitätsklinik, Abt.
I, Otfried-Müller Str. 10, 72076 Tübingen, Germany.
Received 23 July 1996; accepted in final form 3 December 1997.
 |
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