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*
Division of Nutritional Biochemistry, School of Biological Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leics, LE12 5RD, UK and
Department of Molecular Medicine, University of Auckland, School of Medicine and Health Science, Auckland, New Zealand.
2To whom correspondence should be addressed.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • pig hepatocyte • growth hormone-receptor • insulin-like growth factor-I • gene expression • glucose • amino acids
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). Protein or energy deficiency leading to reduced
growth velocity is associated with lowered levels of plasma IGF-I
and is dominant to the positive effects of pituitary GH (Cohick & Clemmons 1993
, Soliman et al. 1986
,
Straus & Takemoto 1990a and 1991
). The dominance of
nutrition over GH was demonstrated in sheep with differing
protein/energy balances, and it was shown that decreased growth
correlates with decreased IGF-I mRNA expression in the liver, but
not in skeletal muscle where no changes occur (Pell et al. 1993
).
In most species (except rats), including pigs (Buonomo & Baile 1991
), nutritional restriction leading to growth arrest is
accompanied by an increase, rather than a decrease, in plasma GH
(Soliman et al. 1986
; Vance et al. 1992
).
This resistance to GH action suggests there are nutritionally sensitive
steps at the level of tissue responsiveness. Hepatic growth
hormone-receptor (GHR) gene expression is decreased by a reduced
energy supply in rats (Straus & Takemoto 1990b
) and pigs
(Dauncey et al. 1994
, Weller et al. 1994
)
and, thus, may partially account for GH resistance in food-deprived
animals. But protein restriction has little effect on GHR expression
(Straus & Takemoto 1990a
). The mechanisms that underlie
the differential regulation of the hepatic GHR gene by energy and
protein are not understood. We have shown that, in pigs, manipulation
of the energy supply alone results in a correlation between growth and
liver, but not longissimus dorsi muscle (LD), IGF-I and GHR
mRNA expression (Weller et al. 1994
). We have also
demonstrated the effects of dietary protein and growth hormone
administration on both IGF-I and GHR mRNA expression in liver,
skeletal muscle and adipose tissue of growing pigs (Brameld et al. 1996
). GH administration increased IGF-I expression in
the liver, all three adipose tissue depots (subcutaneous, perirenal and
omental) and semitendinosus muscle (ST) , but not in LD muscle,
whereas GH increased GHR expression in the liver and muscle, but not in
adipose tissue. Increasing dietary protein intake increased IGF-I
expression only in adipose tissue, whereas it increased GHR expression
in the liver, but decreased it in adipose tissue and muscle. Thus both
tissue- and gene-specific effects were observed. In general,
increasing the growth rate, either by increasing energy availability or
protein intake, is associated with increased GHR expression in the
liver, but decreased GHR expression in muscle and adipose tissue. The
liver is, therefore, a sensor of nutritional and metabolic status in
the animal and a primary site of nutrient-gene interactions.
To identify the mechanism of these observed effects of diet on gene
expression, we have established a primary pig hepatocyte culture system
with serum-free conditions (Brameld et al. 1995
). We
previously demonstrated the stimulatory effects of the synthetic
glucocorticoid dexamethasone (DEX), and the two thyroid hormones,
3,3',5-tri-iodothyronine (T3) and thyroxine, on the expression
of the GHR gene in this culture system (Brameld et al. 1995
). These stimulatory effects on GHR result in an increased
responsiveness of IGF-I expression to GH treatment. GH appears to
be the only positive regulator of IGF-I, increasing the expression
of class 1 transcripts (those transcripts resulting from initiation at
the exon 1 promoter) and initiating the appearance of class 2
transcripts (those resulting from initiation at the exon 2 promoter).
The work described here involved the use of this primary pig hepatocyte
culture system to investigate the possible direct effects of nutrients
(glucose and individual amino acids), in combination with the
stimulatory hormones already mentioned, on the expression of GHR and
IGF-I genes.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Collagenase (type H) was obtained from Boehringer Mannheim (Lewes, East Sussex, UK). Collagen type I; HEPES (sodium salt); EGTA; trypsin inhibitor; bovine serum albumin (BSA); Leibovitz's L15 medium (L15); Hank's balanced salt solution; Dulbecco's modified PBS; penicillin G; streptomycin sulfate; gentamicin sulfate; individual amino acids; porcine insulin; porcine growth hormone; 3,3',5-tri-iodothyronine and dexamethasone were all obtained from Sigma Chemical (Poole, Dorset, UK). Williams' medium E (WE) without any glucose or amino acids was obtained from Imperial Laboratories (Andover, Hampshire, UK). Newborn calf serum (NCS) was obtained from Gibco BRL (Paisley, Renfrewshire, UK). All cell culture plastics were obtained from Becton and Dickenson (Stone, Staffordshire, UK). All other reagents were analytical grade and were obtained from Fisher (Loughborough, Leicestershire, UK).
Porcine hepatocyte isolation and culture.
The animal handling and slaughter procedures met established UK Home
Office guidelines. Pig hepatocytes were isolated following a
modification of the method of Hoogenboom et al. (1989)
, as described
previously (Brameld et al. 1995
), but with a few
modifications. The left lateral lobe of each liver was obtained from
freely fed, 30-d-old, intact male large white x landrace x duroc pigs
(from the University of Nottingham Piggery) following conventional
slaughter, and transported to the laboratory in warm buffer I (8.3 g
NaCl/L, 0.5 g KCl/L, 2.6 g HEPES/L, 0.19 g EGTA/L; pH
7.4). Hepatocytes were isolated by the three-step perfusion
technique, whereby two major blood vessels were connected to an
oxygenator, perfused with 1 L of buffer I, followed by 1 L of buffer II
(buffer I minus the EGTA), and finally 300 mL of buffer III (3.9 g
NaCl/L, 0.5 g KCl/L, 2.6 g HEPES/L, 0.70 g
CaCl2·2H2O/L; pH 7.4) containing 0.25 g
collagenase/L (type H) and 0.25 g trypsin inhibitor/L, which was
recirculated for ~10–15 min. During the perfusion, all buffers were
gassed with 95% O2/5% CO2
("carbogen"—Air Products, Wednesbury, West Midlands, UK).
Following perfusion, the liver was minced with scissors and then filtered through a 250-µm and then a 100-µm nylon mesh (Lockertex, Warrington, Cheshire, UK). Cells were collected by two centrifugations at 50 x g and 4°C for 3 min in HEPES-buffered Hank's balanced salt solution, followed by a wash (and centrifugation) with HEPES-buffered L15 medium containing 2 g BSA/L (L15-BSA). Finally the cells were resuspended in L15-BSA, filtered through a second 100-µm nylon mesh and the viability and cell number calculated via the trypan blue exclusion test. Cell viability ranged from 90 to 98% in the studies described here, with yields averaging 4 x 109 cells.
Monolayer cultures were obtained by plating 3.5 x 107
viable cells in 8 mL HEPES-buffered L15 medium, containing 10%
(v/v) NCS, 5g glucose/L and 100 nmol insulin/L, onto 100-mm, collagen
coated cell culture dishes. The cultures were then maintained at 37°C
and 100% humidity. All cell culture media contained 1 x
105 U penicillin G/L, 0.1g streptomycin sulfate/L and
0.05 g gentamicin sulfate/L. The medium was changed after 2 h
to remove nonviable cells, and then again after another 3 h, at
which stage the medium was changed to HEPES-buffered WE
supplemented with 2 g BSA/L and 100 nmol insulin/L (basal medium),
with or without the various nutrients (pre-incubation step—see Results
section). The basal medium was changed 16 h later, at which stage
cells were maintained on basal medium or in media with differing
concentrations of nutrients (glucose or individual amino acids) in the
presence or absence of T3 (10 nmol/L), DEX (100 nmol/L) and/or GH (1
mg/L). Total RNA was then isolated from the cells 24 h later. This
involved washing with PBS followed by lysing with denaturing solution.
Four plates were pooled together for each treatment, and then frozen at
-20°C prior to extracting the total RNA. In the initial experiments
on the effects of glucose, total RNA was also isolated after 48 h
of treatment, with the medium being refreshed after 24 h. All
glucose experiments were carried out with normal (WE, Table 1
) concentrations of amino acids, and all amino acid experiments were
carried out with glucose present at 5g/L.
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Total RNA was prepared by using the guanidine thiocyanate method
(Chomczynski & Sacchi 1987
) and quantified by measuring
the absorbance at 260 nm. The purity, integrity and equal loading of
total RNA was routinely monitored by agarose gel electrophoresis and
ethidium bromide staining of the two ribosomal RNA bands.
RNase protection assays were performed on 100 µg of total RNA samples
following the methods described previously (Saunders et al. 1991
, Weller et al. 1993 and 1994
). Two
radiolabeled antisense riboprobes were employed, corresponding to the
intra-cellular domain of the pig GHR gene (Weller et al. 1994
), and to class 2 transcripts of the pig IGF-I gene
(exon 3 linked to the exon 2 promoter). The IGF-I riboprobe was
designed so that when hybridized to total RNA, two bands were obtained
corresponding to the homologous mRNA transcript class (class 2
transcripts) as well as to any other IGF-I mRNA transcript class
(class 1 transcripts), which hybridized to the region of the probe
corresponding to exon 3 (Weller et al. 1993 and 1994
).
The relative intensities of the protected bands on the X-ray film
(X-OMAT AR, Kodak, Cambridge, UK) were assessed by image analysis
(Seescan, Cambridge, UK).
Statistical analyses.
At least three pigs were used to provide replication for each
experiment. Cells isolated from each pig were sub divided into groups
to which treatments were randomly applied, with all treatments applied
to cells from each pig. The results of relative optical density,
obtained from the image analysis, were subjected to ANOVA by using the
Genstat 5 for Windows (release 3.2) Statistical Package (Lawes
Agricultural Trust, Rothamsted, Hertfordshire, UK). In the glucose
experiment, the effects of glucose, GH and T3/DEX and their various
interactions were analyzed by three-way ANOVA, with the data
blocked for pig. The dose-response effects of glucose were
similarly analyzed with linear and quadratic trends fitted to the data
for the four glucose concentrations. Removal of individual amino acids
was carried out in three batches of amino acids, each batch with cells
from four pigs to give four replicates for each treatment within a
batch. The effect of amino acid within batches was analyzed by ANOVA,
with the data blocked for the 12 pigs. Means were then compared to
control values within a batch by Dunnett's test (Dunnett, 1955
). The dose-response effects of amino acids were
analyzed in the same way as for glucose, with linear and quadratic
trends fitted to the data for the five amino acid concentrations
(including control as 1x the amino acid). Differences of
P < 0.05 were considered significant, whereas
differences of P < 0.10 were considered as tending
to be significant.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The age of pigs used for the isolation of primary hepatocytes in these
studies differs from our previous studies of the effects of hormones
(Brameld et al. 1995
). We found that the use of younger
pigs allowed for a greater degree of collagenase digestion and,
therefore, an increase in the number of cells isolated (data not
shown). Cell viability was also increased when compared to greater
digestion of the liver from older pigs, which always resulted in
decreased cell viability. The difference in age of the pigs used for
hepatocyte isolation did not affect the stimulatory effects of DEX, T3
and GH on the expression of GHR and IGF-I genes (data not shown).
Thus, the use of younger pigs resulted in greater yields and viability
of isolated hepatocytes and allowed for increased treatment numbers
within an experiment.
Effects of glucose on expression of GHR and IGF-I mRNA.
Initial studies comparing the effects of culturing pig hepatocytes in
normal WE medium (2 g glucose/L) with glucose-supplemented WE (5 g
glucose/L) showed no effect on the expression of either GHR or
IGF-I mRNA in the presence or absence of T3, DEX and/or GH (data
not shown). Similarly, initial studies of the effect of removing
glucose from the culture medium also showed no effect on GHR and
IGF-I expression after 24 h, but there was an effect on GHR
expression when the glucose was removed for 48 h (Fig. 1
), with the medium renewed after 24 h. It, therefore, became
apparent that if the glucose was removed from the medium at the same
time as switching to serum-free medium, and pre-incubated for
16 h prior to the addition of various hormones with or without
glucose, then there was an effect of glucose and the hormones 24 h
later (i.e. a total of 40 h without glucose). As expected, the
combination of T3 and DEX increased GHR expression (P
< 0.001, Table 2
), with a further small increase when GH was added as well (P
= 0.109 for the interaction, Table 2
). The removal of glucose from
the culture medium for 40 h resulted in significant reductions
(P = 0.013, Table 2
) in GHR expression even in the
presence of T3, DEX and GH. There was a significant interaction between
T3/DEX and GH in stimulating the expression of both classes of
IGF-I transcript (P = 0.032 and 0.046 for class 1
and 2, respectively, Table 2
). The removal of glucose resulted in a
significant decrease in class 1 transcripts of IGF-I (P
= 0.011, Table 2
), with a similar effect on class 2 transcripts
(P = 0.068, Table 2
). Thus, the decrease in GHR
expression with removal of glucose from the culture medium resulted in
a decreased response in IGF-I expression to any GH added. The
length of time without glucose necessary for an effect on GHR and
IGF-I expression was thought to reflect the need for the cells to
be depleted of their stored glucose (glycogen) before any effect of
culture medium glucose concentration was observed.
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,
3and
4). The expression of GHR increased with increasing glucose concentration
in all combinations of hormones (P = 0.015 and 0.031
for linear and quadratic trends, respectively), appearing to
reach a plateau at around 1–2 g glucose/L (Fig. 2)
. As before, class 1
transcripts of IGF-I were only observed when GH was added
(P < 0.001, Fig. 3
), with the highest levels detected
when T3, DEX and GH were added together (P = 0.001 for
the interaction, Fig. 3
). Similarly, class 2 transcripts of IGF-I
were only observed when all three hormones were added together
(P = 0.008 for the interaction, Fig. 4
). Increasing
glucose concentrations resulted in increased expression of both classes
of IGF-I transcript, although only class 1 transcripts were
significantly affected (P = 0.007 and 0.145 for linear
trends for class 1 and 2 transcripts, respectively, Figs. 3
and 4
).
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The effects of individually removing all 21 amino acids present in the
culture medium (Table 1)
on the expression of GHR and IGF-I mRNA
were also studied, with the amino acids being removed for the same time
periods (i.e. a total of 40 h) as was necessary to detect the
effects of glucose. However, it should be noted that this time period
may not be optimal. Initial studies in the presence or absence of T3,
DEX and GH indicated that any effect was apparent only when all three
hormones were included in the culture medium (data not shown). As
described in the Materials and Methods section, removing individual
amino acids was carried out in three batches, and the effects were
analyzed statistically by the effect of amino acid within a
batch. In batch 1, removal of tryptophan from the culture
medium inhibited the increase of class 1 transcripts of IGF-I,
which were induced by the inclusion of the three hormones, to ~9%
(P < 0.05) of the control medium (WE plus T3, DEX and
GH) (batch 1, Table 3
). The removal of tryptophan also reduced GHR expression, as did the
removal of lysine or phenylalanine (P < 0.05, batch 1,
Table 3
). Batch 2 demonstrated reductions in the expression of both
classes of IGF-I transcript when arginine was removed (P
< 0.01, batch 2, Table 3
), but with no effect on GHR expression.
The removal of proline, threonine or valine (or tryptophan, again) in
batch 3 reduced the expression of class 1 transcripts of IGF-I mRNA
(P < 0.05, batch 3, Table 3
), with removal of proline
or threonine also reducing GHR mRNA. Thus, the removal of arginine,
proline, threonine, tryptophan or valine appears to inhibit the
stimulation of IGF-I expression induced by the combination of T3,
DEX and GH. There are, in some cases, decreases in GHR expression that
accompany the decrease in IGF-I expression.
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), but had no effect on the expression of class 2 transcripts of
IGF-I nor on GHR (P > 0.10 for all linear and
quadratic trends, Fig. 5
). There was no dose-responsive effect of
either leucine or lysine on the expression of either IGF-I or GHR
mRNA (P > 0.10 for all linear and quadratic trends,
Fig. 5
). Significant dose-response effects of proline and threonine
were observed for the expression of class 1 transcripts of IGF-I
(P = 0.022 and 0.016, respectively, for linear trends,
Fig. 6
), with the dose-response effect of tryptophan tending towards
significance (P = 0.097 for linear trend, Fig. 6
), and
valine not being significant (P = 0.248 for quadratic
trend, Fig. 6
). There was no significant dose-response effect of
any of these amino acids on the expression of GHR or class 2
transcripts of IGF-I (P > 0.10 for all linear and
quadratic trends, Fig. 6
).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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In this study, with the use of younger pigs, there was a greater degree
of collagenase digestion and increased number and viability of cells
isolated agreeing with a recent publication that showed a decrease in
both number and viability of pig hepatocytes with increased pig
bodyweight (Gerlach et al. 1996
). We found no difference
with age on the stimulatory effects of DEX, T3 and GH on the expression
of GHR and IGF-I genes (data not shown). Thus, the use of younger
pigs results in greater yields and viability of isolated hepatocytes
and allows for greater treatment numbers within an experiment.
Effects of glucose on expression of GHR and IGF-I mRNA.
We found direct effects of culture medium glucose concentration on GHR
mRNA, independent of the effects of insulin, thyroid hormones,
glucocorticoids or GH. Thus, increasing glucose concentrations resulted
in an increase in GHR mRNA both in the presence or absence of the
hormones known to stimulate expression, appearing to reach a plateau at
around 1–2 g glucose/L. This demonstrates why in this study, when
supplementing normal WE medium with extra glucose, there was no effect
on GHR expression because these concentrations (2 and 5 g/L) are at the
plateau for the effect of glucose. A previous study demonstrated
stimulatory effects of 5 compared to 2g glucose/L on GHR-binding
(Niimi et al. 1991
), and we observed previously that the
decline in GHR expression in culture with time was less when high
levels (5 compared to 2 g/L) of glucose were included in the medium
(Brameld et al. 1995
). The range of glucose
concentrations shown to have effects on GHR expression are
physiologically relevant because peripheral blood glucose
concentrations in similar pigs are normally around 1g/L (Brameld et al. 1996
), and plasma glucose concentrations in hepatic
portal veins vary from 0.9 to 2.16 g/L (Van Der Meulen et al. 1997
). However, the work described here would suggest a need
for hepatocytes to be depleted of their stored glucose (glycogen)
before an effect on GHR and IGF-I expression is observed. Thus,
further studies including concentrations between 0 and 1 g/L may be
more physiologically relevant to the food-deprived state and would
allow apparent Km values to be
calculated. As a result of the stimulatory effects of glucose on GHR
mRNA, increasing the glucose concentration resulted in an increase in
IGF-I mRNA when GH was added. Thus, class 1 transcripts of
IGF-I increased with glucose concentration either when GH was added
alone or when a combination of T3, DEX and GH was added. Class 2
transcripts also increased with glucose concentration, but were only
present when a combination of T3, DEX and GH was added.
A number of other genes expressed by hepatocytes were also shown to be
regulated by glucose, with many of them also controlled by insulin, DEX
and T3. Firstly, the expression of IGF-binding protein-1 (IGFBP-1)
mRNA by cultured rat hepatocytes was shown to decrease with increased
glucose concentration (Arany et al. 1993
). The same
study showed an interaction between glucose and insulin on the
secretion of IGFBP-1 (Arany et al. 1993
), such that the
inhibitory effects of insulin on IGFBP-1 secretion diminished with
increasing glucose concentration. Genes whose expression was shown to
be positively regulated by glucose include fatty acid synthase
(Foufelle et al. 1995
, Hillgartner & Charron 1998
, Prip-Buus et al. 1995
), L-pyruvate
kinase (Kang et al. 1996
, LeFrancois-Martinez et al. 1994
), glucose-transporter type 2 (Rencurel et al. 1996
, Zheng et al. 1995
) and S14
(Jacoby et al. 1989
, Mariash et al. 1986
). In many of these cases, the effects of glucose
were shown to be effects on gene transcription (Hillgartner & Charron 1998
), with the promoter regions of the DNA involved
having been identified (Foufelle et al. 1995
,
Jacoby et al. 1989
, LeFrancois-Martinez et al. 1994
, Rencurel et al. 1996
, Shih & Towle 1994
). Indeed, a consensus carbohydrate/glucose
response element has been identified (see Vaulont & Kahn 1994
).
Whether the effects of glucose on GHR mRNA described here are
transcriptional or post-transcriptional effects remains to be
established, as does the question of whether a glucose-response
element is present on one or more promoters of the GHR gene. The fact
that GHR mRNA appears to be controlled by the same hormones as these
other glucose-responsive genes suggests that it is likely to be a
similar transcriptional effect. However, the identification of the
structure of the GHR gene in all species is still at a very early
stage. Studies of the GHR gene were carried out both in humans
(Pekhletsky et al. 1992
) and in sheep. The sheep studies
have identified a liver-specific promoter (O'Mahoney et al. 1994
) and also a muscle-derived, but ubiquitously
expressed, promoter (Adams 1995
) for the ovine GHR gene.
However, the total number of promoters for the GHR gene has yet to be
established for any species. There could be as many as seven because
seven distinct 5'-untranslated regions were identified from human GHR
cDNA derived from hepatic poly(A)+ RNA (Pekhletsky et al. 1992
). Thus, identification of the promoter responsible for the
effects of glucose on porcine GHR expression may take some time and is
likely to involve a process of elimination. However, the possibility
that the effects of glucose on GHR mRNA described here could be due to
differences in mRNA stability should not be ruled out. A recent study
identified a glucose-inducible human fatty acid synthase
mRNA-binding protein (Li et al. 1998
), which
stabilizes the mRNA and therefore increases its half-life.
Effects of amino acids on expression of GHR and IGF-I mRNA.
Direct effects of certain individual amino acids on the expression of IGF-I mRNA (both class 1 and 2 transcripts), with little or no effect on GHR mRNA, were observed. The removal of arginine, proline, threonine, tryptophan or valine inhibits the stimulation of IGF-I mRNA induced by the combination of T3, DEX and GH. The effect of some of these amino acids (arginine, proline, threonine and tryptophan) was dose dependent, with only very low concentrations of valine or lysine appearing to be necessary for maximum expression of IGF-I.
An effect of an interaction between amino acid concentrations
and GH on IGF-I mRNA in rat hepatocytes, such that increasing the
concentration of all amino acids from 0.2 times the normal rat plasma
concentration to 1 and 5 times, in the presence of rat GH, resulted in
an amino acid concentration-dependent increase in IGF-I mRNA,
was previously described (Thissen et al. 1994
). The same
study demonstrated an inhibiting effect of increasing amino acid
concentration on IGFBP-1 mRNA. The stimulatory effect of total amino
acids on IGF-I mRNA was associated with increased gene
transcription (Pao et al. 1993
), whereas the inhibitory
effect of amino acids on IGFBP-1 mRNA involved no change in gene
transcription. Studies on the secretion of IGF-I from rat
hepatocytes have shown conflicting results. One study (Phillips et al. 1991
) demonstrated that IGF-I secretion increased
with increasing concentrations of amino acids, whereas another study
(Arany et al. 1993
) showed the opposite (decreased IGF-I
secretion with increasing amino acid concentrations). Harp et al. (1991)
showed decreases in both IGF-I mRNA and IGF-I secretion
with the removal of tryptophan from the medium, with the removal of
lysine having a similar effect on IGF-I secretion. However, the
possible effects on IGF-I mRNA of removing lysine from the culture
medium were not reported. An amino acid-responsive element has
recently been identified in the rat IGF-I gene by using primary rat
hepatocyte transfection studies (Huang & Phillips, 1996
), with expression of this amino acid-responsive
element shown to be particularly sensitive to the availability of
tryptophan.
Removal of individual amino acids from the culture medium was shown to
affect the expression of other genes, both in primary hepatocytes and
also in hepatocyte-derived cell lines. Removal of histidine from
the culture medium decreased both protein synthesis and albumin mRNA
expression in rat hepatocytes (Kimball et al. 1996
).
Similarly, removal of certain amino acids from the culture medium
decreased the expression of fatty acid synthase mRNA in human HepG2
cells (Dudek & Semenkovich 1995
), but increased the
expression of IGFBP-1 mRNA in rat hepatoma cells (Straus et al. 1993
).
Whole animal studies have previously shown effects of dietary protein
on the expression of a number of genes in the liver. Hepatic IGF-I
mRNA content in rats was shown to increase with both the quantity and
nutritional quality of dietary proteins (Miura et al. 1992
), whereas IGFBP-1 mRNA increased in the liver of
protein-restricted rats (Straus et al. 1993
).
Similarly, the hepatic expression of histidase (Torres et al. 1998
) and albumin mRNA in rats (Ogawa et al. 1997
; Oka et al. 1997
) increased with increased
dietary protein or amino acid supply. In the case of albumin,
branched-chain amino acids appear to elicit the effect
(Kuwahata et al. 1998
). Dietary protein restriction or
amino acid supply also affects the quantity or binding activity of a
number of liver-enriched transcription factors (Marten et
al. 1997
, Oka et al. 1997
), including hepatocyte
nuclear factors (HNF)-1, -3 and -4, CCAAT/enhancer-binding
proteins (C/EBP)
and ß and liver-enriched transcriptional
inhibitory protein, as well as the ubiquitous transcription factor Sp1.
Removal of certain amino acids from the culture medium induced the
expression of CHOP (a C/EBP-related gene) by HeLa, HepG2 and Caco-2
cell lines, at both transcriptional and post-transcriptional levels
(Bruhat et al. 1997
). It was also noted that removal of
glucose also increased transcription and that the effect of glucose was
not additive with that of leucine. Thus, a number of transcription
factors appear to be affected by dietary protein/amino acid
availability, but whether these are involved in the observed effects of
amino acids on GH-stimulated IGF-I expression remains to be
established. The fact that IGF-I transcription was shown to be
stimulated by HNF-3ß (Nolten et al. 1996
), Sp1
(Wang et al. 1998
), C/EBP and ß (also called
liver-enriched activating protein) (Nolten et al. 1994
) seems to indicate a possible mechanism for how amino
acids can influence IGF-I gene transcription.
In summary, energy, in the form of glucose, appears to control GHR expression, interacting with the effects of glucocorticoids and thyroid hormones, whereas protein, in the form of certain individual amino acids, appears to control GH-stimulated IGF-I expression. However, the question of whether the observed effects of nutrients on mRNA levels are due to direct effects on gene transcription or differences in mRNA stability remains to be answered.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BSA, bovine serum albumin; C/EBP,
CCAAT/enhancer-binding proteins; DEX, dexamethasone; GH, growth
hormone; GHR, growth hormone receptor; HNF, hepatocyte nuclear factors;
IGF-I, insulin-like growth factor-I; IGFBP-1, IGF-binding
protein-1; L15, Leibovitz's L15 medium; LD, longissimus dorsi skeletal
muscle; NCS, newborn calf serum; OD, optical densities; ST,
semitendinosus skeletal muscle; T3, tri-iodothyronine; WE, Williams'
medium E.
Manuscript received August 5, 1998. Initial review completed January 27, 1999. Revision accepted March 26, 1999.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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