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Department of Physiology, Göteborg University, S-405 30 Göteborg, Sweden
Address all correspondence and requests for reprints to: Dr. Jan Oscarsson, Department of Physiology, Göteborg University, Box 434, Medicinaregatan 1F, S-405 30 Göteborg, Sweden. E-mail: jan.oscarsson{at}fysiologi.gu.se
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Liver fatty acid-binding protein (LFABP) belongs to a family of abundant cytosolic small proteins (14–15 kDa), which includes intestinal, ileal, heart, myelin, and adipocyte FABP as well as retinol- and retinoic acid-binding proteins. These FABPs, except for the last two proteins, are characterized by binding of long chain fatty acids and their coenzyme A esters (17, 18, 19, 20, 21). In contrast to the other members of the FABP family, LFABP binds 2 mol fatty acid/mol protein (22). The fatty acids bound to LFABP are preferentially long chain, unsaturated fatty acids. Moreover, LFABP has been shown to bind heme and a number of eicosanoids with high affinity and to bind a large number of other amphipathic ligands with lower affinity, such as bilirubin, bile salts, lysophosphatidylcholine, steroids, and their metabolites (17, 20, 21). Several enzyme activities, especially those involved in microsomal fatty acid metabolism, were stimulated by LFABP (17). LFABP may play a role as an intracellular acceptor of fatty acids, thereby enhancing fatty acid uptake and facilitating intracellular transport (17, 23, 24, 25, 26, 27).
Rat LFABP complementary DNA (cDNA) was cloned by Gordon and co-workers (28), and the complete nucleotide sequence of the gene was found to contain four exons and three introns (17, 18). LFABP mRNA levels in the liver are developmentally regulated, with a marked increase during the first 24 h after birth and during puberty in the rat (29, 30), whereas LFABP protein levels increase more gradually (31, 32). There is no or very small diurnal variation in LFABP levels (17, 32). LFABP expression is induced by a high fat diet and peroxisome proliferators, such as bezafibrate and clofibric acid (17). LFABP expression has also been shown to be influenced by hormones, but only in vivo. Female rats have higher levels than male rats (17, 26, 27), and this difference was reversed by a combination of gonadectomy and administration of gonadal steroids (26). Other studies on the hormonal regulation of LFABP have relied on measurements of the binding capacity of the cytosol fraction containing LFABP (33, 34, 35). GH has been shown to increase the expression of LFABP mRNA in Hx male rats (30), and the binding capacity of the cytosol fraction containing LFABP was increased in Hx rats given GH compared with that in Hx control rats (35).
In the present study the effects of bovine GH (bGH), and other hormones on the expression of LFABP and its mRNA were studied in vivo in Hx rats. Cytosolic LFABP concentrations were measured with an antibody sandwich enzyme-linked immunosorbent assay (ELISA). Primary cultures of hepatocytes were used to study the direct effect of bGH and other hormones on the expression of LFABP mRNA. Insulin-like growth factor I (IGF-I) mRNA levels were measured as an indication of GH responsiveness and the characteristics of GH regulation of LFABP mRNA. Rat hepatocytes were cultured on a basement membrane matrix (Matrigel) derived from extraction of the Engelbreth-Holm-Swarm sarcoma (36). This system, which allows serum-free conditions, has been used to study in vitro effects of GH on cytochrome P-450 enzymes and IGF-I mRNA (37). Moreover, LFABP mRNA and LFABP expressions have been shown to be induced by peroxisome proliferators when hepatocytes were cultured on Matrigel, but not when they were maintained on collagen-coated plates (38).
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hepatocyte cultures and hormones used in vitro
Hepatocytes were prepared by a nonrecirculating collagenase
perfusion through vena porta of 200- to 300-g female Sprague-Dawley
rats essentially as previously described (14). The rats were
anesthetized by a combination of xylazine (9 mg/kg; Rompun, Bayer,
Lever-Kusen, Germany) and ketamine hydrochloride (77 mg/kg; Ketalar,
Parke-Davis, Detroit, MI). The perfusion started with Hanks’ Balanced
Salt Solution without calcium and magnesium, pH 7.4 (Life Technologies,
Paisley, Scotland) supplemented with 0.6 mM EGTA, 20
mM HEPES, and 10 mM sodium hydrogen carbonate.
This solution was followed by perfusion with Williams’ E medium with
Glutamax (catalogue no. 32551, Life Technologies) supplemented with
penicillin (50,000 IU/liter), streptomycin (50 mg/liter; Life
Technologies), 0.28 mM sodium ascorbate (Sigma Chemical
Co., St. Louis, MO), 0.1 µM sodium selenite (Sigma
Chemical Co.), and 300–400 mg/liter collagenase A (Boehringer
Mannheim, Mannheim, Germany), or 400–500 mg/liter collagenase type IV
(Sigma Chemical Co.). The first medium was given for 5–6 min at a flow
rate of 40–50 ml/min, and the second medium was given for 7–9 min at
a flow rate of 40 ml/min. The perfusion media were kept at 37 C and
infused continuously with 95% oxygen and 5% carbon dioxide. After the
perfusion, the cells were filtered through a 250-µm pore size mesh
nylon filter followed by a 100-µm pore size mesh nylon filter. The
cells were washed by centrifugation at 50 x g three
times for 1 min each time at 4 C in Williams’ E medium with Glutamax
supplemented as described above (except for collagenase), but also
supplemented with 3 g glucose/liter and insulin (16 nM
= 
91 µg/liter = 2600 mU/liter; Actrapid, Novo Nordisk;
28.6 U/mg). The cells were counted in a Burker chamber. The viability
was about 90%, as determined by trypan blue exclusion at the start of
the experiment and about 98–100% at the end of the experiments. The
cells were seeded at a density of approximately 170,000
cells/cm2 in 10 ml medium on plastic dishes (56.7
cm2; Nunclon, Nalge Nunc International, Copenhagen,
Denmark) coated with 500 µl Matrigel (Collaborative Research, Medical
Products, Bedford, MA). The cells were plated during the first 16–18 h
in the same medium as that used for washing the cells. After 16–18 h
of culture, the cells were cultured in this medium but with different
doses of insulin (Actrapid, Novo Nordisk), bGH (American Cyanamide
Co.), or glucagon (Sigma Chemical Co.). Actinomycin D (5 µg/ml; Sigma
Chemical Co.) was dissolved in dimethylsulfoxide (DMSO). When
actinomycin D was used, all culture dishes were cultured in medium
containing 0.15% DMSO. The total culture time was 4–5 days in all
experiments. The medium was changed every day.
Production of antiserum
Rabbits (Russian, Mollegaard Breeding Center) were immunized
with purified recombinant LFABP, which was a gift from Dr. D. Cistola,
Washington University School of Medicine (St. Louis, MO). One hundred
micrograms of LFABP were diluted in saline, mixed with equal volumes of
Freund’s complete or incomplete adjuvant, and injected im. Blood was
collected from the ear vein (20–30 ml every other week). The serum was
shown to contain anti-LFABP antibodies by double immunodiffusion and
Western blot. The IgG fraction was isolated by HiTrap protein G
affinity columns according to the manufacturer (Pharmacia Biotech,
Uppsala Sweden).
Cytosol preparation
Cytosol was prepared from rat liver. The tissue was homogenized
in 2 ml homogenization buffer/g tissue according to the method of
Ockner et al. (27) containing protease inhibitors (Complete
Protease inhibitor cocktail tablets, Boehringer Mannheim). The
homogenate was centrifuged for 20 min at 12,000 x g.
The supernatant was centrifuged for 1 h at 105,000 x
g. The total protein concentration of liver cytosol was
determined by the method of Lowry et al. (42). The cytosol
was stored at -70 C until assay.
Western blot
Two micrograms of cytosol protein were added to each lane
in a buffer containing 62.5 mM Tris-HCl (pH 6.8), 2.3%
SDS, 10% glycerol, 0.001% bromophenol blue, and 5%
ß-mercaptoethanol. The samples were run overnight in 15%
polyacrylamide gels containing SDS. The molecular mass standard See
Blue (Novex, San Diego, CA) was used. The proteins were transferred to
a polyvinyldifluoride membrane (Millipore) by semidry blotting
(Multiphore II, Pharmacia). The membrane was then incubated with LFABP
antiserum (1:200). Immunoreactive protein was visualized by
chemiluminescence using an alkaline phosphatase-conjugated second
antibody and AMPPD (disodium
3-(4-methoxyspiro(1,2-dioxetane-3,2’-tricyclo-[3.3.1.13,7]decan)-4-yl)phenyl
phosphate) as substrate (Tropix, Bedford, MA). The filters were exposed
to ECL film (Amersham, Aylesbury, UK) at room temperature for 5–15
sec, and the films were subsequently developed.
ELISA
An antibody sandwich ELISA was developed to measure soluble
LFABP in liver cytosol. The purified IgG fraction of the antirat LFABP
antiserum was used. Biotinylation of the IgG fraction was performed
using the Protein Biotinylation System (Life Technologies,
Gaithersburg, MD). The ELISA was standardized using recombinant LFABP
dissolved in rat heart cytosol. The protein concentration of
recombinant LFABP was determined according to the method of Lowry
et al. (42). Rat heart cytosol was used as a carrier because
it contains no or very low levels of LFABP. About 1–3 ng/µl or 1
ng/µg of a protein that cross-reacted with the antibody in heart
cytosol were detected. The standard liver cytosol was obtained from a
female rat (400 g) and contained 351 ng LFABP/µl or 63 ng LFABP/µg
protein. The internal standard was obtained from a female rat (200 g)
and contained 54 ng LFABP/µg protein.
Microtiter plates (Costar, Cambridge, MA) were coated with the IgG fraction of the antiserum at a concentration of 10 µg/ml in 50 µl PBS with 0.05% sodium azide. The plates were incubated at room temperature overnight or at 4 C for at least 2 days before use. The plates were rinsed with distilled water and incubated with 200 µl blocking buffer (PBS with 0.05% Tween-20 and 0.25% BSA, pH 7.2) for 30 min at room temperature and then rinsed with distilled water. The samples were added to the plate in a volume of 50 µl diluted in blocking buffer and incubated for 2 h at room temperature. The plates were washed with distilled water, incubated with 200 µl blocking buffer for 10 min, rinsed again before 50 µl secondary antibody was added (biotinylated IgG, 400 ng/ml dissolved in PBS with 0.25% BSA, pH 7.2), and incubated for another 2 h at room temperature. The washing procedure was repeated, and streptavidin-alkaline phosphatase (Life Technologies) was added at a 1:8000 dilution in PBS with 0.25% BSA, pH 7.2, and incubated for 1.5 h. The plates were washed again and incubated with pNPP (Life Technologies) according to the manufacturer’s directions. After 1 h, the plates were analyzed at 405 nm using a microtiter plate reader (model 450, Bio-Rad, Richmond, CA). The detection limit of the assay was 0.15 ng/µl, or 7.5 ng absolute. The intraassay coefficient of variation (CV) was 5%. The interassay CV was 17% when the plates were assayed on different days, and the CV between plates assayed on the same day was 8%.
Probe synthesis
LFABP. The pJG418 plasmid containing a 515-bp insert in the
pBR 322 (28) was a gift from Prof. J. Gordon, Washington University
School of Medicine. The fragment obtained by cleavage of the 515-bp
insert with PvuII and EcoRI (336 bp) was recloned
into the pSP72 vector (Promega, Madison, WI). This new insert was used
for the synthesis of a [32P]CTP cDNA probe (Megaprime,
Amersham) for Northern blots. For solution hybridization assay, the
pSP72 vector was linearized with PvuII, and a
[35S]UTP complementary RNA probe (antisense) was
generated with T7 RNA polymerase (standard transcription protocol,
Promega). The standard (sense) used in the solution hybridization assay
was generated with SP6 RNA polymerase (Promega) using the same vector,
linearized with EcoRI.
IGF-I. A pSP64 vector (Promega) with a 153-bp genomic subclone of mouse IGF-I corresponding to exon 3 (by analogy to human IGF-I) was used (43). The structure of this probe would allow detection of both forms of IGF-I mRNA. The hybridization signal in the solution hybridization assay was compared with that of a synthetic mRNA standard using an another construct in which the 153-bp fragment was inserted in the same vector in the opposite direction (44).
Northern blot
Total RNA was prepared according to the method of Chomczynski
and Sacchi 1987 (45). Twenty micrograms of RNA were electrophoresed in
an agarose (1%)-formaldehyde (0.66 M) gel. The RNA was
transferred to a membrane (Hybond-N, Amersham) with a vacuum transfer
system (LKB, Stockholm, Sweden) and baked at 80 C for 3 h. The
membranes were prehybridized for 4 h at 42 C in a buffer
containing 50% formamide, 25 mM
HNa2PO4, 25 mM
H2NaPO4, 5 x SSC (standard citrate
solution), 0.1% SDS, 1 mM EDTA, 0.05% BSA, 0.05% Ficoll,
0.05% polyvinylpyrrolidone, 200 µg/ml calf liver RNA, and 200
µg/ml salmon sperm DNA and hybridized for 12–14 h at 42 C in the
prehybridization buffer, with the addition of a 32P-labeled
LFABP cDNA probe. Filters were washed once in 2 x SSC-0.2% SDS
at 42 C for 30 min and once 0.1 x SSC-0.2% SDS at 42 C for 30
min. Autoradiography was performed at -70 C using Fuji medical x-ray
film (Fuji Photo Film Co., Tokyo, Japan).
Solution hybridization
Total nucleic acids were prepared according to the method of
Durnam and Palmiter (46), and RNA was prepared according to the method
of Chomczynski and Sacchi (45) from frozen liver and hepatocytes. The
DNA content in the samples was analyzed as described by Labarca and
Paigen (47), and the RNA content was determined spectrophotometrically
at 260 nm.
Aliquots of the prepared total nucleic acids or RNA samples were mixed with hybridization solution containing the 35S-labeled LFABP or IGF-I complementary RNA probes, 0.6 M NaCl, 22 mM Tris-HCl (pH 7.5), 5 mM EDTA, 0.1% (wt/vol) SDS, 0.75 mM dithiothreitol, 7.5% (wt/vol) transfer RNA, and 25% (vol/vol) formamide in a total incubation volume of 40 µl and hybridized at 70 C overnight (46). The samples were then treated with 40 µg ribonuclease A (RNase A) and 2 µg RNase T1 in the presence of 100 µg herring sperm DNA for 45 min at 37 C in a volume of 1 ml. Protected probe was precipitated with 100 µl 6 M trichloroacetic acid (TCA). The precipitate was collected on glass-fiber filters (GF/C, Whatman International, Maidstone, UK) and counted in a scintillation counter. The signal was compared with a standard curve obtained by hybridization of in vitro transcribed LFABP mRNA or IGF-I mRNA. The intraassay CVs calculated from duplicates were 9% for the LFABP mRNA measurements and 14% for the IGF-I mRNA measurements. The results are expressed as attamoles of LFABP or IGF-I mRNA per µg DNA or RNA.
Other methods
Serum insulin concentrations were determined by RIA (Phedabas,
Pharmacia), and serum glucose concentrations were determined by the
glucose-6-phosphate dehydrogenase method (Merck, Darmstadt, Germany)
(40).
Statistics
Values are expressed as the mean ± SEM.
Comparisons between groups were made using one- or two-way ANOVA;
comparisons between individual groups were made using the
Student-Newman-Keuls multiple range test. The values were transformed
to logarithms when appropriate.
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Results |
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1 kb) of LFABP mRNA in the rat liver (Fig 1
). Northern blots of RNA preparations
from adipose tissue, skeletal muscle, or heart did not reveal any
transcripts, but RNA from the intestine contained a single band of
similar size (data not shown). These results were expected from the
known tissue distribution of LFABP mRNA (17, 20, 21, 29). Figure 1
shows that liver from a Hx female rat treated for 7 days with
T4 and cortisol had less LFABP mRNA than liver from a
normal female rat or livers from Hx rats treated with, in addition to
T4 and cortisol, bGH as two daily injections or as a
continuous infusion.
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) and migrated as recombinant rat LFABP
(data not shown). The expression of LFABP was lower in a Hx rat treated
with T4 and cortisol and higher in a Hx rat treated with
bGH as two daily injections for 7 days (Fig. 2).
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, A–C). Three hours after bGH
injection, LFABP mRNA levels had increased significantly. IGF-I mRNA
levels increased in a similar manner as LFABP mRNA, but IGF-I mRNA
levels had decreased to basal levels 24 h after the injection, in
contrast to LFABP mRNA levels (Fig. 3, A and C). No obvious effect of
the bGH injection on the expression of LFABP protein was observed in
liver cytosol (Fig. 3B). The changes in LFABP levels were similar when
the amount of LFABP was calculated as milligrams of LFABP per g liver
(data not shown). Thus, it is not likely that an increased amount of
certain major cytosolic proteins as a result of the bGH injection could
have blunted an increase in LFABP levels.
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). T4 and cortisol
treatment resulted in a small, but significant, increase in LFABP
levels, but had no effect on LFABP mRNA levels. The combination of
T4, cortisol, and bGH increased LFABP levels
nearly 3-fold and increased its mRNA 2-fold (Table 1). To test the
possibility that the effect of bGH on LFABP levels was influenced by
treatment with T4 and cortisol, bGH therapy alone
was compared with combined T4, cortisol and bGH
therapy in Hx female rats (Table 2). GH
treatment alone increased LFABP levels 1.5-fold and mRNA levels
2.7-fold, whereas combined T4, cortisol, and bGH
treatment increased LFABP levels 2-fold and mRNA levels 2.5-fold (Table 2). In summary, these results show that GH has the capacity to increase
LFABP levels without any additional pituitary-dependent hormones, but
it cannot be excluded that T4 and cortisol have
some influence on LFABP protein levels.
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). The lowest dose given (0.1
mg/kg·day) resulted in a 40% increase in LFABP concentrations. The
highest doses (1 and 5 mg/kg·day) resulted in a normalization of the
LFABP concentration (Fig. 4A). As shown in Fig. 4B, an increased bGH
dose resulted in an increased weight gain of the rats.
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). Several sexually dimorphic liver
functions have been shown to be regulated by the sexually dimorphic
secretory pattern of GH (4, 5, 7, 15, 48). To investigate
whether the secretory pattern of GH could influence the levels of
LFABP, Hx female rats were given two daily injections of bGH, mimicking
the male secretory pattern, or a continuous infusion of GH, mimicking
the female secretory pattern. Female rats had 2.3-fold higher levels of
LFABP than male rats (Fig. 5). The effect of Hx was marked in
female rats, but not in male rats. However, in other experiments (data
not shown), a significant decrease in LFABP levels after Hx was
observed in male rats. Two daily injections and a continuous infusion
of bGH had similar effects on the expression of LFABP, resulting in
4.8- and 3.8-fold increases, respectively (Fig. 5).
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). Incubation with 100 ng
bGH/ml increased the LFABP mRNA level 2.3-fold. However, when no
insulin was given during the last 3 days of culture, the level of LFABP
mRNA was low despite bGH treatment, indicating an effect of insulin on
LFABP mRNA levels (Fig. 6A). In a separate experiment (Fig. 6B), a
different dose of bGH was given during the last 24 h to each cell
culture dish. All culture dishes were incubated with 16 nM
insulin during the entire culture period. The result of this experiment
indicated that 100–200 ng bGH/ml results in a maximal effect on LFABP
mRNA expression. Thus, GH has a marked effect on LFABP mRNA levels
in vitro, and this effect was dependent on the presence of
insulin. In a control experiment, the level of LFABP mRNA in fresh
isolated hepatocytes was compared with the level of LFABP mRNA after
16 h and 4 days of culture in the presence or absence of insulin
(data not shown). The level of LFABP mRNA declined to about 60% of the
level in fresh isolated cells after 16 h of culture in 16
nM insulin. In the presence and absence of 3 nM
insulin, the LFABP mRNA levels were 34% and 14%, respectively, of the
level in fresh isolated cells after 4 days of culture. Thus, insulin
lessens the decline in LFABP mRNA gene expression during culture.
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, A and B). bGH treatment had no effect
on LFABP mRNA levels when the cells were not treated with insulin (Fig. 7A), whereas bGH treatment resulted in 2.5-fold higher IGF-I mRNA
levels in the absence of insulin (Fig. 7B). When the data were analyzed
with two-way ANOVA, it was found that both insulin and bGH treatment
had a significant effect (P < 0.05) on LFABP mRNA and
IGF-I mRNA levels. However, insulin had a marked effect on LFABP mRNA
levels, but a small effect on IGF-I mRNA levels. GH, on the other hand,
had a more marked effect on IGF-I mRNA than on LFABP mRNA levels. The
combined action of 100 ng/ml bGH and 3 nM insulin resulted
in a 10-fold increase in LFABP mRNA and a nearly 6-fold increase in
IGF-I mRNA levels (Fig. 7, A and B).
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, A and B.
One group of cultured cells was given 3 nM insulin during
the last 3 days of culture. The fourth day of culture, bGH (100 ng/ml)
was added to the medium for 1, 3, or 24 h. LFABP and IGF-I mRNA
levels increased within 3 h of incubation (Fig. 8). After 3 days
of culture without insulin, other cultured cells were given 3
nM insulin for 1, 3, or 24 h. LFABP mRNA levels
increased between 3 and 24 h of insulin treatment, whereas IGF-I
mRNA levels were not affected by insulin in this experiment (Fig. 8).
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). Incubation with actinomycin D
alone did not affect LFABP mRNA levels, but the effect of bGH on LFABP
mRNA levels was blunted in the presence of actinomycin D.
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).
Hx rats given T4 and cortisol had lower serum
insulin levels than normal rats, and bGH treatment increased serum
insulin levels in the Hx rats. The insulin-treated Hx rats had similar
or higher serum insulin levels than the Hx rats given bGH alone. The Hx
rats given insulin alone had the lowest serum glucose levels. Insulin
treatment and bGH treatment had opposite effects on serum glucose
levels, and when both hormones were given together, intermediate serum
glucose levels were observed. Insulin treatment had no effect on LFABP,
LFABP mRNA, or IGF-I mRNA levels (Table 3). However, bGH treatment (1
mg/kg·day) had the expected effects on IGF-I mRNA and LFABP
expression (Table 3). Although there are few or no IGF type 1 receptors
in the liver (49), the effect of recombinant human IGF-I was tested
in vivo, because IGF-I serum levels increase markedly as a
result of GH therapy and, therefore, might affect other hormonal
systems [such as an altered insulin secretion (50, 51)] that could
influence LFABP expression (Table 4).
However, there was no effect of 6 days of IGF-I treatment of Hx rats on
either LFABP or its mRNA in the liver (Table 4).
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Discussion |
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The normalization of LFABP levels after GH therapy of Hx rats suggests that GH is a major regulator of LFABP expression. The increased concentration of LFABP in liver cytosol after GH therapy seems to at least in part be dependent on an increased amount of LFABP mRNA, as the mRNA and protein levels increased in parallel after a week of GH treatment. There was, however, no increase in the expression of LFABP 24 h after a single dose of bGH, although there was a marked effect on LFABP mRNA levels. Thus, an effect of GH on LFABP expression is dependent upon the presence of GH for a longer period of time. Induction of LFABP by peroxisome proliferators has also been shown to have a slow onset (17, 38).
As GH treatment results in increased insulin secretion (55, 56) and
increased serum concentrations of insulin, the in vivo
effect of GH on LFABP levels may be the result of the additive effects
of GH and insulin on LFABP mRNA expression. The lack of effect of
insulin in vivo argues against this possibility. However,
the rats given insulin had lower serum glucose concentrations,
indicating increased serum concentrations of nonpituitary-dependent
insulin antagonists such as glucagon. The inhibitory effect of glucagon
in vitro indicates that the lack of effect of insulin
in vivo could at least partly be explained by increased
serum glucagon levels. Diabetic rats have been shown to have decreased
cytosolic levels of LFABP, which were partly reversed by insulin
treatment (33). Thus, it is possible that insulin also has effects on
LFABP expression in vivo, but only in insulin-deficient
rats. The insulin concentrations in portal blood have been shown to be
0.34 nM (50 mU/liter) in the fasting rat (57). This
finding indicates that the portal levels of insulin are markedly higher
than those in peripheral blood and that the doses used in the
hepatocyte cultures were within or near physiological levels.
The GH doses used were in the physiological range, as indicated by the normal mean plasma levels of GH (50–100 ng/ml) and the mean pulse heights of GH in plasma (150–300 ng/ml) in the adult rat (58). In the rat, GH secretion has been shown to decrease as a result of food deprivation for a period of 24 h or longer (59). Together with low levels of insulin and high levels of glucagon in serum, decreased GH secretion would result in decreased hepatic LFABP levels. However, parallel decreases in the content of LFABP and total cytosol protein have been observed after prolonged fasting (17), indicating that LFABP is not specifically decreased by fasting.
The continuous and irregular secretion of GH in the adult female rat and the regular episodic secretion of GH in the adult male rat have been mimicked in several studies by sc administration of GH as a continuous infusion and two daily injections of GH, respectively (4, 5, 15, 39, 48). In contrast to many other sexually dimorphic hepatic functions in the rat, the higher levels of LFABP in female rats were not dependent upon the secretory pattern of GH. Therefore, the sex difference in LFABP levels could be dependent on direct effects of the gonadal steroids on the hepatocyte or, alternatively, occur via indirect effects on the A and B cells of the pancreatic islets. Thus, it has been shown that estradiol increases the relative insulin to glucagon molar ratio in the portal vein (57). In line with the present results, this effect of estradiol would result in increased expression of LFABP mRNA in the liver.
The effect of GH on LFABP mRNA was compared with the effect on IGF-I mRNA, as GH is a known regulator of IGF-I mRNA in hepatocytes in vivo and in vitro (37, 43). Insulin has also been shown to play a role in the regulation of IGF-I mRNA in vitro (37, 60). In line with these studies, we observed a 2-fold potentiation of the GH-induced expression of IGF-I mRNA by insulin treatment. We observed small or no effects of insulin alone on the expression of IGF-I mRNA. The half-maximal effective dose of insulin observed by others (0.47 nM) (60) is in line with our results. The lack of effect of insulin therapy of Hx rats on IGF-I mRNA could be due to a relatively small effect of insulin on IGF-I gene expression and also to the fact that the rats were not insulin deficient.
The mechanism of induction of LFABP mRNA by GH is at least partly an increased transcription rate of the gene, as indicated by the lack of effect of GH in the presence of actinomycin D. IGF-I mRNA levels increase after GH therapy as a result of an increased transcription rate (43, 61). The induction of IGF-I mRNA in vivo by a single dose of GH was shown to be transient, in contrast to the induction of LFABP mRNA. This finding may reflect a transient increase in serum GH levels, resulting in an increased transcription followed by degradation of IGF-I mRNA. The maintained LFABP mRNA levels in the same experiment indicates slower turnover of LFABP mRNA than of IGF-I mRNA. The slow induction of LFABP mRNA by insulin treatment will be of interest to study further. It may be that the increase in LFABP mRNA levels caused by insulin treatment reflects an increased stability of the transcript, and the permissive action of insulin on the GH induction of LFABP mRNA may be due to this effect of insulin.
Cytosolic LFABP levels have previously been measured by immunological
methods using radial immunodiffusion (17, 27) and direct noncompetitive
ELISA (32). Similar amounts of LFABP in the liver of mature (60 days
of age) male and female rats were observed in this study and in other
studies using immunological methods (17, 27). We used the Lowry
procedure for determination of protein concentrations. Overestimation
of LFABP levels has been reported to occur when purified LFABP is
measured according to the Lowry procedure (32). Thus, it cannot be
excluded that we overestimated the amount of LFABP, especially if there
was no overestimation of the total protein concentration in cytosol.
Nonimmunological measurements of the amount of LFABP in liver cytosol
have relied on the property of LFABP to bind various ligands. There are
sometimes discrepancies between the nonimmunological and immunological
measurements of the amount LFABP (32). Nonimmunological methods have
several disadvantages compared with the immunological methods, such as
competition with endogenous ligands, nonspecific binding, binding
stoichiometry of LFABP, and the possibility that the ligands bind to
other proteins in the low mol wt protein fraction. However, the results
obtained using a ligand binding assay to determine the amount of LFABP
in liver cytosol after hypophysectomy and GH therapy were similar to
the results obtained in this study (35).
In summary, GH was shown to have a marked effect on LFABP regulation.
Small or no effects of thyroid hormones, glucocorticoids, insulin, and
IGF-I could be detected in vivo using Hx rats. In contrast
to other sexually dimorphic hepatic proteins, LFABP was not differently
regulated by the male- and female-specific secretory patterns of GH.
The induction of LFABP by GH in vivo was accompanied by an
increase in LFABP mRNA, indicating a pretranslational regulation of
LFABP expression by GH. However, a posttranslational regulation cannot
be excluded. The in vitro studies indicate a direct hormonal
regulation of LFABP mRNA expression involving physiological
concentrations of insulin and GH. Thus, in addition to the known
in vitro activation of LFABP via the PPAR
receptor and
its presumed ligands, long chain fatty acids (19, 38, 52, 53, 54), insulin,
and GH receptors are involved in the regulation of LFABP gene
expression. We cannot exclude the possibility that fatty acids
mobilized as a result of GH treatment activate the PPAR receptor and
augment the direct effect of GH on the hepatocyte on LFABP expression.
The role of LFABP in the diverse effects of GH on hepatic lipid and
lipoprotein metabolism (1, 2, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16) is still unclear. It could be
speculated that GH increases intracellular trafficking of fatty acids
in hepatocytes at least partly via a direct effect on the expression of
LFABP.
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Acknowledgments |
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Footnotes |
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Received October 28, 1997.
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