Department of Physiology, Göteborg University, S-405 30 Goteborg, Sweden
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ABSTRACT |
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The aim of this study was to
investigate the interaction between long-chain fatty acids (LCFA) and
growth hormone (GH) in the regulation of liver fatty acid binding
protein (LFABP) and peroxisome proliferator-activated receptor-
(PPAR
). Cultured rat hepatocytes were given oleic acid (OA; 500 µM) and GH (100 ng/ml) for 3 days. LFABP mRNA increased 3.6-fold by
GH and 5.7-fold by OA, and combined incubation with GH and OA increased
LFABP mRNA 17.6-fold. PPAR
mRNA was decreased 50% by GH, but OA had no effect. Hypophysectomized (Hx) female rats were treated with L-thyroxine, cortisol, GH, and dietary fat for 7 days.
PPAR
mRNA levels were three- to fourfold higher in Hx than in normal
female rats. GH decreased PPAR
mRNA 50% in Hx rats. Dietary
triglycerides (10% corn oil) increased LFABP mRNA and cytosolic LFABP
about twofold but had no effect on PPAR
mRNA in Hx rats. GH and
dietary triglycerides had an additive effect on LFABP expression.
Dietary triglycerides increased mitochondrial hydroxymethylglutaryl-CoA synthase mRNA only in the presence of GH. The diet increased serum triglycerides in Hx rats, and GH treatment prevented this increase. Addition of cholesterol to the diet did not influence LFABP levels but
mitigated increased hepatic triglyceride content. In summary, these
studies show that GH regulates LFABP expression independently of
PPAR
. Moreover, GH has different effects on PPAR
-responsive genes
and does not counteract the effect of LCFA on the expression of these
gene products.
peroxisome proliferator-activated receptor-; hepatocytes; mitochondrial hydroxymethylglutaryl coenzyme A synthase; dietary fat; cholesterol; hypophysectomy; serum triglycerides; oleic acid
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INTRODUCTION |
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LIVER FATTY ACID BINDING
PROTEIN (LFABP) is an abundant cytosolic protein expressed in the
liver and the intestine. LFABP binds long-chain fatty acids (LCFA) with
high affinity and their CoA esters with lower affinity. Moreover, LFABP
binds heme and eicosanoids with high affinity, and a large number of
other amphipathic ligands with lower affinity (for review see Refs.
1, 5, 43), but not cholesterol
(46). LFABP may play a role as an intracellular acceptor
of LCFA, thereby enhancing LCFA uptake and intracellular transport
(32, 35) (for review see Refs. 1,
5, 15, 43). Several enzyme
activities, especially enzymes involved in fatty acid metabolism, are
stimulated by LFABP, but it is not clear whether LFABP specifically
enhances esterification or -oxidation pathways (1, 24,
43). Overexpression of LFABP in a fibroblast cell line (L-cells)
produces alterations in membrane phospholipids and intracellular
cholesterol distribution, resulting in increased fluidity of plasma
membranes (23, 49). Moreover, LFABP may have a role in the
trafficking of LCFA to the nucleus (28) and subsequent
activation of peroxisome proliferator-activated receptor-
(PPAR
)
(48), further emphasizing the importance of LFABP in LCFA metabolism.
Activation of both PPAR and classical hormones regulates LFABP
expression in the liver at the mRNA level (1, 8, 9, 36,
37). Growth hormone (GH) increases LFABP mRNA and cytosolic LFABP levels in hypophysectomized (Hx) rats (6, 9).
By use of cultured rat hepatocytes, it was shown that GH increases
LFABP mRNA levels via increased transcription and that the effect of GH
was dependent on the presence of insulin in the culture medium (9).
Peroxisome proliferators, such as fibrates, increase LFABP and LFABP
mRNA levels in vivo (1) and in cultured hepatocytes (8), and the LFABP promotor has been shown to contain a
DR-1 peroxisome proliferator-responsive element
(5). LCFA can activate the peroxisome proliferator
response element via binding to and activation of PPAR (17,
27, 36). Incubation of a rat hepatoma cell line and primary
cultures of rat hepatocytes with LCFA have been shown to increase LFABP
mRNA levels (34). A diet enriched in triglyceride has been
shown to increase the amount of hepatic LFABP and LFABP mRNA in rats
(1, 2, 33, 47) and mice (26, 36), indicating
that an increased hepatic uptake of LCFA in vivo also upregulates
LFABP. Moreover, the use of PPAR
-null mice has clearly indicated the
importance of PPAR
for the effect of LCFA on LFABP gene expression
(18, 26).
PPAR mRNA expression is also increased by dietary triglycerides
(26) and under hormonal control in the liver.
Glucocorticoids have been shown to increase hepatic PPAR
expression
both in vivo and in vitro (29, 42), and the increased
PPAR
expression was shown to increase the responsiveness to a
peroxisome proliferator (29). In contrast, there are a few
reports that indicate that GH may decrease the PPAR
activity. GH may
interfere with PPAR
signaling, as suggested from the finding that
nuclear signal transducer and activator of transcription-5b inhibited
PPAR
-dependent reporter gene expression (52). Moreover,
GH has been shown to decrease PPAR
mRNA levels in cultured rat
hepatocytes (51) and to counteract the increase in
peroxisomal
-oxidation induced by peroxisome proliferators
(44, 50). Thus GH and LCFA alone increase LFABP gene
expression, but the interplay between these factors in the regulation
of LFABP expression is not known.
The primary aim of the present study was to investigate the interaction
between oleic acid (OA) and GH in cultured hepatocytes and the
interaction between GH and dietary triglycerides in Hx rats on LFABP
expression. The second aim was to study the regulation of PPAR mRNA.
Mitochondrial hydroxymethylglutaryl (HMG)-CoA synthase mRNA was
measured as another marker of PPAR
activation. This gene product has
been shown to increase by fat feeding of rats and to be upregulated by
LCFA via PPAR
(20). Serum levels of nonesterified fatty
acids (NEFA) and triglycerides as well as the hepatic content of lipids
were measured as indications of altered LCFA metabolism.
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MATERIALS AND METHODS |
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The Ethics Committee of Göteborg University approved this study. All chemicals used were from Sigma Chemical (St. Louis, MO), if not stated otherwise.
Animals. Female Sprague-Dawley rats from Møllegaard Breeding Center (Ejby, Denmark) were used. Hypophysectomy was performed at 50 days of age by Møllegaard Breeding Center. Intact, age-matched female rats served as controls in two experiments. The rats were maintained under standardized conditions of temperature (24-26°C) and humidity (50-60%) and with lights on between 0500 and 1900. The rats had free access to standard laboratory chow (rat and mouse standard diet, B&K Universal, Sollentuna, Sweden) and water. The standard laboratory chow contains (wt/wt) 2.5% fat (33% saturated fatty acids), 18% protein, and 61% carbohydrates, including 4% fiber. Two different kinds of diets enriched in fat were used. In one experiment, corn oil alone (10% wt/wt; Mazola, CPC Food, Kristianstad, Sweden) was added to the powdered diet, and in the other experiment, cholesterol (2% wt/wt) was added to the same amount of corn oil before it was mixed with the rat and mouse standard diet. The percentage of triglyceride in the diet was increased from 2.5 to ~12% (wt/wt). The fatty acid composition of corn oil triglycerides is 13% saturated, 29% monounsaturated, and 58% polyunsaturated fatty acids.
Hormonal treatment started 7-10 days after hypophysectomy. Hx rats were given cortisol phosphate (400 µg · kgHepatocyte cultures. Hepatocytes were prepared by a nonrecirculating collagenase perfusion through the portal vein of 200- to 300-g normal female Sprague-Dawley rats, as described before (9, 30). The cells were seeded at a density of ~170,000 cells/cm2 in plastic 100-mm dishes (Falcon, Plymouth, England). The dishes were coated with laminin-rich matrigel (Collaborative Research, Medical Products, Bedford, MA), and the cells were plated during the first 16-18 h in Williams E medium, supplemented as described before (9). Therefter, the medium was changed to a medium that differed from the first with respect to the content of hormones, OA, albumin, and DMSO during the following 3 days of culture. bGH was given in a dose of 100 ng/ml (9). Insulin (Actrapid; Novo Nordisk, Denmark) was given in a dose of 3 nM (9). OA (500 µM) was dissolved in DMSO (Merck, Darmstadt, Germany). Essentially fatty acid-free albumin 0.75% (wt/vol) and DMSO 0.15% (vol/vol) were added to all culture dishes (30). The medium was changed every day. The cells were cultured 4 days.
ELISA. An antibody-sandwich ELISA was used for measurements of soluble LFABP in rat liver cytosol (9). Cytosol was prepared as previously described (9, 37), and the total protein concentration of the cytosol was determined according to the method of Lowry (31).
Probes.
Total RNA was prepared according to Chomczynski and Sacchi
(10). LFABP mRNA was measured using a 333-bp fragment, 26 nt upstream from ATG to nt 307 of rat LFABP cDNA (accession no.
J00732.1) inserted in a pSP 72 vector (Promega, Madison, WI)
(9). The pSP 72 vector was linearized with
PvuII, and a [35S]UTP antisense RNA probe or a
[32P]CTP antisense RNA probe was generated with T7 RNA
polymerase (Maxiscript; Ambion, Austin, TX). For PPAR mRNA
measurements, a 249-bp fragment, nt 76 from ATG to nt 324 of rat
PPAR
cDNA (accession no. M88592) was subcloned into a pBluescript II vector (Stratagene, La Jolla, CA). The full-length rat PPAR
plasmid was kindly supplied by Dr. Jan-Åke Gustafsson, Karolinska Institute, Stockholm, Sweden. The pBluescript plasmid was linearized with EcoRI, and a [35S]UTP or
[32P]CTP antisense RNA probe was generated with T7 RNA
polymerase (Maxiscript). A biotin-labeled PPAR
antisense RNA probe
was generated using Biotin-16-UTP (Enzo, Roche) in the reaction.
Gel ribonuclease protection assays.
Gel ribonuclease protection assays with the use of radioactive probes
were performed as described by the manufacturer (RPA III kit, Ambion).
32P-labeled antisense LFABP, PPAR, and GAPDH RNA probes
were used. Protected fragments were separated on denaturing
polyacrylamide 6% TBE-urea gels (Novex, San Diego, CA). Detection and
quantification were performed with a PhosphoImager and Image Quant
software (Molecular Dynamics, Sunnyvale, CA).
Solution hybridization assays.
Measurement of LFABP mRNA and PPAR mRNA levels was performed using
solution hybridization assays as described previously (9,
12). In brief, 35S-labeled antisense RNA probes were
hybridized to aliquots of RNA. After hybridization, the samples were
treated with RNase T1 and A. Protected hybrids were precipitated and
collected on glass fiber filters and counted in a scintillation
counter. The LFABP standard (sense) used in the solution hybridization
assay was generated after linearization with EcoRI. The
PPAR
standard (sense) used was generated after linearization with
Asp718. The hybridization signal obtained with the
antisense probe was compared with the signal of a standard curve of
sense RNA. The results are expressed as picograms per microgram of
total RNA.
Northern blot. Twenty micrograms of RNA were denatured with glyoxal and DMSO and run in a 1% agarose gel according to the protocol of the manufacturer (NorthernMax-Gly; Ambion). The RNA was blotted onto a Bright Star-Plus nylon membrane (Ambion) and covalently linked to the membrane by ultraviolet irradiation (UVC Crosslinker). The membrane was prehybridized at 68°C for 2 h in ULTRAhyb (Ambion). The same solution was used for hybridization for 16 h at 68°C. The final wash of the membranes was carried out in Low Stringency Wash Solution no. 1 (Ambion) for 15 min at room temperature, followed by two washes for 15 min at 68°C in High Stringency Wash Solution no. 2 (Ambion). The detection was carried out using a Bright Star BioDetect Kit, as described by the manufacturer (Ambion). The chemiluminescence was detected and quantified using Fluor-S-Multimager (Bio-Rad). The intensity of the mHMG-CoA synthase mRNA band was divided by the intensity of the 18S band. The amounts of the transcript are expressed as the ratio between these bands.
Other analyses. NEFA concentrations were analyzed with a colorimetric assay according to the manufacturer (NEFA C, ACS-ACOD method; Wako Chemicals, Neuss, Germany). Serum triglyceride levels were determined using a colorimetric enzymatic assay (GPO-PAP-kit; Boehringer Mannheim, Mannheim, Germany). The content of triglyceride and total cholesterol in the livers was determined after homogenization of frozen liver in ice-cold water, followed by extraction of total lipids (7). The chloroform phase containing total lipid was evaporated until dryness under a stream of N2, and the lipid phase was resolved in ethanol. The mass of triglycerides and total cholesterol was measured in duplicate with enzymatic colorimetric assays (triglycerides: GPO-PAP-kit; cholesterol: CHOD-PAP-kit; Boehringer Mannheim).
Statistics. Values are expressed as means ± SE. Comparisons between means were made by analysis of variance (ANOVA), followed by a Student-Newman-Keuls multiple range test (post hoc test) between individual groups. The values were transformed to logarithms when appropriate.
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RESULTS |
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In vitro effects of GH and OA on LFABP mRNA and PPAR mRNA.
In a previous study, it was observed that GH increased LFABP mRNA in
vitro, but this effect was dependent on the presence of insulin
(9). The interaction between GH and OA on LFABP mRNA
expression was therefore investigated in the presence of insulin (Fig.
1A). GH (100 ng/ml) increased
LFABP mRNA levels 3.6-fold and 500 µM OA increased LFABP mRNA levels
5.7-fold. In the presence of GH, incubation with 500 µM OA resulted
in a 4.9-fold increase in LFABP mRNA levels. Incubation with both OA
and GH resulted in a 17.6-fold increase in LFABP mRNA expression
compared with control cell cultures (Fig. 1A). These
findings indicate that GH and OA have an additive effect on LFABP mRNA
expression and that the responsiveness of the cultured hepatocytes to
OA was not markedly affected by GH incubation. In one previous study (51), it was shown that incubation of cultured hepatocytes
with GH resulted in a decrease in PPAR
mRNA levels. Therefore, we also studied the interaction between GH and OA in the regulation of
PPAR
mRNA in the cultured hepatocytes (Fig. 1B). GH
incubation decreased PPAR
mRNA ~50%, but incubation with OA had
no effect (Fig. 1B). Two-way ANOVA (GH and OA incubation as
factors) showed that GH had a significant effect (P = 0.007), but the effect of OA was not significant (P = 0.10; Fig. 1B). To study whether the effect of GH on PPAR
expression was dependent on the presence of insulin, hepatocyte
cultures were incubated with GH (100 ng/ml) the last 3 days of culture
without insulin. The results from three separate experiments showed
that the relative PPAR
mRNA level (divided by the densitometric
value of 18S) was 0.055 ± 0.005 in cell cultures without hormones
and 0.028 ± 0.005 (P < 0.05) in cell cultures
incubated with GH for 3 days. Thus GH decreased PPAR
mRNA to a
similar degree without concomitant treatment with insulin.
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In vivo effects of hypophysectomy and GH on PPAR mRNA.
Next, the effects of hypophysectomy (Hx) and GH on liver PPAR
mRNA were investigated (Fig.
2A). Hx
and combined treatment with T4 and cortisol markedly
increased PPAR
mRNA. GH treatment of Hx female rats given
T4 and cortisol decreased the levels of PPAR
mRNA (Fig.
2A). Because the substitution with T4
(51) and cortisol (29, 42) may have
contributed to the high level of PPAR
mRNA in Hx rats, we
investigated the effects of Hx without any hormone treatment on PPAR
mRNA levels in the liver. Hx of female rats increased PPAR
mRNA
levels four- to fivefold, and treatment with T4 and
cortisol had no further effect (Fig. 2B). To investigate
whether the effect of GH might be enhanced in the absence of
T4 and cortisol, the effect of GH was investigated with and
without T4 and cortisol treatment (Fig. 2C). In
this experiment, one-half of the rats in each group were killed between 0930 and 1030, and the other rats in each group were killed between 1330 and 1430. We did not observe any consistent trends in
PPAR
mRNA levels between rats killed in the morning and those
killed in the afternoon (data not shown); therefore, these groups were pooled. As seen in Fig. 2C, GH decreased PPAR
mRNA levels to a larger degree in the absence of T4 and
cortisol treatment; i.e., T4 and cortisol increased PPAR
mRNA levels in the presence of GH.
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In vivo effects of dietary triglycerides and GH.
Next, the effects of a diet enriched in triglycerides were investigated
in Hx female rats with and without GH therapy (Table 1 and Fig.
3). All Hx rats were given
T4 and cortisol, and the diet and the hormones were both
given for 7 days. The weight gain of the rats was increased by
GH treatment but was not affected by the triglyceride diet (Table 1).
The triglyceride-enriched diet tended to increase PPAR mRNA levels
in Hx rats, but the effect was not significant (Fig. 3A). GH
decreased PPAR
mRNA levels to a similar extent (~50%) in the rats
given a triglyceride diet and in rats given ordinary rat chow (Fig.
3A).
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In vivo effects of GH and dietary triglycerides and cholesterol.
LFABP may be involved in intracellular cholesterol distribution
(23, 49), and we could not exclude that the 30% increase in hepatic cholesterol (Table 1) influenced LFABP expression. Therefore, the effects of a diet enriched in both triglycerides and
cholesterol on LFABP expression were studied in Hx female rats (Table
2 and Fig.
4). As in the previous experiment, all Hx
rats were given T4 and cortisol, and the diet and the
hormones were both given for 7 days. The weight gain of the rats was
increased by GH treatment but was not affected by the fat diet (Table
2). The diet induced a twofold increase in LFABP but had no significant effect on LFABP mRNA levels in the Hx control rats (Fig. 4). GH treatment resulted in a 3.5-fold increase in LFABP mRNA levels and a
1.7-fold increase in cytosolic LFABP levels (Fig. 4). In the group of
rats given GH, the fat diet increased LFABP mRNA 50% and cytosolic
LFABP levels 60% (Fig. 4, A and B).
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DISCUSSION |
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This study showed that GH regulates LFABP in a PPAR-independent
manner. Furthermore, it was shown that GH and LCFA have additive effects on the expression of LFABP. In contrast, PPAR
mRNA
expression was decreased by GH and was not affected by LCFA. Thus LCFA
can induce LFABP mRNA to the same degree when PPAR
mRNA expression is decreased. This finding is in contrast to another study, which indicated that the increased LFABP mRNA expression by dietary triglycerides could be fully explained by increased PPAR
mRNA expression (26). Thus the decreased PPAR
mRNA
expression cannot explain the effects of GH and LCFA on LFABP
expression but may contribute to the understanding of the inhibitory
effect of GH on peroxisomal
-oxidation (44, 50).
Another kind of interaction between GH and the triglyceride content of
the diet was observed when we studied the expression of mHMG-CoA
synthase mRNA. GH treatment decreased mHMG-CoA synthase mRNA levels
when the rats were given ordinary chow. This effect of GH may be
explained by a decreased PPAR
expression. On the other hand, GH
treatment was important for the normal stimulation of mHMG-CoA synthase
mRNA expression by dietary triglycerides. This effect cannot be
explained by the inhibitory influence of GH on PPAR
mRNA expression
or activity (52). There are several reports that GH
stimulates ketogenesis and that this effect is largely dependent on an
increased supply of fatty acids (25). The regulation of
mHMG-CoA synthase mRNA by GH and a triglyceride-enriched diet may
therefore contribute to an explanation at the mRNA level of how fatty
acids and GH interact in the control of ketogenesis.
The finding that hypophysectomy of female rats markedly enhanced the
expression of PPAR mRNA indicates that, at least in female rats, the
sum of the influence of the pituitary-dependent hormones is inhibitory
on PPAR
mRNA expression. In contrast to LFABP expression
(1), PPAR
expression follows a diurnal rhythm in the
intact rat (29). The rats were killed in the morning, when
the hepatic PPAR
mRNA levels are low (29). From the
present results, it may therefore be concluded that the low expression of PPAR
in the morning could not be explained solely by low
glucocorticoid levels but was also dependent on the inhibitory effect
of GH. We observed no effect of combined treatment with T4
and cortisol to Hx rats, but T4 and cortisol increased
expression of PPAR
mRNA when GH was given. The results, therefore,
indicate that the stimulatory effect of T4
(51) and cortisol (29, 42) is dependent on
the presence of GH in vivo. The doses of T4 and cortisol
have previously been shown to be within the physiological range with
respect to longitudinal bone growth (22, 45). Moreover, the serum levels of T4 obtained are about two times the
levels in intact female rats (41). Thus the small effect
of T4 and cortisol is not due to the doses of the hormones
but may be explained by the use of Hx rats as a model.
It must be pointed out that we did not measure PPAR protein levels.
It has been shown in some instances that the PPAR
protein levels
follow the mRNA levels (3, 29), but it remains to show
that the effect of GH on PPAR
mRNA levels results in changed PPAR
protein expression. For example, the half-life of the PPAR
protein
may be too long to decrease significantly during a week of GH therapy.
The diets had to some extent little or no effect on LFABP mRNA levels, whereas the effects on cytosolic LFABP protein levels were more marked. These results indicate that dietary triglycerides also have a posttranscriptional effect on LFABP expression. In fact, it has been shown that LFABP ligand availability is of importance for the protease susceptibility of the protein (21). Thus it may be speculated that increased cytosolic LFABP content may be due to both increased amounts of translatable mRNA and increased binding of LCFA to LFABP, which may stabilize the protein.
The addition of cholesterol to the triglyceride-enriched diet resulted in largely similar effects on the expression of LFABP and its mRNA as the triglyceride-enriched diet, indicating that cholesterol has no major influence on the expression of LFABP mRNA or its protein. Moreover, the addition of cholesterol to the triglyceride-enriched diet counteracted the effect of dietary triglycerides on liver triglyceride content. This result indicates that the increased LFABP expression is not due to increased hepatic triglyceride content per se, but rather to the flux of fatty acids into the liver cells.
The mechanisms behind the effect of dietary cholesterol on the liver triglyceride content can only be speculated on. This effect was surprising and may be due to the fact that we studied Hx rats because treatment of intact rats with cholesterol has been shown to increase hepatic content of triglycerides (14). However, cholesterol feeding of Hx rats may result in another effect on hepatic triglycerides. Thus the hepatic triglyceride content may have been reduced via an effect of cholesterol on the proportion of fatty acids used for very low density lipoprotein (VLDL) assembly. In favor of this hypothesis is the finding that dietary cholesterol increases the expression of microsomal triglyceride transfer protein (4) and VLDL secretion (14). Moreover, the higher serum triglyceride levels when the rats were given a diet enriched in both cholesterol and triglycerides compared with the effect of dietary triglycerides alone indicate a higher VLDL secretion.
In this study, the NEFA levels in serum were increased by the diets but were not affected by GH treatment. In other studies, an increase, a transient increase, and no effect of GH on serum NEFA levels have been observed (16). Most studies that showed elevation of NEFA by GH treatment were performed in fasting animals (16), which was not the case in this study. We have previously shown that GH treatment of Hx rats given ordinary rat chow slightly decreased or had no effect on serum triglyceride levels (38, 39). In this study, GH had no effect on serum triglyceride levels during chow feeding. However, during high-fat feeding, GH treatment resulted in markedly lower serum triglyceride levels. The mechanism behind the different effect of GH on serum triglycerides during chow feeding and fat feeding is unclear but could be due to either a decrease or a prevention of an increase in serum triglyceride levels during fat feeding. Most probably, a decreased production or an increased degradation of triglyceride-rich lipoproteins explains this effect. We have shown, by use of primary hepatocyte cultures, that GH increases VLDL secretion both ex vivo (39) and in vitro (30). Moreover, OA and GH had an additive effect on VLDL secretion (30). Therefore, the lower serum triglyceride levels in GH-treated fat-fed rats are not likely to be due to a decreased VLDL secretion. However, the effect of GH on serum triglycerides may be explained by an increased proportion of VLDL containing apoB-48 (30, 41), because apoB-48 VLDL have a faster turnover than apoB-100 VLDL. Moreover, GH increases the capacity for degradation of triglyceride-rich lipoproteins via increased hepatic lipase activity and increased lipoprotein lipase activity in heart and skeletal muscle (39).
It is unlikely that the effect of GH on LFABP mRNA is mediated via a
decreased metabolism of fatty acids and a buildup of an intracellular
pool of fatty acids. First, we have previously shown that GH increases
LFABP mRNA in vitro within 1-3 h (9). Second, GH has
been shown to increase mitochondrial -oxidation (11)
and specifically the oxidation of OA (13). Moreover, GH
has been shown to increase the incorporation of OA into hepatic triglycerides (13, 30). These findings indicate that, at
least, the intracellular amount of OA does not build up as a result of GH treatment. Because the effect of OA on LFABP mRNA was similar in the
absence and presence of GH, a more specific transportation or
activation is needed to explain the effect of OA on LFABP mRNA expression. However, we cannot rule out the possibility that the intracellular amount of very long chain fatty acids is built up as a
result of the inhibitory effect of GH on peroxisomal
-oxidation (44, 50). It is tempting to speculate that a fatty
acid-activated transcription factor other than PPAR
is involved in
the regulation of LFABP and HMG-CoA synthase by GH and LCFA. One such
candidate is hepatocyte nuclear factor-4
, which has been
shown to be involved in the regulation of both LFABP and PPAR
expression (19) and to be regulated by GH at the mRNA
level (40).
In summary, this study shows different interactions between GH and LCFA
in the regulation of the PPAR-responsive gene products PPAR
,
LFABP, and mHMG-CoA synthase. Moreover, GH has different effects on
PPAR
-responsive genes and does not counteract the effect of LCFA on
the expression of these gene products.
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ACKNOWLEDGEMENTS |
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We thank Dr. Fausto Hegardt (Dept. of Biochemistry and Molecular
Biology, University of Barcelona, Spain) for providing the plasmid
containing rat mitochondrial HMG-CoA synthase cDNA and Dr. Jan-Åke
Gustafsson (Karolinska Institute, Stockholm, Sweden) for providing the
plasmid containing full-length rat PPAR cDNA.
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FOOTNOTES |
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This work was supported by Grant 8269 from the Swedish Medical Research Council, the Novo Nordisk Foundation, the Tore Nilson Foundation, the Åke Wibergs Foundation, King Gustav V and Queen Victoria's Foundation, and the Magnus Bergvalls Foundation.
Address for reprint requests and other correspondence: J. Oscarsson, Dept. of Physiology, Göteborg University, Box 434, S-405 30 Göteborg, Sweden (E-mail: Jan.Oscarsson{at}fysiologi.gu.se).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 8 December 2000; accepted in final form 30 May 2001.
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