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*
Departament de Bioquímica i Biologia Molecular, Facultat de Farmàcia and
Departament d’Ecologia, Facultat de Biologia, Universitat de Barcelona, 08028 Barcelona, Spain
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: • insulin-like growth factor-I • dietary nutrients • diurnal variation • Sparus aurata
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Cohick and Clemmons 1993
, Thissen et al. 1994
). The presence of
IGF-I in fish has been demonstrated unequivocally (Cao et al. 1989
, Duan 1998
, Fine et al. 1997
). IGF-I has been cloned from liver of the teleost fish
gilthead sea bream (Sparus aurata), and the expressed
protein has been found to be two amino acids shorter at its
C-terminus than human IGF-I and to differ from it by a few
amino acids (Fine et al. 1997
). The structure and the
biological potency of IGF-I in fish are very similar to those of
mammalian homologs, and the GH-IGF-I axis seems to be well conserved in
vertebrate evolution (Duan 1998
). Administration of
growth hormone (GH) to rats and to fish results in a rapid rise of the
IGF-I mRNA levels in the liver, which is the major contributor to
circulating IGF-I (Bichell et al. 1992
, Cao et al. 1989
, Duan et al. 1994
, Duguay et al. 1996
, Shamblott et al. 1995
). Indeed,
recently a GH-dependent activation of the salmon IGF-I promoter
in Hep3B cells was caused by a synergistic action of the transcription
factors STAT5 and HNF-1
(Metón et al. 1999a
).
Nutritional, hormonal and genetic factors determine the levels of
circulating IGF-I in mammals and fish (Adamo et al. 1991
, Cohick and Clemmons 1993
, Duan et al. 1994
, Thissen et al. 1994
), but it remains
unclear how the nutritional status regulates hepatic IGF-I mRNA. In
addition, data allowing assessment of the diurnal variation of
IGF-I mRNA following food intake have not been reported to date.
The goal of the present study was to examine the effect of starvation,
diet composition and ration size on the hepatic content of IGF-I
mRNA in Sparus aurata. Diurnal variations of liver IGF-I
mRNA were also studied. |
MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Gilthead sea bream (Sparus aurata) fingerlings with an average weight ± SD of 9.4 ± 1.6 g, obtained from Aquadelt S.A. (Sant Carles de la Ràpita, Tarragona, Spain), were maintained in 250-L aquariums supplied with running seawater at 20°C in a closed system with active pump filter and UV lamps. The photoperiod was regulated as a dark/light cycle of 12/12 h. To obtain tissue samples, fish were anesthetized with MS-222 (1:12500) and killed by cervical section at 0900 h, 24 h after the last meal, unless stated differently. The liver was dissected out, immediately frozen in liquid nitrogen and kept at -80°C until use. The experimental procedures used in this study met the guidelines of the animal use committee of the University of Barcelona.
The effect of diet composition supplied to S. aurata was
studied using the diets shown in Table 1
. Analyses of the diets were performed following standard procedures
(Windham 1997
). Three levels of protein and carbohydrate
were selected, and the ratios of gelatinized starch/fish liver oil were
adjusted to achieve energy levels between 19 and 20 kJ/g. The fish were
fed the different diets, high protein/low carbohydrate diet (HP),
medium protein/medium carbohydrate diet (MP) and low protein/high
carbohydrate diet (LP), daily at 2 g/100 g body weight (BW) for 18 d. The effect of starvation was followed in 19-d food-deprived fish
for each type of diet supplied. Two aquariums were used for each diet
treatment, each stocked with 25 fish.
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To evaluate short-term modulation of IGF-I mRNA, diurnal variations related to feeding were followed for 24 h. To this end, S. aurata were fed once a day (0900 h) diet MP at 2 g/100 g BW for 26 d. Fish livers were dissected out at time 0 h; immediately the rest of the fish received the last meal and sampling was completed at different intervals for a 24-h period. Twelve aquariums were used in this experiment, each containing 6–10 fish.
RNA isolation and Northern blotting analysis.
Frozen pulverized liver samples (100–200 mg) from S.
aurata were homogenized in guanidinium thiocyanate lysis buffer
with a PolytronTM (position 7–8, 30 s) and
centrifuged in a cesium chloride gradient to isolate RNA
(Sambrook et al. 1989
). Total RNA (20 µg/lane) was
separated on 10 g/L agarose/0.67 mol/L formaldehyde gel and transferred
to Nytran-N membranes (Schleicher & Schuell, Dassel, Germany) by
capillary blotting in 5x SSC (1x SSC = 150 mmol/L NaCl, 150 mmol/L
sodium citrate, pH 7.0). Bound RNA was crosslinked to the membranes by
UV irradiation for 3 min. A 201 bp S. aurata IGF-I
cDNA fragment coding for mature IGF-I protein was used as a probe
(Fine et al. 1997
). Prehybridization of blots was at
42°C for 4 h, in 50 mmol/L sodium phosphate, pH 6.5, 1M NaCl,
7.5x Denhardt’s solution (1x Denhardt = 0.2 g/L each of Ficoll
400, poly(vinylpyrrolidone) and bovin serum albumin fraction V), 0.2
mmol/L dextran sulfate, 10 g/L SDS and 0.5 g/L denatured salmon sperm
DNA. The radiolabeled DNA probe was added and hybridization proceeded
at 42°C for 18 h. High stringency washings of membranes were
performed twice for 15 min at 42°C, in 2x SSC, 2 g/L SDS and finally
twice for 30 min at 65°C with 0.1x SSC, 2 g/L SDS. Control
hybridization was carried out reprobing membranes with a rat ß-actin
cDNA probe (data not shown). Prehybridization, hybridization and
washings were all carried out in a Hybaid oven. Quantitation of
IGF-I 3.9 kb mRNA species in autoradiographed films was performed
by scanning densitometry (Vilber Lourmat, Marne-la-Vallée,
France). Data were analyzed by 1 factor ANOVA using a computer program
(SuperANOVA, Abacus Concepts, Berkeley, CA). Differences between
treatments were determined using the Duncan multiple range test, with
significance levels of P < 0.01 and
P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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no significant differences were found among the fish fed different
diets, although a slight tendency to present higher IGF-I mRNA
values was observed in the fish fed diet LP compared to those fed diet
HP (P = 0.08). Sparus aurata deprived of
food for 19 d had significantly lower IGF-I mRNA than fed
fish. Regardless of the diet, the IGF-I mRNA values were 28–38%
of the levels in fed fish.
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). Thus, fish growth depended on the quantity of diet supplied, as we
previously found in S. aurata (Metón et al. 1999c
). The time course of the increase in IGF-I mRNA
levels in refed fish was gradual but independent of the ration size.
After 8 and 22 d of refeeding, the four ration sizes tested
resulted in values significantly greater than those of starved fish.
Thus, after 22 d of refeeding, IGF-I mRNA levels were about
three-fold the values in starved fish, irrespective of the ration
size. However, no significant differences were observed in S.
aurata fed different ration sizes at any of the periods studied
(Fig. 2
).
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).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, Duan 1998
,
Thissen et al. 1994
). IGF-I mRNA decreases in liver
of food-deprived and energy- or protein-restricted rats
(Straus 1994
, Zhang et al. 1998
). We
found that IGF-I mRNA levels in liver of S. aurata were
also reduced in food-deprived fish, and their values increased by
refeeding, although longer periods were required to observe significant
changes than in mammals (Cohick and Clemmons 1993
,
Duan 1998
). Consistent with our data,
Pérez-Sánchez et al. (1995)
reported decreased circulating
IGF-I values in food-deprived S. aurata.
It has been claimed that, as for circulating IGF-I, the quantity
and the quality of dietary protein regulate hepatic IGF-I mRNA
(Miura et al. 1992
, Kanamoto et al. 1994
). The levels of plasma IGF-I were found increased in
S. aurata fed high protein-high energy diets, while GH
binding affinity was not significantly affected by either dietary
protein/energy ratio or ration size (Pérez-Sánchez et al. 1995
). Our findings indicate little or no modulation of
hepatic IGF-I mRNA levels by partial substitution of protein by
carbohydrates and lipids, although the highest carbohydrate/lowest
protein diet promoted slight increased values, which would be
consistent with the reported stimulation of IGF-I expression by
carbohydrate intake (Straus and Burke 1995
). Thus,
variations in plasma IGF-I concentration in S. aurata
fed different diets may be mainly due to changes in plasma IGF-I
clearance or contributions from tissues other than liver rather than
modulation of hepatic IGF-I mRNA. Indeed, Pfaffl et al. (1998)
recently reported that even though liver in growing steers is, as in
fish (Duan et al. 1994
), the main IGF-I producing
tissue, skeletal muscle may considerably contribute to plasma IGF-I.
Our previous studies showed positive correlation between the quantity
of diet supplied to S. aurata, growth and activity of
enzymes involved in liver intermediary metabolism. Besides,
long-term feeding at 0.5 g/100 g BW resulted in loss of weight and
similar or even lower activity of key enzymes for glycolysis and
pentose phosphate pathway than in starved fish (Metón et al. 1999b
, Metón et al. 1999c
). In the
same conditions, IGF-I mRNA levels were regulated by
starvation/refeeding, but not by ration size. Indeed, fish refed at 0.5
g/100 g BW presented negative growth but hepatic IGF-I mRNA values
similar to fish refed at 1 g/100 g, 2 g/100 g or 3.5 g/100 g BW.
Therefore, we found no correlation between body growth and hepatic
IGF-I mRNA. In this regard, the role of liver-derived IGF-I on
body growth is disputed. In contrast to the classical somatomedin
hypothesis, it has been recently proposed that although
liver-derived IGF-I is the principal source of circulating
IGF-I, there is no requirement of hepatic IGF-I for postnatal
growth, suggesting that local IGF-I production becomes more
important than liver-derived IGF-I for body growth
(Sjögren et al. 1999
, Yakar et al. 1999
).
There are no reported studies focused on the influence of feeding on
diurnal variations of hepatic IGF-I mRNA. Our observations indicate
daily variations of IGF-I mRNA in liver of regularly-fed fish,
reaching maximal values after a postpandrial period of about 10 h.
These findings are consistent with data reported in pigs, whose
IGF-I plasma levels increased to a peak in concentration about
8–12 h after feeding (Morovat et al. 1994
). In S.
aurata it appears that the intake of food plays a key role in
short-term modulation of hepatic IGF-I mRNA levels, which in
turn may be of importance to adequate IGF-I expression to its
anabolic functions. In fact, IGF-I mRNA daily variations can be
associated with the classical Specific Dynamic Action effects on
metabolism, which have been related recently to involvement of
metabolic energy for anabolism and protein synthesis (Guinea and Fernández 1997
).
In conclusion, the present study indicates that different mechanisms account for the nutritional regulation of IGF-I mRNA in liver of S. aurata. Particularly, starvation/refeeding and diurnal feeding cycle regulate IGF-I expression at mRNA level; however, changes in diet composition and ration size, in contrast to data reported in mammals, do not promote significant modulation of the hepatic IGF-I mRNA content. Furthermore, no correlation was found between fish growth and the liver IGF-I mRNA, suggesting no dependence of body growth on liver-derived IGF-I.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: BW, body weight; GH, growth hormone; HP, high protein/low carbohydrate diet; IGF-I,
insulin-like growth factor-I; LP, low protein/high carbohydrate diet; MP, medium protein/medium carbohydrate diet.
Manuscript received September 28, 1999. Revision accepted December 16, 1999.
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