PEPTIDE TRANSPORT AND ANIMAL GROWTH: THE FISH PARADIGM
Tiziano Verri, Genciana Terova, Konrad Dabrowski, Marco Saroglia
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1. ANIMALS, FEEDING PROTOCOL AND EXPERIMENTAL SET UP
One hundred and forty sea bass (Dicentrarchus labrax L.) were randomly stocked into four tanks of
2 m3 each, with 35 fish per tank, and allowed to acclimate for 1 month. During the acclimation
period, all fish were fed Hendrix-Skretting Power Excel feed for marine fish. The tanks were
connected to a sea water recirculation system. Other water conditions were: temperature 20±2 °C,
pH 7, and total ammonia <0.2 mg/L. Dissolved oxygen was maintained over 99% saturation by
insufflating pure O2 to the system. At the start of the experiment, two of the tanks were randomly
assigned to each of two treatments. Fish in these two tanks were fed to apparent satiety (fed
control), whereas fish in the other two tanks were deprived of food for 35 days and then refed to
apparent satiety for 21 days with the same type of feed utilized before fasting. Feed consumption
(g) in each tank was estimated from the difference between feed delivered into the tank and uneaten
feed which was collected from the bottom of the tank. Feed intake was converted to grams of feed
consumed per kg body weight (BW) of the fish per day. Five fish from each of the experimental
groups were sampled at the following time points: before fasting (day 0), 4 days after fasting, at the
end of fasting, and then sequentially at 4, 14, and 21 days following refeeding. Fish were sampled
15 min before the scheduled feeding time. For the molecular biology analysis, the whole digestive
tract was dissected out, frozen immediately in liquid N2, and stored at –80 °C.
All the fish in the tanks were weighed at the start of the experiment, at the end of fasting period and
after 21 days of refeeding. They were rapidly anesthetized with tricaine methane sulfonate (MS222, 100 mg/l) and body weight and length (total and standard) were measured.
2. INFLUENCE OF THE FEEDING PROTOCOL ON FISH GROWTH AND FEEDING
RATES
The growth and condition factor data are reported in the manuscript body, figure 1a,b, respectively.
At the onset of fasting period the mean body weight of the sea bass was 119.34 ± 2.38 g for the
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control group and 118.42 ± 3.13 g for the “food deprived” group. After 35 days of fasting the mean
body weight and condition factor of fasted fish were significantly (P<0.05) lower than the fed
control. During the subsequent refeeding period, fasting-associated growth retardation was
completely overcome, and the mean body weight and condition factor of fasted group were the
same as those of the ad libitum fed controls.
Fish that had experienced feed deprivation exhibited higher feeding rates than ad libitum fed
controls during the first two weeks of refeeding (P<0.05) (see manuscript body, figure 1c).
Refeeding of sea bass after 35 days of starvation was marked by hyperphagia as early as the first
day. The hyperphagic period was however shorter than the fasting period.
3. SEA BASS INTEGRATED RESPONSE TO FOOD AVAILABILITY
In our experiments, sea bass nutritional status influenced the expression of key growth-related
genes, such as myostatin in muscle (increase during fasting and decrease during refeeding) (figure
S1), insulin-like growth factor I (IGF-I) and insulin-like growth factor II (IGF-II) in liver and
muscle (decrease during fasting and increase during refeeding) (figure S2a-d) [1,2], and these
regulatory responses correlated highly with changes (decrease during fasting and increase during
refeeding) in fish body weight and condition factor (a morphological indicator of body shape) (see
manuscript body, figure 1a,b). Under the same experimental conditions, at the gastrointestinal level
the stomach responded to fasting by up-regulating ghrelin (figure S3a) and down-regulating
gastricsin (pepsinogen C) (figure S3b) expression as part of an integrated response to food
availability [3,4]. Notably, ghrelin is a well-known appetite-inducing peptide hormone secreted by
the stomach and an important growth hormone (GH) secretagogue1, whereas gastricsin is the
enzyme in charge of the initial and partial hydrolysis of the dietary proteins, a process that is
subsequently completed in the intestine by the combined action of trypsin and chymotrypsin. In the
1
Ghrelin-induced elevations in plasma GH during negative energy balance could have important protein-sparing and
lipolytic effects, while elevated levels of ghrelin may stimulate hypothalamic orexigenic centers responsible for the
hyperphagic responses that occur during refeeding when growth exceeds normal rates (for discussion, see e.g. [6] and
literature cited therein).
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subsequent refeeding phase, ghrelin was down-regulated and gastricsin was up-regulated [3,4]. As
part of an integrated response to food availability in sea bass gastrointestinal tract, PEPT1
expression (see manuscript body, figure 1d) decreased during fasting and increased during
refeeding in the proximal intestine [5].
REFERENCES
1.
Terova, G., Rimoldi, S., Chini, V., Gornati, R., Bernardini, G. & Saroglia, M. 2007 Cloning
and expression analysis of insulin-like growth factor I and II in liver and muscle of sea bass
(Dicentrarchus labrax, L.) during long-term fasting and refeeding. J. Fish Biol. 70B, 219-233.
2.
Terova, G., Bernadini, G., Binelli, G., Gornati, R. & Saroglia, M. 2006 cDNA encoding
sequences for myostatin and FGF6 in sea bass (Dicentrarchus labrax, L.) and the effect of
fasting and refeeding on their abundance levels. Domest. Anim. Endocrinol. 30, 304-319.
3.
Terova, G., Rimoldi, S., Bernardini, G., Gornati, R. & Saroglia, M. 2008 Sea bass ghrelin:
Molecular cloning and mRNA quantification during fasting and refeeding. Gen. Comp.
Endocrinol. 155, 341-351.
4.
Terova, G., Rimoldi, S., Larghi, S., Bernardini, G., Gornati, R. & Saroglia, M. 2007
Regulation of progastricsin mRNA levels in sea bass (Dicentrarchus labrax) in response to
fluctuations in food availability. Biochem. Biophys Res. Comm. 363, 591-596.
5.
Terova, G., Corà, S., Verri, T., Rimoldi, S., Bernardini, G. & Saroglia, M. 2009 Impact of
feed availability on PepT1 mRNA expression levels in sea bass (Dicentrarchus labrax).
Aquaculture 294, 288-299.
6.
Picha, M.E., Strom, C.N., Riley, L.G., Walker, A.A., Won, E.T., Johnstone, W.M. & Borski,
R.J. 2009 Plasma ghrelin and growth hormone regulation in response to metabolic state in
hybrid striped bass: effects of feeding, ghrelin and insulin-like growth factor-I on in vivo and
in vitro GH secretion. Gen. Comp. Endocrinol. 161, 365-372.
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Gene expression
Myostatin (muscle)
Figure S1. Expression levels of myostatin in sea bass (Dicentrarchus labrax) muscle measured by
real-time PCR. Cytoskeletal actin was used as endogenous control. Fish were sampled before
fasting (day 0, before fasting), 4 days after fasting (4 days fasted), at the end of fasting (35 days
fasted), and then sequentially at 4, 14 and 21 days following refeeding (4 days refed, 14 days refed,
and 21 days refed). Means±SEM of three animals in each group are shown. Differences were
determined by ANOVA. Different letters indicate significantly different means (P<0.01) (modified
from [2]).
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Insulin-like Growth Factor I (liver)
Figure S2. Expression levels of insulin-like growth factor I in liver (a) and muscle (b) and insulinlike growth factor II in liver (c) and muscle (d) of sea bass (Dicentrarchus labrax) measured by
real-time PCR. mRNA copy number was normalized as a ratio to 100 ng total RNA. Fish were
sampled before fasting (day 0, before fasting), 4 days after fasting (4 days fasted), at the end of
fasting (35 days fasted), and then sequentially at 4, 14 and 21 days following refeeding (4 days
refed, 14 days refed, and 21 days refed). MeansSEM of five animals in each group are shown.
Differences were determined by ANOVA. Different letters indicate significantly different means
(ANOVA; P<0.05) (modified from [1]).
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Ghrelin (stomach)
Figure S3. Expression levels of ghrelin (a) and gastricsin (b) in sea bass (Dicentrarchus labrax)
stomach measured by real-time PCR. Ghrelin and gastricsin mRNA copy number was normalized
as a ratio to 100 ng total RNA. Fish were sampled before fasting (day 0, before fasting), 4 days after
fasting (4 days fasted), at the end of fasting (35 days fasted), and then sequentially at 4, 14 and 21
days following refeeding (4 days refed, 14 days refed, and 21 days refed). MeansSEM of five
animals in each group are shown. * indicates significant differences between treated and control
groups for each time point tested (ANOVA; P<0.05) (modified from [3] and [4]).