Nutritional assessment of somatolactin function in gilthead sea bream ( Sparus aurata): concurrent changes in somatotropic axis and pancreatic hormones

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Comparative Biochemistry and Physiol

Nutritional assessment of somatolactin function in gilthead sea bream

(Sparus aurata): concurrent changes in somatotropic axis and

pancreatic hormones

Silvia Vega-Rubın de Celisa, Pablo Rojasb, Pedro Gomez-Requenia, Amaya Albalatb,

Joaquim Gutierrezb, Francoise Medalec, Sadasivam J. Kaushikc,

Isabel Navarrob, Jaume Perez-Sancheza,*

aBiology, Culture and Pathology of Fish Species, Instituto de Acuicultura de Torre de la Sal (CSIC), 12595 Ribera de Cabanes, Castellon, SpainbDepartament de Fisiologia, Facultat de Biologia, Universitat de Barcelona, Avda. Diagonal 645, Barcelona, Spain

cLaboratoire de Nutrition des Poissons, Unitee Mixte INRA-IFREMER, 64310 Saint-Pee-sur-Nivelle, France

Received 4 February 2004; received in revised form 22 April 2004; accepted 29 June 2004

Abstract

The role of somatolactin (SL) in the regulation of energy homeostasis in gilthead sea bream (Sparus aurata) has been analysed. First, a

down-regulation of plasma SL levels in response to gross shifts in dietary amino acid profile and the graded replacement of fish meal by

plant protein sources (50%, 75% and 100%) has been observed. Thus, the impaired growth performance with changes in dietary amino

acid profile and dietary protein source was accompanied by a decrease in plasma SL levels, which also decreased over the course of the

post-prandial period irrespective of dietary nitrogen source. Secondly, we examined the effect of SL and growth hormone (GH)

administration on voluntary feed intake. A single intraperitoneal injection of recombinant gilthead sea bream SL (0.1 Ag/g fish) evoked a

short-term inhibition of feed intake, whereas the same dose of GH exerted a marked enhancement of feed intake that still persisted 1 week

later. Further, we addressed the effect of arginine (Arg) injection upon SL and related metabolic hormones (GH, insulin-like growth factor-

I (IGF-I), insulin and glucagon) in fish fed diets with different nitrogen sources. A consistent effect of Arg injection (6.6 Amol/g fish) on

plasma GH and IGF-I levels was not found regardless of dietary treatment. In contrast, the insulinotropic effect of Arg was found

irrespective of dietary treatment, although the up-regulation of plasma glucagon and glucose levels was more persistent in fish fed a fish

meal based diet (diet FM) than in those fed a plant protein diet with a 75% replacement (diet PP75). In the same way, a persistent and

two-fold increase in plasma SL levels was observed in fish fed diet FM, whereas no effect was found in fish fed diet PP75. Taken

together, these findings provide additional evidence for a role of SL as a marker of energy status, which may be perceived by fish as a

daily and seasonal signal of abundant energy at a precise calendar time.

D 2004 Elsevier Inc. All rights reserved.

Keywords: Amino acid profile; Arginine; Feed intake; Gilthead sea bream; Glucagon; Growth hormone; Insulin; Insulin-like growth factor-I; Protein source;

Somatolactin

1. Introduction

Growth hormone (GH) and prolactin (PRL) are single

chain polypeptides that were recognized as members of the

1095-6433/$ - see front matter D 2004 Elsevier Inc. All rights reserved.

doi:10.1016/j.cbpb.2004.06.007

* Corresponding author. Tel.: +34 964 319500; fax: +34 964 319509.

E-mail address: [email protected] (J. Perez-Sanchez).

same protein family many years ago. Subsequently, this

family expanded with the discovery of mammalian placental

lactogens (PLs) (Walker et al., 1991), fish somatolactin (SL)

(Rand-Weaver et al., 1991), and more recently with a trout

SL-like protein (Yang and Chen, 2003) that shares 56%

homology with trout SL. It is now recognized that the rate of

SL evolution is lower than that found for GH and PRL

genes (Forsyth and Wallis, 2002), and the contribution of

ogy, Part A 138 (2004) 533–542

Table 1

Ingredient and chemical composition of the four experimental diets with

different amino acid profiles

Ingredient (g/kg) M MGlu WB WBGlu

CPSPGa 50.0 0 0 0

Fish meal, LT (CP 70%) 499.9 478.1 529.3 484.1

Wheat gluten 0 0 100.0 100.0

Sprouted whole wheat 0 56.1 147.9 138.5

Sprouted peas (Aquatex) 199.1 0 100.0 100.0

Soybean meal (CP 42%) 121.9 300 0 0

Fish oil 83.6 94.5 89.2 94.3

Binder 10.0 10.0 10.0 10.0

Mineral premixb 10.0 10.0 10.0 10.0

Vitamin premixc 10.0 10.0 10.0 10.0

IAA mixd 15.3 16.7 3.5 0

l-Glu 0 24.6 0 53.1

Analysed composition

Dry matter (DM, %) 92.1 93.8 89.3 92.7

Crude protein (% DM) 52.7 53.0 52.1 50.9

Crude fat (% DM) 15.7 16.4 15.9 15.2

Gross energy (kJ/g DM) 22.5 22.5 22.5 22.4

IAA (% DM)d 25.9 26.4 26.1 22.5

DAA (% DM)e 22.8 25.6 26.3 27.9

IAA/DAA 1.13 1.03 0.99 0.80

a Soluble fish protein concentrate from Sopropeche (Boulogne sur Mer,

France).b Mineral premix (NRC, 1993).c Vitamin premix (NRC, 1993).d IAA: indispensable amino acids.e DAA: dispensable amino acids.

S. Vega-Rubın de Celis et al. / Comparative Biochemistry and Physiology, Part A 138 (2004) 533–542534

fish SL in sexual maturation, immune-stress response, and

environmental adaptation has been proposed for both

salmonid and non-salmonid fish (Kaneko, 1996; Calduch-

Giner et al., 1998; Bhandari et al., 2003). Further, there is

increasing evidence for an active role of SL in the regulation

of energy homeostasis in gilthead sea bream (Sparus

aurata): (I) exogenous SL treatment inhibits hepatic

lipogenic enzymes and stimulates lipid mobilisation

(Vega-Rubın de Celis et al., 2003), (II) plasma SL levels

increase with the increase of ration size and adiposity in fast

growing fish (Company et al., 2001), (III) a major peak of

circulating SL occurs following the summer replenishment

of energy stores (Mingarro et al., 2002), and (IV) long-term

feed restriction delays the autumn increase of circulating SL

levels in juvenile fish (Perez-Sanchez et al., 2002). Most of

these findings assume changes in environmental cues and

feeding rates (nutritional status). However, the involvement

of SL in the regulation of feed intake remains unexplored,

and there is no data about the effect of diet composition and

specific nutrients on SL synthesis and release.

For the particular case of gilthead sea bream, the picture

of the nutritional regulation of the somatotropic axis is more

clear, and the general view is that energy deficits in response

to changes in ration size (Company et al., 1999), protein/

energy ratio (Perez-Sanchez et al., 1995; Martı-Palanca et

al., 1996) and nutritive value of nitrogen source (Gomez-

Requeni et al., 2003, 2004) are related to an increase of

circulating GH levels, consequent to a reduced negative

feedback inhibition by hepatic insulin-like growth factor-I

(IGF-I). Several piscine studies have also reported diet

induced changes in circulating insulin and glucagon levels

(Sundby et al., 1991; Mommsen and Plisetskaya, 1991;

Capilla et al., 2002; Novoa et al., 2002). In post-prandial

state, circulating glucagon levels increase in parallel with

plasma amino acid levels in rainbow trout (Navarro et al.,

1993, 1997). Besides, arginine (Arg) is a potent secreta-

gogue of pancreatic hormones in mammals and salmonids

when injected at high doses (Mommsen et al., 2001;

Robertson et al., 2003). Arg also exerts a stimulatory action

upon mammalian GH release (Ghigo et al., 2001), but the

regulatory action of Arg and other amino acids on pituitary

hormones has been poorly studied in teleosts. Accordingly,

the aim of this work is to address in gilthead sea bream the

regulation of plasma SL levels in response to gross shifts in

nutritional status as a result of changes in dietary amino acid

profile and nitrogen source. This was done by means of two

different growth trials (trials I and II) over the course of the

summer replenishment of energy stores (first half of

summer). Subsequently, at the end of summer we addressed

the involvement of peripheral SL in the regulation of feed

intake (trial III), which decreases progressively in juvenile

gilthead sea bream over the course of the second half of the

summer. Finally, also at the end of summer, we examined

the effects of Arg injection on SL, pancreatic hormones and

somatotropic axis activity in fish fed diets with fish meal

and plant protein sources (trial IV).

2. Materials and methods

2.1. Diets

Amino acid profile: indispensable (IAA) and dispensable

(DAA) amino acid content of M and WB diets was based on

the amino acid profile of muscle (M diet) and whole body

(WB diet) of gilthead sea bream (S. aurata), respectively. In

MGlu and WBGlu diets, DAA content was increased by the

incorporation of an excess of l-glutamic acid, changing the

IAA/DAA ratio from 1.1 (M diet) to 0.8 (WBGlu diet)

(Table 1). For more details of diet composition, see Gomez-

Requeni et al. (2003).

Nitrogen source: fish meal was used as the only protein

source in diet FM. A mixture of plant ingredients (corn

gluten meal, wheat gluten, sprouted peas, rapeseed meal and

sweet white lupin meal) was used to replace fish meal at

50% (PP50 diet), 75% (PP75 diet) and 100% (PP100 diet).

Crystalline amino acids were added to plant protein-based

diets to simulate muscle IAA profile, meeting the theoretical

IAA requirement profile of gilthead sea bream (Table 2). For

more details in diet composition, see Gomez-Requeni et al.

(2004).

2.2. Trials I and II: amino acid profile and nitrogen source

In trials I and II, fish of 14–17 g initial body mass were

distributed into experimental tanks (500 l) in groups of 90

Table 2

Ingredient and chemical composition of the four experimental diets with

graded levels of fish meal replacement by plant protein sources

Ingredient (g/kg) FM PP50 PP75 PP100

Fish meal, LT (CP 70%) 703.7 352.0 176.0 0

Corn gluten meal 0 120.0 180.0 250.0

Wheat gluten 0 120.0 180.0 250.0

Sprouted peas (Aquatex) 0 60.0 90.0 120.0

Rapeseed meal (Primor 00) 0 30.9 67.2 27.3

Sweet white lupin meal 0 0 0 6.9

Extruded whole wheat 142.0 94.9 30.6 15.5

Fish oil 124.3 141.3 149.8 158.0

Binder (Na alginate) 10.0 10.0 10.0 10.0

Mineral premixa 10.0 10.0 10.0 10.0

Vitamin premixb 10.0 10.0 10.0 10.0

CaHPO4d 2H2O (18%P) 0 6.5 30.7 51.1

IAA mixc 0 44.3 65.7 91.4

Analysed composition

Dry matter (DM, %) 93.7 94.3 91.9 90.1

Crude protein (% DM) 46.1 47.5 44.8 42.8

Crude fat (% DM) 16.5 16.6 17.2 16.8

Gross energy (kJ/g DM) 20.4 21.3 21.4 21.7

IAA (% DM)c 24.6 25.5 25.7 24.8

DAA (% DM)d 23.2 21.9 22.6 23.2

IAA/DAA 1.06 1.16 1.14 1.07

a Mineral premix (NRC, 1993).b Vitamin premix (NRC, 1993).c IAA: indispensable amino acids.d DAA: dispensable amino acids.

S. Vega-Rubın de Celis et al. / Comparative Biochemistry and Physiology, Part A 138 (2004) 533–542 535

fish each. Day length and water temperature increased over

the course of trials (April–July) following natural changes at

our latitude (4085VN; 0810VE). Water temperature ranged

from 17 to 25 8C, and oxygen content of outlet water

remained higher than 85% saturation.

In trial I, fish were fed experimental diets (M, WB,

MGlu, WBGlu) with changing dietary amino acid profiles.

In trial II, fish were fed experimental diets (FM, PP50,

PP75, PP100 diets) with a graded replacement of fish meal

by plant protein sources. In both trials, each particular diet

was randomly allocated to triplicate groups of fish for 12

weeks, and feed was offered by hand to apparent visual

satiety in two meals per day (9:00 and 14:00 h).

At the end of trial I, 15 overnight fasted fish after the

evening meal were randomly selected and anaesthetised

with MS-222 (0.1 g/l) for blood sampling (10:00 h). At the

end of trial II, 12 animals per dietary treatment were

sampled at 6 h after the morning meal (15:00 h). Following

overnight fasting, 15 additional fish per dietary treatment

(trial II) were withdrawn at 10:00 h. Blood was taken from

caudal vessels with heparinized syringes, and plasma

samples were stored at �30 8C.

2.3. Trial III: SL injection

Juvenile fish grown in our experimental facilities under

natural day length and temperature conditions were main-

tained for more than 2 months with the fish meal based diet

(FM). At the end of August, water temperature still remained

high (23–24 8C) and randomly selected fish of 63 g of initial

body mass were distributed into nine 90 l tanks in groups of

10 fish each. During the acclimation period, fish continued to

be fed with the FM diet for 2 weeks. Then, fish from one

triplicate group were lightly anaesthetised with MS-222

(0.1 g/l), and intraperitoneally injected with a saline solution

(200 Al per fish). The remaining fish (two triplicate tanks)

were injected (0.1 Ag/g fish) with either recombinant trout

GH (Eurogentec, Liege, Belgium) or recombinant gilthead

sea bream SL (Company et al., 2001). After this, diet FM

was offered by hand to visual satiety at 4 and 8 h after saline

or hormone injection. Over the course of the subsequent 6-

day interval, feed was offered to satiety two times per day

(10:00 and 15:00 h). Fish were allowed to eat even at the

bottom of the tank without waste of feed. The amount of

delivered feed was registered at each feeding time. Mass gain

and feed efficiency (mass gain/feed intake) were recorded for

the entire week period.

2.4. Trial IV: Arg injection

Fish of 98–100 g initial body mass were distributed into

500 l tanks in groups of 40 fish each. Over the course of a 6-

week trial (August–September), day length andwater temper-

ature (23–25 8C) followed natural changes at our latitude.

FM and PP75 diets were offered to duplicate groups in

one meal per day (10:00 h) to visual satiety. One week

before Arg treatment, randomly selected fish were allocated

into 90 l tanks in groups of 10 fish each and continued to be

fed with the same diet. Following overnight fasting,

untreated fish (one tank per dietary treatment) were lightly

anaesthetised with MS-222 (0.1 g/l) for blood sampling at

zero time. Fish from remaining tanks (four tanks per dietary

treatment) were anaesthetised and intraperitoneally injected

with either Arg (6.6 Amol/g fish) or a saline solution. A

volume of 0.5 ml was administered, and all fish from the

same tank were sampled quickly (less than 2 min) at 3 or 6 h

after injection. Blood was taken from caudal vessels with

heparinized syringes, and centrifuged at 3000�g for 20 min

at 4 8C. Plasma samples were stored at �30 8C.

2.5. Hormone and metabolite assays

Plasma glucose levels were determined spectrophoto-

metrically using a commercial kit (Sigma 315-100). Plasma

GH and SL levels were assayed by homologous radio-

immunoassays (RIA) (Martınez-Barbera et al., 1995;

Company et al., 1999). Sensitivity and midrange of assays

were 0.1–0.15 and 2–2.5 ng/ml, respectively.

After acid–ethanol cryoprecipitation of plasma (GroPep

Standard Operating Procedure, Adelaide, Australia), the

total amount of circulating IGF-I was determined by RIA.

Recombinant red bream (Pagrus auratus) IGF-I (100%

amino acid similarity with gilthead sea bream IGF-I) was

purchased from GroPep, and used as tracer and standard as

indicated above for standard RIA procedures of gilthead sea

Fig. 1. Curve displacement in the RIA procedure of unlabeled recombinant

red bream IGF-I, recombinant gilthead sea bream IGF-I, recombinant

barramundi IGF-I, and plasma dilutions of gilthead sea bream.

S. Vega-Rubın de Celis et al. / Comparative Biochemistry and Physiology, Part A 138 (2004) 533–542536

bream GH and SL. Briefly, diluted plasma samples (100 Al)were incubated with anti-barramundi (Lates calcarifer) IGF-

I serum [GroPep; 100 Al (1:8000)] for 24 h at 14 8C; 125I-

labeled IGF-I (15,000 cpm, 100 Al) was then added.

Following a further 24-h incubation, goat anti-rabbit IgG

[Biogenesis, Dorset, UK; 100 Al (1:20)] was added as a

precipitating antibody. Bound and free labels were separated

by centrifugation, and the pellets counted in a gamma-

counter. In the RIA procedure, serial dilutions of plasma

parallel well to the standard curve of red bream IGF-I, not

different from that of recombinant barramundi IGF-I

(GroPep) and recombinant gilthead sea bream IGF-I (Fine

et al., 1997) (Fig. 1). Sensitivity and midrange of the assay

were 0.05 and 0.75 ng/ml, respectively.

Plasma insulin levels were measured by RIA, using

bonito insulin as standard and rabbit anti-bonito insulin as

Table 3

Growth performance of gilthead sea bream (S. aurata) fed the experimental diets

Trial I M MGlu

Initial body mass (g) 14.7F0.16 14.7F0.02

Final body mass (g) 74.9F0.68 70.9F1.37

Feed intake (g DM/fish) 78.6F0.96 78.7F5.11

SGR2 (%) 1.99F0.003 1.92F0.02

FE3 0.85F0.01b 0.79F0.04ab

PER4 1.61F0.03 1.51F0.08

Trial II FM PP50

Initial body mass (g) 16.4F0.07 16.7F0.11

Final body mass (g) 73.8F0.86d 69.7F0.52c

Feed intake (g DM/fish) 63.8F1.25d 52.9F0.42c

SGR2 (%) 1.86F0.01c 1.76F0.01b

FE3 0.89F0.003a 0.99F0.01b

PER4 1.91F0.01a 2.09F0.03bc

Each value is the meanFS.E.M. of triplicate groups.1 P values result from analysis of variance. Different superscript letters in ea

Newman–Keuls test, Pb0.05).2 Specific growth rate=[100�(ln final fish mass�ln initial fish mass)]/days.3 Feed efficiency=wet mass gain/dry feed intake.4 Protein efficiency ratio=wet mass gain/crude protein intake.

antiserum (Gutierrez et al., 1984). Glucagon levels were

determined by a mammalian RIA, previously validated for

several fish species including gilthead sea bream (Gutierrez

et al., 1986; Navarro et al., 1995).

2.6. Statistical analysis

Data were analysed by one-way analysis of variance,

followed by Student–Newman–Keuls test at a significance

level of Pb0.05. Tank average values of feed intake and

growth rates were used as experimental units for statistical

analysis.

3. Results

3.1. Effects of amino acid profile and nitrogen source (trials

I and II) on plasma SL levels

Feed intake and growth performance over the course of

trials I and II are summarized in Table 3. In trial I, growth

rates were not significantly different among experimental

groups, but feed efficiency was significantly lower in fish

fed with the lowest dietary IAA/DAA ratio (WBGlu diet).

In trial II, feed intake decreased and feed efficiency

increased with the fish meal replacement by plant protein

sources. Growth rates of fish fed diet PP100 were

significantly lower than in the other three experimental

groups (FM, PP50, PP75).

As shown in Fig. 2, plasma SL levels decreased with

the decrease of the dietary IAA/DAA ratio (trial I).

Thus, in overnight fasted fish, plasma SL levels in fish

fed diet M were significantly higher than in fish fed diet

WBGlu.

for 12 weeks (trials I and II)

WB WBGlu P1

14.6F0.09 14.9F0.11 0.361

72.9F1.35 70.9F1.25 0.127

77.4F1.79 79.63F4.97 0.979

1.96F0.03 1.90F0.02 0.079

0.82F0.02ab 0.74F0.006a 0.043

1.59F0.03 1.50F0.05 0.178

PP75 PP100 P1

16.5F0.19 16.4F0.24 0.52

66.5F0.50b 58.2F0.71a b0.001

47.6F0.80b 41.9F0.83a b0.001

1.72F0.01b 1.56F0.03a b0.001

1.03F0.009b 0.99F0.02b b0.001

2.16F0.02c 2.04F0.04b b0.001

ch row indicate significant differences among dietary treatments (Student–

Fig. 4. Effect of a single peripheral injection (0.1 Ag/g fish) of GH (dark

grey bars) and SL (light grey bars) on feed intake (trial III) in gilthead sea

bream (S. aurata). Saline treated fish (black bars). Each value is the

meanFS.E.M. of triplicate tanks. *Significant differences ( Pb0.05) with

saline-treated fish.

Fig. 2. Plasma somatolactin (SL) levels (overnight fasting) in fish (S.

aurata) fed the four experimental diets having different amino acid profiles

(trial I). For details of diet composition, see Section 2.1 and Table 1. Each

value is the meanFS.E.M. of 12–15 animals. Values with different letters

are significantly different ( Pb0.05).

S. Vega-Rubın de Celis et al. / Comparative Biochemistry and Physiology, Part A 138 (2004) 533–542 537

In trial II, at 6 h after the morning meal, plasma SL levels

decreased with the progressive replacement of fish meal by

plant protein sources, and significant differences were found

between fish fed FM and PP100 diets (Fig. 3A). Following

overnight fasting, the overall plasma SL concentration

decreased regardless of dietary treatment (Fig. 3B). No

significant differences were found among groups, although

the SL pattern was similar to that found at 6 h after the

morning feeding.

Fig. 3. Plasma somatolactin (SL) levels in gilthead sea bream (S. aurata)

fed graded levels of fish meal (FM) replacement by plant protein (PP)

sources from 50% to 100% (trial II). (A) SL levels at 6 h after the morning

feeding; (B) SL levels following overnight fasting (basal measurements).

Each value is the meanFS.E.M. of 12–15 animals. Values with different

letters are significantly different ( Pb0.05).

3.2. Effects of peripheral SL and GH injection on feed

intake (trial III)

As shown in Fig. 4, SL injection evoked a reduction of

feed intake during the first 24-h interval; after which, feed

intake returned to control values without significant changes

in growth rates and feed efficiency over the course of an

entire week period (Table 4). For comparative purposes,

when the same dose of GH was administered, growth rates

were two-fold increased, consequent to a marked enhance-

ment of feed intake and feed efficiency.

3.3. Effects of peripheral Arg injection on hormone plasma

levels (trial IV)

In trial IV, there were no significant differences in growth

rates between fish fed the two selected diets (FM and PP75;

Table 5). However, feed intake was lower in fish fed diet

PP75 than in those fed diet FM, and a light but not

statistically significant increase (Pb0.06) of protein effi-

ciency ratio was found in fish fed diet PP75. Under this

nutritional condition, a two-fold increase in plasma SL

levels (3, 6 h after injection) was found after arginine

injection in fish fed diet FM, whereas no effect was

observed in fish fed diet PP75 (Fig. 5A, B). Irrespective

of dietary treatment, Arg injection did not modify plasma

GH and IGF-I levels (Fig. 5C–F), although both in saline

and Arg-treated fish plasma GH levels increased over the

course of the sampling period.

The effect of Arg injection on plasma glucose and

glucagon levels is summarized in Fig. 6. Irrespective of

dietary treatment, a parallel increase in plasma glucose and

glucagon levels (Arg vs. saline treated fish) was found at 3 h

post-injection (Fig. 6A–D). In fish fed diet FM, glucose and

glucagon levels still remained high at 6-h post-injection. In

fish fed diet PP75, the initial increase of glucose and

glucagon (3-h post-injection) was less pronounced than in

fish fed diet FM, and the return to control values was faster

than in fish fed diet FM. As a result of this, significant

Table 4

Growth and feed intake regulation (trial III) after a single dose (0.1 Ag/g fish) of somatolactin (SL) and GH to gilthead sea bream (S. aurata)

Control SL GH P1

Initial body mass (g) 62.97F0.24 62.37F0.51 63.27F0.08 0.231

Final body mass (g) 66.53F1.65a 66.9F1.75a 75.13F1.55b 0.017

Cumulative feed intake (g DM/fish) 5.95F0.89a 5.68F0.58a 10.68F1.20b 0.015

SGR2 (%) 0.95F0.18a 0.99F0.29a 2.14F0.05b 0.009

FE3 0.86F0.05a 0.94F0.03ab 1.11F0.05b 0.013

Each value is the meanFS.E.M. of triplicate groups.1 P values result from analysis of variance. Different superscript letters in each row indicate significant differences among dietary treatments (Student

Newman-Keuls, Pb0.05).2 Specific growth rate=[100�(ln final fish mass�ln initial fish mass)]/days.3 Feed efficiency=wet mass gain/dry feed intake.

S. Vega-Rubın de Celis et al. / Comparative Biochemistry and Physiology, Part A 138 (2004) 533–542538

differences (Arg vs. saline treated fish) in glucose and

glucagon measurements were found at 6-h post-injection in

fish fed diet FM but not in fish fed diet PP75.

As shown in Fig. 6E,F, Arg injection induced an increase

in plasma insulin levels, although this effect was statistically

significant only at 6-h post-injection. This increase in

plasma insulin levels (Arg vs. saline treated fish) was of

the same order of magnitude with the two diets (FM and

PP75), and the resulting glucagon/insulin ratio was higher in

fish fed diet FM than in fish fed diet PP75.

4. Discussion

The present study is part of a more extensive work where

the effect of dietary amino acid profile and plant protein

supply on N-metabolism and GH-liver axis activity has

already been reported in gilthead sea bream (Gomez-

Requeni et al., 2003, 2004). Nitrogen retention decreased

with the decrease of dietary IAA/DAA ratio, and fish fed the

diet WBGlu showed increased plasma levels of GH, arising

from a reduced concentration of circulating IGF-I (Gomez-

Requeni et al., 2003). Impaired growth performance and

liver GH-desensitisation occurs in response to high plant

protein supply, and fish fed the diet PP100 (free fish meal

diet) showed a depressed hepatic expression of IGF-I and

Table 5

Growth performance of fish (S. aurata) fed FM and PP75 diets for 6 weeks

(trial IV)

FM PP75 P1

Initial body mass (g) 98.7F0.28a 100.9F0.15b 0.021

Final body mass (g) 178.9F1.35 175.4F1.86 0.265

Feed intake (g DM/fish) 96.3F2.64 82.6F1.78 0.05

SGR2 (%) 1.38F0.01 1.29F0.03 0.104

FE3 0.83F0.03 0.90F0.00 0.204

PER4 1.83F0.07 2.12F0.005 0.061

Each value is the meanFS.E.M. of duplicate groups.1 P values result from analysis of variance. Different superscript letters

in each row indicate significant differences among dietary treatments

(Student Newman-Keuls, Pb0.05).2 Specific growth rate=[100�(ln final fish mass�ln initial fish mass)]/

days.3 Feed efficiency=wet mass gain/dry feed intake.4 Protein efficiency ratio=wet mass gain/crude protein intake.

GH receptors that follows the increase in plasma GH levels,

consequent to reduced negative feedback inhibition of

pituitary GH by circulating IGF-I (Gomez-Requeni et al.,

2004). In this context, the first objective of the present work

was to analyse the effects of these feeding treatments (amino

acid profile and plant protein supply) on plasma SL levels at

a precise time of the year with high feeding rates (first half

of summer).

Fig. 5. Effect of Arg injection on plasma SL, GH and IGF-I levels of fish

(S. aurata) fed FM (A, C, E) and PP75 (B, D, F) diets (trial IV). Each value

is the meanFS.E.M. of 10 animals. *Significant differences ( Pb0.05) with

saline-treated fish.

Fig. 6. Effect of Arg injection on plasma glucose, glucagon and insulin

levels of fish (S. aurata) fed FM (A, C, E) and PP75 (B, D, F) diets (trial

IV). Each value is the meanFS.E.M. of 10 animals. *Significant differences

( Pb0.05) with saline-treated fish.

S. Vega-Rubın de Celis et al. / Comparative Biochemistry and Physiology, Part A 138 (2004) 533–542 539

A different regulation of GH and SL by diet can be

deduced from the present study; thus, the decrease in plasma

SL levels with the impairment of dietary amino acid profile

(trial I) and the replacement of fish meal by plant protein

sources (trial II) paralleled with the already reported

increase in circulating GH levels (Gomez-Requeni et al.,

2003, 2004). Previous gilthead sea bream studies also

indicate an inverse relationship between circulating SL and

GH levels, increasing plasma SL availability with the

increase of ration size and adiposity in fast growing gilthead

sea bream (Company et al., 1999). Besides, on a seasonal

basis, the spring–summer peak of GH precedes the autumn

increase of plasma SL levels, which follows the summer

replenishment of energy stores (Mingarro et al., 2002).

Therefore, it appears that fish SL and GH have evolved in

different manners, although both pituitary hormones have

retained a lipolytic action that has been demonstrated in

vivo (Vega-Rubın de Celis et al., 2003) and in primary

cultures of gilthead sea bream adipocytes (Albalat et al.,

unpublished results). If so, this recognized action in energy

mobilisation should be considered of importance in preserv-

ing energy homeostasis in fish under varying growth and

nutritional conditions.

In this regard, plasma SL levels are known to rise in

rainbow trout exposed to stressors (Rand-Weaver and

Kawauchi, 1993), but several attempts in trout (Rand-Weaver

et al., 1995) and gilthead sea bream (Company et al., 1999)

failed to demonstrate consistent changes in plasma SL levels

during long-term fasting. However, after feed deprivation,

Pottinger et al. (2003) outline that refed trout show a temporal

failure for the seasonal rise of circulating SL. This is

consistent with the observation that long-term restricted

feeding in grown up juvenile gilthead sea bream delays the

autumn rise in plasma SL levels (Perez-Sanchez et al., 2002).

To date, there is no information about daily changes in

circulating SL concentration, but the results presented herein

over the course of the post-prandial period of fish fed graded

levels of plant protein sources (trial II) indicate that plasma

SL levels a few hours after feeding are two-fold higher than

following overnight fasting (basal measurements), regardless

of the dietary treatment. According to all this, the daily and

seasonal changes in plasma SL levels may be perceived by

fish as a signal of energy availability, which may serve to

indicate that energy storage for overwintering or other

processes is accomplished by a precise calendar time (critical

window), primed then for the high energy cost processes of

life (e.g. reproductive process or puberty onset). This agrees

with the finding that mammalian leptin, a lipostat signal that

informs the central nervous system about the state of energy

reserves, is able to stimulate in vitro the release of European

sea bass SL (Peyon et al., 2003).

Consistent with the GH growth-promoting action,

exogenous GH treatment increases foraging and swimming

activity in fish (Jfnsson et al., 1996; Herbert et al., 2001).

Fish GH transgenes also show enhanced growth and

appetite (Abrahams and Sutterlin, 1999; Devlin et al.,

2001). However, as indicated by Jfnsson et al. (2003),

these GH effects are not due to a direct effect at the level of

central nervous system, which points out that the stimula-

tory effect of peripheral GH on appetite is mediated via

secondary factor(s) other than GH itself. In agreement with

these findings, we observed in the present study (trial III)

that a single intraperitoneal injection of GH at the end of

summer is able to stimulate feed intake and growth rates in

gilthead sea bream, which is still apparent 1 week later. In

contrast, the same peripheral dose of SL evoked a transient

inhibition of appetite, which is comparable to that reported

for peripheral melatonin in goldfish (Pinillos et al., 2001).

This finding agrees with a role of systemic SL as a signal of

abundant energy, although its short-term inhibitory effect on

appetite suggests that the regulation of feed intake is

sensitive to other factors other than peripheral SL. This

discrepancy illustrates the complexity of the regulation of

feeding, which can only be resolved by integrating different

modulatory mechanisms.

In order to further study the regulation of SL secretion by

metabolites and specific nutrients, we analysed at the end of

S. Vega-Rubın de Celis et al. / Comparative Biochemistry and Physiology, Part A 138 (2004) 533–542540

summer (a critical time for the seasonal up-regulation of

gilthead sea bream SL; trial IV) the effect of Arg

administration on plasma levels of SL and related metabolic

hormones (GH, IGF-I, pancreatic hormones). In mammals,

Arg treatment has a stimulatory effect on pituitary GH

release (Ghigo et al., 2001), but in the present study this

amino acid did not induce a consistent increase of

circulating GH levels. We also failed to demonstrate any

effect on plasma IGF-I levels, and it is likely that the lack of

response of the GH-liver axis is due, at least in part, to the

down-regulation of the somatotropic axis activity precisely

at the time of year when the Arg injection was performed

(September) (Perez-Sanchez et al., 1994; Mingarro et al.,

2002). In contrast, Arg injection was able to induce a fast

and persistent increase in plasma SL levels in fish fed diet

FM. This is in accordance with the fact that the seasonal rise

of plasma SL levels occurs at autumn or just before in

gilthead sea bream (Mingarro et al., 2002). However, the

achieved response depends on the feeding treatment, and no

effect of Arg injection on plasma SL levels was found in

fish fed diet PP75, which always showed a reduced feed

intake (trials II and IV) in comparison to fish fed the FM

diet. This non-responsiveness of SL to Arg treatment would

be explained by some deficit in energy status, although the

fine regulation of both SL and GH needs further research on

the nutritional and seasonal bases.

Arg has a strong insulinotropic action in mammals and

fish (Mommsen and Plisetskaya, 1991), and injections of this

amino acid have been used by several authors as a model of

hyperinsulinemia (six- to nine-fold increase) in salmonids

(Carneiro et al., 1993; Banos et al., 1999; Mommsen et al.,

2001). However, this is the first study in which this Arg has

been tested in gilthead sea bream, and the obtained results

indicate that a relatively high dose of Arg injection only

induced a two-fold increase of circulating insulin levels

regardless of dietary treatment. This poor insulin response is

similar to that found in carp (Parrizas et al., 1994; Banos et

al., 1997), which suggests a low sensitivity of pancreatic h-cells to this amino acid in these fish species.

In salmonids, when the effect of Arg treatment on insulin

and glucagon was studied simultaneously, the insulinotropic

action often predominates over glucagon stimulation (Car-

neiro et al., 1993) and glycemia decreases or does not

change (Plisetskaya et al., 1991). However, the results

presented here show a more pronounced stimulatory effect

over glucagon than insulin. Besides, the resulting increase

of glucagon and insulin ratio would mediate the hyper-

glycemic state in Arg treated fish, showing these animals

parallel plasma profiles of glucose and glucagon over time.

This observation remains valid even when comparisons are

made between fish fed FM and PP75 diets. The lowest

glucagon response in fish fed diet PP75 was concurrent with

a lower and less persistent increase in plasma glucose levels

in fish fed diet PP75. This fact contrasts with a similar

increase of insulin levels in both groups (FM and PP75

diets), which suggests a different effect of dietary treatment

on glucagon and insulin response to Arg. Furthermore, it

could also be possible that the important increase in

glucagon after 3 h would stimulate insulin levels at 6 h in

both groups. In mammals, it is generally accepted that the

up-regulation of glucagon stimulates in turn insulin secre-

tion. Indeed, when different amino acids were tested in

sheep, none was able to specifically increase glucagon

release without increasing at the same time insulin or GH

secretion (Kuhara et al., 1991).

In mammals, a stimulatory effect of insulin over plasma

IGF-I levels has been demonstrated in vivo and in vitro

(Griffen et al., 1987; Bfni-Schnetzler et al., 1991; Hurson et

al., 1995). The stimulatory action of insulin and Arg over

plasma IGF-I levels has also been demonstrated in

salmonids (Banos et al., 1999). Indeed, insulin stimulates,

synergistically with GH, the IGF-I mRNA expression in

hepatocyte cultures of coho salmon (Duan et al., 1992;

Duan and Plisetskaya, 1993). Consequently, it appears that

both GH and insulin act in concert to enhance liver IGF-I

synthesis and release. In the present study, the insulinotropic

effect of Arg treatment was moderate, and irrespective of

dietary treatment a consistent effect of Arg on plasma GH

levels was not found. So, it is not surprising that no effect of

Arg injection over plasma IGF-I levels was observed. The

already mentioned decrease in the somatotropic axis activity

in autumn could contribute to the lack of IGF-I response.

In summary, the response of SL to the reduced nutritive

value of diet is characterized by a down-regulation of

plasma SL levels, which also occurs over the course of the

post-prandial period regardless of dietary treatment. This,

together with the transient but significant reduction of feed

intake by peripheral SL, provides additional evidence for an

active role of SL as a marker of energy surplus (availability)

daily and year round. Regarding Arg injection, measure-

ments of SL, GH-IGF axis and pancreatic hormones suggest

that the achieved response depends on season and past-

nutritional story. Additional research is now underway to

integrate the regulatory action of SL with other modulatory

mechanisms of feed intake and adiposity.

Acknowledgements

This research was funded by European Union (Q5RS-

2000-30068; bPerspectives of Plant Protein Use in Aqua-

cultureQ, PEPPA) and Spanish projects (PB98-672-02 and

AGL 2001-2903). S.V.-R. was recipient of a research grant

from the Spanish National Research Council (CSIC, I3P

Program).

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