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Cysteamine improves growth
and the GH/IGF axis in gilthead
sea bream (Sparus aurata): in
vivo and in vitro approaches
Albert Sa
´nchez-Moya
1
, Sara Balbuena-Pecino
1
,
Emilio J. Ve
´lez
1
†
, Miquel Perello
´-Amoro
´s
1
, Irene Garcı
´a-Meila
´n
1
,
Ramo
´nFontanillas
2
, Josep Àlvar Calduch-Giner
3
,
Jaume Pe
´rez-Sa
´nchez
3
, Jaume Ferna
´ndez-Borràs
1
,
Josefina Blasco
1
and Joaquin Gutie
´rrez
1
*
1
Department of Cell Biology, Physiology and Immunology, Faculty of Biology, Universitat de
Barcelona, Barcelona, Spain,
2
Skretting Aquaculture Research Centre, Stavanger, Norway,
3
Nutrigenomics and Fish Growth Endocrinology Group, Institute of Aquaculture Torre de la Sal (IATS,
Spanish National Research Council (CSIC)), Castello
´n, Spain
Aquaculture is the fastest-growing food production sector and nowadays
provides more food than extractive fishing. Studies focused on the
understanding of how teleost growth is regulated are essential to improve fish
production. Cysteamine (CSH) is a novel feed additive that can improve growth
through the modulation of the GH/IGF axis; however, the underlying
mechanisms and the interaction between tissues are not well understood. This
study aimed to investigate the effects of CSH inclusion in diets at 1.65 g/kg offeed
for 9 weeks and 1.65 g/kg or 3.3 g/kg for 9 weeks more, on growth performance
and the GH/IGF-1 axis in plasma, liver, stomach, and white muscle in gilthead sea
bream (Sparus aurata)fingerlings (1.8 ± 0.03 g) and juveniles (14.46 ± 0.68 g).
Additionally, the effects of CSH stimulation in primary cultured muscle cells for 4
days on cell viability and GH/IGF axis relative gene expression were evaluated.
Results showed that CSH-1.65 improved growth performance by 16% and 26.7%
after 9 and 18 weeks, respectively, while CSH-3.3 improved 32.3% after 18 weeks
compared to control diet (0 g/kg). However, no significant differences were
found between both experimental doses. CSH reduced the plasma levels of GH
after 18 weeks and increased the IGF-1 ones after 9 and 18 weeks. Gene
expression analysis revealed a significant upregulation of the ghr-1, different
igf-1 splice variants, igf-2 and the downregulation of the igf-1ra and b, depending
on the tissue and dose. Myocytes stimulated with 200 µM of CSH showed higher
cell viability and mRNA levels of ghr1,igf-1b,igf-2 and igf-1rb compared to
control (0 µM) in a similar way to white muscle. Overall, CSH improves growth
and modulates the GH/IGF-1 axis in vivo and in vitro toward an anabolic status
through different synergic ways, revealing CSH as a feasible candidate to be
included in fish feed.
KEYWORDS
cysteamine, gilthead sea bream, myocyte, GH, IGF, somatotropic axis, aquaculture,
feed additive
Frontiers in Endocrinology frontiersin.org01
OPEN ACCESS
EDITED BY
Paula Gabriela Vissio,
University of Buenos Aires, Argentina
REVIEWED BY
Fabia
´n Canosa,
CONICET Institute of Biotechnological
Research (IIB-INTECH), Argentina
Pedro Go
´mez Requeni,
BioMar, Denmark
*CORRESPONDENCE
Joaquin Gutie
´rrez
jgutierrez@ub.edu
†
PRESENT ADDRESS
Emilio J. Ve
´lez,
Université de Pau et des Pays de l’Adour,
E2S UPPA, Institut National de Recherche
pour l’Agriculture, l’Alimentation et
l’Environnement (INRAE), UMR1419
Nutrition Métabolisme et Aquaculture,
Saint-Pée-sur-Nivelle, France
RECEIVED 24 April 2023
ACCEPTED 09 June 2023
PUBLISHED 20 July 2023
CITATION
Sa
´nchez-Moya A, Balbuena-Pecino S,
Ve
´lez EJ, Perello
´-Amoro
´sM,
Garcı
´a-Meila
´nI,Fontanillas R,
Calduch-Giner JA, Pe
´rez-Sa
´nchez J,
Ferna
´ndez-Borràs J, Blasco J and
Gutie
´rrez J (2023) Cysteamine improves
growth and the GH/IGF axis in
gilthead sea bream (Sparus aurata):
in vivo and in vitro approaches.
Front. Endocrinol. 14:1211470.
doi: 10.3389/fendo.2023.1211470
COPYRIGHT
©2023Sa
´nchez-Moya, Balbuena-Pecino,
Ve
´lez, Perello
´-Amoro
´s, Garcı
´a-Meila
´n,
Fontanillas, Calduch-Giner, Pe
´rez-Sa
´nchez,
Ferna
´ndez-Borràs, Blasco and Gutie
´rrez. This
is an open-access article distributed under
the terms of the Creative Commons
Attribution License (CC BY). The use,
distribution or reproduction in other
forums is permitted, provided the original
author(s) and the copyright owner(s) are
credited and that the original publication in
this journal is cited, in accordance with
accepted academic practice. No use,
distribution or reproduction is permitted
which does not comply with these terms.
TYPE Original Research
PUBLISHED 20 July 2023
DOI 10.3389/fendo.2023.1211470
1 Introduction
Somatic growth in vertebrates is the result of the positive
balance between metabolic and hormonal stimuli, such as the
ones promoted by diet and the growth hormone (GH)/insulin-
like growth factor (IGF) axis at both systemic and local level, faced
to negative regulators, as somatostatin (SS) and myostatins (1–4).
GH is a peptide hormone synthetized and released by somatotroph
cells located in the adenohypophysis. GH secretion is modulated by
a complex system of stimulators and inhibitors composed of more
than 30 molecules among peptides, hormones and
neurotransmitters. Some of the most important positive
regulators are the growth hormone releasing hormone (GHRH),
ghrelin and some amino acids, and the negative ones the SS and the
IGF feedback system (2,5–7). The pulsatile nature of GH and IGF
secretion on a daily and seasonal basis has been largely established
in fish and other higher vertebrates and the frequency and
magnitude of secretion can be modulated by some factors as age,
environmental conditions, diet, stress, adiposity and exercise,
among others etc (4,7–11). Once the GH is released to plasma, it
can bind to GH binding proteins or its ubiquitous distributed
receptors (GHR), triggering tissue growth, modulating fuel
mobilization and utilization, and promoting the synthesis of
downstream molecules (1).
GH has pleiotropic effects and one of the most important is the
stimulation of the IGFs (IGF-1 and IGF-2) synthesis and secretion
by the liver, the main endocrine IGF source, but also in other tissues
including the muscle, in which IGFs exert both paracrine and
autocrine functions (1,2,4,12). IGF-1 and IGF-2 are delivered
by the IGF binding proteins (IGFBPs), which facilitate their
transport, increase their half-life and modulate the IGF actions,
depending on the physiological context (1,13,14). These IGFs are
then recognized by its receptors (IGF-1Ra and IGF-1Rb), initiating
the signaling by the mitogen activated protein kinase (MAPK)/ERK
and the phosphatidylinositol 3-kinase (PI3K)/AKT pathways (4,15,
16). IGFs modulate nutrient metabolism and promote cell
proliferation and differentiation with a relevant effect on muscular
tissue. These mitogenic an anabolic effects result in an improved
somatic growth (1,14,15,17).
SS family is an antique but diverse cluster of peptides that
presents a key disulfide bond between cysteines, with somatostatin-
14 and -28 as its active forms. Furthermore, different studies suggest
that other members of this family (prosomatostatins) can play a
significant role during zebra fish development (18).It is well known
that SS is one of the main inhibitors of GH production and secretion
in the adenohypophysis (2,19). Furthermore, SS reduces the
production of IGFs as well as the sensitivity of peripheral tissues
to both GH and IGFs by downregulating their corresponding
receptors. SS is mainly produced in hypothalamus and
gastrointestinal tract, carrying out an important role in systemic
and local control of growth. This facilitates the coordination with
other processes such as development, metabolism and
reproduction, resulting in a harmonic growth (19–21).
Cysteamine (2-Aminoethane-1-thiol; CSH) is the simplest
aminothiol endogenously synthesized by animal cells during the
cysteine and coenzyme A degradation, being rapidly metabolized
and excreted by the organism (22,23). CSH is the biosynthetic
precursor of hypotaurine, which is rapidly converted to taurine, a
semi-essential amino acid particularly important in carnivore
nutrition (24–26). In addition, CSH promotes the transport of L-
cysteine into cells, which will be used, among others, to synthesize
glutathione, the main endogenous antioxidant of the organism.
CSH has commonly been administered as a salt form (i.e., as
hydrochloride and bitartrate) for therapeutic purposes including
the treatment of cystinosis, skin lightener and as a radioprotective
agent. Additionally, CSH has been used in higher doses to provoke
digestive ulcers for animal research purposes, since it is capable to
increase the secretion of gastrin, gastric acid and ghrelin, and the
reduction of angiogenesis, enabling ulceration instead of wound
healing (23,27–29). Its properties are mainly due to its thiol group,
which can act as an antioxidant and reducing agent, breaking up the
disulfide bounds as it occurs between cysteines (22,23,27).
Some studies demonstrated that CSH drops SS in different
tissues, being suggested the disulfide bond-breaking mechanism as
the responsible of its depletion, although this model is not well
established yet (30,31). Furthermore, it was observed that the in vivo
administration of CSH increased the levels of GH, IGF-1 and their
receptors in different tissues in mammals and fish. The synergic effect
of this SS reduction and the improvement of the GH/IGF axis is
reflected in a boosted growth in several species (32–34). Nevertheless,
there have also been reported some negative effects in endocrine
control of growth in CSH treated groups in a dose-dependent and
temporal manner, with a reduction of GHRH and depression of the
GH/IGF system in rat, swine and sheep (8,35–38). In this sense, it has
been suggested that the increased levels of plasmatic GH associated to
moderate CSH doses, are consistent with SS depletion, but the
mechanism why GH secretion is disrupted with higher CSH doses
is not well-understood (8,37). Many of the previous studies in
vertebrates were made using in vivo models, where most of the
parameters studied were globally influenced by the interaction among
tissues. Nevertheless, the information about the local and specific
effects of CSH, like those that we could study in vitro, remains scarce.
CSH has been supplemented in cell media for mammalian oocyte
maturation (39), but the effects of CSH on muscle cells is still
unknown. CSH has been proposed as a growth promoter additive
in animal nutrition, including sheep, chicken, yak and pig (38,40,41)
and recently, also in different fish species, such as in red tilapia
(Oreochromis niloticus), orange-spotted grouper (Epinephelus
coioides) and common carp (Cyprinus carpio)(42–44). However,
there is not a consensus about the underlying mechanisms and the
optimal inclusion dose, due to its association with several negative
effects on growth, hormonal regulation, oxidative stress and welfare
(27,44–46).
Overall, it is suggested that CSH could be a useful additive in
animal nutrition since it could improve growth through diverse and
synergic mechanisms. Nevertheless, it is necessary to determine the
most efficient CSH inclusion level in diet considering the
physiological characteristics of each species and the growth stage,
as to the apparition of adverse effects could be close to the
ideal dosage.
In this context, the objective of this study was to investigate for
the first time in gilthead sea bream (Sparus aurata), one of the most
Sa
´nchez-Moya et al. 10.3389/fendo.2023.1211470
Frontiers in Endocrinology frontiersin.org02
important fish in Mediterranean aquaculture, the effects of an in
vivo and in vitro CSH supplementation at different doses on somatic
growth, the GH/IGF axis and myocytes viability.
2 Materials and methods
2.1 Experimental diets
The diets used in this trial were based on a practical commercial
diet for gilthead sea bream at these stages of growth. Five
experimental diets were formulated and produced by Skretting
Aquaculture Research Center (Skretting-ARC, Stavanger,
Norway), by an extrusion process with a 30% of fish meal and 9%
of fish oil to fulfill the essential nutritional requirements. In detail,
for the Phase 1 of the trial, two diets of 1 mm pellet size were used:
an unsupplemented one as control diet (Control), and the same one
but with the addition of 1.65 g of cysteamine hydrochloride (CSH;
Ref. 30080-100G; Sigma-Aldrich, Tres Cantos, Madrid) per Kg of
feed (CSH-1.65). The diets for the Phase 2 were the same Control
and CSH-1.65 diets used in the Phase 1 but with the pellet size
adjusted for bigger fish (1.8 mm). Furthermore, a new experimental
group was added in Phase 2, with the CSH dose doubled at 3.3 g
CSH/Kg of feed (CSH-3.3). All the diets within the same phase were
isolipidic and isonitrogenous. The detailed formulation of diets is
shown in Table 1.
2.2 Animals and ethic statement
Approximately one thousand gilthead sea bream fingerlings
provided by Piscimar (Burriana, Spain) with 1.8 ± 0.3 g of body
weight, were randomly distributed in six circular tanks of 200 L and
three tanks of 400 L at the same initial biomass density of 0.75 g
fish/L, and maintained in the fish facilities of the Scientific and
Technological Centers, Faculty of Biology, University of Barcelona.
The tanks are part of a semi-closed recirculation system with a
constant 36‰salinity, 22 ± 1 °C and under a 13 h light/11 h
dark photoperiod.
For the Phase 1, the fish of two 200 L tanks and one 400 L were
fed with the control diet (Control, n = 3) and four 200 L tanks and
two 400 L with the experimental diet (CSH-1.65, n = 6) for nine
weeks. According to fish requirements, the fish were fed with a daily
3.5% ration distributed in four meals per day during the first five
weeks, and the next four weeks at 3% ration allocated in three meals
per day.
Once the Phase 1 ended and the corresponding biometric data
and samples were obtained as detailed below, the remaining fish
were recovered and returned to their respective tanks maintaining
similar biomass density to later start Phase 2. Pellet composition
and size were slightly adjusted for this Phase 2 (Table 1) and fish
were adapted to the new feed mixing them 50/50 for five days. The
half of the tanks that were fed with the CSH-1.65 diet in Phase 1 had
their CSH dose doubled with the new CSH-3.3 diet, whereas
Control and the half of CSH-1.65 tanks remained in the same
diet that in the Phase 1. The feeding ration was set at 2.5% the first
three weeks, 2% the following three weeks and, finally, 1.75% de last
two weeks, distributed in three meals per day. Hence, each
condition for the Phase 2 (Control, CSH-1.65 and CSH-3.3) had
two 200 L and one 400 L tanks (n = 3).
2.3 Biometric parameters and sampling
AttheendofthePhase1andPhase2allfish were fasted overnight
and properly anesthetized with MS-222 (100 mg/L) (Sigma-Aldrich)
before being measured and weighed. The technical replicate for
biometric data and indexes was the mean of the tank. Besides, three
to five samples/tank were taken from the caudal vein for hormone
analysis. Then, twenty fish of each 200 L tank and forty fish of 400 L
tank were sacrificed by anesthetic overdose (300 mg/L), confirmed by
decapitation, and the tissues were extracted and weighted for the
calculation of somatic indexes: SpecificGrowthRate(SGR)=[ln(final
body weight) –ln (initial body weight)] * (days)
-1
* 100; Condition
Factor (CF) = (body weight/body length
3
) * 100; Viscerosomatic Index
(VSI) = (viscera weight/body weight) * 100; Hepatosomatic Index
(HSI) = (liver weight/body weight) * 100; Mesenteric Fat Index (MFI)
= (mesenteric fat weight/body weight) * 100. For the relative gene
expression analyses of the Phase 2, fourteen fish of each condition (four
TABLE 1 Ingredients and proximal composition of the diets.
Phase 1 (1 mm) Phase 2 (1.8 mm)
Control CSH-
1.65 Control CSH-
1.65
CSH-
3.3
Ingredient (%)
Wheat 19.89 24.89
Corn gluten 9 6
Wheat gluten 9.4 7.13
Soya concentrate 20 20
Fish meal 30 30
Fish oil 9.01 9.31
DL-Methionine 0.17 0.2
Phosphate 1.52 1.69
Antioxidant 0.03 0.03
Vitamin premix 0.02 0.02
Cysteamine HCl 0 0.165 0 0.165 0.33
Proximal composition (%)
Dry matter (DM) 91.99 91.77
Moisture 8.01 8.24
Protein (% DM) 48 45
Lipid (% DM) 15 15
Starch (% DM) 7.49 7.53
Ash (% DM) 12.98 15.33
Sa
´nchez-Moya et al. 10.3389/fendo.2023.1211470
Frontiers in Endocrinology frontiersin.org03
to six samples/tank) were sampled and the white muscle, liver and
stomach were immediately frozen in liquid nitrogen and stored at -80°
C until further analysis.
2.4 Plasma GH and IGF-1
GH concentration in plasma was assayed by a homologous
gilthead sea bream radioimmunoassay in accordance with
Martınez-Barberaet al. (1995) (44). The sensitivity and midrange
(ED50) of the assay were 0.15 and 1.8 ng/ml, respectively. Plasma
IGFs were extracted by the acid–ethanol cryoprecipitation (45), and
the IGF-1 concentration was measured by means of a generic fish
IGF-1 RIA validated for the Mediterranean perciform fish (46). The
sensitivity and mid-range of the assay were 0.05 and 0.7–0.8 ng/
mL, respectively.
2.5 Primary culture of myocytes and
experimental treatments
Seven independent white muscle satellite cell cultures were
performed following the protocol previously described by
Montserrat et al. (2007) (47). Briefly, around 40 juvenile fish (5 to
15 g/fish) supplied by local hatchery were used for each cell culture.
The fish were sacrificed by a blow to the head and their external
surfaces were sterilized. Then, fish were dissected and the epaxial
white muscle tissue was collected in cold buffered Dulbecco’s
Modified Eagle’s Medium (DMEM), containing 1% (v/v)
antibiotic/antimycotic solution and supplemented with 15% (v/v)
horse serum (HS). Subsequently, muscle was minced to small
fragments and centrifuged (3000 rcf, 5 min), washed and
enzymatically digested with 0.2% collagenase type IA. The
obtained suspension was centrifuged and the pellet washed,
resuspended, triturated by repeated pipetting and centrifuged.
After that, the tissue fragments were digested twice with 0.1%
trypsin solution prepared in DMEM and gentle agitation. After
each digestion the remained fragments were pelleted (300 rcf, 1
min) and diluted in complete medium (DMEM supplemented with
15% of HS) to block trypsin activity. Then, the supernatant was
centrifuged (300 rcf, 20 min) and the obtained pellet resuspended,
forced to trituration by pipetting and then, the suspension was
filtered first on a 100 mm, and subsequently on a 40 mm nylon cell
strainer, and finally centrifuged one last time (300 rcf, 20 min).
After that, the obtained cells were diluted in growth media (DMEM
supplemented with 10% fetal bovine serum and 1% of antibiotic-
antimycotic solution) and seeded at a final density of 2105 cells/cm
2
in poly-L-lysine and laminin precoated 6-well plates (9.6 cm
2
/well)
for gene expression or 12-well plates (2.55 cm
2
/well) for the viability
assay. Cells were incubated at 23°C and 2.5% CO
2
in growth
medium and medium was changed every 2 days.
CSH (Ref. 30080-100G; Sigma-Aldrich) was diluted in culture
medium at doses of 50, 200, 400 and 800 µM and applied at day 4
for 96 h (until day 8 of cell culture development) to determine cell
viability (n = 7) and select the most appropriate one. Once the dose
of 200 µM was selected, myocytes at day 4 were incubated with CSH
at 200 µM for 96 h to evaluate gene expression (n = 7). These days
were chosen due to cells retain the ability to proliferate but also have
the capacity to start fusing and differentiating (47). This is
supported by data reported in previous publications (15,47–51).
In the control group, the cells were not incubated with CSH.
2.6 Cell viability assay
The methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay
was used to assess cell viability as explained before (47). Briefly, after
CSH exposure, cells were washed twice and incubated for 5 h in
DMEM with a final concentration of 0.5 mg/mL of MTT (M5655,
Sigma-Aldrich). Cells were washed with PBS and the blue formazan
crystals were allowed to resuspend in DMSO. The viability values
were obtained from the absorbance measured at 570 nm in
duplicate 96-wells, with correction at 650 nm, using a microplate
reader (Tecan Infinite M200, Männedorf, Switzerland). The value
from cells containing PBS instead of MTT was also subtracted. Data
are presented as a fold change relative to the control group (0 µM)
of each independent cell culture (n = 7 independent cultures).
2.7 RNA extraction, cDNA synthesis and
qPCR analysis
Total RNA was extracted from stomach (~30 mg), liver (~30
mg) and white muscle (~100 mg) (n = 14) homogenates as
described in Sanchez-Moya et al. (2022) (52), or from cell
samples collected in triplicate wells of a 6-well plates at day 8
with 1 ml of TRI Reagent®Solution (Applied Biosystems,
Alcobendas, Spain). In the case of the in vivo samples, Precellys®
Evolution Homogenizer cooled at 4°C with Cryolys®(Bertin
Technologies, Montigny-le–Bretonneux, France) was used. RNA
extraction and purification was conducted following the
manufacturer’s recommendations. RNA concentration and purity
was determined using the NanoDrop 2000 (ThermoScientific,
Alcobendas, Spain). RNA integrity was verified in a 1% agarose
gel stained with 3% SYBR Safe DNA gel stain (ThermoScientific)
observing the banding pattern of 28S:18S ribosomal RNA.
Previously to reverse transcription, samples were treated with
DNase I (Life Technologies, Alcobendas, Spain) following the
manufacturer’s recommendations to remove any traces of
genomic DNA. Lastly, cDNA synthesis was carried out from 1 µg
of RNA with the Transcriptor First Strand cDNA Synthesis Kit
(Roche, Sant Cugat del Valles, Spain), using random hexamer
primers and anchored oligo(dT)15.
Genes chosen for gene expression analysis were key
components of the GH/IGF axis, namely GHRs (ghr-1, ghr-2),
IGFs (igf-1a, igf-1b, igf-1c, igf-2), IGF receptors (igf-1ra, igf-1rb)
and IGFBPs (igfbp-1a, igfbp-2a, igfbp-4). Relative mRNA expression
analyses were performed via qPCR from the cDNA samples,
following the MIQUE’s guidelines in Hard-Shell®384-well PCR
plates and a CFX384TM Real-Time System (Bio-Rad, El Prat de
Llobregat, Spain). The analyses were performed in triplicate using
2.5 mL of the iTaq Universal SYBR Green Supermix (Bio-Rad), 250
Sa
´nchez-Moya et al. 10.3389/fendo.2023.1211470
Frontiers in Endocrinology frontiersin.org04
nM of both the forward and reverse primers, and 1 mL of diluted
cDNA for each sample made up to a final volume of 5 mL. The
reactions consisted of an initial denaturation step of 3 min at 95°C,
40 cycles of 10 s at 95°C, 30 s at 60–69°C (primer dependent)
followed by an amplicon dissociation analysis from 55 to 95°C at a
0.5°C increase every 30 s. The sequences, melting temperatures and
GenBank accession numbers of the primers used in the Real-Time
quantitative PCR analysis are displayed in Supplementary Table S1.
The mRNA expression of each target gene was calculated relative to
the geometric mean of the two most stable reference genes from the
four that were determined for each of the tissues and the in vitro
samples according to the geNorm algorithm implemented in the
Bio-Rad CFX Manager v. 3.1. software. The different housekeeping
genes tested were ribosomal protein S18 (rps18), elongation factor 1
alpha (ef1a), beta-actin (b-actin) and ribosomal protein L27 (rpl27).
2.8 Statistical analyses
Statistical analyses were performed with IBM SPSS Statistics
v.22 (IBM, Armonk, USA) software whilst all the figures were
prepared with GraphPad Prism v.7 (GraphPad Software, La Jolla
California USA, www.graphpad.com). Previous to statistical
comparison among treatments, all data was tested for normality
and homoscedasticity by Shapiro-Wilk and Levene’stests,
respectively, and the identification of outliers was assessed by
IQR’s. When the groups compared were two, as in the Phase 1 of
the in vivo trial and the in vitro gene expression analyses, a Student’s
t-test was done. When there were three or more groups, as in Phase
2, tissue relative expression and MTT assay, a one-way analysis of
variance (one-way ANOVA) followed by Tukey’spost hoc test were
carried out. Data is presented as mean ± S.E.M. and statistical
differences were considered significant when p-value < 0.05.
3 Results
3.1 Growth and hormone analysis
Growth performance and somatic indexes are shown in Table 2.
Fish fed with CSH presented larger final body weight compared to
the Control group regardless of the phase and dose. The CSH-1.65
group weighed +16% after Phase 1 and +26.7% after Phase 2,
compared to the Control group respectively. The highest weight
gain was found in the CSH-3.3 fed group, with a +32.2% of
increment respect to the Control fish. Total length also increased
in proportion to weight in all the CSH fed fish, which resulted in no
differences for CF. SGR was higher in Phase 1 for CSH-1.65
compared to Control; but in Phase 2, even though this group
presented a higher value (2.02 vs 2.15), the differences were not
significant. On the other hand, CSH-3.3 got the highest value (2.29),
which was significantly different to that of Control group. No
differences were found for VSI and HSI after Phase 1 and 2. MFI
were increased after Phase 2 in CSH-fed groups in a dose-
dependent manner, showing a proportionally higher fat
deposition in fish fed with CSH-3.3 diet.
The plasmatic levels of GH and IGF-1 were affected by diet and
are shown in Table 2. GH concentration did not show differences
between the Control and the CSH-1.65 group in Phase 1. However,
in Phase 2, GH levels were reduced in fish fed with CSH in a dose-
dependent manner. Contrarily to GH, IGF-1 levels were increased
significantly in Phase 1 and Phase 2 in those groups fed with
CSH-1.65.
3.2 qPCR in white muscle, liver
and stomach
Relative gene expression in liver, stomach and white muscle at the
end of Phase 2 are represented in Figure 1.Inliver,ghr-1 expression
was slightly reduced and increased in CSH-1.65 and CSH-3.3,
respectively, compared to control, showing significant differences
between both treated groups. Regarding to igf-1 expression, igf-1a
was increased in both doses and the expression of total igf-1 was only
significantly raised at 3.3 dose. Moreover, the binding protein igfbp-2a
was reduced in CSH-1.65 group compared to control, whereas CSH-
3.3 had an intermediate value. No differences were foundfor ghr-2, igf-
1b, igf-1c, igf-2, igf-1ra, igf-1rb and igfbp-4.
Concerning stomach, CSH supplementation significantly
enhanced the transcript levels of ghr-1 in CSH-3.3 group in a
similarmannertowhitemuscle.Igf-1b presented significant
differences between the treated groups, with a reduction for the
CSH-1.65 respect to the CSH-3.3. On the other hand, the expression
of the receptors igf-1ra and igf-1rb were lessened, but only
significantly,fortheCSH-1.65dietcomparedtocontrol.No
changes were observed for ghr-2, igf-1a, igf-1c, total igf-1, igf-2,
igfbp-1a and igfbp-4.
Relative gene expression in white muscle was also modulated by
CSH supplementation in diet. The mRNA levels of the receptor ghr-
1and the igf-2 were increased in both groups fed with CSH respect
to the Control group. Interestingly, the igf-1 splice variant igf-1a
doubled its value in the fish fed with the CSH-1.65 dose but not with
the CSH-3.3 one, compared to Control. Contrarily to this profile,
the igf-1ra was downregulated for the CSH-1.65 whereas CSH-3.3
did not change. There were no differences for ghr-2, igf-1b, igf-1c,
total igf-1, igf-1rb, igfbp-1a and igfbp-4 among groups.
3.3 Cell viability
MTT reduction capacity, as indicator of cell viability, of
myocytes exposed to increasing CSH concentrations is shown in
Figure 2. The CSH 200 µM concentration obtained the highest
viability relative value; however, higher doses gradually decreased
cell viability until complete cell death occurs at 2 mM (data
not shown).
3.4 qPCR in myocytes
Relative gene expression of GH/IGF system in primary cultured
myocytes exposed to CSH 200 µM from day 4 to day 8 of cell culture
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is shown in Figure 3. In presence of CSH, the ghr-1, the splice
variant igf-1b, the igf-2 as well as igf-1rb were upregulated compared
to the Control group. Nevertheless, no differences were found for
ghr-2, igf-1a, igf-1c, total igf-1 and igf-1ra.
4 Discussion
Feed is the main expenditure in fish production and its cost is
being raised due to the increase of raw material price. This, together
with the general upward trend to substitute fish meal for more
sustainable alternatives including vegetable, insect and waste-
derived ingredients, has entailed to increase the research for novel
feedstuff without compromising fish growth, quality and
competitiveness (53). The use of natural and functional additives
has particular interest due to its reduced inclusion level in fish feeds
and their attributes as immunostimulants, antioxidants, stress
reducers, digestion facilitators and growth promoters (54–57). In
this context, CSH appears as a potential candidate to become a food
additive to optimize the production of aquaculture species given
that CSH has been proved as a growth promoter in some terrestrial
vertebrates, and to a lesser extent, in fish (e.g., common carp, red
tilapia and orange-spotted grouper) (42–44,58,59).
Notwithstanding, CSH has also been associated to detrimental
effects including digestive ulcers, oxidative stress, reduction of
growth and hormonal downregulation of the GH/IGF axis,
making the inclusion range narrow (8,27,33,37,44). In the
present work we have demonstrated that the inclusion of 1.65 g
of CSH/Kg of feed improved the growth by + 16.1% in 9 weeks
(Phase 1) and + 26.7% after 18 weeks (Phase 2) in gilthead sea
bream, without modifying somatic indexes. On the other hand,
when fish ate the doubled CSH-dose feed (3.3 g/Kg) some
differences appeared, as a slightly increased body weight (+
32.2%), and significantly higher SGR and MFI compared to
Control. Therefore, an increasing dose of CSH boosted growth
and visceral fat deposition but, at the same time, the low one seemed
to have a better relation cost/effect in 9 weeks of trial (Phase 2). It
has to be pointed that those differences could be augmented with
prolonged periods or higher doses. In line with the obtained results,
Li et al. (2013) (44) found that the best CSH inclusion in feed for
orange-spotted grouper in an eight weeks trial was 3 g/Kg, with
significant lower growth for the 1, 2 and the 4 g/Kg doses, although
all of them induced greater growth compared to the control group.
This indicated that the optimal point in this species was between the
3 and 4 g/Kg doses. Tse et al. (2009) (43) found similar results on
common carp with increased growth with 1, 2 and 3 g/Kg doses.
Gonzalez-Plasus et al. (2019) (46) additionally showed marked
detrimental effects as reduced growth, deformities and mortality
at a dose of 10 g/Kg in common carp. On the other hand, Wardani
et al. (2020) (34) found that the optimal dose for tilapia was 0.59 g/
Kg. Overall, there is not a consensus about the optimal and toxic
CSH dosages in fish, which seem to be species-specificand,
consequently, it would depend on their main nutritional
requirements and their digestive tract physiology. Thus, as CSH
effects are mainly dose-dependent, this highlights the need of fine-
tuning CSH inclusion in diets.
TABLE 2 Growth performance, somatic indexes and hormone levels of gilthead sea bream juveniles (Sparus aurata) supplemented with cysteamine
hydrochloride after 9 and 18 weeks.
Phase 1 (week 1 to 9) Phase 2 (week 10 to 18)
Control CSH-1.65 Control CSH-1.65 CSH-3.3
n=3 n=6 n=3 n=3 n=3
IBW (g) 1.77 ± 0.03 1.81 ± 0.03 13.13 ± 0.4 b15.4 ± 0.4 a14.85 ± 0.3 a
IBL (cm) 4.56 ± 0.04 4.63 ± 0.03 8.23 ± 0.07 b8.6 ± 0.02 ab 8.47 ± 0.08 a
FBW (g) 12.84 ± 0.25 14.9 ± 0.1*** 41.4 ± 0.5 b52.45 ± 1 a54.74 ± 1 a
BL (cm) 8.17 ± 0.07 8.5 ± 0.03*** 11.7 ± 0.04 b12.65 ± 0.04 a12.55 ± 0.06 a
CF
1
2.34 ± 0.03 2.39 ± 0.02 2.57 ± 0.03 2.61 ± 0.08 2.75 ± 0.03
SGR
2
3.25 ± 0.05 3.46 ± 0.03** 2.015 ± 0.04 b2.15 ± 0.04 ab 2.29 ± 0.07 a
VSI
3
7.89 ± 0.1 7.76 ± 0.06 6.03 ± 0.31 5.76 ± 0.1 6.18 ± 0.14
HSI
4
1.94 ± 0.12 1.91 ± 0.03 1.37 ± 0.05 1.29 ± 0.08 1.47 ± 0.04
MFI
5
1.04 ± 0.13 1.05 ± 0.06 0.81 ± 0.03 b0.99 ± 0.05 ab 1.11 ± 0.08 a
n = 10 n = 12-13 n = 13 n = 13 n = 14
GH (ng/ml) 1.72 ± 0.24 1.84 ± 0.23 1.66 ± 0.44 a0.96 ± 0.28 ab 0.36 ± 0.14 b
IGF-1 (ng/ml) 13.83 ± 2.01 22.53 ± 3.23* 16.88 ± 1.72 a23.37 ± 1.96 b21.71 ± 1.7 ab
IBW, initial body weight; IBL, initial body length; FBW, final body weight; BL, body length; GH, growth hormone; IGF-1, insulin-like growth factor 1;
1
Condition Factor (CF) = (body weight/
body length
3
) * 100;
2
Specific Growth Rate (SGR) = [ln (FBW) –ln (IBW)] * (days)
-1
* 100;
3
Viscerosomatic Index (VSI) = (viscera weight/body weight) * 100;
4
Hepatosomatic Index (HSI) =
(liver weight/body weight) * 100;
5
Mesenteric Fat Index (MFI) = (mesenteric fat weight/body weight) * 100.
Significant differences were evaluated by a t-test (p-value: **<0.01; ***<0.001) for Phase 1 and one-way ANOVA followed by a Tukey’s post hoc test for Phase 2. Different letters in the same raw
indicate significant differences between groups.
Data are shown as mean ± S.E.M. Tank was used as a technical replicate for the biometric data.
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The mechanism by which the CSH enhances growth has not
been fully understood, although it is generally accepted that CSH
effects are originated at the SS-GH-IGF axis level, the main
hormonal mechanism responsible of somatic growth in
vertebrates (2,14,60). It has been largely demonstrated that CSH
injection or ingestion is associated with the reduction of SS,
probably as consequence of the breakdown of the disulfide bond
of SS by the thiol group of CSH, although this process is not
completely elucidated (23,30,31,61). This is a key point
considering that SS is produced in the hypothalamus but also
along the digestive system, and its depletion can result in multiple
local effects, as the inhibition of the releasing of different endocrine
and paracrine hormones, altering several physiological processes
(62). Dohil et al. (2006, 2014) (63,64) demonstrated that CSH
bitartrate is almost completely absorbed in small intestine, but it
also has effects in the previous pass through the stomach.
FIGURE 1
Relative gene expression in liver, stomach and white muscle of gilthead sea bream (Sparus aurata) supplemented with CSH at the end of the entire
trial (Phase 1 followed by Phase 2). Data are shown as mean ± S.E.M. relativized to Control mean (n = 14). Significant differences between treatments
were determinedby one-way ANOVA and Tukey’spost-hoc test and are indicated with different letters (p < 0.05).
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Furthermore, its absorption and metabolization in mammals takes
place in hours (23,65). A reduction of SS would trigger the secretion
of GH by the adenohypophysis gland as it was previously observed
in the literature, where the administration of CSH by different ways
improved growth and increased the GH and IGF-1 levels (8,32,41,
59). Regarding this, Hu et al. (2016) (41) found a positive
correlation between GHRH, GH and IGF-1 levels in fish fed with
CSH. Interestingly, and contrary to what most bibliography
supports, we report here a CSH dose-dependent reduction of
plasmatic GH levels compared to the control group in the Phase
2 of the trial, while IGF-1 levels were significantly increased by CSH,
but only significant for the CSH-1.65 group. McLeod et al. (1995a,
1995b) (8,66) and McElwain et al. (1999) (37) also observed a
reduction of the GH levels and modifications in the amplitude and
duration of GH release depending on the CSH dose administered.
Those authors explained the GH decrement by the previous low
levels of GHRH, which would be due to a reduction of
catecholamine synthesis in the hypothalamus caused by CSH.
Other explanation could be based on the negative feedback
regulation of the GHRH/GH/IGF axis. In either case, circulating
IGF-1 levels were upraised and the IGF-1/GH ratio was increased in
both phases, indicating an ongoing anabolic condition in agreement
with the biometric data (67,68). The increase of the MFI with the
high dose could be explained, on the one hand, by the anabolic
condition given by the concomitant high IGF-1, promoting
adipocyte proliferation and differentiation and better nutrients
uptake to the cell, and on the other hand, by the reduction of
GH, which has lipolytic effects and plays an important role on
energy management (17,69,70). The improvement of the IGF-1/
GH ratio trough the time could be related with the better response
to CSH with an adaptation period of 4-6 weeks and the increasing
doses (64). Regarding this, during the design of the present study, it
FIGURE 2
Viability of gilthead sea bream (Sparus aurata) myocytes exposed to different concentrations of CSH (50, 200, 400 and 800 µM) and quantified by
methylthiazolyldiphenyl-tetrazolium bromide (MTT) assay. Data are shown as fold change of the mean + S.E.M. (n = 7) with respect to the Control
group (0 µM, dotted line) for each cell culture. Significant differences between treatments were determined by one-way ANOVA followed by a
Tukey’spost hoc test and are indicated with different letters (p< 0.05).
FIGURE 3
Relative gene expression of gilthead sea bream (Sparus aurata) myocytes exposed to CSH (200 µM) from day 4 to day 8 of cell culture development,
presented as the fold change with respect to the Control group mean (dotted line). Data are shown as mean + S.E.M. (n = 7). Significant differences
between treatments were determined by a t-test, and are indicated by asterisks (p< 0.05).
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was proposed to double the dose of CSH during the Phase 2, instead
of reducing it, since both the GH and IGF-1 are gradually reduced
with age (71), and the intensity of the stimulus needed for
increasing their levels would be higher.
Generally, the gene expression results of the GH/IGF axis in the
in vivo model showed two clear patterns in the different tissues. On
one hand, an increase in the mRNA levels of the analyzed genes by
both CSH doses (1.65 and 3.3) compared to control group (e.g., ghr-
1and igf-2 in white muscle). On the other hand, an increase or
decrease of gene expression for the low CSH dose (1.65) but in a
lesser extent for the 3.3-group, which presented similar values to
control group (e.g., igf-1a and igf1-ra in white muscle). GH and
IGFs are recognized by their corresponding receptors (GHR and
IGFR, respectively) widely distributed through the different tissues.
Two different GHR (GHR-1 and GHR-2) have been described in
several fish species, including gilthead sea bream (72,73). The
functional divergence of these two paralogs have not been fully
elucidated yet, though it seems that GHR-1 is upregulated under an
anabolic status whereas GHR-2 is positively related with stress and
energy mobilization signals (1,5,44,60,73–75). This would be in
accordance with the improved growth that we observed in the fish
fed with the CSH diets and the upregulation of the ghr-1 but not the
ghr-2 in white muscle and stomach and interestingly, also in
myocyte cell culture. It appears, thereby, that the in vivo growth-
promoting action of CSH would be partially mediated by the GHR-
1, which increase could be a compensatory mechanism in response
to the lower circulating GH levels.
IGFs play a key role in skeletal muscle growth and
differentiation through the endocrine action of IGF secreted from
liver and its own paracrine function. In our results, we found that
the fish fed with CSH presented an increase in the igf-1a splice
variant for both doses and in total igf-1 for CSH-3.3 in the liver.
However, we did not observe differences in igf-1c transcript levels
among groups, the principal isoform in liver (76). The
overexpression of the total igf-1 observed in liver would explain
the increased levels of the IGF-1 showed in plasma (14,17,34). Tse
et al. (2006) (43) found and exponential increase of the hepatic igf-1
gene expression and, especially, the igf-2 in carps fed with CSH (0, 1,
2 and 3 g/Kg) at day 7. However, at day 63 the igf-1 levels were
equalized among treatments compared to control except for the 3 g/
Kg dose, suggesting that this dose would continue to stimulate the
IGF synthesis. Regarding muscle, Tse et al. (2006) (43)only
observed differences at day 63 and for the 3 g/Kg dose. In our
results the igf-1a was also upregulated in the CSH-1.65 group but
not in the CSH-3.3 compared to Control. This could be due to a
previous peak of the igf-1a in CSH-3.3 group that in the moment of
the sampling was in a downregulation step. Integrating the liver and
the muscle responses to CSH, these suggest a key point between the
1.65 and 3 g/Kg dose that triggers some anabolic signals. It is
interesting to compare the CSH effects on igf-2 expression in
different tissues, emphasizing the significant upregulation
responses in muscle and in vitro myocytes, but not in liver or
stomach. This agrees with the important role of IGF-2 in myocytes
proliferation and myogenesis (48,77) and its overexpression found
in muscle of fast-growing catfish family (78) pointing out the role of
IGF2 in the muscle of these fish species.
Concerning the in vitro experiment, the use of CSH on
myocytes had not been studied yet and there is scarce
information about CSH effects and doses on other cell type
cultures. Besides, the data of this study represents the first
approach to understand its direct effects on primary fish
myocytes. Beyond our study, CSH has been previously used as
antioxidant and maturation-promoter in mammalian oocytes in a
range between 25 to 400 µM (39). Here we reported that the
maximum non-toxic dose of CSH in gilthead sea bream myocytes
under the differentiation phase (day 4 to day 8) is 200 µM, which
agrees with these previous works. Regarding the somatotropic axis-
related genes, myocytes stimulated with CSH showed the same
expression pattern for ghr-1 and ghr-2 as in the white muscle of the
in vivo trial. However, in in vitro conditions, the action of the
circulating GH is not present, and cells are only exposed to GH
traces present within the fetal bovine serum supplementing the
culture media, which in any case will affect equally to control and
CSH incubated myocytes. Therefore, CSH by itself also seems to
modulate the GH sensing in myocytes by an unknown mechanism.
With respect to the IGF family, we observed an increase in the igf-1b
and the igf-2 relative expression after CSH exposure. Similar igf-1
and igf-2 induction was observed in Atlantic salmon (Salmo salar)
and gilthead sea bream cultured myocytes after the exposure to
nutrients, mainly amino acids (15,70,79). This would have sense
since CSH is a natural precursor of taurine, a semi essential amino
acid in carnivorous fish. Thus, these results suggest that together
with the stimulatory effects on GH/IGF axis, CSH can have a direct
effect on GH sensing and IGF-2 expression of muscle cells (14,17).
The modulation of the digestive function by the CSH and the
possible improved entrance of nutrients would be an important
factor that boosted the GH/IGF axis (45,62,80).
The IGFBPs and the IGFR regulate the availability and activity
of the IGFs in the different tissues (14,81). In the CSH-1.65 group
there was a decrease in igfbp-2a in liver, which is the main IGF
carrier in teleost (81). In early-stage zebrafish (Danio rerio),
overexpression of igfbp-2a and igfbp-2b caused a reduction in
body growth and developmental rate (82), suggesting a growth
inhibitory role. In this sense, the hepatic downregulation of this
binding protein in the animals fed with the CSH-1.65 of our study
would be in line with the highest IGF-1 plasma levels observed in
this group. However, variations among studies suggest a complex
role of this binding protein in teleost growth, subjected to the
physiological and species-specific context (81).
Furthermore, we observed a downregulation of the igf-1ra in
white muscle and the igf-1ra and igf-1rb in stomach in fish fed with
CSH-1.65. These reductions could be due to the negative feedback
provoked by the boosted endocrine and paracrine function
represented by the higher levels of IGF-1 in plasma and the
relative expression of the different IGF-1 splice variants,
respectively. Azizi et al. (2016) (15) found similar results in sea
bream cultured myocytes incubated with amino acids, with the
increased igf-1 and igf-2 previously mentioned and a reduction of
the igf1-ra and igf1-rb receptors. However, in our in vitro
experiment we found an upregulation of the igf1-rb instead its
decrease, suggesting that the point of negative feedback had not yet
been reached after 96 h of treatment.
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5 Conclusions
Here we have demonstrated that the inclusion of CSH at 1.65 g/
Kg and 3.3 g/Kg improved the hormonal balance of the
somatotropic axis at both systemic and tissue levels. The
upregulation of markers usually associated with an anabolic state
such as GHR-1, IGF-1 and IGF-2 and the modulation of the IGFR
and IGFBP in liver, white muscle and stomach, resulted altogether
in an enhanced somatic growth. This condition would be mediated
by the direct action on muscle, promoting the paracrine secretion
and sensing of GH/IGF axis, but the effect on hypothalamic and
intestinal somatostatin and the improvement in digestive
absorption cannot be ignored and deserves future investigation.
Thus, the use of CSH as a feed additive in a dose adjusted to the fish
species and growing stage could be a very interesting strategy
in Aquaculture.
Data availability statement
The datasets presented in this study can be found in online
repositories. The names of the repository/repositories and accession
number(s) can be found in the article/Supplementary Material.
Ethics statement
The animal study was reviewed and approved by Ethics and
Animal Care Committee of the University of Barcelona.
Author contributions
AS-M, JG, JB and JF-B conceptualized the study. RF provided
the fish feed. AS-M performed the in vivo trial. AS-M, SB-P, EV,
MP-A, IG-M, JF-B, JB and JG performed the sampling. AS-M and
SB-P performed the in vitro trial. AS-M, SB-P, EV, MP-A, JC-G and
JP-S performed the laboratory and data analyses. AS-M wrote the
original draft. SB-P, EV, RF, JC-G, JP-S, JF-B, JB and JG critically
reviewed the manuscript. JG, JB and JF-B acquired funding and
administrated the project. All authors read and approved the
final paper.
Funding
This publication is part of the R+D+i projects AGL2015-70679-
R and RTI2018-100757-B-I00 to JG and JB, and AGL2017-89436-R
funded by the Spanish “Ministerio de Ciencia e Innovacion”
(MCIN/AEI/10.13039/501100011033/), the “Xarxa de Refèrencia
d’R+D+I en Aqüicultura”(Aqüival Cist-Crec) and the 2017-
SGR1574 from the “Generalitat de Catalunya”. SB-P, EV and
MP-A, were funded by predoctoral fellowships from the
MINECO grants PRE2018-085580, BES-2013-062949 and BES-
2016-078697, respectively.
Conflict of interest
The authors declare that the research was conducted in the
absence of any commercial or financial relationships that could be
construed as a potential conflict of interest.
Publisher’s note
All claims expressed in this article are solely those of the authors
and do not necessarily represent those of their affiliated
organizations, or those of the publisher, the editors and the
reviewers. Any product that may be evaluated in this article, or
claim that may be made by its manufacturer, is not guaranteed or
endorsed by the publisher.
Supplementary material
The Supplementary Material for this article can be found online
at: https://www.frontiersin.org/articles/10.3389/fendo.2023.1211470/
full#supplementary-material
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