Metabolic and mitogenic effects of IGF-I and insulin on muscle cells of rainbow trout.
ABSTRACT The relative function of IGF-I and insulin on fish muscle metabolism and growth has been investigated by the isolation and culture at different stages (myoblasts at day 1, myocytes at day 4, and myotubes at day 10) of rainbow trout muscle cells. This in vitro model avoids interactions with endogenous peptides, which could interfere with the muscle response. In these cells, the effects of IGF-I and insulin on cell proliferation, 2-deoxyglucose (2-DG), and l-alanine uptake at different development stages, and the use of inhibitors were studied and quantified. Insulin (10-1,000 nM) and IGF-I (10-100 nM) stimulated 2-DG uptake in trout myocytes at day 4 in a similar manner (maximum of 124% for insulin and of 142% for IGF-I), and this stimulation increased when cells differentiated to myotubes (maximum for IGF-I of 193%). When incubating the cells with PD-98059 and especially cytochalasin B, a reduction in 2-DG uptake was observed, suggesting that glucose transport takes place through specific facilitative transporters. IGF-I (1-100 nM) stimulated the l-alanine uptake in myocytes at day 4 (maximum of 239%), reaching higher values of stimulation than insulin (100-1,000 nM) (maximum of 160%). This stimulation decreased when cells developed to myotubes at day 10 (118% for IGF-I and 114% for insulin). IGF-I (0.125-25 nM) had a significant effect on myoblast proliferation, measured by thymidine incorporation (maximum of 170%), and required the presence of 2-5% fetal serum (FBS) to promote thymidine uptake. On the other hand, insulin was totally ineffective in stimulating thymidine uptake. We conclude that IGF-I is more effective than insulin in stimulating glucose and alanine uptake in rainbow trout myosatellite cells and that the degree of stimulation changes when cells differentiate to myotubes. IGF-I stimulates cell proliferation in this model of muscle in vitro and insulin does not. These results indicate the important role of IGF-I on growth and metabolism of fish muscle.
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ABSTRACT: In the present study a commercial probiotic Fishery Prime TM , acting as water soluble probiotic, was administered to Yellow Perch (Perca flavescens) for 6 weeks. The probiotic effect on growth and growth regulated genes were evaluated with respect to fish group fed on commercial feed (control group). Measures of body weight were performed to assess the growth performance. In addition, the expression of two related and highly indicative candidate genes involved in growth (Insulin-Like Growth Factor IGF-I and Growth Hormone GH) were quantified through real-time PCR. Fish received the probiotic exhibited higher growth performance than control group at significance level (p < 0.05).Up regulation of GH and IGF-I transcriptions were observed in fish group received with probiotic which revealed higher levels than the control. The results confirmed the positive correlation between growth performance, GH and IGF-I mRNA gene expression in both probiotic treated and control groups. In conclusion, probiotic during early developmental stages can confer maximum beneficial effects resulting in magnitude increase in survivorship.Global Journal of Fisheries and Aquaculture Researches. 11/2014; 1(2):1-15.
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ABSTRACT: In order to elucidate the possible roles of insulin-like growth factors I and II (IGF-I and IGF-II) in the embryonic development of Platichthys stellatus, their cDNAs were isolated and their spatial expression pattern in adult organs and temporal expression pattern throughout embryonic development were examined by quantitative real-time PCR assay. The IGF-I cDNA sequence was 1,268 bp in length and contained an open reading frame (ORF) of 558 bp, which encoded 185 amino acid residues. With respect to IGF-II, the full-length cDNA was 899 bp in length and contained a 648-bp ORF, which encoded 215 amino acid residues. The amino acid sequences of IGF-I and IGF-II exhibited high identities with their fish counterparts. The highest IGF-I mRNA level was found in the liver for both sexes, whereas the IGF-II gene was most abundantly expressed in female liver and male liver, gill, and brain. The sex-specific and spatial expression patterns of IGF-I and IGF-II mRNAs are thought to be related to the sexually dimorphic growth and development of starry flounder. Both IGF-I and IGF-II mRNAs were detected in unfertilized eggs, which indicated that IGF-I and IGF-II were parentally transmitted. Nineteen embryonic development stages were tested. IGF-I mRNA level remained high from unfertilized eggs to low blastula followed by a significant decrease at early gastrula and then maintained a lower level. In contrast, IGF-II mRNA level was low from unfertilized eggs to high blastula and peaked at low blastula followed by a gradual decrease. Moreover, higher levels of IGF-I mRNA than that of IGF-II were found from unfertilized eggs to high blastula, vice versa from low blastula to newly hatched larva, and the different expression pattern verified the differential roles of IGF-I and IGF-II in starry flounder embryonic development. These results could help in understanding the endocrine mechanism involved in the early development and growth of starry flounder.Fish Physiology and Biochemistry 11/2014; · 1.68 Impact Factor
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ABSTRACT: One of the most fundamental biological processes in living organisms that is affected by environmental fluctuations is growth. In fish, skeletal muscle accounts for the largest proportion of body mass, and the growth of this tissue is mainly controlled by the Insulin-like Growth Factor (IGF) system. By using the carp (Cyprinus carpio), a fish that inhabits extreme conditions during winter and summer, we assessed the skeletal muscle plasticity induced by seasonal acclimatization and the relation of IGF signaling with protein synthesis and ribosomal biogenesis. The expression of igf1 in muscle decreased during winter in comparison with summer, whereas the expression for both paralogues of igf2 did not change significantly between seasons. The expression of igf1 receptor a (igf1ra), but not of igf1rb, was down-regulated in muscle during the winter as compared to the summer. A decrease in protein contents and protein phosphorylation for IGF signaling molecules in muscle was observed in winter-acclimatized carp. This was related with a decreased expression in muscle for markers of myogenesis (myoblast determination factor (myod), myogenic factor 5 (myf5), and myogenin (myog)); protein synthesis (myosin heavy chain (mhc) and myosin light chain (mlc3 and mlc1b)); and ribosomal biogenesis (pre-rRNA and ribosomal proteins). IGF signaling, and key markers of ribosomal biogenesis, protein synthesis, and myogenesis were affected by seasonal acclimatization, with differential regulation in gene expression and signaling pathway activation observed in muscle between both seasons. This suggests that these molecules are responsible for the muscle plasticity induced by seasonal acclimatization in carp.Comparative Biochemistry and Physiology Part B Biochemistry and Molecular Biology 10/2014; · 1.90 Impact Factor
Metabolic and mitogenic effects of IGF-I and insulin on muscle
cells of rainbow trout
Juan Castillo1, Marta Codina, Maria Laura Martínez, Isabel Navarro, and Joaquim Gutiérrez*
Departament de Fisiologia, Facultat de Biologia, Universitat de Barcelona,
Av. Diagonal 645, E-08028 Barcelona, Spain.
Short title: IGF-I and insulin effects on trout muscle cells
Key words: IGF-I, insulin, metabolic effects, proliferation, trout muscle cells
* Corresponding author: Departament de Fisiologia,
Facultat de Biologia, Universitat de Barcelona,
Av. Diagonal 645, E-08028 Barcelona, Spain
1Present Address: Faculty of Engineering and Natural Sciences, Sabanci University, 81474
Tuzla, Istanbul, Turkey
Articles in PresS. Am J Physiol Regul Integr Comp Physiol (January 29, 2004). 10.1152/ajpregu.00459.2003
Copyright (c) 2004 by the American Physiological Society.
The relative function of IGF-I and insulin on fish muscle metabolism and growth
has been investigated by the isolation and culture at different stages (myoblasts at day 1,
myocytes at day 4 and myotubes at day 10) of rainbow trout muscle cells. This in vitro
model avoids interactions with endogenous peptides, which could interfere with the
muscle response. In these cells, the effects of IGF-I and insulin on cell proliferation, 2-
deoxy-glucose (2-DG) and L-alanine uptake at different development stages, and the use
of inhibitors were studied and quantified. Insulin (10-1000nM) and IGF-I (10-100nM)
stimulated 2-DG uptake in trout myocytes at day 4 in a similar manner (maximum of
124% for insulin and of 142% for IGF-I), and this stimulation increased when cells
differentiated to myotubes (maximum for IGF-I of 193%). When incubating the cells
with PD98059 and specially cytochalasin B, a reduction in 2-DG uptake was observed,
suggesting that glucose transport takes place through specific facilitative transporters.
IGF-I (1-100 nM) stimulated the L-alanine uptake in myocytes at day 4 (maximum of
239%) reaching higher values of stimulation than insulin (100-1000 nM) (maximum of
160%). This stimulation decreased when cells developed to myotubes at day 10 (118%
for IGF-I and 114% for insulin). IGF-I (0.125-25 nM) had a significant effect on
myoblast proliferation, measured by thymidine incorporation (maximum of 170 %), and
required the presence of 2-5% fetal serum (FBS) to promote thymidine uptake. On the
other hand, insulin was totally ineffective in stimulating thymidine uptake. We conclude
that IGF-I is more effective than insulin in stimulating glucose and alanine uptake in
rainbow trout myosatellite cells, and that the degree of stimulation changes when cells
differentiate to myotubes. IGF-I stimulates cell proliferation in this model of muscle in
vitro and insulin does not. These results indicate the important role of IGF-I on growth
and metabolism of fish muscle.
IGF-I induces, in vertebrates, multiple regulatory functions on growth,
differentiation, reproduction, and metabolism (reviewed in 58). In fish, the IGF-I effects
related to growth are well characterized (15, 22, 26, 34) and abundant literature exists on
the role of IGF-I in fish differentiation (23, 50) and fish reproduction (31), but
information about its metabolic role is scarce.
In mammals, the importance of insulin on muscle metabolic function is well
established in vivo (60) and in vitro. There are many studies on the effects of insulin on
the stimulation of glucose uptake in mammalian muscle cell lines (12, 28, 45) and in
primary culture of cardiac (13) and skeletal muscle, in humans (56) and rats (59). It has
been also reported that IGF-I exerts some of these effects in mammalian established cell
lines (3, 32) in ovine muscle cells (55) and in chicken muscle cells (17).
In addition to the effects of insulin on glucose uptake, its effects on protein
anabolism are also well known and its action on amino acid uptake in L6 cells has been
reported (35) as well as in vivo in neonatal pigs (44). IGF-I caused similar effects in L6
cells (3) and in ovine muscle cells (55). Moreover, IGF-I has been shown to stimulate
protein synthesis in neonatal pigs (10), and in primary cultures of chicken satellite cells
IGF-I is more effective than insulin stimulating amino acid uptake (18).
The mitogenic effects of IGF-I and insulin on muscle have been described in
various studies. Duclos et al (16), working with chicken satellite cells, reported that IGF-
I is more powerful than insulin in stimulating thymidine incorporation in DNA. Hodik et
al (24), using the same model, described the stimulation of DNA synthesis by IGF-I.
IGF-I was also shown to stimulate thymidine incorporation in rat muscle (6). Similar
studies have also been carried out in established cell lines, for example, mouse C2C12
cells, where it was shown that insulin can increase thymidine incorporation in a dose-
dependent manner (8). In mammals, IGF-I is considered to be a more mitogenic and
growth stimulatory molecule than insulin. Insulin plays a more important role in
metabolic processes such as the regulation of carbohydrate metabolism.
Similarly, in fish, IGF-I stimulates proliferation and DNA synthesis in a dose-
dependent manner in zebra fish embryonic cells, while insulin shows a weak mitogenic
activity (53). However, it has been postulated that, in fish, these processes are similar in
both molecules and it is possible that there is an overlapping of metabolic functions
between insulin and IGF-I (51, 52). Our group has previously described the abundance
of IGF-I receptors in skeletal muscle as compared to the number of insulin receptors, in
several species of fish and other poikilotherms (48, 49). This ratio has been observed
during trout ontogeny and in adult fish muscle (38) and also in a primary culture of
rainbow trout muscle cells (5), but at the present time there is a lack of evidence on the
role of IGF-I in fish muscle. Drakenberg et al (14) and Degger et al (11) showed that
IGF-I in vivo administration increased glucose incorporation to fish muscle glycogen.
Negatu and Meier (42) and Degger et al (11) reported that IGF-I and insulin stimulate
amino acid uptake in this tissue in Gulf Killifish and in barramundi.
These data taken together suggest an important role for IGF-I in fish skeletal
muscle, which remains to be established. In addition, fish is a very good model in which
to study the role of IGF-I, since, in contrast to other vertebrates, the skeletal muscle mass
grows continuously throughout their life. By using a primary culture of trout muscle
cells, which is a well-defined technique for physiological and functional studies (20, 54)
the purpose of this study was to try to understand the role of IGF-I and to compare the
effects of both insulin and IGF-I on metabolic (glucose and amino acid uptake) and
mitogenic processes (thymidine uptake during cell proliferation).
Material and Methods
2-deoxy-D [2,6-3H] glucose (cat # TRK672), with a specific activity of 43
Ci/mmol, L-[2,3-3H] alanine, with a specific activity of 52 Ci/mmol, and [methyl-3H]
thymidine, with a specific activity of 25 Ci/mmol, were purchased from Amersham
Pharmacia Biotech Europe GmBH (Barcelona, Spain). Recombinant trout IGF-I was
purchased from GroPep (Adelaide, Australia), salmon insulin was kindly supplied by Dr.
E. Plisetskaya; porcine insulin was purchased from Lilly (Indianapolis, IN, USA) and
human recombinant IGF-I was from Peninsula Laboratories, Inc. Europe Ltd.
(Merseyside, UK). Other reagents were obtained from Sigma Aldrich Química, S. A.
Animals and cell culture
Animals (Oncorhynchus mykiss) were obtained from Piscifactoria Truites del
Segre (Oliana, Barcelona) and maintained in the facilities of the Servei d’Estabulari of
the Faculty of Biology at the University of Barcelona, in a closed-water flow circuit with
water at a temperature of 12 ºC. Fish were fed ad libitum with a commercial diet and
fasted for 24 h previously to the experiments. Fish (30 to 50 for each culture) with an
approximate weight of 5 g, were killed by a blow to the head and immersed for 30
seconds in 70% ethanol to sterilize the external surfaces. Cells were isolated, pooled and
cultured following the protocol described previously (5, 54). All experiments were
conducted with cells seeded at a density of 1.5 to 2 X 106 per well in 6-well plastic plates
(9.6 cm2 /well, NUNC). Observations on morphology were regularly made to control the
state of the cells, which were used at day 1 (mononucleated cells) for thymidine uptake
experiments, and at day 4 (mostly small myotubes) and day 10 (big myotubes) for 2-DG
and L-alanine uptake assays. All experiments were performed in triplicate; each
experiment was performed in triplicate (three wells). Cells were incubated at 18ºC, the
optimal temperature for growth of the culture.
2-DG uptake assays
For 2-DG assays 30 to 50 fish, with an approximate weight of 5 g, were used for
each culture. After pooling cells from all the animals of the same culture, the
experiments were conducted with cells seeded at a density of 1.5 to 2 X 106 per well in
6-well plastic plates. The cells, after 4 or 10 days of culture, were incubated for 4 h with
DMEM without FBS and after this period preincubated (30 or 60 minutes) in the
presence or absence of insulin or IGF-I in DMEM+0.5% BSA (concentrations ranging
from 10 to 100 nM for IGF-I and 100 nM to 1 µM for insulin). After preincubation the
cells were rinsed two times with ice-cold PBS and incubated with unlabeled 2-DG 50
µM together with labeled 2-DG (2 µCi/mL) in HEPES-saline buffer. The incubations
with labeled and cold 2-DG, except for the time-course experiments, were routinely of
30 minutes. The contents of the wells were aspirated and rinsed 3 times with ice-cold
PBS, and the cells were lysed with NaOH 0.5 N. The contents of the wells were removed
and placed into scintillation vials, and the radioactivity was quantified with a β counter
(Packard Bioscience Company, Meriden, CT, USA). Preliminary studies showed that
porcine insulin had very similar effects to fish insulin for the in vitro studies with fish
muscle cells (data not shown). Porcine insulin was used in the next experiments due to
the limitation in fish insulin.
In order to better characterize glucose transport and the role of both peptides, the
effects of several compounds on glucose uptake stimulation by IGF-I or insulin were
analyzed. PD98059 is an inhibitor of the MEK1 protein, a component of the MAPK
pathway; wortmanin is an inhibitor of the PI3K-Akt pathway; and cytochalasin B a
specific inhibitor of the facilitative glucose transporters. Cells were preincubated for 30
minutes with wortmanin (1 µM) or PD98059 (50 µM), and peptides (IGF-I or insulin)
were added for 30 additional minutes. Experiments to assess the effects of DMSO
(diluent of the PD98059 and the wortmanin) were performed (data not shown),
demonstrating that at the concentration used, 0.1% (v/v), it did not interfere the glucose
uptake. The cytochalasin B (20 µM) was added and incubated simultaneously with the
labeled 2-DG for 30 minutes.
L-alanine uptake assays
For L-alanine uptake assays, fish number and weight, and cell culture and cell
density was equivalent to that used for 2-DG uptake assays. After 4 or 10 days of culture,
the culture medium (90% DMEM/ FBS 10%) was aspirated, and the cells were rinsed
with ice-cold PBS and maintained in DMEM+ 0.5% BSA (DMEM/BSA) without FBS
for 2-3 hours. After preincubation with DMEM+0.5% containing different
concentrations of peptides (from 10 to 100 nM for IGF-I and 100 nM to 1 µM for
insulin) at different times (1 h or 2 h), the medium was aspirated, rinsed two times with
ice-cold PBS and the cells incubated with 1 µCi/ mL of L-alanine (except for the time-
course experiments, the incubations were routinely of 20 minutes). The amino acid
uptake was stopped by aspiration of the supernatant, followed by three rapid washes with
ice-cold PBS. Finally cells were solubilized with NaOH 0.5 N, samples were placed in
scintillation vials and the radioactivity was counted (Packard Bioscience Company,
Meriden, CT, USA).
Cell Proliferation assays (Thymidine uptake)
For proliferation assays, fish number and weight, and cell culture and cell density
was equivalent to that used for 2-DG or L-alanine uptake assays. The proliferation assays
were performed with 1 day cells (myoblasts). The medium was aspirated, and the cells
were incubated with DMEM + 0.02 % FBS for 24h to restrict the cell growth. After this
period, the medium was changed to DMEM containing different concentrations of FBS
and peptides (from 0.125 nM to 25 nM). Cells were maintained for 24 additional hours
under these conditions, and cell proliferation was analyzed by quantifying the 3H-
thymidine uptake (0.2 µCi/ mL) for 24 additional hours.
Finally, the supernatant was removed and the cells washed 3 times with ice-cold
PBS. By addition of 1 mL of TCA (10% w/v) after 20 minutes at 4º C, the soluble
fraction was eliminated, and the insoluble fraction remained. The assay was terminated
and the radioactivity associated with the cells determined as described above for 2DG
and L-alanine uptake.
The treatment was performed in triplicate for each experiment. Data are presented
as mean ± standard error of at least three experiments. Statistical differences between
conditions were analyzed by one-way analysis of variance and the Tukey test.
Differences were considered statistically significative at P< 0.05
IGF-I and insulin effects on 2-DG uptake
Different concentrations of cold and labeled 2-DG were tested in preliminary
experiments and a concentration of 50 µM of cold 2-DG and 2 µCi/mL of labeled 2-DG
glucose were selected for the following experiments. Our preliminary time-course
experiments, performed by incubation of cells at day 4 with 50 µM of unlabeled 2-DG
and 2 µCi/mL of labeled 2-DG, showed that basal 2-DG uptake was linear between 0
and 60 minutes (r2= 0.99) (from 1645 cpm/well at 10 min to 14537 cpm/well at 60 min).
Additionally, preincubation of cells at day 4 with IGF-I for 60 minutes and subsequent
incubation with 50 µM of cold 2-DG together with 2 µCi/mL 2-DG for 10 or 30 minutes
resulted in an increase of glucose uptake for both times when compared to basal levels
(in absence of IGF-I). Very similar results were obtained when preincubating IGF-I for
30 minutes (data not shown).
Fig. 1 compares the effects of IGF-I and insulin on glucose uptake, preincubating
with these peptides for 30 or 60 minutes and fixing glucose uptake for 30 minutes. IGF-I
showed the highest stimulation of glucose uptake after 60 minutes (Fig. 1A), while
maximum insulin stimulation was observed after 30 minutes (Fig. 1B). Insulin exerted
this stimulation at a concentration 10 times higher (1 µM) than that for IGF-I (100 nM).
The stimulatory effect of IGF-I (preincubated for 30 minutes) on glucose uptake
was also studied on day 10 of culture (Fig. 2) resulting in a maximum stimulation of 193
± 6 %, a higher level of stimulation than that in cells at 4 days (Fig. 1A). In addition, in
myotubes at 10 days and at equimolar concentrations, IGF-I was more effective than
insulin in stimulating glucose uptake (data not shown).
Figure 3 shows the effects of inhibitors of glucose uptake in IGF-I and insulin
stimulated 4 days cultured myocytes. Although not significant, wortmanin reduced the
basal glucose uptake and also reduced the stimulatory effect of IGF-I and insulin.
PD98059 significantly inhibited the effects of IGF-I on glucose uptake. Cytochalasin B
provoked a clear and significant inhibition in glucose uptake in all conditions: basal, and
IGF-I and insulin stimulation.
IGF-I and insulin effects on L-alanine uptake
Time-course experiments of L-alanine uptake, performed by incubation of cells at
day 4 with 1 µCi/mL of labeled L-alanine, showed the linearity of the uptake between 0
and 30 minutes (r2 = 0.84) ( from 11011 cpm/well at time 5 min to 19279 cpm/well at
time 30min). A period of uptake of 20 minutes was adequate to measure the effects of
IGF-I and insulin in these cells, and it was used routinely in the subsequent experiments.
Figure 4 shows the effects of IGF-I and insulin preincubation for 2 hours on
alanine uptake. Both peptides stimulated alanine uptake over the basal values, IGF-I
being more potent than insulin, even with a concentration 10 times lower.
In day 10 of in vitro development, values of stimulation for both peptides in cells
(Figure 5) were significantly lower than the data obtained for the same cells in day 4
(Figure 4). Stimulation was equivalent for both peptides, although insulin was 10 times
more concentrated than IGF-I.
IGF-I and insulin effects on thymidine uptake
Several experiments were performed to verify the incubation conditions for the of
proliferation study of the cells in culture. Cells (in day 1) were incubated with peptides
(IGF-I and insulin) for 24 h and labeled 3H-thymidine was added for an additional 24 h.
The first experiments were performed by incubation of the cells in DMEM without FBS
and with different concentrations of IGF-I and insulin. However, we could not observe
any effect on proliferation. The following incubations were done using different
concentrations of FBS (ranging from 0% to 10%), in order to determine which was the
optimal FBS concentration to quantify the proliferation. Only in the presence of 2% or
5% FBS proliferation was detected when IGF-I was added (stimulation of 153 % and
172% over basal levels, respectively).
Figure 6 shows an IGF-I dose response experiment, although with a dosage of 25
nM at 5 % FBS the thymidine uptake was lower. (Basal levels were measured in the
presence of 2% FBS and 5% FBS without peptides). None of the treatments with insulin
were able to stimulate the 3H-thymidine uptake above the basal levels of uptake.
This is the first study to describe the IGF-I and insulin-stimulated uptake
of metabolic substrates (2-DG and L-alanine) and cell proliferation in trout
skeletal muscle cells, and as far as we know is the first time that this kind of
study has been done in a primary culture of muscle of any fish species. Our
results point to a key role (both metabolic and mitogenic) for IGF-I in fish
muscle, which is important considering the fact that fish have continuous
growth, and therefore IGF-I could act as a key regulatory factor of this growth
through the fish life cycle.
Here we describe for the first time the effects of insulin and IGF-I on 2-DG
uptake in a primary culture of trout muscle cells. IGF-I is more effective than insulin in
stimulating glucose uptake and this stimulation was higher when cells developed in vitro
to form differentiated myotubes. So far, only a few studies have reported the effects of
insulin and IGF-I upon glucose uptake in fish muscle in vivo (11, 14), and knowledge of
the relative importance of both peptides in this tissue in fish is still scarce. The absence
of established cell lines of fish muscle has been a very important factor in choosing a
primary culture in which to test the biological effects of insulin and IGF-I in fish muscle
in a model exempt of systemic influences (liver, pancreas, etc). Besides it has been
proposed that primary myoblast cultures from other vertebrates recapitulate muscle
development more precisely than immortal myogenic lines (4). Our results suggest an
important metabolic role for IGF-I in fish muscle, which is different to the predominant
role of insulin in mammalian cells, either in whole muscle (60) or cultured muscle cells
(3, 7, 32), where insulin is more potent or equipotent to IGF-I in stimulating glucose
In our results, both peptides stimulated glucose uptake at short incubation times
(between 30 and 60 minutes), and in concentrations (1 µM for insulin and 100 nM for
IGF-I) similar to that described for mammalian, in primary cultures of human muscle (7)
and in C2C12 cells (12, 45). Other authors have also observed glucose uptake when
longer (8-18 hours) incubations with the peptides were performed (3, 33).
Results in this study are similar to those reported by Duclos et al (17) where, in a
primary culture of chicken muscle cells, IGF-I stimulated more than insulin glucose
uptake, both peptides being incubated for 4 hours and at equimolar concentrations. In
addition, insulin needed to be 10 times more concentrated than IGF-I to have the same
effect, which supports our results in 4-and 10 day trout muscle cells.
In our primary culture of trout myotubes at 10 days and at equimolar
concentrations, IGF-I was more effective than insulin in stimulating glucose uptake,
although Kelley et al (27), in goby skeletal muscle explants, a system that can be
analogous to trout myotubes, found no differences in 2-DG uptake between IGF-I and
insulin at equimolar concentrations.
In our trout muscle cells the glucose uptake stimulated by IGF-I in day 10 was
almost 70% higher than in day 4, a situation also described by Beguinot et al (3) in L6
cells (where IGF-I stimulated glucose uptake increased by 30% when developing from
myoblasts into myotubes) and more recently by Niu et al (43) in the same model. From
these results we can conclude that the stimulation of glucose uptake by IGF-I in trout
muscle cells increase with differentiation, and this could be explained by the reported
increase in the IGF-IR levels (5). In addition, the mRNA levels (57) and protein levels
(1) of the glucose transporter GLUT4 increase during the differentiation of myoblasts to
myotubes in culture, with a higher effect on glucose uptake.
By incubating trout muscle cells with wortmanin and PD98059 a decrease on the
glucose uptake was observed, especially on IGF-I stimulated cells, in agreement with
that observed by other authors in isolated rat cardiomyocytes (13) and C2C12 cells (45).
These findings suggest that further studies should go deep into the possible role of
MAPK and PI3K pathways in glucose transport in fish muscle. Cytochalasin B blocked
the glucose uptake (of basal and also stimulated cells), which has been confirmed in
several in vitro models of muscle (17, 28, 56), indicating that glucose uptake takes place
through specific facilitative transporters in these cells. Our results in trout muscle cells
contrast with those from Legate et al (30), that working with skeletal muscle membrane
vesicles of rainbow trout, found no effect of cytochalasin B on glucose uptake. However,
Krasnov et al (29) reported the existence of a functional glucose transporter in isolated
rainbow trout hepatocytes, which could be inhibited by cytochalasin B.
In this study we also analyze the effects of insulin and IGF-I on the L-alanine
uptake in a primary culture of trout muscle cells. Interestingly, in these cells, IGF-I
stimulated the L-alanine uptake more than insulin did, at equimolar concentrations, and
this stimulation of uptake decreased when cells differentiated from myoblasts to
myotubes. Previous studies demonstrated that insulin or IGF-I stimulate amino acid
uptake in fish muscle in vitro (25, 42) and in vivo (11), although none of those studies
have compared simultaneously the effects of both peptides. The conditions we have used
for L-alanine uptake in trout muscle cells are similar and equivalent to that used by other
authors in different in vitro muscle models of mammals and birds (3, 18). Duclos et al
(18) observed in a primary culture of chicken muscle cells that IGF-I was more effective
than insulin at equimolar concentrations stimulating the amino acid uptake, and more
recently, Gallardo et al (21), working with isolated trout cardiomyocytes, found that
IGF-I stimulates alanine uptake and protein synthesis, while insulin showed no effects.
All these data are in agreement with our results and point out the important role of IGF-I
in fish muscle. However, the stimulation of both peptides was lower in cells at day 10,
and suggests that once the cells are differentiated, it decreases the capacity of stimulation
of amino acid uptake by IGF-I and insulin. Pan et al (47) described in intestinal cells that
the L-alanine transport decreases when the cells differentiate in culture, because the
amino acid requirements to proliferate and grow are much higher than when cells have
already differentiated. In addition, it has been described that the increase in protein
synthesis in skeletal muscle of pig neonates induced by IGF-I reduces with development
(10). Therefore, a similar effect may take place in our cultured trout muscle cells, and
IGF-I may play a more important role in amino acid uptake when cells are still
proliferating and new cells are being generated.
Thymidine uptake (Cell proliferation)
The effects of insulin and IGF-I on the cell proliferation have been compared for
the first time in primary cultures of rainbow trout skeletal muscle cells. Only IGF-I
caused an increase in thymidine uptake above of the basal uptake levels in the cells, and
we did not observe any stimulation when they were incubated with insulin, even at very
high concentrations. In addition, we did not detect any cell proliferation when the cells