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 current dairy production systems, an average of 25% of dietary N is captured in milk, with the remainder being excreted in urine and feces. About 60% of total N losses occur postabsorption. Splanchnic tissues extract a fixed proportion of total inflow of each essential AA (EAA). Those EAA removed by splanchnic tissues and not incorporated into protein are subjected to catabolism, with the resulting N converted to urea. Splanchnic affinity varies among individual EAA, from several fold lower than mammary glands' affinity for the branched-chain AA to similar or higher affinity for Phe, Met, His, and Arg. On average, 85% of absorbed EAA appear in peripheral circulation, indicating that first-pass removal is not the main source of loss. Essential AA in excess of the needs of the mammary glands return to general circulation. High splanchnic blood flow dictates that a large proportion of EAA that return to general circulation flow through splanchnic tissues. In association with this constant recycling, EAA are removed and catabolized by splanchnic tissues. This results in splanchnic catabolism equaling or surpassing the use of many EAA for milk protein synthesis. Recent studies have demonstrated that EAA, energy substrates, and hormones activate signaling pathways that in turn regulate local blood flow, tissue extraction of EAA, and rates of milk protein synthesis. These recent findings would allow manipulation of dairy diets to maximize mammary uptake of EAA and reduce catabolism by splanchnic tissues. Dairy cattle nutrient requirement systems consider EAA requirements in aggregate as metabolizable protein (MP) and assume a fixed efficiency of MP use for milk protein. Lysine and Met sufficiency is only considered after MP requirements have been met. By doing so, requirement systems limit the scope of diet manipulation to achieve improved gross N efficiency. Therefore, this review focuses on understanding the dynamics of EAA metabolism in mammary and splanchnic tissues that would lead to improved requirement prediction systems. Inclusion of variable individual EAA efficiencies derived from splanchnic and mammary responses to nutrient and hormonal signals should help reduce dietary protein levels. Supplementing reduced crude protein diets with individual EAA should increase gross N efficiency to more than 30%, reducing N excretion by the US dairy industry by 92,000 t annually.Journal of Dairy Science 04/2014; · 2.57 Impact Factor
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ABSTRACT: Soybeans and other legumes investigated as fishmeal replacements in aquafeeds contain phytoestrogens capable of binding to and activating estrogen receptors. Estradiol has catabolic effects in salmonid white muscle, partially through increases in protein turnover. The current study determines whether phytoestrogens promote similar effects. In rainbow trout (Oncorhynchus mykiss) primary myocyte cultures, the phytoestrogens genistein, daidzein, glycitein, and R- and S-equol reduced rates of protein synthesis and genistein, the phytoestrogen of greatest abundance in soy, also increased rates of protein degradation. Increased expression of the ubiquitin ligase fbxo32 and autophagy-related genes was observed with high concentrations of genistein (100μM), and R- and S-equol (100μM) also up-regulated autophagy-related genes. In contrast, low genistein concentrations in vitro (0.01-0.10μM) and in vivo (5μg/g body mass) decreased fbxo32 expression, suggesting a potential metabolic benefit for low levels of genistein exposure. Phytoestrogens reduced cell proliferation, indicating effects of phytoestrogens extend from metabolic to mitogenic processes. Co-incubation of genistein with the estrogen receptor (ER) antagonist, ICI 182,780, ameliorated effects of genistein on protein degradation, but not protein synthesis or cell proliferation, indicating effects of genistein are mediated through ER-dependent and ER-independent mechanisms. Collectively, these data warrant additional studies to determine the extent to which dietary phytoestrogens, especially genistein, affect physiological processes that impact growth and nutrient retention.Comparative biochemistry and physiology. Toxicology & pharmacology : CBP. 05/2014;
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ABSTRACT: Skeletal muscle growth and development is controlled by nutritional (amino acids, AA) as well as hormonal factors (insulin-like growth factor, IGF-I); however, how its interaction modulates muscle mass in fish is not clearly elucidated. The purpose of this study was to analyze the development of gilthead sea bream cultured myocytes to describe the effects of AA and IGF-I on proliferating cell nuclear antigen (PCNA) and myogenic regulatory factors (MRFs) expression, as well as on the transduction pathways involved in its signaling (TOR/AKT). Our results showed that AA and IGF-I separately increased the number of PCNA-positive cells and, together produced a synergistic effect. Furthermore, AA and IGF-I, combined or separately, increased significantly Myogenin protein expression, whereas MyoD was not affected. These results indicate a role for these factors in myocyte proliferation and differentiation. At the mRNA level, AA significantly enhanced PCNA expression, but no effects were observed on the expression of the MRFs or AKT2 and FOXO3 upon treatment. Nonetheless, we demonstrated for the first time in gilthead sea bream that AA significantly increased the gene expression of TOR and its downstream effectors 4EBP1 and 70S6K, with IGF-I having a supporting role on 4EBP1 up-regulation. Moreover, AA and IGF-I also activated TOR and AKT by phosphorylation, respectively, being this activation decreased by specific inhibitors. In summary, the present study demonstrates the importance of TOR signaling on the stimulatory role of AA and IGF-I in gilthead sea bream myogenesis and contributes to better understand the potential regulation of muscle growth and development in fish.General and Comparative Endocrinology 05/2014; · 2.82 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