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.
- metabolic effects
igf-i induces, in vertebrates, multiple regulatory functions on growth, differentiation, reproduction, and metabolism (reviewed in Ref. 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 also been 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 zebrafish embryonic cells, whereas 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 compared with 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, because, 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
Chemicals. 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 (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); and human recombinant IGF-I was from Peninsula Laboratories, Europe (Merseyside, UK). Other reagents were obtained from Sigma Aldrich Química (Alcobendas, Madrid).
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°C. Fish were fed ad libitum with a commercial diet and fasted for 24 h before the experiments. Fish (30-50 for each culture) with an approximate weight of 5 g were killed by a blow to the head and immersed for 30 s 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 × 106 per well in six-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-deoxyglucose (2-DG) and l-alanine uptake assays. All experiments were performed in triplicate; each condition was performed in triplicate (3 wells). Cells were incubated at 18°C, the optimal temperature for growth of the culture.
2-DG uptake assays. For 2-DG assays, 30-50 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 × 106 per well in six-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 min) 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 50 μM 2-DG 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 min. The contents of the wells were aspirated and rinsed three 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, Meriden, CT). 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.
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. PD-98059 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 is a specific inhibitor of the facilitative glucose transporters. Cells were preincubated for 30 min with wortmanin (1 μM) or PD-98059 (50 μM), and peptides (IGF-I or insulin) were added for 30 additional minutes. Experiments to assess the effects of DMSO (diluent of the PD-98059 and the wortmanin) were performed (data not shown), demonstrating that at the concentration used, 0.1% (vol/vol), it did not interfere with the glucose uptake. The cytochalasin B (20 μM) was added and incubated simultaneously with the labeled 2-DG for 30 min.
l-Alanine uptake assays. For l-alanine uptake assays, fish number and weight and cell culture and cell density were 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 h. 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 or 2 h), the medium was aspirated, rinsed two times with ice-cold PBS, and the cells were incubated with 1 μCi/ml of l-alanine (except for the time course experiments, the incubations were routinely of 20 min). 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).
Cell proliferation assays (thymidine uptake). For proliferation assays, fish number and weight and cell culture and cell density were 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 24 h to restrict the cell growth. After this period, the medium was changed to DMEM containing different concentrations of FBS and peptides (from 0.125 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 were washed three times with ice-cold PBS. By addition of 1 ml of TCA (10% wt/vol) after 20 min at 4°C, the soluble fraction was eliminated, and the insoluble fraction remained. The assay was terminated, and the radioactivity associated with the cells was determined as described above for 2-DG and l-alanine uptake.
Statistical analysis. The treatment was performed in triplicate for each experiment. Data are presented as means ± SE of at least three experiments. Statistical differences between conditions were analyzed by one-way analysis of variance and the Tukey's test. Differences were considered statistically significant 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 min (r2= 0.99) (from 1,645 cpm/well at 10 min to 14,537 cpm/well at 60 min). Additionally, preincubation of cells at day 4 with IGF-I for 60 min and subsequent incubation with 50 μM of cold 2-DG together with 2 μCi/ml 2-DG for 10 or 30 min resulted in an increase of glucose uptake for both times compared with basal levels (in absence of IGF-I). Very similar results were obtained when preincubating IGF-I for 30 min (data not shown).
Figure 1 compares the effects of IGF-I and insulin on glucose uptake, preincubating with these peptides for 30 or 60 min and fixing glucose uptake for 30 min. IGF-I showed the highest stimulation of glucose uptake after 60 min (Fig. 1A), whereas maximum insulin stimulation was observed after 30 min (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 min) 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-day cultured myocytes. Although not significant, wortmanin reduced the basal glucose uptake and also reduced the stimulatory effect of IGF-I and insulin. PD-98059 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 min (r2 = 0.84) (from 11,011 cpm/well at time 5 min to 19,279 cpm/well at time 30 min). A period of uptake of 20 min 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 h 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 (Fig. 5) were significantly lower than the data obtained for the same cells in day 4 (Fig. 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 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%), to determine which was the optimal FBS concentration to quantify the proliferation. Only in the presence of 2% or 5% FBS was proliferation 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 was 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.
2-DG uptake. 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 on 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.). In addition, 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 from 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 uptake.
In our results, both peptides stimulated glucose uptake at short incubation times (between 30 and 60 min), 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 h) 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 h 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 ∼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 increases with differentiation, and this could be explained by the reported increase in the IGF-I receptor 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 PD-98059, 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 deeper 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), who, 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.
l-Alanine uptake. 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 stimulates amino acid uptake in fish muscle in vitro (25, 42) and in vivo (11), although none of those studies has 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, whereas 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 and Stevens (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 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 were incubated with IGF-I or insulin in a medium without FBS. It was necessary to prolong the exposure of the cells to peptides (48 h) to observe any response. Other authors have also described incubation times with IGF-I up to 48 h to stimulate cell proliferation in L6 cells (2, 19, 40). To detect cell proliferation, it was necessary to perform the experiments by incubating the cells in a serum-supplemented medium, which suggests that there is some component in serum necessary for IGF-I to stimulate cell proliferation. McWade et al. (36) called this component (or components) MCF (mitogenic competence factor), and it was shown that it is required by IGF-I to stimulate proliferation (41). Its composition still remains unclear, but it was shown that it is not transforming growth factor-β, platelet-derived growth factor, transferrin, IGF binding proteins, or fibroblast growth factor (36, 37).
Our results are in accordance with Pozios et al. (53), in ZF-4 cells from zebrafish embryos, who detected that IGF-I and IGF-II were stimulators of cell proliferation and DNA synthesis, whereas insulin had a very low mitogenic activity. There are other works showing the proliferative effects of IGF-I in mammalian models such as porcine muscle cells (46), human cells (9), and L6 rat cells (40).
In conclusion, IGF-I stimulates, more than insulin, glucose uptake in a primary culture of rainbow trout myosatellite cells, and this stimulation is higher when cells develop in vitro to form differentiated myotubes. IGF-I also stimulates l-alanine uptake more potently than insulin, and this stimulation decreases as the cells differentiate. Finally, IGF-I stimulates cell proliferation in this model of muscle in vitro and insulin does not. These results are in agreement with the abundance of IGF-I receptors in trout skeletal muscle myosatellite cells previously reported (5) and also with the high total tyrosine kinase activity of IGF-I receptors compared with insulin in fish tissues (39). They also highlight the important role of this peptide in the growth and metabolism of this tissue in fish. Considering that IGF-II seems to exert its biological effects through the IGF-I receptor it will be interesting to study the effects of this growth factor in metabolism and proliferation of these cells. Further studies of our group will also focus on understanding the main steps on signal transduction mechanisms of the insulin and IGF-I receptors in trout skeletal muscle.
This study was supported by grants from the European Union (QLRT-1999-30068), Direccíon General de Investigacíon, Ciencia y Tecnología, Spain (AGL-2001-2903 ACU), and Comissió Interdepartamental de Recerca i Innovació Tecnològica (2001 SGR-00122, 1998 FI 00634).
We thank J. Baró of the Piscifactoria Truites del Segre (Lleida) for providing the rainbow trout and facilities to conduct the sampling and for assistance. We also thank P. Y. Le Bail, C. Weil, B. Fauconneau, and P. Y. Rescan from SCRIBE, INRA, Rennes, for help and suggestions with myocyte culture. The English text was corrected by the Language Advisory Service of the University of Barcelona.
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Present address of J. Castillo: Faculty of Engineering and Natural Sciences, Sabanci University, 81474 Tuzla, Istanbul, Turkey.
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