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.

Expression of rainbow trout glucose transporters GLUT1 and GLUT4 during in vitro muscle cell differentiation and regulation by insulin and IGF-I

“…Interestingly, in the trout muscle cell culture, IGF-I caused a marked increase in GLUT1 expression at day 2 and a more modest increase at day 10. Since IGF-I is also known to stimulate the proliferation of trout myoblasts (9), it is possible that the increased expression of GLUT1 could contribute to a greater fuel intake to face the increased energy demand imposed by the proliferative activity of IGF-I.…”

“…Moreover, insulin accelerates the rates of protein synthesis and amino acid uptake to promote muscle growth (31). Recently, insulin has been shown to directly stimulate the uptake of glucose and amino acids by trout muscle cells in culture (9) and IGF-I shares the same effects, supporting the notion that insulin and IGF-I may directly regulate carbohydrate and protein metabolism in trout skeletal muscle. In brown trout, skeletal muscle (red and white) accounts for approximately half of the total glucose uptake by tissues despite having the lowest glucose uptake rates and is the only tissue that increases its glucose uptake rate in response to a glucose load (5).…”

“…To address this issue we used a primary culture of trout muscle satellite cells, a system that can reproduce the differentiation process taking place in skeletal muscle, shows insulin and IGF-I binding (10), and has been used to study the direct metabolic effects of insulin and IGF-I in trout muscle (9). The results from our study indicate that the basal expression of GLUT4, but not of GLUT1, markedly increases throughout the differentiation process of trout skeletal muscle cells and that the expression of both GLUT4 and GLUT1 is stimulated by insulin and IGF-I in myoblasts and myotubes.…”

Regulation of proliferation and differentiation of adipocyte precursor cells in rainbow trout (Oncorhynchus mykiss)

“…IGF-I is a mitogenic and differentiation factor for various mammalian cell lines, and primary stromalvascular cell cultures, including pre-adipocytes (Smith et al 1988, Schmidt et al 1990, Richardson et al 1998. IGF-I has mitogenic and anabolic effects in fish myocytes in culture (Castillo et al 2004). This growth factor induces proliferation, measured as thymidine incorporation, in primary cell cultures of trout myocytes (Castillo et al 2004) as well as DNA synthesis in zebrafish embryonic cells (Pozios et al 2001).…”

Unidentified cells reside in fish skeletal muscle

“…Many of the earliest efforts to isolate fish satellite cells utilized juvenile fish of small size and required large sample numbers to gather enough tissue for culturing (Matschak and Stickland 1995;Fauconneau and Paboeuf 2001;Castillo et al 2004). Mulvany and Cyrino (1995) did attempt to culture satellite cells from catfish ranging from 1.9 to 330 g in weight, but they only propagated the cultures to 48 h and experienced poor replating efficiency of the cells.…”