IGF-I binding and receptor signal transduction in primary cell culture of muscle cells of gilthead sea bream: changes throughout in vitro development

Abstract

We examined the possibility of culturing muscle cells of gilthead sea bream in vitro and assessed variations in insulin-like growth factor-I (IGF-I) binding during myocyte development. The viability of the cell culture was determined by fluorescence-activated cell-sorting analysis, which showed that the percentage of dead cells decreased with cell differentiation. The intracellular reduction of MTT into formazan pigment was preferentially carried out as cells differentiated (from day 4) indicating an increase in metabolic activity. IGF-I-binding assays demonstrated that the number of receptors increased from 190 +/- 0.09 fmol/mg protein in myocytes at day 5 to 360 +/- 0.09 fmol/mg protein in myotubes at day 12. The affinity of IGF-I receptors did not change significantly during cell development (from 0.89 +/- 0.09 to 0.98 +/- 0.09 nM). The activation of various kinase (ERK 1/2 MAPK and Akt/PKB) proteins by IGFs and insulin was studied by means of Western blot analysis. Levels of MAPK-P increased after IGF and insulin treatment during the first stages of cell culture, with a low response being observed at day 15, whereas IGFs displayed a stimulatory effect on Akt-P throughout the cell culture period, even on day 15. This study thus shows that (1) gilthead sea bream myocytes can be cultured, (2) they express functional IGF-I receptors that increase in number as they differentiate in vitro; (3) IGF signalling transduction through IGF-I receptors stimulates the MAPK and Akt pathways, depending on the development stage of the muscle cell culture.

Adiponectin effects and gene expression in rainbow trout: an in vivo and in vitro approach

“…In trout myocytes, it was revealed that adiponectin activates the PI3K/Akt pathway but not the p44/42 MAPK pathway, similar to the process that occurs in C2C12 myocytes. These observations indicate that the conservation of the signalling pathways of adiponectin along the vertebrates does occur, as previously reported for insulin, and also that IGF signalling pathways are conserved in myocytes in culture, which was previously described by our group (Montserrat et al, 2007;Codina et al, 2008). In the present study, wortmannin blocked the stimulatory effect of adiponectin on Akt phosphorylation, demonstrating the specificity of the detected molecule.…”

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

“…For this purpose we used a well-characterized primary culture of trout skeletal muscle cells (10,15,36,37) that provides a useful tool to reproduce the muscle differentiation process in vitro and study the biology of fish muscle cells. Importantly, trout muscle cells have receptors for insulin and IGF-I and IGF-I binding has been shown to gradually increase during myogenesis (10,33).…”

Transient up- and down-regulation of expression of myosin light chain 2 and myostatin mRNA mark the changes from stratified hyperplasia to muscle fiber hypertrophy in larvae of gilthead sea bream (Sparus aurata L.)

“…IGF-1 is a molecule with a pleiotropic action in gilthead sea bream and appears to mediate the effects of growth hormone in seasonal growth, to have a pivotal role in compensatory growth following starvation, to induce osteoblast and myoblast proliferation in primary cell cultures and to increase glucose and amino acid uptake in cultured myoblasts through both MAPK and PI3K-Akt signal transduction pathways (Mingarro et al 2002;Montserrat et al 2007;Capilla et al 2011;Rius-Francino et al 2011;Montserrat et al 2012). Igf1 transcripts have been detected in unfertilized gilthead sea bream eggs and its later expression appears to be tissue-and age-dependent, with skeletal muscle displaying high immunoreactivity (Perrot et al 1999).…”