Insulin-like growth factors effects on the expression of myogenic regulatory factors in gilthead sea bream muscle cells Vanesa Jiménez-Amilburu, Cristina Salmerón, Marta Codina, Isabel Navarro, Encarnación Capilla, Joaquim Gutiérrez ⇑ Departament de Fisiologia i Immunologia, Facultat de Biologia, Barcelona Knowledge Campus, Universitat de Barcelona, 08028 Barcelona, Spain article info Article history: Available online 14 March 2013 Keywords: Primary cell cultures Myogenic regulatory factors Insulin-like growth factors Growth hormone Muscle differentiation abstract Gilthead sea bream (Sparus aurata) is a widely cultured fish; however, muscle development regulation is poorly known. Myogenesis can be activated by the myogenic regulatory factors (MRFs: MyoD, Myf5, myogenin and MRF4) and by endocrine signals from the growth hormone (GH)/insulin-like growth fac- tors (IGFs) axis. We cultured gilthead sea bream myocytes to better understand the role of IGFs in muscle growth and differentiation through the regulation of MRFs expression. First, we studied the expression pattern during culture development of IGFs and IGF-I splice variants. The expression of igf-II was highest at the beginning of the culture and decreased when the cells started to differentiate, similarly to that observed for total igf-I. Igf-Ib showed a paralleled expression pattern as that of total igf-I, whereas igf- Ic was more stable during culture progression. Next, we analyzed the expression of IGFs and MRFs after incubation of cells at day 4 with GH, IGF-I, IGF-II and combinations of them at 3, 6 and 18 h. IGF-II increased myod2 and myf5 expression, genes involved in early muscle cell proliferation. Moreover, IGF- I caused an increase on mrf4 and myogenin expression, both involved in the later stages of development corresponding to differentiation. Regarding the regulation of IGFs expression, igf-I was stimulated by GH and IGF-II alone and combined, whereas igf-II expression was increased in response to IGF-I, suggesting a nice model of crossed regulation. Overall, the present model could be very useful to understand the dif- ferent regulatory roles of these endocrine and transcription factors on fish myogenesis. Ó 2013 Elsevier Inc. All rights reserved. 1. Introduction In recent years, the study of fish’s physiology has been im- proved with the development of aquaculture, as this practice has been found as an alternative to overfishing. The white muscle in some fish species represents up to 60% of total weight, and thus it is involved in most fish growth; so it is one of the most important tissues from the commercial viewpoint. Gilthead sea bream, Sparus aurata is one of the most important farmed marine species pro- duced in southern Mediterranean countries, and has a high com- mercial value since it is very important in the European diet. The endocrine regulation of gilthead sea bream growth has been abun- dantly studied by different authors (Funkenstein et al., 2006, 2002; Mingarro et al., 2002; Saera-Vila et al., 2009); however, there is less literature on skeletal muscle development and the improvement of the flesh quality of this species for aquaculture. Myogenesis is the generation of muscular tissue during embry- onic development from stem cells, by fusion of myoblasts into multinucleated fibers (myotubes) (Johnston, 2006). This process is highly conserved in all vertebrates and requires the synchro- nized participation of four myogenic regulatory factors (MRFs): MyoD, Myf5, myogenin and MRF4. These MRFs are transcription factors with a basic helix-loop-helix (bHLH) domain (Edmondson and Olson, 1993; Massari and Murre, 2000), which is responsible for recognizing the E-box in the promoter of the target genes (Buckingham et al., 2003) to carry out growth and differentiation of numerous cell types. MyoD and Myf5 are involved in myogenic lineage determination, whereas myogenin and MRF4 play a key role initiating and maintaining the myogenic differentiation pro- gram (Holterman and Rudnicki, 2005; Rescan, 2001; Rudnicki et al., 1993). Tissue development and muscle growth in teleost fish, as well as in mammals (Velloso, 2008), is also regulated by the growth hormone (GH)/insulin-like growth factors (IGFs) axis (Castillo et al., 2004, 2002; Green et al., 1985; Le Bail et al., 1998; Mor- iyama et al., 2000; Peterson et al., 2004). In this axis are in- volved GH, IGF-I and IGF-II. GH is synthesized by the anterior pituitary and stimulates the secretion of IGF-I from the liver, 0016-6480/$ - see front matter Ó 2013 Elsevier Inc. All rights reserved. http://dx.doi.org/10.1016/j.ygcen.2013.02.033 ⇑ Corresponding author. Address: Departament de Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 643, 08028 Barcelona, Spain. Fax: +34 934110358. E-mail address: jgutierrez@ub.edu (J. Gutiérrez). General and Comparative Endocrinology 188 (2013) 151–158 Contents lists available at SciVerse ScienceDirect General and Comparative Endocrinology journal homepage: www.elsevier.com/locate/ygcen