ORIGINAL ARTICLE The growth-promoting activity of egg white proteins in the C2C12 myoblast cell line Wataru MIZUNOYA, Ayumi TASHIMA, Yusuke SATO,* Ryuichi TATSUMI and Yoshihide IKEUCHI Department of Bioresource Sciences, Graduate School of Agriculture, Kyushu University, Fukuoka, Japan ABSTRACT In this study, we examined the effects of several egg white proteins (ovalbumin, ovomucoid, ovotransferrin and lysozyme) on proliferation and myotube growth in C2C12 murine myoblast cells. Cell proliferation was measured using a water- soluble tetrazolium salt (WST-8)-based assay and then validated using Giemsa staining. Significant proliferative activities of C2C12 cells were observed in response to the addition of 10 −5 –10 −4 mol/L ovalbumin or ovomucoid. Ovotransferrin decreased C2C12 cell proliferation and lysozyme showed no significant effects on the proliferation of C2C12 cells. In contrast, the proliferative effects of ovalbumin and ovomucoid were not observed in 3T3-L1 murine preadipocyte cells. We also measured the effects of ovalbumin and ovomucoid on C2C12 myotube diameters by using histological analysis. In comparison to control cells, myotube diameters were significantly increased in cells cultured in 10 −6 –10 −4 mol/L ovalbumin or ovomucoid, suggesting that ovalbumin and ovomucoid stimulate the growth of myotubes. Thus, our results clearly demonstrated that ovalbumin or ovomucoid stimulated the proliferation of myoblasts and growth of myotubes. Key words: C2C12, egg white, ovalbumin, ovomucoid, skeletal muscle. INTRODUCTION Eggs are one of the most economical animal protein sources, possessing the highest nutritional content. Egg white proteins contain many kinds of proteins, including ovalbumin, ovotransferrin, ovomucoid and lysozyme that constitute 54%, 13%, 11% and 3.5% of the egg white, respectively (Besler & Mine 1999), and have unique biological activities. Several egg white proteins affect cell growth. Ovotransferrin reportedly inhibits the proliferation of bacteria (Valenti et al. 1983), whereas it increases the proliferation of lymphocytes (Otani & Maenishi 1994). Similarly, lysozyme shows both antimicrobial (Ibrahim et al. 1996) and immunostimulatory activities (Sugahara et al. 2002). The skeletal muscle is the largest organ in the human body and provides the strength and support required for body movement. Skeletal muscle mass is known to decline with aging (Short et al. 2004), bed rest (Bodine 2013) or space flight (Fitts et al. 2010). Currently, resistance training is the most effective method to prevent muscle wasting and weakness. Satellite cells are resident myogenic stem cells that are positioned beneath the basal lamina of mature muscle fibers. Under sedentary conditions, satellite cells are in a quiescent state; however, they become active, proliferate, differentiate and fuse into multinucleated myofibers in response to stimuli such as exercise (Peterson et al. 2008), muscle stretching (Tatsumi et al. 2006) or muscle injury (Snow 1977). Thus, satellite cells play a critical role in skeletal muscle hypertrophy and regeneration. Stimulated proliferation of satellite cells is expected to increase skeletal muscle hypertrophy and regeneration. We speculated that dietary intake of egg white pro- teins could stimulate the proliferation of muscle satel- lite cells because egg white proteins affect cell growth. In that case, egg white proteins can be quite useful dietary component for the prevention of muscle wasting and weakness. In this study, we examined the effect of several egg white proteins on proliferation of the C2C12 murine myoblasts cell line, a model of activated satellite cells. We found that ovalbumin and Correspondence: Wataru Mizunoya, Department of Bioresource Sciences, Graduate School of Agriculture, Kyushu University, 6-10-1 Hakozaki, Higashi, Fukuoka 812- 8581, Japan. (Email: wataru_m@agr.kyushu-u.ac.jp) *Present address: Department of Animal Science, Utsunomiya University, Utsunomiya, Japan. Received 16 April 2014; accepted for publication 8 May 2014. Animal Science Journal (2015) 86, 194–199 doi: 10.1111/asj.12257 © 2014 Japanese Society of Animal Science