The growth and development of vertebrates are mainly mediated through the GH-IGF system. Especially in teleost fish, the multiple forms of GHRs, and IGFRs have been described. However, they have distinct functions and expression patterns in various fish tissues [22]. In tilapia, GHR2 participates in growth and metabolism and is highly expressed in liver and muscle,while GHR1 expression is higher in liver [23]. In seabream and rainbow trout, GHR2 expression is much higher than GHR1 in numerous tissues such as muscle, pituitary, kidney and gonad [24, 25]. Interestingly, although GHR expression is affected by GH in many other fish, the opposite effects are found may due to the different expression levels. The growth responses are enhanced in loaches [26] and GH-transgenic salmon [27] and restrained in tilapia [28] and carp [29]. The weight and thick muscle fibers of GH/GHR-transgenic zebrafish is lower than the other genotypes [30]. In our experiments, however, both the enhancement and inhibition of growth occurred under different conditions, respectively. With the change of time, the expression of GHR2 among different sizes of individuals showed no significant difference (Fig. 5 GHR2), indicating that the stable expression of GHR2 was crucial to promoting growth and development. During starvation, high expression of GHR on the surface of muscle cells can save protein consumption [23], which also has been reported in gilthead sea bream and rainbow trout [22, 31]. Therefore, in S.chuatsi, GHR2 appears to inhibit growth through significant high expression during starvation (Fig. 7 GHR2) and plays a physiologically significant role in muscle metabolism, involving in the mobilization of muscle energy stores.
IGFRs can bind to IGF-1, IGF-2 and insulin, and the affinity for insulin is much lower than for IGFs in all species [32]. Furthermore, in contrast to the situation in mammals, the IGFR1 of fish muscle is greater abundance than insulin receptors (IR) [32], indicating that IGFR1 is more relevant to the regulation of muscle function than IR in fish [33], involving promoting growth, enhancing protein, increasing cell proliferation and reducing protein degradation [34]. IGFR1 was highly expressed in S at 3-month old (Fig. 5 IGFR1) (P < 0.05), suggesting IGFR1 played a more essential role for S over L in muscle, such as participating in and promoting the growth of muscle. Interestingly, prolonged fasting results in significantly reduced IGF-1 mRNA in the liver and muscle of numerous fish species [35–37], whereas IGFR1 increased significantly [13, 35, 38]. In this study, the IGFR1 expression markedly rose during fasting (Fig. 7 IGFR1), illustrating that IGFR1 expression will increase to restrict IGF-I during starvation, which is probably related to the anti-apoptotic effects reported in mammals [38].
Phosphorylated of protein kinase B (PKB) is controlled by the metabolic pathway regulated by IGFR1, and PKB can mediate the AKT/mTOR/p70S6K pathway to facilitate protein synthesis and cell growth [39–41]. Meanwhile, mTOR can regulate translation by 4ebp [42], and the phosphorylation of 4ebp releases eIF4E to stimulate translation initiation [43]. eIF4E is able to enhance the translation of mRNAs, implicating in cell proliferation and growth [44], but it is hypothesized that overexpression of eIF4E leads to the deregulation of translational and cellular homeostasis [45]. However, the family of 4ebps can inhibite the assembly of eIF4F complex [45]. Thus, 4ebp inhibits cell growth and revert the transformed phenotype of eIF4E-overexpressing cell [45], which may save energy and prevent the malignant transformation of cells during starvation by significant expression (Fig. 7 4ebp). In conclusion, we speculate that the expression of upstream IGFR1 will promote the expression of downstream 4ebp through the above-mentioned pathways in the case of starvation, so as to cope with the food restriction. In compensatory growth experiments, the expression of IGFR1 and 4ebp was consistent (Fig. 7 IGFR1 and 4ebp), proving the hypothesis, but further research is needed on specific regulatory mechanisms. It has been reported that fasting promotes the metabolic actions of GH rather than the growth-promoting actions [46], however, relevant aspects of researches in GHR2, IGFR1 and 4ebp are less. In the study, the functions of GHR2, IGFR1 and 4ebp also changed from growth promotion when food was abundant to growth inhibition in starvation, thus highlighting the metabolic functions of them.
Mhc expression is significantly related with growth rate [47–49], which also affects myofiber hyperplasia and indeterminate growth [50]. In this study, Mhc was highly expressed in S at 5-month old (Fig. 5 Mhc) (P < 0.05), and transcriptome results also showed that the growth rate of small fish was high, suggesting that Mhc can promote the growth of S through its high expression. In larval stage of S.chuatsi, the high expression of Mlc regulates muscle formation and early development [51], which also influences the tail shaft swimming character at posterior area, needing more muscle fiber and protein [52]. The significant high expression of Mlc at 5-month old in S (Fig. 5 Mlc) proved that muscle protein and muscle fiber synthesize quickly to meet physiological requirement. Taken together, as important components of myosin, Mhc and Mlc played a more positive role in S comparing to L at 5-month old. Interestingly, although they all enhanced the growth of S.chuatsi, the function time and action modes of myosin (Mlc, Mhc), GHR2, IGRF1 and 4ebp were different. GHR2 was expressed stably to promote growth (Fig. 5 GHR2), and IGFR1 had a more obvious promotion effects in S at 3-month old (Fig. 5 IGFR1). Furthermore, both myosin (Mlc, Mhc) and 4ebp facilitated more markedly in S at 5-month old (Fig. 5 Mlc, Mhc and 4ebp), showing all of them had their own temporal and spatial expression patterns in S.chuatsi.
Myosin, the primary protein of muscles, amounts to 50% of the muscle proteins [53]. Troponin as a complex protein is largely expressed in muscle and plays a vital role in muscle contraction [54, 55]. During fasting conditions, the markedly down-regulated of Mhc and Mlc indicated that muscle protein synthesis decreased (Fig. 7 Mhc and Mlc), and muscle proteins may be used as the major energy source to maintain basic metabolism which limited growth and was consistent with the observed reduced weight (Fig. 6). In the meantime, because muscle breakdown and muscle protein synthesis were inhibited, muscle contraction and motor function were restrained, which was manifested as a significant decrease in troponin expression (Fig. 7 Troponin). After re-feeding, significant up-regulation of Mhc and Mlc was observed (Fig. 7 Mhc and Mlc), showing that muscle protein was no longer used as the major energy source, and synthesis of muscle and rapid compensatory growth occurred, which resulted in increased weight [56]. As the normal synthesis and growth of muscle, muscle contraction was no longer inhibited and expression levels returned to normal (Fig. 7 Troponin).
MyoD is associated with myogenesis during developmental condition which plays a regulatory role in muscle hypertrophy and muscle mass [57, 58]. Myf6 participates in myofiber differentiation by recruiting structural proteins [59]. From 3-month old to 5-month old, MyoD and Myf6 expression had a downward trend (Fig. 5 MyoD and Myf6), which agreed with the experiment phenomenon of Zhu et al [60].It suggested that 3-month old may be a rapid growth stage for S.chuatsi, accompanying with an intense myoblast differentiation, which primarily contributed to the muscle growth [60]. What puzzled us is that both MyoD and Myf6 belong to the MRFs family, but the trends of expression results were opposite in L and S (Fig. 5 MyoD and Myf6), and there was no significant difference, which may be influenced by the complex regulatory systems in vivo. It has been reported that the reduction in muscle weight may stimulate MRFs transcription [61]. And in mammals, satellite cells can express MyoD and Myf5 in response to muscle damage [62, 63]. Interestingly, during the starvation period, the expression of MyoD fell to the lowest level and then increased following the weight loss. After re-feeding, the expression gradually decreased from the highest level, and during the whole process, the expression had a certain delayed (Fig. 7 MyoD). But, the expression of Myf6 was stable throughout the process. These phenomena indicated that MyoD had a sensory and regulatory effects on the weight loss and was conducive to the muscle growth and recovery for S.chuatsi.