Role of serum myostatin during the lactation period

Myostatin, also known as GDF-8 (Growth/Differentiation Factor-8), is a member of the TGF-beta superfamily that negatively regulates skeletal muscle mass in mammals. Mutation of the myostatin gene in mice, cattle, and humans causes a massively developed skeletal muscle, characterized by muscle hypertrophy and hyperplasia. Although myostatin is predominantly expressed in skeletal muscle tissue, several recent studies have shown the presence of myostatin protein in blood and suggested a possible role for circulating myostatin in the regulation of skeletal muscle mass. In the present study, we examined changes in the levels of active form myostatin (13 kDa) in serum after birth by Western blot analysis to predict the role of serum myostatin in early postnatal muscle growth in the rat. Interestingly, the amount of active form myostatin in serum increased after birth and then decreased along with ageing after weaning. To clarify the role of increased serum myostatin during the postnatal period, we administrated follistatin, an inhibitor of myostatin activity, into postnatal rats intraperitoneally just after birth. Follistatin-administration during the postnatal period caused selective hypertrophy of type II muscle fibers in the soleus muscle. These results demonstrate that myostatin in serum acts on skeletal muscle and negatively regulates early postnatal muscle growth.     

Analysis of myostatin and its related factors in various porcine tissues

Myostatin is expressed in skeletal muscle tissue where it functions to suppress myoblast proliferation and myofiber hypertrophy. Recently, myostatin was detected in the tendon, mammary gland, and adipose tissue of mice. We sought to determine whether myostatin is expressed in the liver, spleen, lung, and kidney of pigs. Real-time PCR and Western blots demonstrated that myostatin, follistatin, decorin, and activin receptor IIB (ActRIIB) mRNA and proteins were expressed in skeletal muscle, heart muscle, and adipose tissue, and also in liver, spleen, lung, kidney, and cultured fibroblasts. The relative abundance of myostatin was closely related to follistatin and decorin in porcine tissues. Immunohistochemical analysis further demonstrated the presence of myostatin, follistatin, and decorin in the skeletal muscle, adipose tissue, heart muscle, liver, spleen, lung, and kidney of pigs. These results suggest that myostatin could be associated with certain functions of the internal organs, such as energy metabolism or fibrosis. We conclude that myostatin is a factor broadly expressed in the internal organs and muscle tissues of pigs.     

肌生成抑制素基因研究进展

肌生成抑制素是 1 997年发现的骨骼肌生长发育负调控因子 ,肌生成抑制素不仅在骨骼肌中表达 ,还可在心肌和浦肯野纤维等多种组织中表达。研究表明 ,大鼠骨骼肌在体内的再生 ,对 MSTN m RNA的表达表现出明显的时间依赖性 ,并且 m RNA的表达不必由功能性神经支配。通过对不同品种双肌表型牛 MSTN基因的研究 ,发现其骨胳肌增大的共同特点是肌生成抑制素基因发生了突变 ,如布鲁牛 MSTN基因在编码区外显子 3发生 1 1 bp的缺失 ,导致MSTN此位点后的阅读框架改变 ,并在此点后的 1 4个密码子处终止了阅读框架 ,从而使MSTN被截短 ,MSTN蛋白活性区域消失 ,活性丧失 ,结果肌肉大量增加。目前 ,已分别对人、牛和猪的 MSTN基因进行了定位研究 ,猪肌生成抑制素基因定位研究表明 ,猪 MSTN基因位于1 5号染色体上。     

肌肉抑制素与“双肌”表型关系的研究进展

肌肉抑制素是特异性表达于骨骼肌的一种细胞因子,属于TGF-β超家族成员,可以抑制骨骼肌的发育,并同多种疾病有关。肌肉抑制素在不同物种间高度保守,其抑制功能的丧失可以导致骨骼肌显著增生,即动物产生"双肌"表型。具有"双肌"表型的动物则表现出肌肉产量显著增加、饲料转化率高、肌肉嫩度增加、脂肪含量少、而脂肪酸组成中不饱和脂肪酸比例显著增加等优秀性状,这些性状是动物育种计划中的理想育种目标。本文从"双肌"表型的起源及特点、肌肉抑制素的发现及功能等方面对肌肉抑制素与"双肌"表型关系的研究进展进行了综述。     

Expression profile of myostatin mRNA during the embryonic organogenesis of domestic chicken (Gallus gallus domesticus)

Myostatin is a potent growth and differentiation factor involved in skeletal muscle tissue formation in vertebrates. However, recent studies in chicken embryo suggested that the myostatin was expressed even before the establishment of myogenic lineage. No studies have thus far been reported in birds to define the role of myostatin during the embryonic organogenesis. The present experiment was designed for studying the expression profiles of myostatin mRNA in the chicken liver, heart, brain, and intestine during their morphogenesis, using real-time PCR. The myostatin mRNA expression was significantly upregulated in liver during E15-E18. Similar results were observed during the development of chicken heart. In brain, the expression of myostatin was upregulated from E4 onwards. In intestine, the expression of myostatin was significantly increased many folds on E9-E18. Therefore, the increase in myostatin expression might be related to the growth of liver and heart on days E12-E18; morphogenesis and growth of brain during E15-E18; and morphogenesis and differentiation of intestine during E9-E18. In the present study, the tissue-specific expression of myostatin gene in chicken is similar to fishes, but different from that in mammals. Further, the inspection of chicken genome also suggested that there is no differentiation of GDF-8 and -11. A recent finding suggests that the chicken myostatin gene is closely related to mammals than fishes. Therefore, we propose that the chicken myostatin gene might have diverged in its function between teleosts and mammals. Indeed it is possible that its function might have only become fully differentiated to serve as a control of muscle mass in mammals.     

山羊myostatin基因慢病毒RNA干扰载体的构建及效果验证

myostatin基因是肌肉生长和发育的关键负调控因子之一,该基因突变或失活的物种都呈现肌肉增大的双肌表型,暗示在家畜中使该基因失活可以增加其产肉量。为了进一步揭示myostatin在山羊胎儿成纤维细胞中的生物学功能以及制备可有效失活myostatin基因的工具,将筛选的myostatin基因潜在RNA干扰靶位点连接到慢病毒干扰载体的转移质粒中,构建慢病毒介导的RNA干扰载体,验证其干扰效果。结果表明,干扰序列与转移质粒连接正确,成功构建myostatin基因慢病毒干扰载体,慢病毒三质粒共转293T细胞,获得的慢病毒颗粒可高效转染山羊胎儿成纤维细胞,Real-time PCR检测发现慢病毒干扰载体Lv322有效减少山羊胎儿成纤维细胞中myostatin基因mRNA 75%的表达(P<0.01),Western blotting检测显示myostatin蛋白则有效降低了94%(P<0.01)。本试验为研究myostatin基因的功能及制备失活转基因动物提供有效工具以及理论基础。     

Laminin binds to myostatin and attenuates its signaling

Myostatin is a growth and differentiation factor and acts as a negative regulator of skeletal muscle mass. Although the mechanism whereby myostatin controls muscle cell growth is mostly clarified, the regulation of myostatin activity after its secretion into the extracellular matrix (ECM) is still unclear. In the present study, we investigated the interaction between laminin and myostatin and the effect of laminin on myostatin signaling in vitro. The surface plasmon resonance assay showed that laminin bound to mature myostatin and activin receptor type IIB (ActRIIB), but did not bind to latency‐associated protein, which remains non‐covalently linked to mature myostatin. Furthermore, kinetic analysis demonstrated that the affinity of mature myostatin for laminin was similar to that for ActRIIB. Next, we examined the action of laminin on the myostatin signaling pathway using a conventional reporter assay. The luciferase activity of myostatin‐treated cells was repressed significantly (P < 0.05) by coincubation of laminin. These results suggest that laminin has a potential to regulate myostatin activity through binding to mature myostatin and/or its receptor ActRIIB.     

牛肌肉生长抑制素基因的研究进展

肌肉生长抑制素,属TGF-β超家族,是一种骨骼肌生长发育的负调控因子。其生物活性的丧失或受到抑制,会导致骨骼肌增生从而引起动物肌肉的过度发育,表现为双肌性状。本文综述了MSTN基因的发现、结构特点、作用机制、在牛科物种中的研究进展及其应用前景。     

Genetic models for the study of insulin-like growth factors (IGF) and muscle development in birds compared to mammals.

IGFs are important positive modulators of overall body and muscle growth in different species. Genetic variation in IGF-I gene expression exists as shown by the possibility of genetic selection for high or low circulating IGF-I concentrations in mice and the associated variations in growth potential. Targeted over-expression of IGF-I in transgenic mice results in muscle hypertrophy, but it is yet unknown whether genetic variability in muscle IGF-I gene expression exists. Much less data are available in birds. This review is focussed on the potential role of IGFs on chicken muscle development. Apart from the absence of a type 2 IGF receptor (IGF-II receptor), the general characteristics of the chicken IGF system seem to be similar to mammalian species. In different genetic models with altered growth rates or body composition, differences in the IGF system are observed suggesting its importance in regulating body growth in those species. The components for a paracrine action of IGF are also present in chicken muscle but it has not yet been demonstrated if they contribute to differences in muscle development.     

Nucleotide sequence of myostatin gene and its developmental expression in skeletal muscles of Japanese Black cattle

Myostatin, a member of the transforming growth factor‐β superfamily, is a well known negative regulator of skeletal muscle growth. In the present study, the 6660 bp nucleotide sequence of the myostatin gene in Japanese Black cattle (JBC), including the entire coding region of 1128 bp, was determined. The amino acid sequence deduced from the nucleotide sequence of JBC was well conserved with its sequence of other cattle, although it was found that an Α→G transition at nucleotide position 641 results in the substitution of asparagine by serine at amino acid position 214. In order to examine the expression pattern of the myostatin gene in the skeletal muscles of JBC, its expression in three skeletal muscles, Semitendinosus (ST) muscle, Biceps femoris muscle and Longissimus lumborum muscle, of fetal and calf stages was analyzed by real time polymerase chain reaction. The highest level of the myostatin expression was observed in the fetal stage. In calf stages the highest expression was observed in ST muscle compared with the other two muscles. These results suggest that a higher expression of myostatin gene, especially in the fetal stage and in ST muscle during calf stages, is involved in the arrest in skeletal muscle growth and that its functional domains and genomic structure in JBC are well conserved with those in other mammals.     

猪各组织器官内肌生成抑制素基因mRNA表达谱的研究

用Trizol试剂法提取军牧一号猪的骨骼肌、心肌、肝、肾、肺、脾、胰、脂肪的总RNA做RT-PCR和嵌套PCR,结果在军牧一号猪骨骼肌、心肌和脂肪组织内检测到了肌生成抑制素mRNA的表达,而在其他组织中未检测到.     

cDNA encoding sequences for myostatin and FGF6 in sea bass (Dicentrarchus labrax, L.) and the effect of fasting and refeeding on their abundance levels

Fish have the ability to compensate for set-backs in growth as a result of fasting. When food levels are restored, growth in these fish can increase over and above normal rates. This phenomenon, known as “compensatory growth”, has been studied with respect to enhancing food conversion efficiency. However, the mechanisms by which food intake activates an increase in somatic growth, and especially in muscle growth, are not well understood. In this study, we report first on the isolation of two complete cDNAs sequences encoding sea bass (Dicentrarchus labrax) myostatin and fibroblast growth factor 6 (FGF6), which have been shown to be major genetic determinants of skeletal muscle growth. The open reading frames of myostatin (376 amino acids) and FGF6 (209 amino acids) showed 97–63% and 87–62% sequence identity with other vertebrate myostatins and FGF6s, respectively. We also report on the expression profile of myostatin and FGF6 in sea bass skeletal muscle in response to different feeding regimens, as quantified by real-time RT-PCR. Nutritional status significantly influenced the myostatin expression levels in muscle, inducing an up-regulation during fasting and a down-regulation during the recovery from fasting, whereas the muscular FGF6 mRNA levels were not significantly affected by the feeding status of the animals. These findings suggest that myostatin has an inhibitory role in muscle growth in response to different feeding regimens, whereas FGF6 is not involved in the muscle compensatory growth induced by refeeding.     

Growth factors controlling muscle development.

The enlarged muscles of certain breeds of cattle, such as the Belgian Blue, have been shown to result from a marked increase in the number of normal sized muscle fibers. Originally insulin-like growth factors (IGFs) were implicated in this myofiber hyperplasia, as IGFs have been shown to stimulate myoblast proliferation as well as maintain fiber differentiation. Recently it has been reported that mice lacking a myostatin gene, a member of the TGFbeta superfamily, have enhanced skeletal mass resulting from increased muscle fiber number and size. Mutations in this gene have been found in double-muscled cattle, indicating that myostatin is an inhibitor of muscle growth. Myostatin is expressed early in gestation and then maintained to adulthood in certain muscles. Myostatin expression in bovine muscle is highest during gestation when muscle fibers are forming and some of the myogenic regulatory factors have elevated expression over the same period as myostatin. Molecular expression of the IGF axis does not differ between Belgian Blue and normal muscled cattle, and IGF-II mRNA is increased throughout formation of secondary fibers in both breeds. However, myostatin and MyoD expression in muscle differ between normal and hypertrophied muscle cattle breeds. This evidence strongly suggests that lack of myostatin is associated with an increase in fiber number which then results in a marked increase in potential muscle mass in double-muscled cattle.     

Regulation of protein metabolism by insulin: value of different approaches and animal models

Insulin induces protein accretion by stimulating protein synthesis and inhibiting proteolysis. However, the mechanisms of regulation of protein metabolism by insulin are complex and still not completely understood. The use of approaches combining hyperinsulinemic clamp and isotopic methods, or measurement of the activation of intracellular kinases involved in insulin signaling, in addition to the use of different animal models in a comparative physiology process, provide better understanding of the potential regulation of protein metabolism by insulin. Studies using the clamp technique in lactating goats have shown a clear inhibitory effect of insulin on proteolysis, with an interaction between the effects of insulin and amino acids. Such studies revealed that the insulin-inhibited proteolysis is improved in lactating goats, this adaptative process limiting the mobilization of body protein under the conditions of amino acid deficit which occurs during early lactation. Insulin signaling studies in growing chickens have also provided some interesting features of insulin regulation compared to mammals. Refeeding or insulin injection leads to the activation of the early steps of insulin receptor signaling in the liver but not in the muscle. Muscle p70 S6 kinase, a kinase involved in the insulin activation of protein synthesis, was found to be markedly activated in response to insulin and to refeeding, suggesting that other signaling pathways than those classically described in mammalian muscles may be involved in signal transduction. Finally, although the role of insulin has been doubtful and has long been considered to be minor in ruminants and in avian species, this hormone clearly regulates protein metabolism in both species.     

Myostatin expression and possible functions in animal muscle growth

Myostatin (also known as growth/differentiation factor-8) is a recently identified member of the transforming growth factor-β family of secreted regulatory factors. Mice having targeted disruption of the myostatin gene displayed a marked increase in muscle mass, up to three times normal size. Additionally, a myostatin mutation has been linked to double muscled cattle breeds characterized by a visible, generalized increase in muscle mass. Therefore, it is suggested that myostatin in muscle may be one of the long sought inhibitors that specifically control the growth of individual tissues or organs. In the present paper, we review involvement of myostatin in muscle growth of different species.     

Salmonella enterica Typhimurium infection causes metabolic changes in chicken muscle involving AMPK,fatty acid and insulin/mTOR signaling

Salmonella enterica serovar Typhimurium (Salmonella Typhimurium) infection of chickens that are more than a few days old results in asymptomatic cecal colonization with persistent shedding of bacteria. We hypothesized that while the bacterium colonizes and persists locally in the cecum it has systemic effects, including changes to metabolic pathways of skeletal muscle, influencing the physiology of the avian host. Using species-specific peptide arrays to perform kinome analysis on metabolic signaling pathways in skeletal muscle of Salmonella Typhimurium infected chickens, we have observed key metabolic changes that affected fatty acid and glucose metabolism through the 5''-adenosine monophosphate-activated protein kinase (AMPK) and the insulin/mammalian target of rapamycin (mTOR) signaling pathway. Over a three week time course of infection, we observed changes in the phosphorylation state of the AMPK protein, and proteins up and down the pathway. In addition, changes to a large subset of the protein intermediates of the insulin/mTOR pathway in the skeletal muscle were altered by infection. These changes occur in pathways with direct effects on fatty acid and glucose metabolism. This is the first report of significant cellular metabolic changes occurring systemically in chicken due to a Salmonella infection. These results have implications not only for animal production and health but also for the understanding of how Salmonella infection in the intestine can have widespread, systemic effects on the metabolism of chickens without disease-like symptoms.