Chapter 2 Regulation of skeletal muscle protein metabolism in growing animals

PART II Protein metabolism

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Regulation of skeletal muscle protein metabolism in growing animals T. A. Davis and M. L. Fiorotto United States Department of Agriculture/Agricultural Research Service, Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030, USA

The skeletal musculature is not only of great significance to the physiological function and long-term well-being of the growing animal, but, by virtue of its large mass, has tremendous impact on the overall rate of protein metabolism in the whole body. Protein deposition is very rapid during early life and this is largely driven by the high fractional rate of protein synthesis in skeletal muscle. A number of factors regulate the growth, development, and metabolic activity of the skeletal musculature, and these include the intrinsic or genetic factors that influence muscle differentiation as well as the extrinsic factors such as nutrients, hormones, and activity that influence muscle hypertrophy.

1. INTRODUCTION In the mature adult, the skeletal musculature constitutes the largest single protein pool in the body, and comprises approximately 60% of the body’s metabolically active mass. Thus, despite its relatively low basal rate of metabolism, skeletal muscle mass is such that changes to its composition and/or its size have implications for the overall metabolism of the body. Until relatively recently, the primary interests in skeletal muscle metabolism were related to its functional role in dictating locomotor performance, specifically speed, strength, and endurance, and its influence on the quantity and quality of meat products that constitute a primary source of protein and micronutrients in the human diet. More recently, however, a renewed interest in the contribution of the muscle metabolism to the overall health of the human individual has emerged. This interest, together with the developments in our understanding of the regulation of gene expression and cellular signalling, have spurred substantial amounts of research to advance our understanding of how muscle mass, metabolism, and function respond to nutrients, hormones, growth factors, activity, and other anabolic agents. The fully differentiated skeletal muscle is made up of multinucleated myofibres. The postnatal growth rate of muscle mass is a function of the total number of fibres, and the growth

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Biology of Metabolism in Growing Animals D.G. Burrin and H. Mersmann (Eds.) © 2005 Elsevier Limited. All rights reserved.

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T. A. Davis and M. L. Fiorotto

rate of each fibre. Thus, understanding the regulation of myofibre formation during prenatal life, and the rate of muscle protein accretion in postnatal life, are critical for evaluating the animal’s maximal capacity for muscle growth. The mature myofibre is composed primarily of the three basic systems required for muscle contraction: the myofibrillar proteins composed primarily of the contractile elements and the associated structural proteins; an extensive membrane system that regulates the release and uptake of ions in response to the neural inputs; and the mitochondrial and cytoplasmic system of enzymes involved in the generation of the ATP required to drive functional processes. These components vary in their relative abundance, as well as in their level of activity, thereby giving rise to substantial diversity in fibre function and size. The combination of functional and metabolic properties of fibres is used as the basis for the standard classification of muscle fibres in the adult. Muscle fibres can be divided into two main categories on the basis of their twitch characteristics, that is, slow-twitch (S) or fast-twitch (F), which correspond with the Type I and Type II nomenclature, respectively. The contractile property of the myofibrils is largely determined by the ATPase activity of the myosin heavy chain (MHC) isoform expressed within each fibre (Schiaffino and Reggiani, 1996). Fibres are also identified in a variety of ways by their metabolic properties: slow-twitch fibres, and a subcategory of fast-twitch fibres, generate ATP predominantly by oxidative metabolism (O). All fast-twitch fibres also can generate limited amounts of ATP by glycolysis and, hence, they are categorized as glycolytic (FG or Type IIB) or fast-twitch, oxidativeglycolytic (FOG or Type IIA). The capacity for oxidative metabolism is supported by a relatively high abundance of mitochondria, enzymes for fatty acid oxidation, and oxygen delivery and uptake. The latter is effected by a high capillary density, and the presence of large amounts of myoglobin in the sarcoplasm, both of which impart a red tint to the muscle, so that SO and FOG fibres also are referred to as red muscle. Because these metabolic properties render to the fibres a greater degree of fatigue-resistance, they constitute primarily those muscles that undergo prolonged periods of sustained slow isometric contraction, such as postural muscles (e.g. soleus and rhomboides), or muscles that are required for continuous episodes of isotonic contraction (e.g. diaphragm and jaw muscles). In contrast, FG fibres have a paucity of these components and thus, muscles where these fibres predominate (e.g. longissimus dorsi) are much lighter in colour and are referred to as white muscles. The origin of myofibre heterogeneity is complex and not entirely understood. There is evidence that the various fibre types are derived from distinct myoblast lineages. However, within an organism, the relative abundance of different fibre types varies between muscles according to their physiological function. There is also variability between individuals of the same breed, between different breeds of the same species, and between different species (Rehfeldt et al., 1999). Fibre-type proportions are of relevance with respect to livestock, as they are a key determinant of meat quality (Koohmaraie, 2003). Fibre diversity has its origins in the embryo, is amplified during the process of differentiation during fetal and early postnatal life, and thereafter is fine-tuned in response to the functional demands placed on the muscle. By and large, however, fibre-type composition is a genetically determined, inherited trait. Muscle fibre hypertrophy, on the other hand, occurs after the fibres have differentiated. Hypertrophy is largely a postnatal event and metabolically is dominated by the accretion of muscle-specific proteins. Unlike the early processes of determination, commitment, and differentiation that are normally orchestrated by signalling molecules and intracellular pathways inherent to the developing organism and are primarily under genetic control, muscle hypertrophy is highly responsive to external cues, such as nutrient availability, muscle use, and various hormones. The aim of this review is to consider the factors that influence muscle protein metabolism in the growing organism. We shall address the “heritable/congenital” component, i.e. the

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developmental aspects of muscle growth that principally determine fibre number and fibretype diversity, and the role of external influences that modulate muscle hypertrophy. Owing to space limitations, this review cannot cover all aspects of muscle metabolism in equal depth. Rather, we have selected those areas in which substantial progress has been made recently, and have focused largely on early life, when the most marked changes occur.

2. MUSCLE DIFFERENTIATION 2.1. From myoblast to myofibre Much of our understanding of early myogenesis is derived from studies on the chick. However, the overall pattern is similar in mammals as indicated by the extensive study of the mouse in which the advent of transgenic technology has permitted the function of individual genes to be identified. Myogenesis begins in early embryonic life and, with the exception of craniofacial muscles, the skeletal musculature develops from mesodermal progenitor cells in the somites (Perry and Rudnicki, 2000; Buckingham, 2001). The somites develop in pairs from aggregates of epithelial cells on either side of the neural tube and notochord and mature in a rostro-caudal direction under the regulation of positive signals and negative regulators, in the form of diffusible molecules, produced by the tissues adjacent to the somites (Buckingham, 2001; Buckingham et al., 2003; Francis-West et al., 2003) (fig. 1). Cells of the dorsal surface of the somite are compartmentalized into the dermomyotome and are specified to form myogenic and dermal progenitors, whereas signals to cells on the ventral aspect of the somite specify the formation of the sclerotome which gives rise to the ribs and axial skeleton (Buckingham, 2001). Myogenic precursor cells originate from the dorso-medial (epaxial) and ventro-lateral (hypaxial) edges of the dermomyotome. The epaxial precursors delaminate and translocate ventrolaterally to form the myotome, under the dermomyotome, and eventually they expand and differentiate to form the deep back muscles. The hypaxial myogenic precursors of the dermomyotome migrate ventrally to form the ventral body wall muscles, tongue, and diaphragm, or delaminate and migrate into the limb buds to give rise to the limb musculature. Specification of myogenic cells occurs upon the activation of myogenic regulatory factors (MRF), specifically those that encode the basic helix–loop–helix transcription factors Myf5, MyoD, MRF4, and myogenin (Molkentin and Olson, 1996; Perry and Rudwicki, 2000).

Fig. 1. Schematic representation of somite structure and key molecules responsible for myogenic specification, determination, delamination, and migration.

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Myf5 and MyoD are the earliest MRFs to be expressed in the dermomyotome, and their patterns of expression appear to be spatially and temporally regulated. Wnt-1 produced in the dorsal neural tubes induces Myf5 expression in the epaxial region, whilst Wnts produced by the dorsal ectoderm induce MyoD expression by the dermomyotome. Sonic hedgehog (Shh), produced from the notochord and floor plate of the neural tube, also appears to play a role in the early activation of Myf5 in the determination of the epaxial dermomyotome (Buckingham et al., 2003). Cells committed to the muscle lineage express the homeobox gene, Pax-3, and its expression is most likely activated by Wnts and Shh. During early myogenesis, however, the differentiation of cells that express Pax-3 and are committed to the muscle lineage is blocked by the expression of inhibitory factors. These include the growth factor, bone morphogenic protein-4 (BMP-4), and fibroblast growth factors, both of which are produced by the lateral plate mesoderm. Inhibition of differentiation is critical as it enables the cells to continue proliferation, delaminate, and migrate. In the regions of the limbs, migrating cells do not express MRFs until they reach the limbs where, after some delay during which they undergo several rounds of replication, they give rise to the appendicular muscles (Buckingham, 2001; Buckingham et al., 2003; Francis-West et al., 2003). Delamination and migration of muscle precursor cells are crucial steps in myogenesis (Birchmeier and Brohmann, 2000; Francis-West et al., 2003) and require the activation of the c-met receptor by hepatocyte growth factor (HGF, also known as scatter factor) (Scaal et al., 1999; Birchmeier and Brohmann, 2000). Transcription of the c-met gene is activated by Pax-3, and in its absence no limb muscles form, whereas when a constitutively active form is expressed, there is an overproduction of hypaxial muscles (Epstein et al., 1996). Lbx1 is another transcription factor required for migration of the somitic cells, with particular importance for those cells that give rise to the dorsal muscle masses of the limbs (Brohmann et al., 2000). Once commitment of cells to the myogenic lineage has occurred, the cells are prevented from differentiating further by a variety of gene products produced by the cells themselves, as well as the cells’ matrix, and a variety of mitogens that promote proliferation (Perry and Rudnicki, 2000; Fuchtbauer, 2002). The maintenance of the committed, but not fully differentiated, state is a critical determinant of myofibre number and size because it permits myoblast proliferation to continue and, hence, to expand the population of cells that can give rise to myofibres (Fuchtbauer, 2002). Indeed, all instances of muscle hypertrophy that are associated with increased myofibre number have their origin in embryonic and fetal life, and by birth, fibre number is fixed (Rehfeldt et al., 1999). The degree of myoblast proliferation in vivo is the product of the balance between activities of stimulatory and inhibitory factors (Fuchtbauer, 2002). The former include Msx1, basic fibroblast growth factor (bFGF), HGF, and the insulin-like growth factors (IGF)-I and -II. The transforming growth factor (TGF-β) super-family of peptides exert a variety of effects which appear to be species-dependent, but by and large, they inhibit terminal differentiation with either little effect or even inhibition of proliferation (Fuchtbauer, 2002). Members of the TGF-β family that have been shown to regulate myogenesis include TGF-β itself, activin, BMPs, and growth differentiation factor 8, also known as myostatin (McPherron et al., 1997; Fuchtbauer, 2002). Their precise role and the incurred responses vary between muscle beds and according to the net balance between positive and negative regulators. The in vivo significance of these factors in regulating myoblast hyperplasia and fibre formation has been demonstrated in studies with an extensive variety of transgenic animals. Notable among these has been the development of transgenic mice in which the myostatin gene is inactivated (McPherron et al., 1997). Myostatin is expressed beginning early in embryonic life and inhibits cell cycle progression, thereby limiting myoblast proliferation

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(Lee and McPherron, 1999). Myostatin also inhibits differentiation by down-regulating MyoD and myogenin expression and activity (Langley et al., 2002). Hence, in its absence, there is greater myoblast proliferation and this ultimately results in the formation of a larger number of muscle fibres. The characterization of myostatin and its mechanism of action pointed to the identification of the genetic basis for the muscle hypertrophy in some breeds of cattle, commonly known as “double-muscling” (Grobet et al., 1997). In vivo, myostatin is regulated by follistatin, and follistatin overproduction increases muscle mass, whereas impaired production reduces muscle mass. The IGFs are unique in their actions in that they can stimulate both proliferation and differentiation by activating the type 1 IGF receptor; these effects, however, are temporally separated. The switch from proliferative to myogenic effects is associated with changes in the intracellular signalling pathways from primary activation of the mitogen-activated protein kinase (MAPK) pathway during the proliferative phase, to signalling through the phosphatidylinositol 3-kinase (PI 3-kinase) pathway for differentiation (Coolican et al., 1997). In cell culture, the proliferative phase is associated with IGF-I-stimulated expression of cell cycle markers and cell proliferation and reduced expression of myogenic markers, whereas during differentiation IGF-I promotes expression of myogenin and muscle-specific gene expression (Engert et al., 1996). The exact trigger responsible for switching the response from proliferation to differentiation is unclear, especially in vivo. Studies in the chick embryo have demonstrated that increased local expression of IGF-I in the limb at an early stage of development before cells have differentiated increases myoblast number with the formation of larger muscles containing an increased number of myofibres (Mitchell et al., 2002), in much the same way as, although independently of, decreased myostatin expression. Similarly, administration of growth hormone (GH) to pregnant sows in early gestation (10–24 days) indirectly stimulates fetal IGF secretion and results in greater myofibre number at birth (Rehfeldt et al., 1993). This response contrasts with the response to over-expression of IGF-I in the differentiated muscle, where muscle hypertrophy is not associated with increased fibre number (Musaro et al., 2001; Fiorotto et al., 2003). The stimulation of muscle differentiation by the MRFs entails not only up-regulation of their expression within presumptive muscle cells, but also the co-ordinated orchestration of a series of events that enables them to be transcriptionally active. These include: the downregulation of Id proteins which mitigate the binding of MRFs to their E-box consensus sequence (Benezra et al., 1990); the association with the myogenic enhancer factor 2 (MEF2) family of transcription factors that bind to both their own DNA site and form protein–protein interactions with the MRFs (Molkentin and Olson, 1996); and the dissociation of histone deacetylases from the transcription factors and subsequent recruitment of histone acetylases to E-boxes associated with muscle-specific genes. The resulting acetylation of histones produces the chromatin relaxation necessary to permit transcription of the underlying gene (McKinsey et al., 2001, 2002). This differentiation step is associated concomitantly with inhibition of cell cycling, and the alignment and subsequent fusion of the adhered myoblasts to form myotubes. Fibres form in two waves: the first wave occurs during early embryogenesis and results in the formation of primary myotubes that shape and position the orientation of individual muscles (Ontell and Dunn, 1978; Ontell, 1982). These primary fibres are originally in clusters, but progressively become separated by the basal lamina. Subsequently, a secondary population of myoblasts located under the basal lamina of the primary fibres begins to proliferate using the plasma membrane of the primary fibres as scaffolding, but without fusing to them. These then fuse among themselves to form secondary myotubes, and gradually separate from the

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primaries by forming their own basal lamina. There is a third population of myoblasts, the satellite cells, that remain quiescent, sandwiched between the plasma membrane and basal lamina. The expression of the homeobox protein, Pax-7, distinguishes satellite cells from other lineages of myoblast, and appears to be essential for the formation of satellite cells (Seale et al., 2000). Satellite cells proliferate to enlarge the myonuclear population present in the myofibres to which they are attached, and contribute only to myofibre hypertrophy, not new fibres (except to repair muscle damage). To this point in the process of skeletal muscle development, the primary effect of perturbations in the growth process are likely to manifest themselves as variations in the number of myofibres that form. The timing of the perturbation will determine if primary or secondary fibres are affected. It is unclear if impairment of satellite cell replication during the terminal phase of muscle formation can permanently impact the size of a muscle’s reserve of satellite cells and, hence, postnatal muscle growth potential. 2.2. Compositional development Once myoblasts have fused, the expression of muscle-specific proteins dominates muscle growth. The cells undergo a complex set of transformations to create the highly structured myofibrils which have the capacity to perform contractile work. This process of maturation is critical for the developing offspring as it is essential for postnatal survival: skeletal muscles are a prerequisite for independent breathing and suckling. Indeed, the offspring die at birth in all transgenic animals in which normal skeletal muscle development is impaired by targeted disruption of key regulatory molecules (e.g. Venuti et al., 1995). However, there is a wide variation in the level of muscle maturity at birth among species, and between muscle groups within the same individual. From these observations, it is evident that birth occurs at different stages of muscle development across species, and that within an individual, maturation proceeds in a rostral to caudal direction, and from proximal to distal in the appendages. Hence, altricial species, like rabbits, rodents, and most carnivores, are born with functional head and thoracic muscles, whereas their lower abdominal and limb muscles, especially those of the hindlimbs, are still immature. In contrast, precocial species, such as ungulates, have relatively longer gestations and the newborn muscle is at a fairly advanced stage of maturity. Indeed, locomotor function is attained very soon after birth. The maturation of myotubes into fully functional myofibres involves the co-ordinated development of the metabolic machinery of the cells, the ion transport membrane system, and the contractile elements. The complex sarcoplasmic reticulum and T-tubular system responsible for coupling excitation and contraction followed by muscle relaxation develop in a co-ordinated fashion and attain their mature configuration at approximately 2 weeks of age in the rat. At this point, the membrane system constitutes approximately 40% of the nonmyofibrillar compartment in the muscle fibre (Schiaffino and Margreth, 1969). Contractile proteins, not present in the myotube at fusion, comprise 55−65% of total muscle protein by 2 weeks of age in the rat (Yates and Greaser, 1983; Fiorotto et al., 2000a). The accretion of myofibrillar proteins, therefore, is a major determinant of whole-body protein accretion during this developmental period. The diversity in fibre-type characteristics results from the combination of the inherent properties of the myoblast lineage from which the fibres were derived and extrinsic signals from the organism. Thus, in the development of a myofibre from a myotube, in addition to the first-order general pattern of compositional development that occurs (described above),

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significant changes also occur in the isoform composition of muscle proteins and the supporting metabolic machinery. These result in a second order of compositional changes that produce the development of the adult phenotype of individual fibre types (Schiaffino and Reggiani, 1996). A significant proportion of the proteins that constitute the thick, intermediate, and thin filaments initially are expressed as immature isoforms and subsequently are replaced by the adult isoforms during maturation (table 1). In the thick filaments, all primary fibres initially express a combination of embryonic and slow MHC isoforms, or neonatal and slow MHC isoforms. The associated myosin light chains (MLC) also differ according to the MHC isoform with which they are associated and, therefore, their expression changes during maturation. Secondary fibres, in contrast, initially express the embryonic and neonatal MHC isoforms, which are replaced usually by any of the adult fast MHC isoforms according to the functional characteristics of the individual muscle. This process of terminal maturation is amenable to regulation by hormones, activity pattern, and neural inputs, and contrasts with the earlier differentiation of myoblast lineage which proceeds independently of extrinsic factors. A fundamental conundrum regarding the differentiation among fibre type relates to the mechanism that co-ordinates the appropriate expression of the plethora of proteins responsible for the metabolic and contractile characteristics of muscle. Recent studies have focused on the

Table 1 Contractile protein isoforms expressed in the developing and mature muscles of the rat Developing muscles

Adult muscles

Embryonic

Neonatal

Fast

Slow

Myosin heavy chains embryonic β-slow

neonatal (embryonic)

2B 2X 2A

β-slow

MLC-1fast (MLC-3fast)

MLC-1fast MLC-3fast

MLC-1slow/ventricular (MLC-1slow-α)

MLC-2fast

MLC-2fast

MLC-2slow

α-skeletal (α-cardiac)

α-skeletal

α-skeletal

TnC-fast

TnC-fast

TnC-slow/cardiac

TnI-fast TnT-fast, fetal isoforms

TnI-fast TnT-fast, adult isoforms

TnI-slow TnT-slow

TM-β TM-αfast

TM-αfast (TM-β)

TM-αslow TM-αfast TM-β

Myosin light chains (MLC) MLC-1embryonic MLC-1slow-α MLC-1fast MLC-2fast Actin α-cardiac α-skeletal Troponins (Tn) TnC-fast TnC-slow/cardiac TnI-slow TnT-cardiac TnT-slow Tropomyosins (TM) TM-β TM-αfast TM-αslow

Minor isoforms are indicated in parentheses. Isoform profile indicated for neonatal developing muscles is that of the majority of hindlimb muscle fibres, which are secondary generation fibres destined to become fast-type fibres. Adapted from Schiaffino and Reggiano (1996).

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role of calcineurin (CaN), a calcium and calmodulin-dependent serine/threonine phosphatase, in the regulation of expression of those genes responsible for the slow muscle phenotype (Schiaffino and Serrano, 2002; Spangenburg and Booth, 2003). CaN is activated when there are high intracellular steady-state levels of Ca2+, typical of slow fibres that are subjected to chronic, low-frequency nerve stimulation. Once activated, CaN dephosphorylates the transcription factors, nuclear factor of activated T-cells (NFAT) and MEF-2, so that they can then be translocated into the nucleus where, in conjunction with MRFs, they effect changes in gene expression (McKinsey et al., 2001, 2002). These factors bind to their respective DNA consensus sequences which form a characteristic motif, the slow upstream element (SURE), present in the promoter of a variety of slow muscle genes such as slow troponin I and myoglobin (Calvo et al., 2001). Despite the ability of CaN to sense and transduce changes in cell calcium levels into changes in gene expression, it is by no means a global regulator of the SO fibre phenotype as was initially proposed. For example, it has been observed that expression of MHC-IIa, the predominant MHC isoform in FOG muscles, is also highly responsive to CaN activation (Allen and Leinwand, 2002). Considerable progress has been made in identification of the mechanisms that co-ordinate the contractile and metabolic characteristics of muscle. Again, the sustained elevation of intracellular calcium appears to be a central factor in signalling not only the fast-to-slow shift in muscle gene expression (Allen and Leinwand, 2002), but also an increase in mitochondrial biogenesis (Ojuka et al., 2003). In addition to CaN, calcium activates calcium/calmodulindependent protein kinases (CaMK) which catalyse a series of reactions that result in the transcription of a coactivator of nuclear receptors, peroxisome proliferator-activated receptor coactivator-1α (PGC-1α) (Handschin et al., 2003). PGC-1α plays a pivotal role in glucose metabolism, mitochondrial biogenesis, and adaptive thermogenesis by activating various transcription factors. Specifically, in muscle PGC-1α has been shown to stimulate mitochondrial DNA replication, mitochondrial abundance, cytochrome c and cytochrome oxidase levels, GLUT4 expression, and uncoupling protein expression. PGC-1α also enhances its own transcription (Handschin et al., 2003). Consequently, once activated, an autoregulatory loop is set up which sustains PGC-1α expression and its downstream effects, and thereby maintains stable expression of the oxidative phenotype. In addition to calcium, PGC-1α expression is regulated by thyroid hormone and AMPactivated protein kinase, an enzyme that is activated by chronic reductions in the cellular ATP/AMP ratio, for example, with energy deprivation (Irrcher et al., 2003; Ojuka et al., 2003; Spangenburg and Booth, 2003). These mechanisms that co-ordinate the metabolic properties of a muscle with its contractile properties and pattern of use, however, are pertinent primarily to the development of slow-twitch and/or oxidative properties, presumably in muscles where these characteristics are not present. This suggests that fast-twitch, glycolytic properties are the default phenotype of skeletal muscle and there is, indeed, some evidence to support this suggestion. During terminal maturation, the loss of polyinnervation and acquisition of single innervation from a nerve with a low-frequency firing pattern is necessary for the development and maintenance of slow-twitch characteristics. Moreover, if the soleus is denervated at birth in the rat, slow myosin isoenzymes are gradually replaced by fast myosins (Gambke et al., 1983). Thus, the replacement of the immature isoforms of myosin specifically by adult slow myosin occurs only with the appropriate neural input. In the mature muscle, cross-innervation of a mature fast-twitch muscle with the nerve from a slow-twitch muscle gradually transforms the entire contractile phenotype and metabolic properties to those of a slow muscle (Barany and Close, 1971). Thyroid hormone also plays a critical role in the maturation of skeletal muscle. Moreover, because thyroid hormone is sensitive to changes in energy balance, it may serve as a signal

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to the muscle to produce adaptive changes in muscle metabolism. The importance of thyroid hormone has been studied extensively with respect to the regulation of MHC expression where it is required for down-regulation of the neonatal isoform of MHC in muscles destined to become either fast or slow (Gambke et al., 1983; Adams et al., 1999). Furthermore, in the absence of thyroid hormone, the accumulation of slow MHC is accelerated, whereas that of IIA MHC is down-regulated. Thus, variations in energy balance that produce changes in thyroid hormone might be anticipated to alter the postnatal pattern of muscle maturation. Our studies in suckling rats suggest that the changes in thyroid hormone have to be relatively severe in order to effect changes at the level of gene expression. However, changes in muscle use and protein turnover also occur as a consequence of alterations in food intake and growth rate. These changes serve to mitigate the effect of altered gene expression and, consequently, the maturation of muscle phenotype is preserved (Fiorotto and Davis, 1997; Fiorotto et al., 2000a). The suckling pig responds to mild hypothyroidism during the suckling period by increasing slow MHC expression, although the effect is somewhat mitigated by increases in nuclear thyroid hormone receptor (Harrison et al., 1996). Relatively severe energy restriction in post-weaned pigs has similar effects, increasing the abundance of slow MHC at both the protein and mRNA level, and increasing the oxidative properties of the muscles, but with substantial muscle-to-muscle variability (White et al., 2000). Although the increase in slow MHC expression is compatible with the known effects of hypothyroidism on MHC expression, the enhanced oxidative properties are the opposite of those anticipated on the basis of PGC-1α regulation by thyroid hormone. However, they are compatible with a change in AMP kinase activity, which increases with a chronic deficit in energy intake, and thereby promotes mitochondrial biogenesis and fatty acid oxidation. Overall, these responses to a chronic deficit in energy intake represent beneficial adaptations by the muscle to enhance its metabolic efficiency: a slow muscle requires less energy than a fast-twitch muscle to generate the same amount of tension, and it is able to derive more of its energy by fatty acid oxidation and oxidative phosphorylation (Henriksson, 1990). The regulation of the fast-twitch, glycolytic phenotype of muscles is much less clearly understood than that of slow-twitch muscle. Some genes expressed in fast fibres contain a characteristic binding motif, the fast intronic regulatory element (FIRE), analogous to the SURE motif in slow fibre genes (Nakayama et al., 1996). Myoblast lineage established during fetal life appears to be a primary determinant of whether a fibre matures into a fast fibre. As noted previously, gene mutations that promote secondary myoblast proliferation result primarily in fast fibre hypertrophy. During terminal maturation, thyroid hormone is required for the down-regulation of neonatal MHC and, if present at high levels, thyroid hormone tends to drive the expression of fast MHC in muscle fibres that normally would be slow (Nakayama et al., 1996). The role of innervation also appears to be less critical in the development of fast-twitch fibres than for slow fibres. In both rodents and chickens, denervation delays the elimination of immature MHC isoforms, but does not prevent the development of the fast phenotype. Inactivity also tends to promote the fast phenotype, although this is attributable in part to the preferential atrophy of slow fibres. In post-weaned pigs (Katsumata et al., 2000), but possibly not neonatal pigs (Louveau and Le Dividich, 2002), mild undernutrition up-regulates the expression of the growth hormone receptor (GHR) on FOG and FG fibres which normally express the lowest level of GHR. Together with the reduction in thyroid hormone expression, the resulting changes in hormone responsiveness may be responsible for the metabolic shift that occurs in muscle during undernutrition and which enables it to derive more of its energy from lipid oxidation. A broader implication of these findings, however, relates to the anti-insulin effects of GH in the undernourished animal which serve to divert

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nutrients from muscle towards the visceral organs. This is demonstrated by the differential response in rates of tissue growth in protein-malnourished piglets where body protein partitioned into gastrointestinal tissue is preserved, while that of skeletal muscle is reduced (Ebner et al., 1994). 2.3. Role of protein synthesis and degradation in the regulation of compositional development As the above discussion of the developmental changes in muscle composition indicates, protein synthetic rates in the immature muscle must sustain not only the de novo accretion of myofibrillar proteins and membrane structures, but also their continuous and co-ordinated replacement as the tissue develops its mature compositional and functional characteristics. The faster accumulation of myofibrillar proteins compared to sarcoplasmic proteins is explained almost entirely by their higher fractional synthesis rate compared to other protein components (Fiorotto et al., 2000a). As compositional maturity is attained, the synthesis rate of myofibrillar proteins decreases to a greater extent than sarcoplasmic proteins, and in the mature muscle, sarcoplasmic protein synthesis rates are higher than for myofibrillar proteins. However, once the mature composition is attained, the rates of degradation also differ in parallel and results in the maintenance of constant composition. In altricial mammals such as rodents and rabbits, the differential regulation of protein synthesis in the different protein pools occurs in the immediate postnatal period. In precocial animals, the full complement of myofibrillar proteins in fibres is mostly completed by birth, although they still undergo some limited, second-order compositional maturation postnatally. Nonetheless, the intrauterine pattern of development and mechanisms of regulation at comparable stages of maturity are likely to be similar across species. In the mature muscle, there are fibre-type differences in the rate of protein turnover that reflect their compositional differences; slow fibres have higher rates of protein turnover than fast-twitch fibres (Bark et al., 1998; Dardevet et al., 2002), and this diversity emerges only upon maturation (Davis et al., 1989). These phenotypic differences are attributable to the differences in the synthesis rate of muscle proteins in combination with the variation in their relative abundances among muscles. In skeletal muscles from mature pigs, the average synthesis rate of mitochondrial proteins is higher than for sarcoplasmic proteins and this, in turn, tends to be slightly higher than for the myofibrillar proteins (Balagopal et al., 1997; Fiorotto et al., 2000a). Although the ratio of myofibrillar to sarcoplasmic proteins tends to be greater in slower muscles (Hemel-Grooten et al., 1995), the difference in synthesis rates is substantially lower than that of mitochondrial proteins, which are more abundant in the slower, oxidative muscles. The greater overall protein synthetic activity of the slow muscles is supported by a higher ribosomal abundance and entails minimal changes in protein synthetic efficiency. In addition to the inherent variation in synthesis rates, the myofibre protein components can also differ in their responses to extrinsic stimuli. For example, in adult porcine muscle, stimulation of protein synthesis by insulin appears to be limited to the mitochondrial proteins (Boirie et al., 2001); the developmental decline in muscle protein synthesis rates is dominated by myofibrillar proteins (Fiorotto et al., 2000a). Clearly, these differences among muscle protein components have to be factored into our understanding of the overall regulation of skeletal muscle protein metabolism. In the newly differentiated muscle, the high ribosomal abundance is the principal factor that enables high rates of protein synthesis to be attained, and its reduction with maturation is one mechanism that may underlie the general reduction in fractional synthesis rates

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observed for all muscle proteins. However, this cannot explain the differences in composition of proteins synthesized, and some regulation must occur at the level of gene transcription. During the transition from myoblast to myotube, genes encoding non-muscle proteins are repressed, while those specific for muscle proteins are induced in a co-ordinated manner. There is then a commensurate change in the composition of proteins expressed (Devlin and Emerson, 1978, 1979; Shani et al., 1981). In vivo, it has been demonstrated that the stoichiometry of the total mRNAs encoding all isoforms within a protein family is maintained accurately, and that production of individual myofibrillar proteins in appropriate stoichiometric amounts, therefore, is regulated at the message level (Wade et al., 1990). However, such changes would not explain the differential responses of sarcoplasmic and myofibrillar proteins even if the decrease in ribosomal abundance were accompanied by a reduction in the proportion of myofibrillar mRNAs. Such a change in mRNA composition would increase the translational efficiency of sarcoplasmic proteins, but decrease the translational efficiency of myofibrillar proteins. Translational efficiency, however, increases for all proteins in the immature muscle. Clearly, therefore, although differences in mRNA abundances are involved, as we have demonstrated in newborn pigs (Fiorotto et al., 2000b), this also cannot be entirely responsible for the compositional differences in protein synthesis, suggesting that there must also be regulation of mRNA at the translational and post-translational levels.

3. POSTNATAL MUSCLE GROWTH 3.1. Satellite cells and hyperplasia Postnatal growth of skeletal muscle is driven by hypertrophy of the existing fibres. This requires both an increase in myonuclear content, and the accretion of muscle proteins. Myonuclei are postmitotic and, thus, satellite cells are entirely responsible for the postnatal increase in muscle fibre DNA. This is clearly demonstrated in mice in which the expression of Pax-7 is abolished and consequently no satellite cells form (Seale et al., 2000). These muscles contain both primary and secondary fibres but they fail to hypertrophy postnatally. Indeed, in a variety of circumstances normally associated with accelerated postnatal muscle growth, the inhibition of satellite cell replication will prevent the growth response (Rosenblatt and Parry, 1992). Recent evidence suggests that subpopulations of satellite cells may exist that can be distinguished by their proliferative potential (Perry and Rudnicki, 2000; Seale and Rudnicki, 2000). There is a reserve population of quiescent, non-differentiated cells that retains its mitogenic potential and has the capacity for self-renewal. Under the appropriate stimulation, these satellite cells become activated, migrate as necessary, and undergo a limited number of replications before they terminally differentiate. These cells can no longer divide and undergo fusion into the myofibre. In the rat, satellite cells comprise approximately 32% of muscle nuclei at birth and decrease to 10% at 4 weeks of age and less than 5% at sexual maturity when the cells are largely mitotically quiescent (Allbrook et al., 1971). A similar pattern is seen in the pig, in which satellite cells constitute approximately 20% of total muscle nuclei at birth, and 4% at 64 weeks of age (Campion et al., 1981; Mesires and Doumit, 2002). These values vary according to the metabolic properties of the muscle. The significance of myonuclear number in the context of muscle growth relates to the observation that in skeletal muscle, “myonuclear domain” size, i.e. the quantity of cytoplasm regulated by a single myonucleus (and reflected by the protein:DNA ratio), is tightly regulated (Allen et al., 1999). This implies that the amount of protein that can be deposited without further addition of

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myonuclei is limited. Nuclear domain size under “steady state” conditions appears to vary according to the metabolic activity of the fibre. It is smaller for oxidative than glycolytic fibres, and for any given fibre type, values increase with age. Although satellite cells become quiescent as growth rate plateaus, their proliferation can be reactivated in response to muscle injury, denervation, or increased muscle stretch and it is essential for muscle repair and hypertrophy (Bischoff, 1994). The close similarity between the developmental changes in satellite cell replication and protein synthesis strongly suggests that these processes may be linked. This is further supported by the differential response of immature and mature skeletal muscles to suboptimal nutrient intakes. Alterations in food intake (greater or less than average) in the neonatal animal, provided they are not severe, alter DNA and protein accretion proportionally as indicated by the maintenance of relatively normal, age-appropriate protein:DNA ratios despite a wide range of growth rates (Fiorotto and Davis, 1997). In transgenic animals, sustained over-expression of IGF-I in the skeletal muscle promotes satellite cell replication and transiently increases the accretion of total muscle DNA; this increase precedes the enhanced accumulation of muscle protein and results in protein:DNA ratios that temporarily are lower than normal (Fiorotto et al., 2003). These ultimately increase to age-appropriate values, but never surpass those in wild-type control animals. A potential link between satellite cell replication and the capacity for protein synthesis is through the regulation of ribosomal production, ribosomal abundance being the primary determinant of a cell’s maximal capacity for protein synthesis. Regulation of ribosome biogenesis is achieved in the majority of cells by altering the rate of rRNA synthesis by rDNA transcription (Zahradka et al., 1991), the regulation of which is coupled to cell cycling via the retinoblastoma gene product, pRb (Hannan et al., 2000). We have demonstrated that the enhanced replicative capacity of satellite cells from muscles that overexpress IGF-I is associated with increased phosphorylation of pRb upon mitogen stimulation (Chakravarthy et al., 2001). Thus, when rates of satellite cell division are high, pRB is phosphorylated, and in this form it enables a key transcription factor for rDNA transcription, UBF, to transactivate rDNA genes to promote rRNA synthesis. Thus, accretion of ribosomes is necessarily correlated to the rate of cell division. 3.2. Role of protein synthesis in the regulation of muscle growth A rapid increase in the absolute rate of growth occurs during early postnatal life and a majority of this growth is comprised of skeletal muscle protein (Young, 1970). The more rapid accretion of muscle protein than other tissue proteins results in an increase in the proportion of the body protein pool that is represented by muscle protein from ~30% in the newborn to ~50% in the adult (fig. 2). However, the fractional rate of growth, i.e. the amount of weight gained in relation to the existing mass, is extremely high at birth and decreases with development, with the most rapid change in the fractional rate of growth occurring during the neonatal period. This developmental decline in the fractional rate of growth is largely explained by a developmental decline in the fractional rate of protein deposition in skeletal muscle (Shields et al., 1983; Mitchell et al., 2001). Changes in the rate of protein deposition are driven by changes in the rates of protein synthesis or protein degradation such that a decline in the fractional rate of protein deposition can be due to a decline in the fractional rate of protein synthesis, an increase in the fractional rate of protein degradation, or both. The developmental decline in protein deposition in skeletal muscle is due to a developmental decline in the fractional rate of muscle protein synthesis

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Fig. 2. Relative changes in the proportion of whole-body protein mass attributable to skeletal muscle in the rat between birth and weaning (Fiorotto et al., unpublished observations).

(Kelly et al., 1984; Denne and Kalhan, 1987; Davis et al., 1989) (fig. 3). In fact, the fractional rate of muscle protein synthesis in the pig and rat is about 3-fold higher in the newborn than at weaning, and the rate of decline is attenuated as development proceeds (Kelly et al., 1984; Davis et al., 1989, 1996; Baillie and Garlick, 1992; Fiorotto et al., 2000a). This developmental decline in skeletal muscle protein synthesis is more profound in muscles containing predominately FG fibres than in those containing primarily SO fibres (Davis et al., 1989). By contrast, fractional protein degradation rates in skeletal muscle decline modestly with development. The rate of protein synthesis is determined by the abundance of ribosomes, the efficiency of the translational process, and potentially, the concentration of translatable mRNA (Kimball and Jefferson, 1988). Because the majority of RNA in tissues is rRNA, ribosomal abundance can be estimated from the RNA to protein ratio, or can be measured more precisely from the amount of 18S rRNA expressed per unit protein. The efficiency of the translation process can

Fig. 3. Relationship between the postnatal decline in the rate of muscle protein accretion and the fractional synthesis rate of skeletal muscle proteins in the hindlimbs of rats. (Data compiled from Davis et al., 1989; Fiorotto et al., 2000a.)

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be calculated from the amount of protein synthesized per unit RNA and reflects how well the protein synthetic machinery is functioning. Chronic changes in protein synthesis are thought to be a result of a change in ribosome number. Thus, the high rate of protein synthesis in immature muscle and its overall decline with development are driven largely by an elevated number of ribosomes at birth and a developmental decline in ribosome concentration as the musculature matures (Kelly et al., 1984; Davis et al., 2001). Rapid changes in the rate of protein synthesis, including those due to food ingestion, are generally regulated by changes in the efficiency of translation process secondary to modulation of the rate of translation initiation (Harmon et al., 1984; Kimball and Jefferson, 1988; Kimball et al., 1994), the rate-limiting step in protein synthesis. One of the best characterized steps involved in the regulation of translation initiation, depicted in fig. 4, is the binding of initiator methionyl-tRNA (met-tRNA) to the 40S ribosomal subunit to form the 43S preinitiation complex via mediation of eukaryotic initiation factor (eIF) 2 (Pain, 1996; Kimball et al., 1997; Webb and Proud, 1997). The eIF2-mediated met-tRNA binding to the 40S subunit is further regulated by the activity of eIF2B, which exchanges GDP for GTP on eIF2 (Kimball et al., 1996). A second well-characterized step in translation initiation, shown in fig. 4, is the binding of mRNA to the 43S preinitiation complex via mediation of the assembly of the eIF4F complex of proteins (Lin et al., 1994; Rhoads et al., 1994; Sonenberg, 1994). The three proteins comprising the eIF4F complex are eIF4A, an RNA helicase, eIF4E, the protein that binds to the m7GTP cap structure at the 5′-end of the mRNA, and eIF4G, a scaffolding protein that binds to the 40S ribosomal subunit. Thus, mRNA binds to the 40S ribosomal subunit through the association of eIF4E with eIF4G. The availability of eIF4E for binding to eIF4G is regulated by its association with 4E-BP1, a repressor protein that competes with eIF4G for binding to eIF4E (Pause et al., 1994). Upon stimulation by an anabolic agent, such as insulin, 4E-BP1 becomes phosphorylated, resulting in reduced affinity of eIF4E for 4E-BP1, release of eIF4E, and enhanced binding of eIF4E to eIF4G to form the active eIF4E:eIF4G complex (Gingras et al., 1999). Activation of translation initiation is mediated through a signal transduction pathway involving a protein kinase referred to as the mammalian target of rapamycin (mTOR) which,

Fig. 4. Regulation of translation initiation. Abbreviations: eIF, eukaryotic initiation factor; 4EBP1, eIF4E binding protein; Met-tRNA, initiator methionyl tRNA; S6K, 70 kDa ribosomal protein S6 kinase 1; 43S, 43S ribosomal subunit; 48S, 48S ribosomal subunit.

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in addition to phosphorylating 4E-BP1, also phosphorylates and activates the 70 kDa ribosomal protein S6 kinase, S6K1 (Jefferies et al., 1994; von Manteuffel et al., 1997). These phosphorylation events lead to an increase in the rate at which most proteins are synthesized and, in addition, the preferential increase in translation of mRNAs encoding elements of the translational apparatus, including ribosomal proteins and elongation factors. Recent studies in growing pigs suggest that the overall developmental decline in the response of skeletal muscle protein synthesis to feeding involves regulation by eIF2B (Davis et al., 2000). Availability of eIF4E for 48S ribosomal complex formation follows a similar pattern. This response is primarily modulated by the developmental change in the feedinginduced activation of the factors involved in the binding of mRNA to the 43S preinitiation complex. 3.3. Role of protein degradation in the regulation of muscle growth Less is known about the mechanisms that regulate protein degradation than those that regulate protein synthesis. It is known that there are multiple pathways in mammalian tissues for the degradation of proteins and that these pathways are highly controlled and selectively degrade specific protein substrates. These pathways include the lysosomal–autophagic system, the calpain–calpastatin system, and the ubiquitin–proteasome system (Goll et al., 1989; Attaix et al., 1999). The lysosomal–autophagic systems involve primarily cathepsins. Most evidence suggests that this pathway of degradation is unselective and may be of special importance under conditions in which cellular proteolysis is maximally activated. The calpain–calpastatin system is the major calcium-activated pathway of protein degradation. At least two main calpain isoforms, μ− calpain and m-calpain, have been identified and the system is subject to inhibition by the protein, calpastatin. The proteases play an important role in muscle myofibrillar protein turnover by catalysing initial disruption of the structure via proteolysis at the Z-disc. Released myofilaments can then be degraded into amino acids by the proteasome and/or lysosomal enzymes (Goll et al., 1992). The ubiquitin–proteasome pathway is widely distributed among tissues and has a relatively broad protein specificity. It consists of a recognition system involving the protein ubiquitin, which is responsible for targeting the protein substrates towards degradation by forming a polyubiquitin complex, and a multifunctional protease, referred to as the proteasome, which degrades the proteins. The role of these proteolytic pathways in the regulation of muscle growth and development remains to be explored.

4. REGULATORS OF PROTEIN SYNTHESIS 4.1. Feeding Dietary protein is utilized very efficiently for the deposition of whole-body protein during early postnatal life (Pellett and Kaba, 1972; McCracken et al., 1980; Fiorotto et al., 1991; Davis et al., 1993a). The accumulated evidence suggests that young animals utilize their dietary amino acids more efficiently for growth because they are capable of a greater increase in muscle protein synthesis in response to feeding than older animals (Davis et al., 1991, 1996). Feeding stimulates protein synthesis in the whole body of the newborn human (Denne et al., 1991); in skeletal muscle of the suckling lamb (Oddy et al., 1987; Wester et al., 2000); and in skeletal muscle of the post-weaned, but still growing, rat (Garlick et al., 1983). However, the stimulation of muscle protein synthesis by feeding is blunted or absent in adult mammals (Melville et al., 1989; Baillie and Garlick, 1992; Tessari et al., 1996).

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Stimulation of protein synthesis by feeding decreases with development in neonatal pigs.

In the suckling pig (Davis et al., 1996, 1997; Burrin et al., 1997a) and rat (Davis et al., 1991, 1993b), protein synthesis in skeletal muscle is maximally stimulated after eating. Figure 5 shows that the postprandial rise in protein synthesis in skeletal muscle of the neonatal pig declines sharply during the first 4 weeks of life. Although feeding stimulates protein synthesis in all tissues of the neonatal animal, the magnitude and the developmental decline in the response to feeding are most pronounced in skeletal muscle (Burrin et al., 1991, 1995, 1997a; Davis et al., 1991, 1993b, 1996). This enhanced ability of skeletal muscle protein synthesis to respond to the provision of nutrients in young growing animals should not be surprising, because the rate of protein deposition during the postprandial period must be higher than the rate of protein loss during the postabsorptive period to permit growth of skeletal muscle. Recent studies have examined the developmental changes in the expression and activation of factors that regulate the feeding-induced stimulation of protein synthesis in skeletal muscle of young, growing pigs. The results show that eIF2B activity, which regulates the binding of met-tRNA to the 40S ribosomal subunit, is unaffected by feeding but decreases with development. The stimulation of muscle protein synthesis by feeding, and the developmental decline in this response, involve regulation by the eIF4F complex (Davis et al., 2000; Kimball et al., 2002). In skeletal muscle of the neonatal pig, feeding increases the phosphorylation of 4E-BP1, resulting in dissociation of the inactive 4E-BP1 . eIF4E complex, and increased association of the active eIF4E . eIF4G complex. This response leads to a global increase in the rate of muscle protein synthesis. These feeding-induced changes in the activity of factors that regulate eIF4F formation decrease with development in parallel with the developmental change in the feeding-induced stimulation of muscle protein synthesis. A response to feeding has been observed in “teenage” rats (Yoshizawa et al., 1997). However, the magnitude of the response is smaller than that in neonatal pigs, thus further supporting a developmental decline in the feeding-induced formation of the eIF4F complex. The developmental changes in the feeding-induced eIF4F activation occur in parallel with increased phosphorylation of S6K1, which is involved in the translation of mRNAs encoding specific proteins that regulate translation initiation (Davis et al., 2000; Kimball et al., 2002). An increased phosphorylation of both 4E-BP1 and S6K1 suggests involvement of the mTOR signalling pathway in this process. Furthermore, rapamycin, a specific inhibitor of mTOR,

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strongly attenuates the feeding-induced assembly of both eIF4F and S6K1 activation (Kimball et al., 2000). Thus, the enhanced activation of the eIF4F complex following food consumption likely plays an important role in the postprandial stimulation of muscle protein synthesis in growing animals and the efficient use of dietary amino acids for muscle protein deposition in the neonate. 4.2. Insulin Studies performed in incubated muscles and in perfused hindlimbs of growing animals clearly demonstrate that insulin stimulates protein synthesis (Jefferson et al., 1977; Davis et al., 1987; Kimball et al., 1994). The infusion of physiological concentrations of insulin in fasted, weaned rats stimulates muscle protein synthesis in vivo to rates similar to those found in the fed state (Garlick et al., 1983). This response to feeding can be blocked by co-administration of anti-insulin serum (Preedy and Garlick, 1986). Furthermore, insulin has been shown to stimulate whole-body amino acid utilization and protein synthesis in the fetal sheep (Liechty et al., 1992; Thureen et al., 2000), protein synthesis in hindlimb of the young lamb (Wester et al., 2000), and skeletal muscle protein synthesis in the weaned rat (Garlick et al., 1983). In marked contrast to studies conducted in growing animals, most studies in adult animals (Baillie and Garlick, 1992; McNulty et al., 1993) and humans (Gelfand et al., 1987; Heslin et al., 1992; Louard et al., 1992) show little, if any, response of muscle protein synthesis to physiological increases in insulin. This suggests that the response of muscle protein synthesis to insulin is developmentally regulated. Insulin plays a key role in the increased response of skeletal muscle protein synthesis to feeding, and thus the increased rate of protein deposition, during the early postnatal period. In fasted and fed neonatal pigs, there is a positive curvilinear relationship between the postprandial increase in fractional muscle protein synthesis rates and circulating insulin concentrations (Davis et al., 1997). Studies using a hyperinsulinemic–euglycemic–euaminoacidemic clamp technique show that when amino acids and glucose are maintained at fasting levels, insulin infusion increases amino acid disposal, and that the insulin sensitivity and responsiveness of amino acid disposal decrease with development (Wray-Cahen et al., 1997). This response suggests that the developmental change in the insulin sensitivity of whole-body amino acid disposal may underlie the developmental change in the efficiency of utilization of dietary amino acids for protein deposition. Furthermore, raising insulin concentrations in the neonatal pig to levels typical of the fed state increases the rate of skeletal muscle protein synthesis to within the range normally present in the fed state, even when amino acids and glucose are maintained at fasting levels (Wray-Cahen et al., 1998). This response to insulin, like the response to feeding, is attenuated with development and is greater in muscles that are composed primarily of FG fibres, and is not specific to myofibrillar proteins (Davis et al., 2001). The insulin signalling cascade (fig. 6) leading to the stimulation of protein synthesis is initiated by insulin binding to its receptor. This leads to autophosphorylation of the receptor, the activation of insulin receptor tyrosine kinase, and the subsequent phosphorylation of several cytosolic substrates including insulin receptor substrate (IRS)-1 and -2 (Sun et al., 1991; White and Kahn, 1994). IRS-1 and -2 serve as “docking proteins”, transmitting insulin signals to several proteins that contain Src-homology 2 (SH2) domains (Backer et al., 1992; Sun et al., 1993) including phosphatidylinositol (PI) 3-kinase, which catalyses the phosphorylation of PI. The activation of PI 3-kinase triggers the activation of components of the insulin signalling pathway leading to translation initiation, i.e. protein kinase B (Akt) and mTOR.

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Fig. 6.

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Insulin signalling pathway leading to translation initiation.

Studies focusing on the developmental changes in the insulin signalling pathway that leads to translation initiation have shown that in the pig the abundance of insulin receptor protein in muscle during the early suckling period is 2-fold higher than at weaning (Suryawan et al., 2001). Although the abundance of IRS-1 and IRS-2 does not change with development, the abundance of the downstream signalling proteins, protein kinase B and mTOR, decreases with development (Kimball et al., 2002). This developmental decline in the abundance of insulin receptor, protein kinase B, and mTOR in skeletal muscle likely contributes to the overall decline in the responsiveness of muscle protein synthesis to feeding that occurs over the course of development. Because insulin mediates the postprandial elevation in skeletal muscle protein synthesis and this response decreases with development (Wray-Cahen et al., 1998; Davis et al., 2001), it is not surprising that the feeding-induced activation of the insulin signalling pathway that regulates protein synthesis decreases with development. Thus, the feeding-induced activation of the insulin receptor, IRS-1, IRS-2, PI 3-kinase, and protein kinase B in skeletal muscle decreases with development (Suryawan et al., 2001; Kimball et al., 2002), in parallel with the developmental decline in the feeding-induced activation of translation initiation factors and protein synthesis (Davis et al., 1996). This suggests that the developmental decline in the postprandial stimulation of protein synthesis in skeletal muscle results from a reduction in the capacity of the intracellular insulin signalling pathway to transduce to the translational apparatus the stimulus provided by the feeding-induced rise in insulin and/or amino acid concentrations. A number of studies performed in cell culture, in the perfused hindlimb, and in intact growing rats have demonstrated that the stimulation of protein synthesis by insulin involves increased phosphorylation of the translational repressor protein, 4E-BP1, reduced interaction with eIF4E, and increased assembly of the mRNA cap-binding complex, eIF4G:eIF4E (Kimball et al., 1994, 1997). Furthermore, insulin increases phosphorylation of S6K1, thereby increasing the translation of specific proteins involved in the regulation of translation. Recent in vivo studies performed in neonatal pigs support these findings and further show that the insulin-induced

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changes in factors regulating translation initiation as well as the upstream components of the insulin signalling pathway occur in a dose-response manner within the physiological range (Suryawan et al., 2001; O’Connor et al., 2003). Recently, however, studies in adult rats suggest that while insulin increases the phosphorylation of S6K1, insulin does not alter 4E-BP1 phosphorylation (Long et al., 2000). This lack of effect of insulin on 4E-BP1 phosphorylation and, by inference, eIF4F formation, is not surprising as physiological hyperinsulinaemia has no effect on muscle protein synthesis in adults. Thus, insulin plays an important role in the regulation of protein synthesis in muscle of growing animals, but its importance during adulthood is less apparent. 4.3. Amino acids Although amino acids are the precursors for the synthesis of proteins, they also play a key role as nutritional signals in the regulation of muscle protein synthesis. Amino acids have the capability to stimulate muscle protein synthesis throughout a substantial part of the life cycle, in contrast to the developmental decline and loss of the capability of insulin to stimulate muscle protein synthesis with age. In weaned but still growing rats (Preedy and Garlick, 1986), adult humans and rats (Bennet et al., 1990; McNulty et al., 1993; Vary et al., 1999), and elderly people (Volpi et al., 1998), acute amino acid infusion, either alone or concurrent with insulin infusion, stimulates protein synthesis in skeletal muscle. Recent studies suggest, however, that the magnitude of the stimulation of muscle protein synthesis by amino acids may decrease in the early postnatal period (Davis et al., 2002a). When a balanced amino acid mixture is infused into fasted, growing pigs, muscle protein synthesis increases and this response to amino acid infusion decreases with development, in parallel with the developmental decline in the feeding-induced stimulation of skeletal muscle protein synthesis (Davis et al., 2002a). In young pigs, the stimulation of skeletal muscle protein synthesis by amino acids is greater in muscles that contain predominately FG muscle fibres than in those that contain primarily SO fibres, and is similar for myofibrillar and sarcoplasmic proteins. The response to amino acid infusion occurs when insulin levels either remain at the fasting level or are raised to the fed level by infusion (O’Connor et al., 2003). Indeed, the magnitude of the increase in muscle protein synthesis with amino acid stimulation is similar to that which occurs with insulin stimulation alone, implying that insulin and amino acids may be interacting with the same signalling pathway within skeletal muscle. Studies performed in cell culture have shown that amino acid availability modulates protein synthesis by regulating both the met-tRNA and mRNA binding steps of translation initiation (Fox et al., 1998; Hara et al., 1998; Kimball et al., 1998; Patti et al., 1998; Jefferson and Kimball, 2001). In vivo studies in mature, food-deprived rats in which a large oral dose of leucine was administered suggest that in muscle, leucine promotes the binding of eIF4G to eIF4E, increases the phosphorylation of 4E-BP1, and represses the association of eIF4E with 4E-BP1 (Anthony et al., 2000). In neonatal pigs, raising amino acids from the fasting to the fed levels in the presence of insulin produced a similar response (O’Connor et al., 2003). In the absence of insulin, amino acids do not affect either the phosphorylation of S6K1 and 4E-BP1, or the association of eIF4E with 4E-BP1 and eIF4G, even though they stimulate muscle protein synthesis. This suggests that amino acids stimulate muscle protein synthesis in growing animals by modulating the availability of eIF4E for 48S ribosomal complex formation, and by processes that do not require enhanced assembly of the mRNA cap-binding complex.

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4.4. Insulin-like growth factors Many (Douglas et al., 1991; Fryburg et al., 1995; Bark et al., 1998; Vary et al., 2000; Davis et al., 2002b), although not all (Oddy and Owens, 1996; Boyle et al., 1998), studies have demonstrated an anabolic effect of IGF-I on protein synthesis in skeletal muscle. However, in some studies reductions in circulating concentrations of amino acids, insulin, and/or glucose during the administration of IGF-I may have limited the ability of IGF-I to stimulate protein synthesis. Thus, when amino acids, glucose, and insulin are maintained at fasting levels, infusion of IGF-I to the level seen in the fed state stimulates muscle protein synthesis in growing swine (Davis et al., 2002b). IGF-I, however, is unlikely to play a role in the feeding-induced stimulation of muscle protein synthesis. First, in contrast to insulin, the rise in circulating IGF-I after feeding is not immediate (Buonomo and Baile, 1991; Goldstein et al., 1991; Davis et al., 1993b, 1996; Svanberg et al., 1996). Second, the postprandial changes in muscle protein synthesis in young animals are positively correlated with changes in circulating insulin, but not IGF-I, concentrations (Davis et al., 1997, 1998). Third, with development, circulating IGF-I levels increase, whereas skeletal muscle protein synthesis rates decrease (Davis et al., 1996). Although circulating IGF-I is unlikely to be a physiologically significant regulator of the feeding-induced stimulation of skeletal muscle protein synthesis, this does not negate the potential role of IGF-I as a long-term regulator of growth, as has been suggested by others (Buonomo and Baile, 1991; Donovan et al., 1991; VandeHaar et al., 1991), or the potential usefulness of IGF-I as an anabolic agent to enhance protein deposition as discussed previously. IGF-I likely stimulates protein synthesis in skeletal muscle by acting on the same signalling pathway as insulin that leads to translation initiation (Dardevet et al., 1996; Vary et al., 2000). The receptors for both IGF-I and insulin share considerable homology of structure and function (Ullrich et al., 1986; Cheatham and Kahn, 1995; LeRoith et al., 1995) and both hormones act on some of the same intracellular signalling pathways (Dardevet et al., 1996; Suryawan et al., 2001). Furthermore, both insulin and IGF-I stimulate protein synthesis by increasing the formation of the active eIF4E . eIF4G complex that regulates the binding of mRNA to the ribosome (Kimball et al., 1997; Vary et al., 2000).

4.5. Growth hormone Growth hormone treatment increases protein deposition, improves nitrogen retention, and enhances the efficiency with which dietary protein is utilized for growth (Campbell et al., 1990; Caperna et al., 1991; Vann et al., 2000a). Furthermore, GH treatment profoundly decreases the synthesis and excretion of urea, and the oxidation of amino acids. Whole-body protein balance is improved in response to GH treatment due to the minimization of protein loss during fasting, and maximization of protein gain during meal absorption (Vann et al., 2000b). GH treatment in GH-deficient (Bier, 1991; Russell-Jones et al., 1998) and normal, mature animals and adult humans (Eisemann et al., 1989; Pell et al., 1990; Fryburg et al., 1991; Bell et al., 1998) increases protein deposition by stimulating whole-body and skeletal muscle protein synthesis. Chronic GH treatment in cattle and swine increases amino acid uptake by the hindquarter (Boisclair et al., 1994; Bush et al., 2003a) and protein synthesis in muscle (Eisemann et al., 1989; Seve et al., 1993) with no change in protein degradation across the hindlimb (Bush et al., 2003a).

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In young, growing swine, GH treatment increases skeletal muscle protein synthesis in the postprandial state, but not in the fasting condition. This increase is due to modulation of translational efficiency by GH and not by ribosome number (Bush et al., 2003b). The GH-induced increase in translation initiation is attributable to modulation of the factors associated with the binding of both mRNA and met-tRNA to the ribosomal complex, that is, the phosphorylation of 4E-BP1, association of eIF4E with eIF4G, and eIF2B activity. Because GH increases circulating IGF-I and insulin concentrations and this increase is greater in the fed than in the fasting state, the GH-induced increase in protein synthesis may involve mediation by IGF-I and/or insulin, or may be due to a direct effect of GH. In fact, GH indirectly activates some of the same signalling components as insulin and IGF-I, i.e. IRS-1 and -2, PI 3-kinase, protein kinase B, and S6K1 (Anderson, 1993; Yenush and White, 1997). In addition, the increased substrate availability, i.e. amino acids, provided in the fed condition may be permissive for the GH-induced increase in muscle protein synthesis. 4.6. Colostrum Colostrum provides a rich source of nutrients for the newborn mammal that supports the rapid growth and accretion of body protein during the first few days of postnatal life (Burrin et al., 1997b). In addition to nutrients, colostrum also contains maternal immunoglobulins that for many species are essential for passive immunity, and a variety of bioactive components that include insulin, IGF-I, IGF-II, and epidermal growth factor. Although the benefits of the consumption of nutrients and immune factors are readily apparent, the functional significance of the numerous hormones and growth factors present in colostrum is unclear. Studies that have compared the growth of newborns have demonstrated an enhanced anabolic response in association with the feeding of colostrum, especially of the visceral organs (Widdowson and Crabb, 1976; Widdowson et al., 1976). Given their mitogenic and anabolic properties, this response was often attributed to the presence of trophic factors in the colostrum. However, it must also be considered that the consumption of colostrum entails the ingestion of a larger quantity of nutrients than that typically provided by mature milk or, indeed, many formulas. Studies designed to distinguish between the trophic effects of macronutrient intake and those due to factors in colostrum (Burrin et al., 1995; Fiorotto et al., 2000b) showed that in newborn pigs, feeding stimulates protein synthesis in all tissues, but the stimulation of protein synthesis in skeletal muscle is greater when colostrum, as opposed to a nutrient-matched formula or mature sow’s milk, is fed. This suggests that the enhanced stimulation of skeletal muscle protein synthesis in newborn pigs fed colostrum, as opposed to other feeds, is not due solely to the provision of macronutrients. Furthermore, the stimulation of protein synthesis by colostrum feeding was restricted specifically to the myofibrillar proteins, unlike the general stimulation of protein synthesis by feeding which incurred a proportional stimulation of the synthesis of both sarcoplasmic and myofibrillar proteins (Fiorotto et al., 2000b). Feeding also resulted in a general increase in muscle mRNA concentration, but in the colostrum-fed piglets the enhanced synthesis rate of myofibrillar proteins was associated with a disproportionate increase in the abundance of myofibrillar mRNA, as exemplified by total MHC mRNAs. Additionally, colostrum augmented the effect of feeding on protein synthesis by promoting a greater accretion of ribosomes. Thus, feeding colostrum has both quantitative consequences for the anabolic process in the skeletal musculature of the newborn animal and qualitative consequences, with potential implications for the development of muscle function. Improvement of skeletal muscle

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function is advantageous insofar as it is critical for the development of the newborn’s ability to survive independently from its mother. The effects observed are likely attributable to nonnutritive factors present in colostrum, although these have not yet been identified. However, a number of potential factors, including insulin, IGF-I, thyroid hormone, and growth hormone, have been excluded. Identification of the mechanisms underlying this phenomenon will be critical for advancing our understanding of the biological role of early mammary secretions in the regulation of neonatal growth and in establishing how diet contributes to the regulation of skeletal muscle growth in early postnatal life.

5. FUTURE PERSPECTIVES There are numerous issues concerning the regulation of skeletal muscle growth and metabolism that need to be explored further. From the point of view of agriculture, the relative significance of these is determined by the economic benefits to be gained. The ultimate aim is to enhance feed efficiency. This, however, needs to be accomplished without compromising meat quality, especially tenderness and fat content. However, it is becoming increasingly evident that consumers are becoming more resistant to the use by the livestock industry of anabolic agents, growth promoters, and antibiotics, and frequently are prepared to pay a premium for products in which they have not been used. Although one may question the validity of these concerns, it must be acknowledged that they are widespread and, therefore, should not be ignored. In this regard, the application of genomics and proteomics to select for breeding stock with desirable traits, and improved husbandry practices to reduce mortality and morbidity in the birth to weaning period, are likely to be the most productive approaches. Given the large increase in fish consumption, research on the growth and composition of muscle of different fish varieties deserves substantially more attention. Enhancement of muscle growth can be accomplished either by increasing the number of myofibres, or by promoting myofibre hypertrophy. As should be evident, the former is a prenatal event and is dictated by maternal and genetic factors. Thus, continued research on the regulation of cell cycle progression and withdrawal of individual myoblast lineages, as well as the factors that control terminal differentiation, are likely to yield relevant information, especially when this can be merged with genomic trait analysis. Because mechanistic studies are difficult to conduct in vivo, in normal animals, much of the basic research on these mechanisms must be performed in cell and tissue culture. However, the widespread use of genetically engineered mice has been most productive and helpful in this regard because, although far removed from livestock animals, transgenic mice provide an important and apposite tool with which to assess the relevance of specific cellular events in the context of the whole animal. “The Myostatin Knockout Story” presents an excellent example of the usefulness of this approach. For some mammalian species, the ability to increase muscle fibre number has limitations, however, because the enhanced fetal growth may increase maternal morbidity and/or compromise maternal lactational capacity. In principle, therefore, enhanced postnatal muscle hypertrophy would be preferable. As we have presented, postnatal hypertrophy is dictated by two key factors, satellite cell number and protein accretion. Satellite cell number is the balance between the continued division of these cells acquired during the third phase of myoblast determination, and their loss, either by terminal differentiation and fusion into the myofibre, or by apoptosis. Although substantial progress has been made in understanding the factors that regulate satellite cell division, there are still many unanswered questions. Satellite cells have their origins in the latter part of fetal life and, therefore, are likely to be influenced

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by maternal variables; however, relatively little is known about the influence of maternal physiology and metabolism on the satellite cells of the progeny. The factors, especially environmental factors, that dictate terminal differentiation and apoptosis of satellite cells, and the extent to which these processes can be manipulated through husbandry practices and diet, are much less clearly understood, and warrant a closer examination as they are likely to have long-term consequences. More recently it is has been demonstrated that under certain in vitro conditions, satellite cells can change lineage and form adipocytes. Clearly this has significant consequences not only for overall muscle growth potential, but also for the composition of meat. The extent to which this occurs in vivo, and the conditions that would favour such a change, merit further attention. The rate of protein accretion is the balance between protein synthesis and degradation. We have demonstrated that in young animals the rate of protein synthesis is the principal regulatory factor. This unique feature is attributable to the ability of the immature muscle to markedly increase translation when food is available. The latter is critical because it enables amino acids to be diverted towards protein synthesis rather than to be oxidized. Thus, dietary protein can be used with greater efficiency, provided the composition of amino acids and energy intake are optimal. Thus, from the nutritional standpoint, the ability to meter the amino acid composition of dietary protein to meet the needs for growth versus maintenance during development can enable maximal exploitation of this high synthetic capacity of the immature muscle. The characteristics of the immature muscle that enable this synthetic response are primarily its high ribosomal content and an enhanced sensitivity and responsiveness of muscle protein synthesis to insulin. Clearly, therefore, further understanding of those factors that are responsible for these unique features of the immature muscle, and their down-regulation with maturation, would be warranted. Moreover, it is equally important that the impact of environmental variables such as infection, temperature, activity (duration, type, and intensity), and dietary nutrients other than protein and energy (e.g. micronutrients, modified lipids, and various non-nutritive factors present in foodstuffs) on protein synthesis during this anabolic phase of growth be investigated. Our emphasis on protein synthesis rather than degradation does not negate the importance of the latter in the regulation of protein accretion. Indeed, the regulation of protein degradation potentially represents a much more energetically efficient approach for improving the efficiency of muscle protein deposition (Goll et al., 1989) beyond the early postnatal period. However, much less is understood about the in vivo regulation of protein degradation, especially the factors that regulate myofibrillar breakdown, and the variability in these mechanisms between muscles and among different species. In addition to the consequences for protein deposition, protein degradation has consequences for meat quality because those enzymes that are responsible for the degradation of muscle myofibrillar proteins are also important determinants of post-mortem meat tenderisation. The evidence would suggest that in domestic animals muscle hypertrophy resulting from suppression of protein degradation in vivo can compromise meat tenderness. Examples of this negative consequence of suppressing protein degradation is the callipyge lamb in which the degree of hypertrophy of certain muscles is positively correlated to calpastatin expression, and increased toughness. The effects of some β-adrenergic agonists in certain species is similar to the effect of the callipyge gene. Thus, a clear understanding of the interplay between the structural characteristics of a muscle, the relative contribution of protein synthesis versus degradation to its overall growth, the variation among species, and how these aspects of muscle structure and metabolism are influenced by environmental factors and husbandry practices, are subjects that merit further study.

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Recently there has been much effort refocused on skeletal muscle metabolism in humans with the recognition that there is an inevitable depletion of skeletal muscle with ageing (sarcopenia). The consequence of this loss is quite severe as it results in a loss of strength, flexibility, and overall mobility, which thereby compromises the individual’s quality of life. The resulting decrease in activity not only exacerbates the muscle loss, but also decreases basal and activity-related energy expenditure, which therefore enhances the propensity for excessive fat deposition and glucose intolerance. The causes of sarcopenia appear to be extensive and include the loss in the replicative capacity of satellite cells, age-related increases in factors that are antagonistic to muscle growth, such as myostatin and Id factors, and a loss in the body’s capacity to produce anabolic agents such as growth hormone and testosterone. The relative importance of these, however, is far from clear. Additionally, or possibly in consequence to these changes, skeletal muscle loses its regenerative capacity with ageing. As the average life expectancy of humans increases, understanding the causes of sarcopenia and the development of therapies and modalities to mitigate its occurrence has enormous economic implications. Importantly, from the metabolic perspective, a better appreciation of the nutrient needs and dietary regimens that are required to sustain optimal muscle metabolism are warranted.

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