14 Protein Metabolism and Turnover ´ D Attaix,1 D Remond1 and I.C Savary-Auzeloux2 ´ Institut National de la Recherche Agronomique, Unite de Nutrition et ´tabolisme Prote ´ique, Theix, 63122 Ceyrat, France; 2Institut National Me ´ de la Recherche Agronomique, Unite de Recherches sur les Herbivores, Theix, 63122 Ceyrat, France Introduction All cellular proteins are in a continuous state of turnover in which they are synthesized and degraded (Waterlow et al., 1978) Thus, the intracellular concentration of any protein, and the tissue, organ or whole-body protein mass, are determined by the relative synthetic and degradation rates It should be pointed out that a change in the size of a given protein pool only depends on the imbalance between both processes of protein turnover In other words, an increase or a decrease in such a protein pool does not necessarily correlate with only an enhanced rate of either protein synthesis or protein breakdown, respectively For example, the anabolic agent trenbolone acetate decreased rates of both protein synthesis and breakdown and resulted in net muscle protein gain (Vernon and Buttery, 1976) The cyclical nature of protein turnover also implies that rates of protein synthesis and degradation are considerably greater than the net flux (protein deposition or loss) through the protein turnover cycle For example, a large proportion of free amino acids arising from protein breakdown is reutilized for protein synthesis, so that the rate of whole-body protein synthesis is much greater than the rate of dietary influx of amino acids Both protein synthesis and breakdown require energy (see below) However, the process of protein turnover provides the organism with several adaptive mechanisms that clearly outweigh the metabolic costs: Growth and mobilization of tissue/organ and whole-body protein mass is easily achieved, depending on the physiological status Large amounts of free amino acids can be mobilized from skeletal muscle and used to provide energy and precursors for protein synthesis in vital organs (brain, heart, etc.) and synthesis of specific sets of proteins (e.g acute phase ß CAB International 2005 Quantitative Aspects of Ruminant Digestion and Metabolism, 2nd edition (eds J Dijkstra, J.M Forbes and J France) 373 374 D Attaix et al proteins by the liver) in stress situations, even when dietary amino acid supply is deficient Abnormal (e.g miscoded or misfolded) proteins can be broken-down and not accumulate in cells Both endogenous and exogenous proteins, including bacterial and viral proteins, are hydrolysed into peptides and presented on major histocompatibility complexes to eventually activate the immune system The intracellular abundance of key proteins (e.g enzymes, cyclins or transcription factors) is tightly regulated so that major biological processes are precisely controlled A major challenge is to understand both general and tissue/organ-specific mechanisms, which are responsible for these adaptations In vitro studies have provided detailed information on the regulatory mechanisms of protein turnover In vivo studies are inevitably more descriptive, and experiments in animal production are mostly designed to optimize protein deposition efficiency in skeletal muscle (meat) or milk production Furthermore, the cost of research in large animal species has clearly impeded our understanding of protein metabolism in ruminants, so that most available information remains fragmentary Mechanisms of Protein Turnover The precise mechanisms of protein synthesis, which include transcription, translation and post-translational modifications, have been extensively studied and are detailed in many textbooks of biochemistry The mechanisms that regulate protein breakdown are much more obscure First, there are several proteolytic pathways within cells (e.g lysosomal, Ca2ỵ -dependent, ubiquitin proteasome-dependent (see Fig 14.1), etc.), and many proteases remain to be discovered or characterized In addition, the relative contribution of proteolytic pathways to the rate of overall proteolysis is tissue specific The lysosomal pathway plays a prominent role in liver (Attaix et al., 1999), while the ubiquitin–proteasome system has a major importance in skeletal muscle (Attaix and Taillandier, 1998; Jagoe and Goldberg, 2001) Second, there are many alternative routes within a given proteolytic process (Attaix et al., 1999) Third, in vivo, different proteolytic systems may either independently degrade a given protein substrate (Attaix et al., 1999), or sequentially participate to its complete hydrolysis into free amino acids (Attaix et al., 2002) Protein synthesis requires the hydrolysis of both ATP and GTP However, the actual cost of protein synthesis is much higher than the theoretical cost of peptide bond formation, presumably because many proteins involved in translational control are G-proteins, which are activated in the presence of GTP Direct measurements of oxygen consumption in the presence of cycloheximide have yielded values of 5.4 and 7.5 kJ/g protein synthesis when measured in vivo in chickens, and in vitro in sheep muscle, respectively (see Lobley, 1994) Protein breakdown also requires energy For example, ATP hydrolysis is required in many steps of ubiquitin–proteasome-dependent proteolysis Protein Metabolism and Turnover 375 n Ub + protein Free Ub E1 + ATP E3 + Protein E2 E1~Ub (1) Protein-(Ub)n E2~Ub (2) DUB (4) (3) 26S Proteasome (5) nUb + peptides TPP II (6) + AP Free AA Fig 14.1 Schematic representation of the ubiquitin (Ub)–proteasome-dependent proteolytic pathway Polyubiquitination of the substrate is achieved in sequential steps (1) to (3) (1) The Ub-activating enzyme, E1, forms a thiol–ester bond with Ub (2) The activated Ub is then transferred to an Ub-conjugating enzyme, E2, which also forms a thiol–ester linkage with Ub (3) In the presence of an Ub–protein ligase, E3, that specifically recognizes the substrate, the E2 and / or E3 covalently binds a polyUb chain (Ub)n to the target protein (4) A huge family of deubiquitinating enzymes (DUB) can remove the polyUb degradation signal, so that the substrate is not degraded and free ubiquitin is recycled (5) More generally, the polyUb degradation signal is recognized by the 26S proteasome, and the substrate is cut into peptides with recycling of free Ub (6) The peptides generated by the proteasome are finally hydrolysed into free amino acids (AA) by the tri-peptidyl peptidase II (TPP II) and several associated aminopeptidases (AP) (see Attaix et al., 2002 for more detailed information) (Attaix et al., 2002) It has been suggested that 10% of the cellular energy requirements are linked to proteolysis (Lobley, 1994) This estimation must be taken with caution The amount of energy required to degrade g of protein is unknown, cannot be assessed experimentally, and presumably largely depends on numerous factors, which include the nature of the substrate, the proteolytic system(s) involved in its breakdown, the site of proteolysis, etc Measurement of Protein Synthesis and Degradation Whole-body protein turnover The constant infusion technique has been widely used to estimate both components of whole-body protein turnover A labelled amino acid is infused intravenously until the plasma specific radioactivity or enrichment (for a radio- or a stable isotope, respectively) of the free amino acid used as a marker reaches a plateau This is achieved within a few hours (Fig 14.2a) The ratio, rate of isotope infusion/isotopic activity at the plateau, gives the flux or irreversible loss rate (ILR) of the amino acid from the plasma If the labelled amino acid infused into the blood/plasma free amino acid pool is an essential amino acid, and if this pool has a constant size (steady state) the total input through this pool is equal to the total output, so that: 376 D Attaix et al Plasma Muscle Liver Free label specific activity Fig 14.2 Schematic representation of the specific activity of the tracer following the administration of a constant infusion (a) or of a flooding-dose (b) of a labelled amino acid In (a) the ratio of the isotopic activity of the label at the end of the infusion crucially depends on the rate of protein turnover in the tissue (e.g the tissue homogenate/plasma isotopic activity is high (0.9 to 0.7) in skeletal muscle, where the intensity of protein turnover is low, and is low (0.6 to 0.3) in tissues where protein turnover is rapid (liver, gut)) In (b), this problem is minimized over a short period of time, and this ratio is usually over 0.7, including when protein turnover is a rapid process (see Attaix and Arnal, 1987) h (a) Plasma Muscle Liver 40 10 20 30 50 Time after label administration (b) ILR ẳ Synthesis(S) ỵ Oxidation(O) ẳ Breakdown(B) ỵ Intake(I) Amino acid oxidation (O) can be determined by using a 1-14 C or 1-13 C tracer amino acid, and collecting expired 14 CO2 or 13 CO2 that should be corrected for an apparent CO2 fixation in the body The whole-body protein synthesis rate (S) is then deduced from S ¼ ILR À O Alternatively, the whole-body rate of protein breakdown (B) is equal to B ¼ ILR À I in the fed state, or to B ¼ ILR (I) Tracer (S) Free AA Protein (B) (O) Fig 14.3 Two-pool model used for the estimation of the whole-body irreversible loss rate (ILR) and tissue protein fractional synthesis rate (FSR) in vivo, see text Amino acid (AA) fluxes, which are inputs into the free amino acid pool (e.g intake (I) and protein breakdown (B)), and outputs from this pool (e.g protein synthesis (S) and amino acid oxidation (O)) are shown The tracer, usually an essential amino acid, is infused or injected into the blood/plasma free amino acid pool, which is assumed to be the precursor pool for protein synthesis A third pool (e.g the intracellular free amino acid pool in equilibrium with the blood/plasma free amino acid pool and the protein pool) is often used to calculate the fractional rate of protein synthesis in a given tissue or organ (see Waterlow et al., 1978 for detailed explanations) Protein Metabolism and Turnover 377 in the fasted state In ruminants I (absorption) is particularly difficult to estimate, and fasting is not easily achieved The technique is simple, non-destructive, allows different measurements in the same animal, but has some major flaws, which have been extensively discussed elsewhere (Waterlow et al., 1978; Lobley, 1994) First, whole-body data are difficult to interpret and the ILR technique totally obscures changes in both rates of protein synthesis and breakdown in various tissues Second, the technique provides only a minimum estimate of the rates of protein turnover and of amino acid oxidation since the isotopic activity is much higher in the plasma than in tissues, where the tracer is diluted by unlabelled free amino acid from protein degradation (Fig 14.2a) Third, there is some recycling of the tracer from tissues where protein turnover is rapid (e.g liver, gastrointestinal tract (GIT), see below), and this also causes underestimation of the ILR Regional estimations of protein turnover Another closely related technique involves selective catheterization of an artery and a vein draining a hind limb bed An index of both the rates of protein breakdown and synthesis is calculated by measuring the concentration of the label and its isotopic activity in arterial and venous blood, and the blood flow Labelled phenylalanine (Barrett and Gelfand, 1989) and other amino acids can be used (Hoskin et al., 2001) Amino acid oxidation can also be determined by following the fate of the C-1 moiety of essential amino acids The arteriovenous approach has the same limitations as the ILR technique, and there is some contamination from the other tissues within the hind limb, e.g skin and bone Amino acid mass transfers have been also quantified by arteriovenous procedures across the portal-drained viscera (PDV) and liver in sheep (Lobley et al., 1996) Such procedures require extensive surgery, but they allow repeated measurements within the same animal Tissue and organ protein turnover Protein synthesis To measure fractional rates of protein synthesis (FSR, usually expressed in % per day) in vivo the specific radioactivity (or enrichment) of the labelled amino acid must be measured in both the precursor and the protein pools (Waterlow et al., 1978) Except for skeletal muscle and skin, in which biopsies can be easily performed, slaughter is usually required to collect internal samples Two techniques have provided most of the data available in ruminants The most commonly used is the constant tracer infusion analysis, as in the ILR technique (see above and Fig 14.2a) The difficulty is to estimate the activity of the precursor pool for protein synthesis The activity of the actual pool, the charged aminoacyl-tRNAs, is technically very difficult to determine Based on experiments performed in vitro and in vivo, it is generally assumed that aminoacyl-tRNAs are charged from both extracellular (plasma) and intracellular 378 D Attaix et al (tissue homogenate) free amino acid pools (Waterlow et al., 1978) However, as the label is diluted by the unlabelled amino acid used as a marker, which arises from protein breakdown, there are large differences between the isotopic activities in these pools (Fig 14.2a) This is especially true when protein turnover is high (liver, GIT) Consequently there are also large differences between FSR calculated by using the isotopic activity of the free label in the plasma and the tissue homogenates In addition, since the label is infused during several hours, secreted or export proteins, which are for example synthesized in the liver and the intestines, are not taken into account in the measurements To overcome all these problems, the label can be injected with a large or flooding dose of the same unlabelled amino acid This results in nearly constant and close isotopic activity of the tracer, both in the plasma and in tissue homogenates within a short period of time (Fig 14.2b) To meet these goals the large dose of unlabelled amino acid should ideally represent several times the wholebody free amino acid content For example, when [3 H]valine was used as a tracer in 1-week-old lambs the flooding dose was very efficient with an unlabelled amount of valine that represented about ten times the whole-body free valine content (Attaix, 1988) In such conditions, FSR calculated from the isotopic activity of the free label either in the plasma or the tissue homogenates are quite similar Although the technique is potentially interesting for measuring protein synthesis in tissues where FSR are high, there are some potential problems First, the injection of a large amount of amino acid may affect amino acid transport and/or hormonal secretions (e.g insulin) Second, the procedure is rather expensive Consequently, there are very few measurements in adult ruminants, and all published data have been obtained for only the ovine species Finally, the procedure may favour the measurement of FSR in short-lived proteins Protein breakdown Methodological problems associated with reliable measurements of in vivo proteolysis impede the understanding of its regulation In addition, all techniques that can be used in vivo not provide any information on proteolytic systems that are responsible for changes in proteolysis In tissues and organs from growing animals, the fractional rate of protein breakdown (FBR) can be calculated as the difference between FSR and the fractional rate of protein deposition (FGR) (Waterlow et al., 1978) Such estimations are very imprecise because FGR must be estimated over several days, FSR being measured over a few minutes or hours However, FSR and FGR are not necessarily constant over the period of measurements For example, they may fluctuate largely with the feeding pattern In addition the technique requires slaughter and cannot be used in tissues that secrete or export proteins 3-Methylhistidine is formed by a post-translational methylation of histidine residues in actin and in myosin heavy chains of fast-twitch glycolytic skeletal muscles In the rat and cattle, but not all species (see below), the urinary excretion of 3-methylhistidine provides an index of myofibrillar protein breakdown Unfortunately, the visceral smooth muscles of the GIT and other tissues such as skin contain significant amounts of actin These tissues contribute disproportionately for their size to 3-methylhistidine urinary excretion, because of Protein Metabolism and Turnover 379 their high rates of protein turnover In addition, changes in renal clearance of 3methylhistidine may affect the interpretation of the data (see Attaix and Taillandier, 1998) Finally, in some species (e.g in pigs and to a lesser extent in sheep), a high proportion of 3-methylhistidine is retained in muscle as a dipeptide, balenine (Harris and Milne, 1987) A compartmental model of 3-methylhistidine metabolism has been developed, which involves the assessment of muscle proteolysis and 3-methylhistidine kinetics without the collection of urine (Rathmacher and Nissen, 1998) However, due to the numerous limitations of the 3methylhistidine approach, caution must be exercised Non-quantitative approaches Non-quantitative approaches may be of special interest in ruminant tissues, due to the costs of experiments with isotopic amino acids As a very crude rule, the control of protein synthesis occurs mainly at the transcriptional level Therefore the quantification of the mRNA(s) of a given protein by molecular biology techniques is often used as an index of protein synthesis However, many mRNAs are also subject to translational control, and the relative amount of any mRNA depends on both rates of transcription and of mRNA breakdown Finally, there are frequent discrepancies between mRNA levels and the corresponding protein levels and/or activities Similarly, changes in mRNA levels for many proteolytic genes, in particular within the muscle ubiquitin–proteasomedependent pathway, closely mimic variations of proteolytic rates measured with incubated rodent muscles (see Attaix and Taillandier, 1998) These observations, together with the use of specific inhibitors of lysosomal and Ca2ỵ dependent proteases and of the proteasome, lead to the concept that most muscle proteins, and in particular myofibrillar proteins, are degraded in an ubiquitin–proteasome-dependent fashion (Attaix and Taillandier, 1998; Jagoe and Goldberg, 2001) However and again, elevated mRNA levels for proteolytic genes only reflect increased transcription in a few instances (see Attaix and Taillandier, 1998), and not always strictly correlate with rates of proteolysis (see Combaret et al., 2002) Measuring proteolytic gene expression may be of interest in small muscle biopsies from ruminants, with complementary approaches (e.g measurements of protein levels for some enzymes of the ubiquitination machinery and proteasomal subunits, of the rate of ubiquitination of protein substrates, and of proteasome activities) Whole-body Protein Metabolism The age of animals and the level of nutrition are the best described factors that regulate whole-body protein metabolism in ruminants When expressed on a metabolic liveweight basis, whole-body protein synthesis in lambs increases during the first days following birth, declines very rapidly within months (without any major effect of weaning), and thereafter remains stable with increasing age (Fig 14.4) 380 D Attaix et al WB protein synthesis (g/day/kg BW0.75) 70 60 50 40 30 20 10 −10 10 20 30 Birth 40 50 60 70 Age (weeks) Milk-fed Weaned 80 90 100 Fig 14.4 Effect of age on whole-body (WB) protein synthesis in sheep (Data from Patureau Mirand et al., 1985; Attaix, 1988; Harris et al., 1992; Neutze et al., 1997; Adams et al., 2000; Yu et al., 2000; Savary et al., 2001.) Whole-body protein synthesis (g/day/kg BW 0:75 ) increases with metabolizable energy (ME) intake (kJ/day/kg BW 0:75 ) (Fig 14.5) This increase is linear in sheep (Harris et al., 1992; Yu et al., 2000; Savary et al., 2001), but not in steers (Dawson et al., 1998; Lapierre et al., 1999) In both species fed above maintenance (based on an energy maintenance requirement of 400 and 500 kJ/day/kg BW 0:75 for sheep and steers, respectively) the slope of the relationship is very similar (e.g 13–14 g of protein synthesized per MJ ME) However, below maintenance, protein synthesis decreases in sheep but is not altered in steers (Lapierre et al., 1999) Above maintenance requirements, the calculated whole-body protein degradation rate (protein synthesis minus deposition) increases in both sheep and steers (Harris et al., 1992; Lapierre et al., 1999) Below maintenance protein breakdown decreases in sheep (Harris et al., 1992), but increases in steers (Lapierre et al., 1999) Besides species differences, the duration of the underfeeding period, the composition of the diet and the age of animals may account for these discrepancies Nevertheless, whole-body protein loss was similar (about g/day/kg BW 0:75 ) in both underfed (0.6Â maintenance) steers and sheep Tissue Protein Metabolism Portal-drained viscera On average, the portal net release of essential amino acids accounts for only two-thirds of their apparent disappearance from the small intestine (MacRae Protein Metabolism and Turnover 381 WB protein synthesis (g/day/kg BW0.75) 40 35 30 25 20 Lobley et al (1987) Hammond et al (1987) Dawson et al (1998) Lapierre et al (1999) Lobley et al (2000) (Angus) Lobley et al (2000) (Charolais) 15 10 200 400 600 800 1000 1200 0.75 (a) Metabolizable energy (kJ/day/kg BW ) WB protein synthesis (g/day/kg BW0.75) 30 25 20 15 Harris et al (1992) Neutze et al (1997) Savary et al (2001) Adams et al (2000) 10 (b) 200 400 600 800 Metabolizable energy (kJ/day/kg BW0.75) 1000 1200 Fig 14.5 Effect of metabolizable energy intake on whole-body (WB) protein synthesis in cattle (a) and sheep (b) et al., 1997b, Berthiaume et al., 2001) Moreover, the sequestration of individual essential amino acids in the PDV may account from one- to two-thirds of their whole-body flux, and the majority ($80%) of the amino acids sequestrated arose from the arterial supply (MacRae et al., 1997a) Thus, first-pass PDV 382 D Attaix et al metabolism of dietary amino acids as well as PDV use of systemic amino acids significantly impact the quantitative and qualitative supply of amino acids to other tissues or organs The portal vein drains heterogeneous tissues (GIT, pancreas, spleen, omentum), but the GIT is by far the major contributor to PDV protein synthesis For this reason, only GIT protein metabolism is reviewed below Gastrointestinal tract The mass of the GIT increases with intake, and the importance of its different compartments varies according to the composition of the diet Protein mass of the ruminant GIT accounts for 4–6% of whole-body proteins (Lobley et al., 1980; Attaix, 1988; MacRae et al., 1993) However, because of the high FSR in these tissues, the GIT contributes $25–35% of whole-body protein synthesis (Lobley et al., 1980, 1994; Attaix, 1988) compared to $12% in pre-ruminant animals (Attaix and Arnal, 1987) The large dose procedure is best suited for measuring protein synthesis in the GIT (see above), and data reported in this section are derived from studies using this technique (export proteins being included in synthesis) Whatever the age, the pattern of FSR along the GIT is very similar to the highest values in the small intestine (Fig 14.6) Rumen growth is rapid and stimulated by the initiation of solid food intake and the concomitant establishment of microbial fermentation Thus, the reticulorumen represents $7% and 30% of the GIT protein mass in 1-week-old milk-fed and 8-week-old weaned lambs, respectively (Attaix, 140 120 100 80 60 40 20 on ol C ae cu m C Ile um m nu Je ju nu m uo de D as um as u O m um R Ab om m en Protein fractional synthesis rate (% per day) FORESTOMACHS 1-week-old 8-week-old 8-month-old Fig 14.6 Protein fractional synthesis rates in the gastrointestinal tract from milk-fed (1-week-old) and weaned lambs (Data from Attaix, 1988; Lobley et al., 1994.) 384 D Attaix et al ` from chronically underfed ewes (Noziere et al., 1999), suggesting that such changes are only seen following acute manipulation of dietary intake LARGE INTESTINE In 8-week-old weaned lambs the large intestine accounts for 13% of the protein mass of the GIT, and for 9% of its absolute rate of protein synthesis (Attaix, 1988) In mature sheep, corresponding values are 22% ` (Noziere et al., 1999) and 18% (Lobley et al., 1994) The mass of the large `re intestine increases with the level of intake (Nozie et al., 1999), owing to a tendency for enhanced FSR (Lobley et al., 1994) However, several lines of evidence suggest a role of proteolysis in the control of large-intestinal protein mass (Attaix et al., 1992; Samuels et al., 1996) Liver FSR in the liver follows the general pattern observed for whole-body protein synthesis (Fig 14.7) FSR increases during the first days following birth, and thereafter declines exponentially with increasing age This decline is linked to a decrease in both ribosomal capacity and protein synthesis efficiency In ruminants, FSR in the liver is not affected by the level of intake (Lobley et al., 1994; Adams et al., 2000) Absolute protein synthesis in the liver accounts for about 35–40% of the PDV protein synthesis (Attaix, 1988; Lobley et al., 1994) Assuming that all plasma proteins are of hepatic origin, it has been estimated that export proteins accounted for 38–51% of total hepatic protein synthesis, Protein fractional synthesis rate (% per day) 120 100 80 60 40 20 1-day 1-week 5-week 8-week 13-month 20-month Age Fig 14.7 Effect of age on protein fractional synthesis rate in sheep liver (Data from Patureau Mirand et al., 1985; Attaix, 1988; Lobley et al., 1992; Adams et al., 2000.) Protein Metabolism and Turnover 385 and that albumin represented 15–22% of export protein production (Connell et al., 1997) These proteins act as a mobile protein reservoir, and synthesis in this fraction (but not in the constitutive protein fraction) is particularly sensitive to acute change in nutritional status such as fasting (Connell et al., 1997; Lobley et al., 1998) In contrast, changes in liver protein mass in response to ` intake (Burrin et al., 1992; Noziere et al., 1999) seem mainly related to alterations in protein degradation (Lobley and Milano, 1997) Peripheral tissues Skeletal muscle Proteins in skeletal muscle account for about 30–45% of whole-body protein mass (Attaix, 1988; Lobley et al., 1994) Although this is the largest protein reservoir in the body, muscle contributes only 15% to 22% to whole-body protein synthesis because of its low FSR (Attaix, 1988; Adams et al., 2000; Lobley et al., 2000) In lambs, FSR declines exponentially between birth and months of age (Fig 14.8) This decline is fully related to a decrease in the capacity for protein synthesis and is not confounded by nutritional effects (Attaix et al., 1988) In sheep, muscle FSR increases linearly with the level of intake from 0.6 to 1.8 Â maintenance (Fig 14.9) Similar data were obtained in the perfused hind limb between 0.5 and 2.5 Â maintenance (Boisclair et al., 1993; Thomson et al., 1997; Hoskin et al., 2001; Savary et al., 2001) Data on protein degradation in the perfused hind limb are more confusing An Protein fractional synthesis rate (% per day) 20 18 16 14 12 10 0 20 40 60 Age (weeks) 80 100 Fig 14.8 Effect of age on protein fractional synthesis rate in sheep skeletal muscle (Data from Patureau Mirand et al., 1985; Attaix, 1988; Lobley et al., 1992; Adams et al., 2000.) 386 D Attaix et al Muscle protein fractional synthesis rate (% per day) 3.5 2.5 1.5 Liu et al (1998) 0.5 Lobley et al (1992) Adams et al (2000) 0 0.5 1.5 Metabolizable energy intake (ϫ maintenance) Fig 14.9 Effect of metabolizable energy intake on protein fractional synthesis rate in sheep skeletal muscle increased intake above maintenance resulted in a concomitant increase in both protein synthesis and breakdown in two studies (Harris et al., 1992; Lobley et al., 2000) In contrast, protein breakdown was unchanged in two different experiments where the level of intake increased up to 2.5 Â maintenance (Thomson et al., 1997; Savary et al., 2001) Below maintenance, protein loss in the hind limb depends on the duration and of the severity of underfeeding In restricted animals (0.5–0.6 Â maintenance for week), proteolysis tended to increase (McDonagh et al., 1999; Hoskin et al., 2001) Conversely, after weeks of underfeeding, muscle proteolysis was unchanged (Harris et al., 1992; Boisclair et al., 1993) or eventually decreased (Thomson et al., 1997), and muscle protein loss totally resulted from an impairment in protein synthesis Skin FSR in skin from cattle (4–6% per day; Lobley et al., 1980) and goats (2.5% per day; Champredon et al., 1990) are much lower than in sheep (6–25% per day; Attaix, 1988; Lobley et al., 1992; Liu et al., 1998; Adams et al., 2000), evidencing the production of wool in the latter species, which accounts for 10–20% of skin protein synthesis (Liu et al., 1998; Adams et al., 2000) During the first week of life, FSR are roughly equivalent in skin and muscles of lambs (Patureau Mirand et al., 1985; Attaix et al., 1988) After weaning, FSR in skin are three- to ninefold higher than in muscle (Attaix, 1988; Lobley et al., 1992; Liu et al., 1998; Adams et al., 2000) Consequently, the contribution of skin to whole-body protein synthesis in ruminants is about Protein Metabolism and Turnover 387 1.5-fold greater than that of skeletal muscle (Attaix, 1988; Adams et al., 2000) FSR in skin increases with the level of intake (Liu et al., 1998; Adams et al., 2000), and is greater in sheep fed rapeseed meal than in sheep receiving lupin seed, which contains less methionine, the primary limiting amino acid for wool growth (Liu et al., 1998) Mammary gland Protein synthesis in the mammary gland is a negligible part of whole-body protein synthesis in dry animals, but becomes a major contributor in lactating animals (Champredon et al., 1990; Baracos et al., 1991; Bequette et al., 1996) The mammary gland synthesizes not only milk proteins but also structural proteins and enzymes These constitutive proteins may account for 40–45% of total mammary gland protein synthesis (Oddy et al., 1988; Champredon et al., 1990; Baracos et al., 1991) Degradation of newly synthesized milk proteins could account for about one-third of milk protein synthesis (Oddy et al., 1988) Control and Manipulation of Protein Metabolism Insulin This polypeptide hormone secreted by the pancreatic b-islets plays a key role in the regulation of growth and nutrient utilization in ruminants (see Lobley, 1994, 1998; Grizard et al., 1999; Nieto and Lobley, 1999) Insulin inhibits whole-body protein breakdown in lambs (Oddy et al., 1987) or adult goats (Tesseraud et al., 1993) and decreases plasma amino acid concentrations, but does not stimulate whole-body protein synthesis (Tesseraud et al., 1993; Tauveron et al., 1994) Insulin increases protein synthesis and decreases proteolysis in cell cultures or muscle explants, leading to a strong anabolic effect (see Lobley, 1998; Grizard et al., 1999) Insulin infusion stimulates protein synthesis in various muscles from young piglets and rats, but has no effect in young and adult fasted ruminants (Douglas et al., 1991) Actually, protein breakdown and even protein synthesis were inhibited in skeletal muscle from insulin-infused fasted lambs (Oddy et al., 1987) In fed ruminants, insulin had no effect on muscle protein synthesis (Oddy et al., 1987; Tauveron et al., 1994) The lack of the effect of insulin on in vivo protein synthesis in the majority of studies was attributed to hypoaminoacidaemia and hypoglycaemia However, the utilization of eu- or hyperaminoacidaemic and euglycaemic clamps after insulin infusion in fed or fasted ruminants (Tesseraud et al., 1993; Tauveron et al., 1994) failed to demonstrate any stimulatory effect of insulin on both whole-body and muscle protein synthesis These data suggest that the effect of insulin is only seen: (i) in fasted or restricted animals where the endogenous insulin concentration is low, because basal insulinaemia is already stimulating protein synthesis at its maximum rate in the fed state; and (ii) in young growing animals exhibiting a high insulin 388 D Attaix et al sensitivity The former hypothesis has been recently confirmed in vivo using fed rats submitted to an acute hypoinsulinaemia induced by diazoxide, which decreased muscle protein synthesis by 40% (Sinaud et al., 1999) Recent data from Wray-Cahen et al (1998) also confirmed the latter hypothesis, since the stimulation of muscle protein synthesis by insulin was greater in 7- than in 26-day-old pigs Therefore, the sensitivity of muscle to insulin decreases with age in monogastrics and presumably in ruminants (Eisemann et al., 1997) The lack of effect of insulin on muscle protein synthesis in ruminants, even at an early stage of development, is presumably due to their digestion pattern, which results in relatively high insulin circulating levels that prevent any further anabolic effect (Tesseraud et al., 1993; Tauveron et al., 1994) Insulin regulates protein synthesis by several mechanisms A long-term insulin deficiency decreases ribosomal capacity, but acute insulin deficiency impairs the translation efficiency through changes in phosphorylation/dephosphorylation of initiation factors (Kimball et al., 1997) In ruminant species, and possibly in pre-ruminants, one or several components of the translational apparatus are presumably insulin-resistant In vitro, insulin inhibits the muscle lysosomal and ubiquitin–proteasomedependent proteolytic pathways For example, insulin decreased both the mRNA levels for the 14-kDa ubiquitin-conjugating enzyme E2 and proteasome activity in cultured cells (see Larbaud et al., 2001) A role of insulin on in vivo muscle proteolysis has also been suggested in euglycaemic hyperinsulinaemic ruminants (Larbaud et al., 1996) and rats (Larbaud et al., 2001) In contrast with in vitro data, insulin had no effect on the mRNA levels for the 14-kDa E2, but decreased ubiquitin expression in fast-twitch or mixed muscles, without any effect on the amount of ubiquitin conjugates However, alterations in the expression of regulatory subunits of the 26S proteasome may contribute to explain the antiproteolytic effect of insulin in vivo (Larbaud and Attaix, unpublished data) In lactation, insulin increases (15% to 30%) the partition of the amino acids towards the mammary gland (Bequette et al., 2001) Although insulin infusion results in hypoaminoacidaemia, the utilization of amino acids for milk protein synthesis, and mammary amino acid net extraction is not altered or even increased (Mackle et al., 2000) This can be explained by an increase in both the mammary blood flow and the udder sensitivity to insulin (Bequette et al., 2001) Growth hormone/IGF-1 axis Growth hormone or somatotropin (ST) is a polypeptide hormone secreted by the anterior pituitary gland that stimulates, directly or indirectly, anabolic processes such as cell division, skeletal muscle growth and protein synthesis ST increases nutrient partitioning between skeletal muscle and adipose tissue, and alters the growth of lean tissues and bones (see Etherton and Bauman, 1998) In ruminants, bovine ST induces a 40% increase in protein accretion in lean tissues, including muscle (Boisclair et al., 1994) An even Protein Metabolism and Turnover 389 more impressive effect has been reported in pigs where porcine ST infusion resulted in a 90% increase in protein accretion (Etherton and Bauman, 1998) An ST treatment for several weeks tends to induce a less important effect on growth and nitrogen retention than a short treatment (e.g several hours to several days) (Boisclair et al., 1994), possibly due to the development of a relative ST resistance state However, there is a positive effect of a long-term ST treatment on whole-body nitrogen retention in well-fed animals and when nitrogen supply is adequate for maximal growth (Spencer et al., 1994) Rausch et al (2002) reported that cattle fed at two levels of energy and protein intake (0.75 Â ad libitum and ad libitum) and submitted to a bST treatment for 14 days, grew more rapidly and efficiently with the highest level of intake An increased amino acid requirement during ST treatment is consistent with the increased protein accretion, since more amino acids are required to sustain the rapid growth of the animals Except in early lactation, ST has pronounced effects on milk output and milk protein yield, which increased by about 10–20% (Faulkner, 1999) An increased blood flow and an increased amino acid utilization explain this improvement by the mammary gland The possible mechanisms by which ST increases milk production have been extensively reviewed elsewhere and may involve an increased local production of IGF-1 (see Etherton and Bauman, 1998) In ruminants, ST infusion increases whole-body (Eisemann et al., 1989) and muscle (Eisemann et al., 1989; Boisclair et al., 1994) protein synthesis In contrast, no strong effect of ST on protein degradation was reported (Boisclair et al., 1994) ST administration results in elevated hepatic insulin-like growth factor-1 (IGF-1) mRNA levels, an increased release of IGF-1 by the liver and a global increase in IGF-1 circulating levels (Rausch et al., 2002) A direct effect of ST cannot be excluded since acute ST infusion stimulated protein synthesis without any alteration in IGF-1 levels, which commonly occurs within 10–12 h However, a comparison of in vivo studies using close arterial infusions of IGF-1 to systemic/close arterial infusions of ST in humans or ruminants led to the conclusion that ST has a direct effect on protein accretion in lean tissues and milk synthesis, and an indirect effect via IGF-1 Indeed, IGF-1 is not only synthesized in the liver but also in other target tissues such as skeletal muscle Thus the hormone exhibits both endocrine and paracrine effects In addition, IGF-1 binds non-covalently to carrier proteins (IGFBPs), which extend its halflife and can regulate its action (see Lobley, 1998 for review) In vivo, IGF-1 increased muscle protein synthesis in lambs (Douglas et al., 1991; Koea et al., 1992) In mice, this stimulatory effect is localized to muscle, and does not prevail in other tissues or organs However, IGF-1 infusion in the sheep hind limb artery for 24 h increased transiently skin and wool protein synthesis (Lobley et al., 1997) In the IGF-1-infused hind limb of lambs, Oddy and Owens (1996) have reported a pronounced reduction in protein breakdown, regardless of feed intake A strong inhibition of whole-body protein breakdown was also observed in fasted IGF-1 infused lambs, but this antiproteolytic effect was potentialized by total parental nutrition (Koea et al., 1992) The proteolytic systems 390 D Attaix et al downregulated by IGF-1 are not fully identified but a decrease in m-calpain activity in muscle (McDonagh et al., 1999) and in the expression of some components of the ubiquitin-dependent proteolytic system have been observed after IGF-1 treatment (see Attaix and Taillandier, 1998) b-Agonists b-Agonists currently used as growth promoters are clenbuterol, cimaterol, ractopamine and L644,969 These molecules present chemical similarities with natural catecholamines and can bind to b-adrenergic receptors Like ST, b-agonists alter nutrient partitioning and promote the growth of lean tissues at the expense of the fat stores, but they act independently An increased heart rate and blood flow increases nutrient supply to the target tissues in b-agonisttreated ruminants (Eisemann et al., 1988) In contrast with ST, b-agonists are more effective in ruminants than in monogastrics, and may induce an up to 65% increase in muscle mass (Byrem et al., 1998) The direct or indirect role of b-agonists in protein deposition has been extensively studied, because they induce changes in circulating levels of anabolic hormones A consistent anabolic effect of b-agonists prevails in many catabolic situations (e.g diabetes, denervation) or in castrated and adrenalectomized animals and suggests a direct effect of b-agonists on protein metabolism Indeed, close arterial infusion of cimaterol in the hind limb of young steers, both in acute (6 h) or chronic (1–20 days) conditions, increased muscle protein content by 9–11% (Byrem et al., 1998) However, these results not exclude an indirect effect of b-agonists For example, clenbuterol administration increased muscle mass by 10–13% in rodents and this effect was associated with increased IGF-1 mRNA and protein levels, and IGF-binding protein mRNA levels (Awede et al., 2002) Furthermore, clenbuterol increased in rat muscle the phosphorylation of eIFBP-1 and P70S6k, involved in the signal transduction pathways of insulin and IGFs (Sneddon et al., 2001) The precise mechanisms responsible for the positive effect of b-agonists on muscle protein deposition are not fully understood The elevated protein synthesis is consistent with an increased capacity for protein synthesis in some experiments, but ractopamine seems to increase myosin mRNA in steers and a-actin mRNA in pigs (reviewed by MacRae and Lobley, 1991) In addition, in muscle showing clenbuterol-induced anabolism, Sneddon et al (2001) have also recently reported an increased phosphorylation of eukaryotic initiation factor 4E-BP1, suggesting a stimulation of translation b-Agonists also suppressed muscle proteolysis (e.g Bohorov et al., 1987), possibly by inhibiting Ca2ỵ -dependent proteases (Navegantes et al., 2001) The interaction of b-agonist treatment with amino acid utilization is not very clear However, clenbuterol is more effective in well-fed than in underfed cattle (Sillence et al., 1993) Consequently, animals treated with b-agonists present greater protein requirements Protein Metabolism and Turnover 391 Anabolic steroids These molecules include the natural androgens, oestrogens and some biosynthetic compounds with similar activities (e.g zeranol or trenbolone acetate, which exhibit an oestrogenic or an androgenic activity, respectively) A combination of an oestrogenic and an androgenic molecule is usually used in ruminants This makes it difficult to understand the actual effect of each steroid on protein metabolism Furthermore, steroid administration induces an alteration in the concentration of ST, IGF-1, insulin and thyroid hormones Therefore, the direct and indirect effect of steroids on protein metabolism are still not well understood Entire males have higher whole-body and muscle growth rates and less body fat than castrated animals Consequently, the involvement of sex steroids in the control of muscle growth has been extensively studied in farm animal species The role of steroids with androgenic activity such as trenbolone acetate has also been studied in female ruminants where they also improve protein deposition (Sinnett-Smith et al., 1983) Trenbolone acetate and estradiol are commonly used as growth promoters in the USA They act synergetically and increase the growth rate and feed efficiency by about 15–20%, even after 115 days of treatment in steers (Johnson et al., 1996) Most studies indicate a decrease in protein breakdown after trenbolone acetate or testosterone treatment in ruminants (MacRae and Lobley, 1991), with no effect or a small decrease in both whole-body (Sinnett-Smith et al., 1983; Lobley et al., 1985) and muscle protein synthesis (Lobley et al., 1990) Conclusions As pointed out at the beginning of this chapter, our knowledge of protein turnover in ruminants remains fragmentary Although species differences may explain some discrepancies between monogastrics and pre-ruminants/ ruminants, more information is obviously needed on farm species In this respect, the recent development of new approaches such as cDNA macroand microarrays will certainly contribute to solving many questions and to providing more information about the precise mechanisms that regulate protein turnover in ruminants (e.g to identify signalling pathways or crucial genes that have a major influence on protein synthesis and breakdown) However, protein turnover in the GIT from adult ruminants has probably some important effects on whole-body and peripheral tissue protein turnover An important unresolved question is to determine whether protein turnover in the GIT should be stimulated or inhibited to optimize protein deposition efficiency in skeletal muscle This clearly requires new experiments that aim to understand the relationships between protein turnover in different tissues 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tract leucine metabolism and reduces availability of leucine for other tissues Journal of Animal Science 78, 380–390 This page intentionally left blank ... of the ubiquitination machinery and proteasomal subunits, of the rate of ubiquitination of protein substrates, and of proteasome activities) Whole-body Protein Metabolism The age of animals and. .. rate (% per day) FORESTOMACHS 1-week-old 8-week-old 8-month-old Fig 14.6 Protein fractional synthesis rates in the gastrointestinal tract from milk-fed (1-week-old) and weaned lambs (Data from Attaix,... Thissen, J.P and Lebacq, J (2002) Role of IGF-1 and IGF-BPs in the changes of mass and phenotype induced in rats soleus muscle by clenbuterol American Journal of Physiology, Endocrinology and Metabolism