15 Interactions between Protein and Energy Metabolism T.C Wright1, J.A Maas2 and L.P Milligan1 Department of Animal and Poultry Science, University of Guelph, Guelph, Ontario N1G 2W1, Canada; 2Centre for Integrative Biology, University of Nottingham, Sutton Bonnington, Leicestershire LE12 5RD, UK Introduction The corresponding chapter in the previous edition of this book concluded by describing protein and energy metabolism as a unity instead of an interaction of separate components of metabolism This edition will examine some of the recent knowledge generated about this subject with an emphasis on those metabolites and tissues that serve important roles for biochemical reactions in which carbon and nitrogen are, in effect, equal partners Animals encounter numerous challenges during their lives, and respond to achieve maximum advantage for their welfare and survival in meeting those challenges This does not imply, however, that the response will necessarily be measured as the most efficient possible in terms of agricultural animal performance It is possible to make estimates of the stoichiometry of numerous reactions for many metabolic pathways involving protein and energy intermediates The opportunity for nutritionists is to develop a better understanding of the fate of nutrients under differing circumstances and of the regulatory system that determines an end point The energetic costs associated with disposing of an amino acid (AA) can differ from tissue to tissue Current models have advanced nutritional efficiency, in terms of product per unit animal, but it is appropriate now to explore those pivot points and signals that may determine nutrient fate and associated energetic costs of protein and energy metabolism It will become clear that a better comprehension of the unity of protein and energy metabolism follows from the further development of quantitative models that reflect metabolic mechanisms Rumen Aspects The initiation of ruminant protein and energy metabolism begins in the rumen where the energetic efficiency of the rumen microbes within their anaerobic ß CAB International 2005 Quantitative Aspects of Ruminant Digestion and Metabolism, 2nd edition (eds J Dijkstra, J.M Forbes and J France) 399 400 T.C Wright et al environment compares unfavourably with the aerobic environment of the host The anaerobic state of the rumen dictates that the microbes must metabolize greater amounts of carbon substrates than the host to derive equal energy (see Chapter 9) Recent advances in protein and carbohydrate nutrition for ruminant animals have produced some estimates for AA requirements in ruminants (e.g NRC, 2001) as well as a better understanding of the fermentation of nitrogen and carbohydrate sources in relation to each other (see Chapter 10) The proportions of fermentation end-products, principally AA, protein, volatile fatty acids (VFA), carbon dioxide and methane can dictate in large part the subsequent metabolic efficiencies for the host Nutritional manipulations that affect the end-products of rumen fermentation in a sustained manner are often difficult to achieve Asanuma et al (1999) investigated the contributions to ruminal H2 production from the major cellulolytic bacteria Ruminococcus albus and R flavefaciens and the potential benefits of enhanced electron accepting reactions in vitro Asanuma et al (1999) concluded that there was potential to reduce ruminal methane production and enhance energy efficiency of the animal through the use of fumarate and malate as feed additives that would serve as electron acceptors The importance of AA, peptides and ammonia as substrates for microbial protein synthesis should be quantitatively described in terms of both the ruminal environment they contribute to, and as the major source of protein for the host, as microbes pass from the rumen to the small intestine Oldick et al (1999) and Clark et al (1992) both reported that the profile of microbes passing to the small intestine from the rumen changes depending on the diet, and therefore the AA profile of microbial protein is not constant, as is commonly assumed in several models The availability of AA in the animal can be increased by increasing dry matter intake, which increases the synthesis of microbial protein, and by providing dietary proteins that are resistant to ruminal digestion but are digested by the animal One of the most important variables associated with abomasal protein flow is the level of feed intake VFA represent the principal form of energy substrate for ruminant animals (Sutton, 1985) Considerable proportions (30%, 50% and 92% of acetate, propionate and butyrate, respectively) are subjected to first-pass absorptive metabolism and never reach the venous blood (Reynolds, 2002) Fermentation imbalances in the rumen (e.g resulting from excess supply of degradable nitrogen) can be minimized by using current feeding recommendations, that will benefit animal performance as well as reduce the negative impact on the environment, whether measured locally (e.g on-farm balance of nitrogen and phosphorus) or in a more global sense (e.g greenhouse gases) Further improvements to mechanistic models of metabolism will result in more effective strategies to minimize the potential for negative environmental impact Energetics and Protein Metabolism The synthesis and degradation of protein in the body continues to be the subject of most research Energetically costly, the estimate for ATP-equivalent Interactions between Protein and Energy Metabolism 401 cost per peptide bond formed remains at ATP However, the true cost of peptide bond formation in vivo remains unknown Various experimental estimates for peptide bond formation cost are presented in Table 15.1 Of interest from the study of Storch and Portner (2003; see Table 15.1) was their determination of peptide bond formation cost in cold-adapted or eurythermal fish species; the authors reported no difference in bond formation cost between these two types of fish, and noted that cold adaptation may be achieved at the level of protein stability A problem with all peptide bond cost estimates is the absence of accounting for protein specific pre- and post-translational energy costs The general acceptance of ATP/bond is based on ATP for AA activation, ATP for bond formation, ATP for translocation and ATP for AA transportation, RNA production and associated errors (Fuery et al., 1998) Protein degradation costs have been estimated to be less than 25% of the cost of protein synthesis (Lobley, 2003), which in tissues with rapid protein turnover rates, such as the small intestine, still represents a significant energy expenditure for the animal Protein turnover estimates should also include the indirect costs such as RNA turnover and the cost of metabolic regulation (Storch and Portner, 2003), to provide a more accurate picture of total energy cost Experiments designed to examine the regulation of protein turnover in the body have the potential to increase our understanding of metabolism, beyond an appreciation of protein turnover costs The concept of nutrients, including AA, functioning in the dual role of nutrient signal and biochemical substrate is well established (Grizard et al., 1995) Amino acids have been shown to affect protein synthesis and degradation through their role as metabolic signals A complex regulatory framework interacts to govern independent protein synthesis and degradation rates in different tissues, including hormones, neural signals, physical activity, nutritional status and environmental conditions There have been studies of protein and energy metabolism in humans that have explored a variety of conditions (e.g such as normal man, burn trauma and ageing), which have increased knowledge of protein metabolism and energy expenditures Wolfe (2002) noted that in burn patients in whom protein Table 15.1 Energy cost estimates of protein synthesis (revised from Kelly et al., 1993) Method Inhibition (reticulocytes) Inhibition (chicks) Inhibition (fish) Stoichiometry Stoichiometry Stoichiometry Regression (swine) Regression (chicks) Energy cost (mole ATP per molar peptide bond synthesized) 3.0 7.5 4.3–5.6 4.0 5.0 6.3–7.0 30.2 18.8 References Siems et al (1984) Aoyagi et al (1988) Storch and Portner (2003) Buttery and Boorman (1976) Millward et al (1976) Webster (1981) Reeds et al (1980) Muramatsu and Okumura (1985) 402 T.C Wright et al degradation rates were elevated above protein synthesis rates, supplementation of AA had the effect of reducing protein degradation without an offsetting effect on protein synthesis rate The results of this study led the author to ask the question as to whether or not there is independent regulation of protein degradation and protein synthesis (Wolfe, 2002) The answer to this question has important implications for nutritionists who must consider that a variety of results can be achieved from intake of the same AA The outcome of a set AA intake will depend on the dynamics of the governing factors in play in the metabolic situation being studied We concur with the conclusion of Wolfe (2002) that it may be more beneficial in the long run to determine the mechanisms by which AA and energy affect muscle protein synthesis and degradation rather than seeking a particular value for a ‘requirement’ There is potential for direct regulation of proteolysis by AA (Kadowaki and Kanazawa, 2003) The regulation of protein synthesis by AA in human skeletal muscle (Liu et al., 2002) has recently been reviewed (Wolfe and Miller, 1999; Yoshizawa, 2004) While there are likely to be similarities between humans and ruminants in the underlying mechanisms for AA signalling to quantitatively alter protein synthesis and degradation rates, this remains to be confirmed Sarcopenia, the condition of muscle protein wasting in ageing humans, presents an interesting model to examine the factors that control muscle protein turnover Volpi et al (2001) conducted a large study of young and elderly men to examine the basis for muscle protein loss observed in the elderly Earlier studies had suggested that sarcopenia results from a decreased muscle protein synthesis rate (Volpi et al., 2001) However, Volpi et al (2001) concluded that older men had slightly higher protein synthesis and degradation rates in leg muscle than younger men, but that the basal protein turnover rate in muscle was unlikely to account for the muscle loss associated with ageing This suggests that additional factors that determine muscle protein loss with ageing (e.g hormonal or nutritional) play an important role in controlling muscle protein mass, and that individually, neither synthesis nor degradation rates can explain the net balance of protein turnover Integration of the myriad factors that control the balance between protein synthesis and degradation into a mathematically based description is likely the most effective approach to arrive at accurate predictions of the synthesis and degradation balance that will result from changes in nutritional or hormonal status Non-essential Amino Acids Non-essential AA such as alanine, glutamine and glutamate are direct metabolic links between energy and protein metabolism Some of the inter-organ relationships for alanine and glutamine are illustrated in Fig 15.1 Olde Damink et al (1999) summarized the important metabolic functions provided by glutamine as: the inter-organ transfer of nitrogen and carbon; to provide energy for rapidly dividing cells; as a precursor for nucleic acid biosynthesis; and the regulation of acid/base homoeostasis Peripheral tissues synthesize glutamine and alanine as a way of partially oxidizing AA and yet supplying nitrogen and Interactions between Protein and Energy Metabolism Liver 403 Intestine Muscle Glutamine Diet Glutamine Amino acids CO2 + H2O Gluconeogenesis Alanine Alanine TA Alanine NH3 TA Glutamine Pyruvate Glucose BCAA Kidney Urea Glucose Glutamine NH3 H + Glucose + NH4 BCAA − branched chain amino acids Glucose−alanine cycle TA − transamination Fig 15.1 Inter-organ relationships in the metabolism of alanine and glutamine (from Kelly et al., 1993) carbon to the tissues of the gut and the liver The compromise of incomplete oxidation leaves the nitrogen in a non-toxic form that can be transported back to the liver Because the tissues of the gut almost completely metabolize the supply of glutamate, aspartate and glutamine during first-pass absorption, the supply of these AA for protein synthesis in other tissues must be met almost completely from de novo synthesis (Reeds et al., 1996) These are likely to be synthesized by transamination from glutamate at a cost of ATP per molecule of non-essential AA Thus diets balanced for non-essential as well as essential AA could have an energy sparing effect for the animal Lobley et al (2001) provided an interesting perspective whereby the metabolism of glutamine was described with respect to its contribution to whole-body protein and energy metabolism Glutamine has many metabolic roles, but responses to glutamine supplementation have been inconsistent and it is not considered to be limiting for growth or lactation For example, glutamine is the most abundant free AA in tissues of most animals, which Van Milgen (2002) noted is energetically favourable compared with protein storage Previously, researchers have focused on the extensive use of glutamine and glutamate as energy substrates by the tissues of the gut 404 T.C Wright et al Glutamine and glutamate, respectively, constitute 6.5–12.5% and 7.2– 10.0% of AA residues in bovine caseins, therefore uptake and synthesis of glutamine by the mammary glands must be considerable in a high-producing dairy cow In addition, the uptake of many non-essential AA by the mammary glands is below that required for milk synthesis, and glutamine is likely the source of both carbon and nitrogen for mammary synthesis of other nonessential AA Glutamine also appears to have a role in mediating intracellular activity through transport-mediated changes in cell volume Reeds et al (2000), using the neonatal pig as a model, suggested mechanisms exist that allow pigs to sense an imbalance in the AA supply from milk so they can make acute metabolic changes to ensure AA are still used with high efficiency These mechanisms may also be present in more mature animals Data from both the rat and the neonatal pig suggest that the number of ribosomes decreases but the translational activity of each ribosome increases as the animal approaches weaning The reduction in efficiency of protein utilization in neonatal pigs from birth to 26 days of age is mirrored by changes in sensitivity and responsiveness of protein deposition to insulin concentration Lobley (1992) suggested that in lambs the conversion of dietary nitrogen to body nitrogen was only 13% Data from isotopic studies suggest that 50% to 100% of oxidized glucose was synthesized from glutamate, glutamine and alanine The incremental efficiency for protein gain of absorbed AA ranges from 40% to 80% (Lobley, 1992) Tracer approaches suggest that in fasted sheep, daily protein synthesis amounted to approximately 8% of the wholebody protein pool There is some suggestion that gluconeogenesis from AA occurs even under supramaintenance conditions, which may explain the low efficiency of incremental AA use as supply increases (Lobley, 1992) The use of non-essential AA as a fuel source in visceral tissues is, intuitively, energetically more expensive than the direct use of glucose Van Milgen (2002) presented a useful framework to examine the energetics of intermediary metabolism, wherein this efficiency was re-examined in some detail The additional net cost of converting glucose to glutamate and then oxidizing the glutamate and regenerate ATP (in muscle and viscera, respectively), relative to using glucose as an ATP precursor, is the equivalent of 1.25 ATP, which Van Milgen (2002) indicated is less than the energy cost involved in glycogen turnover The benefits of deriving energy from non-essential AA presumably outweigh the better theoretical energetic efficiency of direct use of glucose as a fuel source Glutamine may also have benefits to visceral tissues in terms of modulating ă protein turnover, with a resulting economy for energy expenditure Coeffier et al (2003) used enteral infusion of glutamine into human subjects to examine effects on protein metabolism Two noteworthy findings resulted from their experiment The first was that glutamine stimulated non-specific protein synthesis as has been demonstrated in other mammals The second, based on the analysis of duodenal biopsies, indicated a decrease in ubiquitin mRNA level compared with either a saline control or an isonitrogenous AA mixture infuă sion Coeffier et al (2003) concluded that mucosal protein degradation through the ATP-ubiquitin dependent proteolytic pathway might be limited Interactions between Protein and Energy Metabolism 405 via a glutamine-specific mechanism These authors also raised the possibility that glutamine could regulate the inflammatory response in the intestinal mucosa of humans These possibilities are worthy of investigation in ruminant animals in which glutamine supplementation may be useful to support animal well-being during periods of physiological and metabolic stress, for example the periparturient dairy cow, which can experience metabolic disorders and which mobilizes significant body reserves to support milk production Portal-drained Viscera (PDV) The PDV in mature ruminant animals comprises those tissues whose venous drainage is combined and flows into the hepatic portal vein, including the rumen, reticulum, omasum, abomasum, small intestine, large intestine, spleen, pancreas, caecum and mesenteric and omental fat tissue Some small anatomical differences exist between ruminant species but they are generally quite similar (Seal and Reynolds, 1993) The PDV tissues differ from other tissues of the body because of their exposure to dual sources of nutrient supply, namely digesta and arterial blood supply Ruminant PDV tissues utilize glucose, volatile or short-chain fatty acids, ketones and AA as oxidative substrates (Reynolds et al., 1990) The absorption of free AA and peptides across the small intestine is achieved by specific transporters, some of which require energy This, and the high turnover rate of gut tissue, are two significant contributions of the small intestine to whole-body energy expenditure Maintenance of Naỵ , Kỵ , ATPase activity, substrate cycling, urea synthesis, protein synthesis and degradation in the gastrointestinal tract and liver were estimated together to account for 22.8% of whole-body oxygen consumption in growing steers (Huntington and McBride, 1988) and, more recently, Reynolds (2002) estimated that the total splanchnic tissues usually account for 40–50% of total body oxygen consumption The energetic cost to the animal for maintenance and turnover of gut tissues and for nutrient absorption is, therefore, considerable and a large proportion of this energy expenditure is directly linked to protein and AA metabolism Coordination of nutrient use by the whole animal is an important part of protein/energy metabolism, particularly in the PDV Ebner et al (1994) conducted an experiment with 2-week-old pigs to examine the effects of a lowprotein diet (15% crude protein (CP)) compared with a control, isocaloric protein diet (30% CP) on PDV tissue growth and metabolism In their experiment, feed intake was not different (P ¼ 0:76) between the experimental groups, but after weeks there was evidence of protein malnutrition including reduced carcass weight and higher circulating concentrations of 3-methylhistidine in the pigs fed low protein diet These piglets had PDV blood flow and O2 consumption rates approximately 50% and 22% higher, respectively, than control pigs on a lean body mass basis, under fasting conditions Ebner et al (1994) suggested that under conditions of protein malnutrition, gastrointestinal tissues and their metabolic rate were preserved at the expense of peripheral tissues Reduced concentrations of insulin were measured in the low protein 406 T.C Wright et al group, which may have helped to coordinate a response to reduce the use of AA for protein synthesis in skeletal muscle Understanding the mechanisms in ruminant animals that serve to prioritize tissue nutrients to cope with situations of protein malnutrition (e.g disease, parasitic infection, low feed quality, etc.) would improve our understanding of whole animal nutrient use The energetic cost of protein synthesis in the small intestine of lambs in response to level of feed intake was quantified by Neutze et al (1997a,b) As in other studies of this type, the choice of pool to represent the actual AA-specific radioactive pool had a dramatic impact on fractional synthesis rate calculations Use of the tissue-free phenylalanine-specific radioactivity gave a fractional synthesis rate of approximately 130% per day, while the use of the arterial blood phenylalanine-specific radioactivity gave estimates of approximately 30% per day The small intestine accounted for approximately 13% of whole-body protein synthesis, which accounted for 18–27% of total energy use by that tissue, depending on the true precursor pool Neutze et al (1997a,b) accounted for the production of exported proteins and their results suggested that, in growing lambs, exported proteins such as sloughed cells and secretory proteins might account for the largest component of total protein synthesis in the small intestine The energy expended for the synthesis of exported proteins is noteworthy because the opportunity for energetically efficient reuse of their carbon and nitrogen metabolites is reduced The important role of the PDV and the liver to modulate the quantity and concentration of nutrients supplied to peripheral tissues was reported by Lapierre et al (2000) using multi-catheterized animals These authors used growing steers and achieved three different levels of intake of a single diet, calculated to provide 0.6, 1.0 and 1.6 times the estimated requirements for ME and CP Their experiment examined in detail the uptake and release of AA, hormones and key metabolites across tissues and provided a better understanding of nutrient fluxes in total splanchnic metabolism The information gained from this intricate type of research provides important data on nutrient use and systemic regulation that will ultimately permit the development of diets that improve efficiency of the conversion of dietary nitrogen to animal protein Further improvements in our understanding of PDV metabolism might be achieved if the luminal nutrients that can directly signal protein synthesis or degradation were determined Identification of these nutrients through the use of normal feeding trials is difficult because as the luminal nutrient supply changes, both basolateral nutrient concentrations and hormonal changes will result The kinetics of AA use by the PDV are complex, in part because the use of AA of arterial origin appears to increase concomitantly with increases in luminal AA supply (Reynolds, 2002) The sensitivity of intestinal protein synthesis to the avenue of nutrient supply is unique Discerning systemic effects from the direct effects of increased luminal nutrient concentration is difficult because techniques to distinguish these two events are a challenge to develop, and, invariably, increased luminal nutrient concentrations lead to systemic responses for growth factors and hormones that can stimulate protein synthesis Interactions between Protein and Energy Metabolism 407 Recently, a technique has been validated in piglets to determine the acute effects of luminal nutrient supply on intestinal protein synthesis (Adegoke et al., 1999a) using multiple cannulation of the small intestine to permit luminal nutrient perfusion of short, discrete intestinal segments Multiple segments of small intestine within the same animal can be perfused, which together account for less than 4% of total small intestinal absorptive surface area This multiple perfusion approach, combined with the luminal flooding dose technique, resulted in a method that measured the acute effects of luminal nutrient concentration on intestinal protein synthesis in the absence of systemic responses such as increased plasma insulin, AA or glucose concentrations (Adegoke et al., 1999a) Several interesting findings were reported with the application of this technique in an experiment designed to examine the acute effects of luminal nutrients on intestinal protein synthesis and mRNA abundance of m-calpain and components of the ATP-ubiquitin protein degradation system (Adegoke et al., 1999b) A 20–25% suppression of mucosal protein fractional synthesis rate (Ks ) occurred with luminal perfusion of a 30 mmol/l mixture of AA or a 30 mmol/l perfusion of glutamine compared with a saline perfusion A second experiment examined the perfusion of mucosal energy substrates (50 mmol/l glucose, 50 mmol/l short-chain fatty acids or 20 mmol/l b-hydroxybutyrate) without added AA and there was no effect on the fractional rate of protein synthesis in the mucosa (Adegoke et al., 1999b) Analysis of the abundance of mRNA for the protein for degradation systems revealed that while there was no effect of AA perfusion on m-calpain expression, there was a 28% reduction in ubiquitin mRNA abundance and a 20% reduction in the ubiquitin-conjugating ă enzyme, which agrees with the data of Coeffier et al (2003) in which enteral glutamine in humans reduced gut mRNA abundance of ubiquitin The effectiveness of AA compared with ammonia to suppress protein synthesis was also tested by perfusing intestinal segments with buffer, 30 mmol/l mixture of AA or two concentrations of ammonium chloride Their results (Table 15.2) indicated that there was a 26% reduction in Ks when the AA mixture was perfused, while ammonium chloride perfusion had the effect of raising tissue ammonia levels to those that resulted with AA perfusion, but without an equivalent effect on Ks Thus, the signal for protein synthesis is mediated by AA Adegoke et al (1999b) noted the rapid (90 min) time frame for the changes detected in Table 15.2 Effect of buffer, an AA mixture or ammonium chloride on mucosal protein fractional synthesis (Ks) in piglets (from Adegoke et al., 1999b) Ammonium chloride Treatment Buffer (PBS) Amino acids 30 mmol/l Tissue ammonia, mg/g wet weight Ks, % PBS 6.30 + 0.17a 8.42 + 0.29b 7.46 + 0.28ab 8.39 + 0.28b 100 + 3.8a 74 + 3.7b 98 + 4.6a 102 + 3.4a 0.5 mmol/l 1.0 mmol/l Values are mean + SEM for n ¼ Different superscripts within a row are different from one another (P