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Extent and Physiological Implications of Protein Turnover

In the human adult, approximately 300 g of protein turnover occurs each day (4g/kg/day)—that is, three or four times the daily dietary intake. Rates vary between tissues with rapid turnover in visceral tissues and those with slow turnover in muscle. Liver and intestine account for approximately 8% of the lean body mass (LBM) and up to 50% of whole body protein turnover, with skeletal muscle, at 55% of the LBM, accounting for only approximately 25% of total protein turnover. Thus, whole body protein turnover varies with body composition and this largely explains developmental changes. In the infant, turnover rates are much higher, ranging between 10 and 20g protein turnover per kilogram per day, consistent with the higher proportion of metabolically active tissue and lower muscle mass. However, animal studies indicate a developmental decline in protein turnover in skeletal muscle that may be an additional component of the marked decline in protein turnover with age. In the elderly there is little evidence of any change other than that associated with the decline in LBM.

Protein turnover constitutes an appreciable fraction of the maintenance energy expenditure. On the basis of 5 mol ATP/GTP per mole of protein turnover (4mol per peptide bond with 1 mol for amino acid transport, RNA turnover, and proteolysis), an energy cost of 22 kcal/mol ATP, and a molecular weight of 110 per mole of peptide bond, this is equivalent to approximately 1 kcal/g protein turnover. Thus, in the normal adult, protein turnover at 300 g/day accounts for approximately 20% of the basal metabolic rate. Therefore, changes in the protein turnover and metabolic rate would be expected to occur in parallel to some extent, and this is observed. Thus, the decline with age in both protein turnover and metabolic rate from birth to adulthood involves a factor of three or four in each case.

Regarding protein turnover and protein requirements, there is no a priori reason for any interrelationship, and there is little evidence of any. Thus, turnover does not consume amino acids, and amino acid catabolism and oxidation is not linked to turnover. Maintenance protein requirements decrease relatively little with age (<20%) compared with the 3- or 4-fold decrease in turnover.

Regulatory Mechanisms of Protein Turnover Control

The physiological importance of protein turnover is undoubtedly the regulatory flexibility it allows. With opportunities for control of both synthesis and proteolysis, the number of potential control sites is increased. In addition, because of the continuing turnover in the steady state, changes in the amount of protein can be achieved with low energy costs through inhibition of proteolysis to allow growth or through inhibition of synthesis to allow mobilization.

At the molecular level, regulation of protein synthesis is necessarily complex at both transcrip-tional and translational levels. Advances in molecular biology have revealed many examples of transcriptional control to the extent that changes in specific mRNA concentrations have become a surrogate measure of changes in rates of synthesis for specific proteins. Notable nutritional examples include control of hepatic export protein synthesis. Thus, the downregulation of albumin synthesis in response to either protein deficiency or the proinflammatory cytokine-mediated acute phase response is mediated largely at the level of tran-scriptional control of mRNA levels, with reductions in mRNA for albumin and other hepatic export proteins and increases in mRNA for acute phase proteins.

The concentration of ribosomes in tissues determines the capacity for protein synthesis and in this way controls overall tissue protein turnover rate and the changes associated with postnatal development. Cellular ribosome concentrations can change both acutely (e.g., during the diurnal cycle of feeding and fasting) and chronically in response to protein and energy intakes, increased functional demand, and hormones such as insulin, thyroid, growth hormone, and the glucocorticoids. Furthermore, these influences are tissue specific, with glucocorticoids, for example, increasing hepatic ribosome concentrations (as part of the hepatic acute phase response) and decreasing ribosome concentrations in muscle. In contrast, thyroid hormones increase ribosomes (and proteolytic enzymes) in both muscle and liver in association with a generalized increase in protein turnover.

Acute regulation of translation is exerted mainly through initiation, with reversible phosphorylations known to regulate at least four separate steps of the initiation cycle enabling very rapid changes in protein synthesis. Peptide hormones (insulin and insulinlike growth factor-1 (IGF-1)), glucocorticoids, and amino acids have all been implicated in such regulation, although the specific targets of control remain uncertain. Furthermore, there are major differences between the mechanisms observed in the young, rapidly growing animal and the adult animal. Thus, in skeletal muscle in the young rat, an insulin-mediated stimulation occurs. In adult human, muscle insulin is relatively ineffective, with amino acid levels the main stimulatory influence. Indeed, because insulin inhibits proteolysis and lowers amino acid levels, insulin alone appears to inhibit protein synthesis in human muscle.

Regarding the nutritional regulation of proteo-lysis, most is known about lysosomal proteolysis, especially hepatic autophagy, with both amino acids and insulin having inhibitory roles. Leucine, alanine, and insulin interact to regulate this pathway, with a leucine-sensitive receptor-mediated inhibitory pathway identified in liver. In the case of the ubiquitin-proteasome system, its activation in skeletal muscle during fasting and following gluco-corticoid treatment supports a role in the physiological regulation of protein turnover. On the other hand, both lysosomal and calcium-activated proteo-lysis are activated under the same conditions. Similarly, in response to protein deficiency when protein turnover rates generally decrease in tissues, in part through the decline in thyroid hormone levels, the activities of all three systems decrease. One control mechanism involves changes in cell volume. Thus, swelling acts like a proliferative anabolic signal, inhibiting proteolysis, whereas cell shrinkage is cata-bolic, stimulating proteolysis. These effects have been shown in liver, and there is evidence for such a mechanism in skeletal muscle.

Postprandial Protein Utilization

Overall nitrogen homeostasis within the LBM is maintained within a diurnal cycle of postprandial protein gain and postabsorptive loss. The amplitude of these diurnal changes increases as habitual protein intakes increase, with implications for the qualitative nature of the metabolic demands for amino acids and hence dietary protein. The key questions are how both acute and chronic protein intakes mediate such responses and, most important, what influences the efficiency of postprandial protein utilization and consequent protein requirements. Nutritional regulation of dietary protein utilization, protein turnover, and amino acid oxidation involve both hormonal responses to food intakes and direct substrate influences. Whereas interactions between insulin, thyroid hormones, and IGF-1 mediate the anabolic drive of dietary protein on muscle and bone growth in the growing animal, the control mechanisms involved in the transient gains and losses of protein during diurnal cycling differ since neither thyroid hormones nor IGF-1 levels vary from meal to meal or in relation to habitual protein intakes. On the basis of several studies on either insulin or amino acids alone or variations in meal protein levels, it appears that insulin and amino acids act as main acute regulators.

The mechanisms involved are best understood in the context of the interrelationships between the free and protein-bound amino acid pools. Many indispensable amino acids are potentially toxic and are maintained at very low concentrations in tissues, so rapid and regulated postprandial disposal is important. After a protein meal there are two pathways for amino acid disposal. The first comprises the various high-capacity, finely regulated oxidative pathways activated by a protein meal. In most cases, rates of amino acid oxidation are influenced by their tissue concentrations (generally similar to the Km of the rate-limiting enzymes), together with substrate activation and covalent enzyme modification. Examples are phenylalanine hydroxylase and branched-chain a-keto acid dehydrogenase, which are both regulated by substrate binding and reversible phosphorylation and dephosphorylation. The second pathway is net protein deposition. This can be achieved by stimulation of protein synthesis or inhibition of proteolysis so that a regulatory link between postprandial hyper-amino acidemia and protein synthesis and proteolysis can be expected and does indeed exist.

For protein synthesis, amino acids cannot exert simple kinetic concentration-related influences since the low Km of amino acyl tRNAs synthesis means that they are usually fully charged. Nevertheless, there is ample evidence for regulatory stimulation by amino acids. This remains poorly understood but in some cells is known to involve signaling events linked to the mammalian target of rapamycin, mTOR, which in turn regulates S6 (an initiation factor) as well as eEF2 (an elongation factor). However, stimulation of protein synthesis through increased amino acid levels may also stimulate amino acid oxidation pathways, as discussed previously. Although this allows effective removal of amino acids, in the context of an efficient protein utilization this would not be a preferred mechanism.

For proteolysis, amino acids exert an inhibitory influence as described previously, and this inhibition will reduce endogenous amino acid supply. This will prevent undue increases in amino acid levels and will therefore minimize amino acid oxidation and maximize dietary protein utilisation. Furthermore, since inhibition of proteolysis and lowering of intra-cellular amino acid levels can be achieved by receptor-mediated mechanisms involving insulin as well as specific amino acids (e.g., leucine), this allows the postprandial increases in plasma amino acids to mediate substantial amino acid transport into cells, resulting in protein deposition without any increase in intracellular amino acid levels and with minimal increases in amino acid oxidation. Thus, as a strategy for mediating postprandial protein utilisation, inhibition of proteolysis is predicted to be more efficient.

13C leucine studies have provided clear experimental support for such a mechanism. The meal protein-dependent responses of protein synthesis, proteolysis, and leucine oxidation are shown in Figure 3 based on measurements in adult subjects fed isoenergetic meals of increasing protein intake

Change rate t

Increase i 0

Decrease

Energy (insulin) effects

Amino acid effects I

Figure 3 Feeding-induced responses of leucine kinetics shown as protein synthesis, proteolysis, and leucine oxidation. Patterns of responses reflect actual changes observed in 13C-1 leucine infusion studies of feeding responses to frequent small meals of increasing protein intake, equivalent to daily intakes between 0.3 and 2.0gkg~1 day-1. (Adapted from Pacy PJ, Price GM, Halliday D et al. (1994) Nitrogen homeostasis in man: 2. The diurnal responses of protein synthesis, degradation and amino acid oxidation to diets with increasing protein intakes. Clinical Science 86:103-118; and Gibson NR, Fereday A, Cox M et al. (1996) Influences of dietary energy and protein on leucine kinetics during feeding in healthy adults. American Journal of Physiology 33: 282-291.)

50 60 70 80 90 100

Efficiency of protein utilization %

Figure 4 Relationships between the efficiency of protein utilization (Abalance/Aintake) and responses of leucine turnover and oxidation to protein feeding observed in 24 normal adults fed frequent small meals containing protein intakes similar to the habitual intakes of the subjects. (Adapted from Fereday A, Gibson NR, Cox M et al. (1998) Variation in amino acid mediated, insulin activated inhibition of proteolysis determines the efficiency of protein utilization. Clinical Science 95: 725-733.)

Change rate t

Increase i 0

Decrease

Energy (insulin) effects

Amino acid effects I

Figure 3 Feeding-induced responses of leucine kinetics shown as protein synthesis, proteolysis, and leucine oxidation. Patterns of responses reflect actual changes observed in 13C-1 leucine infusion studies of feeding responses to frequent small meals of increasing protein intake, equivalent to daily intakes between 0.3 and 2.0gkg~1 day-1. (Adapted from Pacy PJ, Price GM, Halliday D et al. (1994) Nitrogen homeostasis in man: 2. The diurnal responses of protein synthesis, degradation and amino acid oxidation to diets with increasing protein intakes. Clinical Science 86:103-118; and Gibson NR, Fereday A, Cox M et al. (1996) Influences of dietary energy and protein on leucine kinetics during feeding in healthy adults. American Journal of Physiology 33: 282-291.)

from 0.36 to 2.07gprotein/kg/day. Inhibition of proteolysis occurs at all levels of protein intake, but it increases with intake. However, the direction and magnitude of the response of protein synthesis reflect the level of dietary protein intake, with slight inhibition or no change at low intakes and stimulation at high intakes. Such studies clearly establish the importance of proteolysis as a regulator of tissue protein balance in the postabsorbtive and postprandial state. These and other 13C leucine studies of postprandial protein utilization have allowed the separate influences of dietary energy and amino acids protein to be identified as shown in Figure 3. The response to energy alone involves insulin-mediated changes allowing leucine balance to becomes less negative through inhibition of pro-teolysis with minimal changes in protein synthesis or amino acid oxidation. In fact, because this tends to lower amino acid levels, there is a decrease in protein synthesis. With increasing amino acid supply as dietary protein intake increases, there is further inhibition of proteolysis by amino acids with increases in protein synthesis and, to some extent, amino acid oxidation, allowing net protein deposition as tissue protein.

The increase in amino acid oxidation with protein feeding is an unwanted response that reduces the efficiency of protein utilization. Although utilization

50 60 70 80 90 100

Efficiency of protein utilization %

Figure 4 Relationships between the efficiency of protein utilization (Abalance/Aintake) and responses of leucine turnover and oxidation to protein feeding observed in 24 normal adults fed frequent small meals containing protein intakes similar to the habitual intakes of the subjects. (Adapted from Fereday A, Gibson NR, Cox M et al. (1998) Variation in amino acid mediated, insulin activated inhibition of proteolysis determines the efficiency of protein utilization. Clinical Science 95: 725-733.)

of proteins such as milk is higher than that of wheat, as would be expected because of the lysine limitation of wheat gluten utilization, there is variability between individuals in the efficiency of postprandial protein utilization, ranging from 50 to 100% with milk protein. Figure 4 shows the results of studies that have examined this variation. Efficient utilization involves maximal inhibition of proteolysis by protein feeding with minimal increases in free amino acid concentrations and consequent amino acid oxidation and stimulation of protein synthesis, indicating that the efficiency of protein utilization in individuals is determined by the sensitivity of the insulin-mediated inhibition of proteolysis to amino acid supply.

Current understanding suggests a mechanism indicated in Figure 5, in which the major target of insulin is inhibition of proteolysis, with amino acids acting to both enhance the inhibition of pro-teolysis and stimulate synthesis and oxidation. With tissue amino acid levels controlled by both diet and endogenous supply from proteolysis, inhibition of proteolysis will minimize any increase in amino acid levels, minimize oxidation, and maximize protein utilization. Since protein synthesis and amino acid oxidation appear to be stimulated in parallel, the optimum strategy for maximum efficiency of postprandial protein utilization appears to involve maximal inhibition of proteolysis and minimal postprandial increases in tissue amino acid levels.

Dietary protein and energy

Figure 5 Scheme for the action of insulin and amino acid supply on postprandial protein utilization. Insulin and extracellular amino acids exert inhibitory influences on proteolysis and protein synthesis through receptor-mediated mechanisms (a and b), whereas amino acid uptake (c) and proteolysis regulate intracellular amino acid levels, amino acid oxidation, and protein synthesis in parallel. Maximal inhibition of proteolysis and maintenance of low intracellular amino acid levels is the optimal response. (Modified from Millward DJ, Fereday A, Gibson NR et al. (1996) Postprandial protein metabolism. Baillier's Clinical Endocrinology and Metabolism 10: 533-549.)

Figure 5 Scheme for the action of insulin and amino acid supply on postprandial protein utilization. Insulin and extracellular amino acids exert inhibitory influences on proteolysis and protein synthesis through receptor-mediated mechanisms (a and b), whereas amino acid uptake (c) and proteolysis regulate intracellular amino acid levels, amino acid oxidation, and protein synthesis in parallel. Maximal inhibition of proteolysis and maintenance of low intracellular amino acid levels is the optimal response. (Modified from Millward DJ, Fereday A, Gibson NR et al. (1996) Postprandial protein metabolism. Baillier's Clinical Endocrinology and Metabolism 10: 533-549.)

In summary, postprandial protein utilization appears to be mediated by an insulin-mediated, protein-conserving influence of dietary energy that inhibits proteolysis, lowers amino acid levels, and reduces oxidation, with dietary amino acids augmenting the inhibition of proteolysis. The response of protein synthesis is primarily determined by the resultant intracellular amino acid levels that reflect the balance between the decreasing endogenous supply following insulinmediated inhibition of proteolysis and the increasing exogenous supply as dietary protein intake increases, stimulating protein synthesis and increasing oxidation when amino acid dietary supply exceeds the capacity for its net deposition.

See also: Amino Acids: Metabolism. Protein: Requirements and Role in Diet; Digestion and Bioavailability; Quality and Sources.

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New Mothers Guide to Breast Feeding

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