|Protein-Energy Interactions (International Dietary Energy Consultative Group - IDECG, 1991, 437 pages)|
|Amino acid oxidation and food intake|
* Department of Biochemistry and Molecular Biology, University of Massachusetts Medical Center, Worcester, MA 01655, U.S.A.
Proteins, but not carbohydrates and fats, contain substantial amounts of nitrogen (N). By measuring nitrogen intake and excretion, it has therefore been possible to monitor nitrogen (and hence protein) balances and to assess amino acid (AA) oxidation rates, a task facilitated by the fact that most of the N liberated by amino acid degradation in mammals is eliminated in the form of urinary urea. Thanks to such measurements, it has long been recognized that the organism strives to maintain approximate nitrogen balance. In the absence of disease and as long as protein intake is above a certain minimum, nitrogen balances are indeed quite effectively maintained by adjustment of AA degradation to protein intake, regardless of the relative proportions of carbohydrate and fat provided by the diet to cover the body's energy needs. Protein metabolism provides an example of a situation where problems of considerable practical importance can be dealt with so well, that one often forgets that the mechanisms by which the oxidation of the various amino acids is coordinated and adjusted to match protein intake are still poorly understood.
The manner in which nitrogen balances vary as a function of protein-N intake in adults is illustrated in the upper panel of Figure 1. Three typical conditions are represented: (A) When common foods are consumed, so that dietary protein is accompanied by carbohydrates and fats to the tune of about 150 kcal/g protein-N (i.e., 17% of dietary energy as protein). When no food is consumed, urinary nitrogen excretion is shown here to be about 10 g/d; this amount varies somewhat, depending in particular on the length of the fasting period. (B) When foods high in carbohydrate, but containing essentially no protein, are consumed in amounts sufficient to cover daily energy needs (i.e., - 1500 kcal/d), nitrogen losses are reduced to some 5 g N/d. This corresponds to the minimal or 'obligatory N loss'. Consumption of increasing amounts of protein in addition to the protein-free foods causes the nitrogen balance to become less negative. With an intake of about 12 g of nitrogen/d, the situation is similar to that where usual foods are consumed in amounts covering daily needs, N balance being achieved with intakes of about 12 g of protein-N/d and about 1800 kcal/d. (C) When only protein is consumed, nitrogen balances are less negative than during total food deprivation, but ingestion of 12 g of protein-N per day is not sufficient to achieve N balance. Nitrogen balance can be approached even while the energy balance remains markedly negative, but substantially higher protein intakes are required. This situation is commonly encountered during a 'protein-sparing modified fast', where, after one week of adaptation, a dose of 1.5 g protein/kg body weight/d is generally sufficient to maintain N balance (LINDNER and BLACKBURN, 1976). In subjects affected by trauma or disease, the three curves are all shifted downward, as these conditions bring about increased protein breakdown to amino acids as well as a more rapid amino acid oxidation (KINNEY and ELWYN, 1983). In protein-depleted individuals, on the other hand, N balances are generally less negative and they become more markedly positive during reconvalescence than in well-fed subjects, so that the three curves shown in the upper panel of Figure 1 are shifted upward in such conditions (WATERLOW, 1987). However, even then, N balances do not rise much above a few grams of protein-N retained per day, regardless of the fact that protein and other nutrients may be consumed (or infused parenterally) in amounts greatly exceeding protein and energy requirements (KINNEY and ELWYN, 1983).
Upper panel: Nitrogen balances in adults as a
function of protein nitrogen intake: (A) when consuming mixed foods (-),
(B) 1500 kcal/d of non-protein energy plus variable amounts of protein (-), or
(C) only protein (---).
Lower panel: Nitrogen excretion rates as a
function of protein-N intakes, as implied by the nitrogen balances shown in the
upper panel. (Nitrogen balance is shown by.....)
Restatement of these quite familiar N-balance patterns allows us to examine what they imply about rates of amino acid degradation (lower panel of Figure 1), of which one is generally less well aware. The thin dotted line shows the conditions for which N excretion matches N intake, i.e. when N balance is equal to zero. It can be seen that on high protein and energy intakes, nitrogen excretion increases in direct proportion to further increments in protein intake. On the other hand, amino acid oxidation rates vary substantially when food intake is restricted, as they are then markedly influenced by the availability of other fuels, i.e., glucose, free fatty acids (FFA) and ketone bodies (FLATT and BLACKBURN, 1976). This reflects the fact that maintenance of ATP levels takes priority in all cells, so that any available substrates will be used to regenerate ATP from ADP and phosphate. Consuming carbohydrate to maintain glucose availability reduces the need to obtain energy by amino acid oxidation. Ingestion of some 100 g of carbohydrate per day reduces N excretion by about half, a phenomenon well known as the 'protein-sparing effect of dietary carbohydrate' (GAMBLE, 1946). Unfortunately, influx of exogenous carbohydrates is much less effective in curtailing N losses in the face of disease, even in high doses (KINNEY and ELWYN, 1983). During prolonged starvation, mobilization of endogenous fat reserves can yield enough substrates to meet almost all of the body's energy needs, thanks to the production of ketone bodies by the liver which can be used by the brain instead of glucose (CAHILL, 1970). However, about two weeks of starvation elapse before circulating ketone body levels rise enough for the combined availability of FFA, ß-hydroxy-butyrate and acetoacetate to allow maximal curtailment of amino acid oxidation. Having learned to recognize the importance of energy metabolism on the nitrogen balance, much attention has been focused on the interactions between metabolic fuels, hormone levels and the protein economy (CAHILL, 1971; FLATT and BLACKBURN, 1974). However, the concepts which have evolved and which shape much of our thinking in this area are based primarily on observations made under conditions of protein and/or energy deprivation. They are of little help in explaining the control of amino acid oxidation under conditions of plenty.
Differences between hypocaloric and hypercaloric situations are summarized schematically in Figure 2 which shows the regulatory interactions in the 'metabolic fuel regulatory system' (FLATT and BLACKBURN, 1976). As suggested by the 'metabolic funnel', the main pathways of intermediary metabolism have the effect of channelling various substrates toward the mitochondrial system where their ultimate conversion to CO2 by the reaction of the citric acid cycle is accomplished. The first equation states the fact that overall substrate oxidation is dictated by energy expenditure, but also that determination of overall substrate oxidation (e.g., by indirect calorimetry) provides a measure of total energy expenditure. In hypocaloric situations (second equation), the extent to which amino acid oxidation will rise above the minimum obligatory rate is determined primarily by the extent to which energy needs can be covered by oxidation of glucose, FFA and KB. In hypercaloric situations (third equation), amino acid oxidation increases in direct proportion to further increments in protein intake, regardless of carbohydrate and fat substrate availability (Figure 1). Many key features of metabolic control in hypocaloric situations are thus obviously not applicable in hypercaloric situations. For instance, while the availability of carbohydrate and high circulating insulin levels are believed to be particularly critical in limiting amino acid oxidation (CAHILL, 1971), amino acid oxidation in hypercaloric situations will proceed as rapidly as needed to match protein intake, in spite of high glucose availability and elevated insulin levels. In fact, the increase in amino acid oxidation encountered following trauma, and to an even greater extent during periods of sepsis (KINNEY and ELWYN, 1983) occurs in spite of the hyperglycemia and the high insulin levels which generally prevail under such conditions.
In trying to make sense of these seeming inconsistencies, it is useful to remember that most enzymes catalyzing the transformation of substrates along the main pathways of intermediary metabolism are present in considerable excess. Such enzymatic 'reserve capacity' allows the organism to digest and store occasional, unusually large nutrient influxes, and to handle the exceptional physical activity demands encountered from time to time. When such demands remain altered for some time, induction and repression of protein synthesis bring about appropriate increases or decreases in enzyme levels and in the muscle mass, so as to assure the maintenance of an adequate, but not excessive reserve capacity for the new set of circumstances. The availability of such excess enzymatic activities is what enables the organism to strive for a desirable overall integration of substrate oxidation, without being impeded by lack of catalytic power at some particular steps in the main metabolic pathways. As a consequence of such relatively high enzyme levels, many metabolic intermediates are maintained at near equilibrium conditions, a situation where substrate influxes and drainage, as well as KM and competition for coenzymes can markedly influence flux rates. When metabolic fuel availability is not a factor, variations in amino acid concentrations can thus play a major role in bringing about changes in amino acid degradation rates (HARPER et al., 1984). This may appear to be a rather primitive mechanism, potentially associated with a good measure of instability, though this is presumably not too threatening in this instance because protection against undue depletion is offered by the constant influx of amino acids liberated by degradation of endogenous proteins. Such a mechanism readily accounts for the fact that an influx of dietary amino acids enhances amino acid oxidation in spite of the anabolic conditions prevailing post-prandially. One can, of course, also expect that the rate of amino acid oxidation will be influenced by changes in the rate of protein breakdown, as well as by the avidity with which amino acids are used for protein synthesis (KINNEY and ELWYN, 1983; WATERLOW, 1986).
The various pathways of intermediary metabolism have the effect of a 'metabolic funnel', channelling the various intermediates toward common steps leading to terminal oxidation to CO2 in the mitochondria. The captions in the lower part of the figure show that the overall rate of energy expenditure determines total substrate oxidation; that the availability of glucose, free fatty acids (FFA) and ketone bodies (KB) to meet energy demands is an important factor in determining amino acid oxidation rates when food intake is restricted (hypocaloric situations); and that amino acid oxidation in hypercaloric situations is largely determined by protein intake, a condition where the rate of amino acid oxidation influences carbohydrate plus fat oxidation, rather than the reverse.
Substrate level interactions between amino acid, carbohydrate and fat oxidations are bound to occur, notably because pyruvate, fatty acids, ketone bodies and the a-keto derivatives of the branched-chain amino acids (BCAA) all need to react with Coenzyme A (CoA) to enter the pathways for their oxidative degradation. When glucose is available, insulin facilitates glucose transport and activates pyruvate dehydrogenase (PDH) (DENTON et al., 1987) so that pyruvate can capture most of the CoA made available by oxidation of acetyl-CoA, thereby restraining the degradation of amino acids needing CoA for their oxidation (Figure 3). During prolonged starvation, once starvation ketosis is fully induced, FFA and KB appear together to be almost as effective in preventing the a-keto derivatives of the BCAA from reacting with CoA as a plentiful supply of glucose. When food and protein intake are limiting, losses and failure to replenish the BCAA pool appear to assume special importance in determining overall amino acid oxidation and N balance (WALSER, 1983). During disease, the supply of carbohydrate is diminished due to spontaneous or forced reduction in food intake. The release of 'catabolic hormones' causes mobilization of fuels as well as insulin resistance. The increases in insulin secretion needed to control blood glucose levels in such situations is not sufficient to counteract the lipolytic effect of catecholamine on FFA mobilization when stress is high (KINNEY and ELWYN, 1983), but when stress is minor, FFA levels may be lower than during equivalent food deprivation in the absence of disease (NEUFELD et al., 1980). However, insulin levels are sufficient to markedly inhibit ketogenesis even in the face of elevated circulating levels of catabolic hormones (WATTERS and WILMORE, 1986). As demonstrated by various observations, ketone bodies have an effect in reducing amino acid oxidation which goes beyond that accomplished by high FFA availability (SHERWYN et al., 1975; FLATT and QUAIL, 1981) (Figure 4). Given the limited availability of glucose and the effect of insulin resistance on the control of PDH and pyruvate conversion to acetyl-CoA, stress plus food deprivation create a situation for increased amino acid oxidation. Indeed, rapid protein-wasting is induced in the absence of disease, if the mobilization of the fat reserves and ketogenesis are inhibited by administration of antilipolytic agents, though these agents have no effect on N excretion in fed animals (TALKE et al., 1973). It is noteworthy that the increase in amino acid degradation which occurs as a result of lack of other fuel leads to a rise in blood glucose levels, indicating that increased amino acid degradation is not driven by the need to provide glucose (TALKE et al., 1973). The effects of sepsis and trauma in inhibiting ketogenesis thus appear to be important factors in explaining protein-wasting (FLATT and BLACKBURN, 1974, WEDGE et al., 1976), and competition for CoA provides a widely applicable concept in helping to understand how rate-setting interactions between intermediates of carbohydrate, fat and protein metabolism can occur at the substrate level.
High free fatty acid (FFA) and ketone body (KB) availability under fasting conditions, or a plentiful supply of pyruvate in the fed state assure that the a-keto derivatives of the branched-chain amino acids (BCKA) are not rapidly drawn into irreversible degradation by reaction with Coenzyme A (CoA), unless their concentrations are sufficiently high to compete more intensely for CoA.
When changing from situations of high to low protein intake, or vice-versa, a few days are required before N balance is again achieved, during which small amounts of protein are lost or gained. Since these are a prerequisite for the reestablishement of N balance, small changes in protein content appear to play an important role in adjusting amino acid oxidation rates to amino acid intake This can be envisioned to result from the expansion or contraction of rapidly turning-over protein pools, which would be expected to influence the concentration of intracellular amino acids between meals. Coupled with the evidence that amino acid degradation is greatly influenced by their availability when metabolic fuel availability is not a factor (HARPER et al., 1984), the role of rapidly turning-over proteins (perhaps a part of the so-called 'labile protein pool'), may be seen as exerting a function analogous to that of a 'fly wheel' in making amino acid oxidation commensurate with amino acid intake.
Intraperitoneal injection of CC14 (1 mL/kg body weight) to 24-hour fasted rats (·-·), which does not reduce FFA mobilization, causes marked lowering of ketone body levels and a concomitant increase in urinary-N excretion relative to fasting controls (o--o). (Adapted from FLATT and QUAIL, 1981).
It is often believed that gluconeogenesis from amino acids is induced during starvation to provide glucose and that the need to provide glucose is a driving factor in the increased gluconeogenesis taking place after trauma and during disease (KINNEY and ELWYN, 1983). This view may or may not include the belief that gluconeogenesis from amino acids is a minor process in the fed state. In fact, conversion of the glucogenic moieties of the degraded amino acids to glucose occurs even in the fed state. A detailed quantitative analysis of the energy exchanges associated with the degradation of amino acids in man (JUNGAS et al., 1992) demonstrates that gluconeogenesis and export of glucose is essential, because complete oxidation of the amino acid mixture by the liver would provide much more ATP than needed by this organ. Gluconeogenesis from amino acids must thus be regarded as a normal process associated with amino acid degradation, occurring at higher rates under conditions of normal food intake than during fasting.
The activation of pyruvate dehydrogenase by insulin is one of the mechanisms allowing insulin to promote carbohydrate oxidation (DENTON et al., 1987). The first irreversible steps in the degradation of the branched-chain amino acids are catalyzed by a-ketoacid oxidizing enzyme complexes which are similar to PDH and can also be activated by insulin (HARPER et al., 1984). It seems that glucose availability and the readiness to oxidize pyruvate which are enhanced by insulin explain the carbohydrate-sparing effect of exogenous carbohydrate (Figure 3). However, high insulin levels also open the pathway for the irreversible degradation of the BCAA, notably in skeletal muscle. Since the BCAA are indispensable amino acids, their loss by the irreversible oxidation of their a-keto derivatives condems the other amino acids to being degraded as well, since they cannot be used for protein synthesis without enough BCAA.
The concepts which have evolved to explain the interactions between protein and energy metabolism are based primarily on observations made under conditions of protein and/or energy deprivation. Though they shape much of our thinking, they are not appropriate to explain the control of amino acid oxidation when food intake is high. The situation is analogous to the irrelevance of concepts such as 'biological value' or 'net protein utilization' for conditions where protein and energy intakes are high.
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