Wasting and the substrate-to-energy controlled pathway: a role for insulin resistance and amino acids

Wasting and the Substrate-to-Energy Controlled Pathway: A Role for Insulin Resistance and Amino Acids Francesco S. Dioguardi,

MD

Amino acids contained in proteins can be transformed either in glucose precursors or in acetate, the end product of free fatty acid (FFA) oxidation. The dynamics of glucose, FFA, and amino acid competition for entry into the citric acid cycle (tricarboxylic acid [TCA] cycle) are very complex and not fully understood. Conditions where glucose is insufficiently driven to full oxidation are characterized by lowest efficiency in energy production per mole of oxygen consumed. Moreover, acetate provided by oxidation of FFA increases consumption of amino acids as precursors of the oxaloacetate required for condensation with acetate and for maintenance of citrate synthesis. Increased consumption of amino acids in the TCA cycle, if not matched by adequate intake, leads to muscular wasting and cachexia. Therefore, amino acid needs are very complex, and their intake

must provide a balanced ratio of glucogenic and ketogenic precursors suitable to trigger entry of glucose to full oxidation and blunt the level of FFA utilization. Optimization of substrate entry into energy production must also be coupled with sufficient availability of amino acids in ratios suitable for maintaining protein synthesis, inhibiting the catabolic drive, and promoting integrity of cellular proteic structures. Alimentary proteins have a content of amino acids that is far from the stoichiometric ratios of essential amino acids required by humans. An amino acid formulation suitable to match energy needs, control carbohydrate and lipid flow into the TCA cycle, and promote protein synthesis in contracting cells is detailed in this article. 䊚2004 by Excerpta Medica, Inc. Am J Cardiol 2004;93(suppl):6A–12A

ammals derive energy from the carbon skeleton of macronutrients like proteins, carbohydrates, M and lipids. Metabolism breaks the carbon-to-carbon

lysis produces a relatively small amount of energy (8 mol adenosine triphosphate [ATP] for 1 mol D-glucose), and it is controlled mostly by the availability of reducing equivalents (nicotinamide adenine dinucleotide [NAD⫹]/reduced NAD [NADH]) and pyruvate/ lactate. The respiratory chain and availability of oxygen control these ratios. Pyruvate can enter the mitochondria, where it is oxidized and consumes oxygen by a chain sequence of enzymes known as the tricarboxylic acid (TCA), or citric acid, cycle. This process produces the largest amount of energy: 2 mol pyruvate gives origin to 30 mol ATP. In turn, the TCA cycle releases 2 carbon atoms, linking them to O2. Thus, a 2-carbon molecule, acetate, must be continuously reintroduced to maintain the effectiveness of this cycle. The TCA cycle starts with the condensation of oxaloacetate with acetate, forming citrate. Oxaloacetate is a symmetrical molecule and is the final product of the cycle. Theoretically, the TCA cycle can infinitely recycle this molecule. Pyruvate is a refueling source for oxaloacetate, and this step is under the control of pyruvate carboxylase, an enzyme allosterically activated by acetate. Oxaloacetate can also be synthesized by the TCA cycle, starting from those amino acids that are transformed in intermediates of the cycle that enter at a committed step, particularly as ␣-ketoglutarate and succinate. Acetate can be derived from pyruvate by decarboxylation, from free fatty acid (FFA) oxidation, or by ketogenic amino acids. Either FFAs or amino acids may compete for acetate production with pyruvate, sparing glucose-derived pyruvate from decarboxylation to acetate. This reduces the need of glucose

bonds and provides or stores the energy that cells use to sustain life. The interrelation between the metabolism of proteins, carbohydrates, and lipids (ie, the macronutrients) is displayed in Figure 1. Whereas carbohydrates and lipids contain carbon, oxygen, or hydrogen, only proteins contain nitrogen. Thus, carbohydrates and/or most lipids can be synthesized from proteins, illustrating the importance of proteins in sustaining life functions. The ultimate fate of any nutrient, if not used for energy, is to be transformed either into glucose (and deposited as glycogen) or into lipids (which are deposited in adipose tissues, mostly as triglycerides [TGs]). Because lipids are a product of metabolism suitable only for energy production, they cannot be transformed back into carbohydrates by mammals. Only negligible amounts of carbohydrates and lipids may be transformed into essential amino acids.

ENERGY PRODUCTION AND SUBSTRATE ORIGIN In the absence of oxygen, glucose is oxidized outside of the mitochondrion (anaerobic glycolysis). In this process, glucose is split into 2 pyruvate moieties (1 mol glucose in 2 mol pyruvate). Anaerobic glycoFrom the Department of Internal Medicine, University of Milan, Milan, Italy. Address for reprints: Francesco S. Dioguardi, MD, Department of Internal Medicine, University of Milan, via Pace 9, Milan 20122, Italy. E-mail: [email protected].

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©2004 by Excerpta Medica, Inc. All rights reserved.

0002-9149/04/$ – see front matter doi:10.1016/j.amjcard.2003.11.002

FIGURE 1. Relation between proteins, lipids, and carbohydrates. Proteins can be dismantled and the resultant amino acids transformed into carbohydrates and lipids. Carbohydrates can be transformed into lipids, but neither carbohydrates nor lipids can produce essential amino acids, because only proteins contain nitrogen.

oxidation to pyruvate, which is used to a limited degree to refuel oxaloacetate to the TCA cycle. Entry of pyruvate in the TCA cycle is controlled by the extramitochondrial pyruvate decarboxylating enzyme, pyruvate dehydrogenase (PDH), which is inhibited by elevated acetate concentrations. The key to understanding the mechanisms by which amino acids control energy metabolism and entry of other substrates into the TCA cycle lies in the end result of oxaloacetate metabolism, rather than in the origin of acetate.

ENERGY PRODUCTION, PROTEIN METABOLISM, AND WASTING: THE ROLE OF INSULIN Myocardial contractility is dependent on a constant rate of ATP production. Glycolysis produces only a minor fraction of the ATP necessary for contraction in cytoplasm, but the majority of ATP is produced in mitochondria by the TCA cycle. Energy production by the TCA cycle is dependent on adequate oxygen and acetate refueling. Acetate is condensed with oxaloacetate regenerated by the TCA cycle, forming citrate and thus initiating the cycle. Two molecules are involved in the regulation of inflow capacity of substrates in energy production: oxaloacetate and acetate (or acetyl-coenzyme A [CoA]). Export of TCA cycle intermediates from the mitochondria or energy-rich compounds (ATP and NADH) control glycolysis, glyconeogenesis, and fatty acid synthesis. In the liver, gluconeogenesis is also initiated. Insulin is indispensable for the entry of glucose in muscles and the myocardium, and it activates GLUT [glucose transporter]–1 transporters normally found in sarcolemma. Insulin also increases GLUT-4 translocation and, as a result, the number of glucose transporters in membranes. GLUT-4 translocation is also increased by physical exercise. Glucose enters independently into the liver, stimulated by insulin and GLUT-2. Insulin modulates enzyme activity and drives glucose-derived pyruvate to oxidation, glycogen, or FFA synthesis, depending on the energy needs of the liver. Either circulating glucose or insulin concentrations independently regulate

glucose entry into glycolysis. In physiologic conditions, most glucose undergoing oxidation comes from glycogen and not from plasma (⬍32% of total glucose molecules undergoing glycolysis come from plasma). In turn, pyruvate originating from circulating glucose comprises 14% of the pyruvate entering the TCA cycle through PDH. McNulty et al1 showed that, even at largely supraphysiologic levels, nonglucose substrates continue to supply ⬎40% of myocardial TCA cycle flux. Exogenous pyruvate and acetate were found to provide up to 86% of acetyl-CoA for citrate synthesis. In combination, up to 65% came from acetate and up to 30% from pyruvate; no more than 15% was produced from glycogen or TGs present in the heart. These data are important, because they indicate that a significant portion of energy production would be lost if glycogen synthesis is impaired. Consistent with this finding, glycogen synthase activity is decreased and glucose-6 phosphate level is increased at elevated plasma FFA concentrations. Amino acids can activate glycogen synthase-kinase 3, even in the absence of insulin, although this action prevents further increase of glyconeogenesis rates when insulin rises.2 Generally, it is reported that FFA oxidation could account for 60% to 90% of ATP resynthesis in normal conditions.3 A shift toward increased FFA and amino acid oxidation is necessary to maintain constant energy production. ␤-Oxidation of FFAs may contribute significantly to energy production; however, overflow of acetate derived from FFA oxidation is a potent inhibitor of the PDH complex, thus preventing further glucose oxidation in the TCA cycle. The hypothesis that glucose entry into the TCA cycle could be inhibited at this level was initially proposed by Randle et al4 as the “glucose-fatty acid cycle.” Intracellular accumulation of fructose- and glucose-6 phosphate found in cytoplasm of muscle cells in patients with peripheral resistance syndrome is consistent with this hypothesis.5 The intracellular availability of glucose-6 phosphate has been identified as the predominant factor in determining the rate of glucose oxidation. Wolfe6 has focused attention on the concept that glucose oxidation may be much more efficient in reduc-

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FIGURE 2. The aspartate– glutamate and ␣-ketoglutarate (␣-KG)–maleate antiporters. Amino acids can enter the tricarboxylic acid (TCA) cycle either as oxaloacetate (Oxal-Ac) precursors or as acetate. When Oxal-Ac enters the TCA cycle by condensation with acetate, adenosine triphosphate (ATP) production is increased. The newly produced ATP inhibits free fatty acid ␤-oxidation and acetylcoenzyme A overproduction. This, in turn, stimulates pyruvate dehydrogenase and reactivates pyruvate entry into the acetate pool and glycolysis. Adaptation of aspartate– glutamate and ␣-KG–maleate antiporter activity controls the type and amount of intermediates that enter the TCA cycle. Through glutamate, 2 nitrogen and 5 carbon molecules are exported into plasma. Glutamate exported from the muscle is taken mostly by the splanchnic bed, where it is either transformed and released as alanine and lactate or where it becomes the substrate for the synthesis of nonessential amino acids, such as arginine and glutamine. NAD ⴝ nicotinamide adenine dinucleotide; NADH ⴝ reduced NAD.

ing FFA entry into mitochondria (and therefore limiting FFA oxidation) than FFA oxidation in inhibiting intracellular oxidation of glucose. In normal subjects, the lowest FFA plasma concentrations correlate with and increase in pyruvate oxidation and PDH activation, as well as a more elevated respiratory exchange ratio. Low ATP and NADH/NAD⫹ ratios seem to be very important factors in controlling activation of PDH.7 NAD⫹ is also indispensable for lactic dehydrogenase– driven transformation of lactate to pyruvate, and its availability is dependent on maleate and aspartate mitochondrial antiporter activity. Whereas ATP is exported from the mitochondrion by selective transporters, NADH cannot cross the mitochondrial membrane; therefore, intermediates of the TCA cycle are exported for further metabolism and production of NADH in cytoplasm, which is suitable for producing ATP in the respiratory chain. In contrast to FFAs, which provide acetate as a unique end product of their metabolism, amino acids can replenish TCA at virtually any step, according to their chemical structure. In conditions in which acetate accumulates in the mitochondria and inhibits glycolysis and pyruvate entry into oxidation, amino acids can become the prevalent source of oxaloacetate. Increasing ATP production by increasing the synthesis 8A THE AMERICAN JOURNAL OF CARDIOLOGY姞

of citrate through amino acid– derived oxaloacetate may have different positive effects on cellular metabolism. ATP is produced and readily inhibits ␤-oxidation of FFAs. The resulting reduction in acetate concentration releases PDH and reactivates the entry of glucose in the oxidative pathway. This condition is more favorable than that of unmatched acetate production because any eventual excess accumulation of intermediates of the cycle and regulation of oxaloacetate concentrations can be tightly ruled by mitochondrial antiporter activity (Figure 2). Aspartate is derived by transamination of oxaloacetate by mitochondrial transaminase. It can leave the mitochondria and enter the cytosol with a maleate molecule reentering the mitochondrion. This process occurs in parallel with ␣-ketoglutarate extrusion, using specific antiporters. Transamination of ␣-ketoglutarate is the origin of glutamate synthesis in the cytosol. In turn, glutamate is either able to move back into the mitochondrion, maintaining aspartate outflow by the proper antiporter, or glutamate is exported into plasma. In cytosol, aspartate readily regenerates oxaloacetate, losing the aminic group that is used for the ␣-ketoglutarate–glutamate coupled reaction. Subsequently, maleate is formed from oxaloacetate in par-

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allel with lactate retransformation to pyruvate, forming NAD⫹ from NADH. Pyruvate is the end product of glycolysis, and, as a result, its fate is determined by the oxidative status. If NADH/NAD⫹ is low, PDH is activated, and pyruvate will enter the TCA cycle as acetate, condensing with oxaloacetate to form citrate. If NADH/NAD⫹ is high, PDH is inactivated, the lactic dehydrogenase reaction equilibrium will be shifted toward lactate, and the TCA cycle will stop. Excess acetate will activate pyruvate carboxylase in the mitochondria, forming oxaloacetate from pyruvate. Oxaloacetate will condense with acetyl-CoA, producing citrate. Citrate is exported to the cytoplasm if ATP is increased in the mitochondria, such as when ␤-oxidation of FFAs is fully active. If liver gluconeogenesis is not suppressed by insulin, cytoplasmic oxaloacetate is preferentially transformed into phosphoenolpyruvate by pyruvate-carboxykinase and funneled to gluconeogenesis. This transformation results in rising plasma levels of glucose, and it occurs in situations such as preventing hypoglycemia in fasting states. These events may be improperly activated even when insulin is elevated, particularly in patients with peripheral insulin resistance. This process explains the hyperglycemic and hyperinsulinemic fasting state observed in obesity and in type 2 diabetes mellitus. If significant amounts of oxaloacetate are driven to cytoplasm and subsequently to gluconeogenesis in the liver, progressively less oxaloacetate will be available for citrate synthesis in the mitochondria. Reduction in citrate synthesis may cause acetate concentrations to increase. Accumulation of acetate further inhibits PDH, maintaining reduced pyruvate entry into the acetate pool. These considerations about interactions between biochemical pathways are important in that they indicate that if amino acid metabolism drives a further increase of acetate in cells, worsening of glucose metabolism may be expected.8 Conversely, it could be hypothesized that amino acid formulations aimed at balancing metabolic requirements of oxaloacetate precursors, thereby balancing proper refueling of the TCA cycle, may have a positive effect on energy production efficiency and glucose utilization in the heart. Disproportionate acetate originating from FFA oxidation inhibits pyruvate entry into the TCA cycle, exerting a negative effect on energy production efficiency. This process has an “oxygen-wasting” effect compared with carbohydrates because there is an increased myocardial oxygen demand for any ATP produced and for any given level of cardiac work.9 Existence of a glucose–FFA–amino acid cycle has already been postulated, as an expansion of Randle’s hypothesis.10 Insulin also has a role in directly regulating the balance between anabolism and catabolism. Experimental models using perfused heart and skeletal muscle have shown that insulin in both cardiac and skeletal muscle inhibits protein degradation. In the presence of glucose and amino acids, insulin maintains the rate of protein synthesis and ribosomal ag-

gregation. In the absence of insulin, protein synthesis diminishes and polysomes disaggregate, indicating that insulin is required for peptide chain initiation. The combined effects of insulin on synthesis and degradation reduce the release of free amino acids from muscle proteins. In the presence of FFAs, normal rates of peptide chain initiation were maintained in the heart, but not in the skeletal muscles, of rats.11 Increased concentrations of FFAs are also a protective mechanism for the rate of protein synthesis, a compensatory metabolic mechanism present during fasting and in diabetes. The role of muscle as a substrate reservoir to be used as fuel in conditions of poor nutritional support or enormous metabolic requirements has been recognized only since the 1970s.12 Energy and substrate utilization for protein synthesis is secondary to ensuring that the energy needs are met for the survival of cells. Therefore, a continuous competition between substrates for energy requirements and substrates for synthesis is present. Nutritional input must match both requirements; otherwise energy needs will drive glucose toward catabolism, with loss of muscles and adipose tissue, resulting in wasting and cachexia. Consistent with these concepts, Swan et al13 found insulin resistance detectable in a large percentage of patients with chronic heart disease and no diabetes. Insulin resistance may be the key to understanding muscular wasting, a major life-threatening problem in patients with chronic heart disease that is often present in patients with a normal intake of macronutrients. Humans are efficient in muscle wasting.14 Although energetically costly, exercise is fundamental for muscle trophism. In normal subjects, muscles normally responding to insulin undergo rapid atrophy if immobilized, as exemplified by extremities plastered as a result of bone fractures.15 Exercise is an effective therapy in different pathologic conditions16; however, the type and extent of exercise that should be prescribed remains unknown.17,18 Insulin’s effects on protein breakdown or amino acid utilization for protein synthesis might be modulated by the intensity and endurance of physical activity. Interestingly, the balance between protein degradation and synthesis may differ even within the same muscular territory.19 Protein catabolism provides amino acids either for gluconeogenesis and maintenance of glycemia or for use as intermediates of the TCA cycle in the mitochondria. This mechanism is the main pathway resulting in muscle wasting, but through this survival is sustained as long as possible in conditions of imbalance between nutritional input and energy requirements associated with metabolic overload. A convincing body of evidence exists to suggest that elevated available amounts of amino acids can improve and drive protein synthesis. Some amino acids are required as protein precursors, whereas others also serve to fulfill energy requirements.20 Determination of which role amino acids play may depend on differences in the way ingestion of nitrogen-containing food modifies plasma patterns of amino acids.

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From a nutritional and metabolic point of view, the proposed distinction between “fast” and “slow” alimentary proteins has been innovative. The fast proteins are rapidly digested and absorbed and preferentially will induce an anabolic drive. Conversely, the slow absorption of most other proteins promotes the use of the derived amino acids toward energy precursors.21 Thus, it can be argued that amino acid plasma concentrations sufficiently high for promoting protein synthesis will not occur for most alimentary proteins that are slowly digested. It can be hypothesized that the higher the concentration variation, the stronger the anabolic drive, independent of the initial plasma concentration. This concept may also explain the failure of slow infusion of amino acids in promoting protein synthesis.22 Periodic induction of rapid and clinically relevant amino acid concentrations may be a powerful tool in counteracting muscle wasting. A specific role in controlling protein synthesis has been attributed to leucine, an essential ketogenic amino acid of the branched-chain amino acid family. Elevated amounts of leucine blunt catabolism and promote protein synthesis.23,24 Leucine is metabolized mostly for energy production in peripheral muscles rather than in the liver. An increase in leucine plasma concentrations may be perceived as an indicator of rich availability of energy and synthetic substrates for both myocardial and muscle cells. This hypothesis is supported by the observation that leucine and other branched-chain amino acids are not the most prevalent amino acids in intracellular proteins,25 and, accordingly, requirements for other amino acids during active synthesis may be similar or greater.26 Other amino acids must also be present to maximally exert a cooperative messenger role in promoting availability of energy substrate.23 An imbalanced supply of gluconeogenic nonessential amino acids, like alanine, glycine, or glutamine,27 may differently influence glucose-dependent energetic metabolism because an overflow of these amino acids may blunt pyruvate entry into the acetate pool, triggering the oxidation of acetate from FFAs. In some insulin-deficient conditions, the family of branched-chain amino acids can be increased in plasma. Although the branched-chain amino acids have different metabolic pathways, the rise in the plasma concentrations of these amino acids may reflect a potentially compensatory event similar to that of glucose or FFAs.28 Interestingly, insulin resistance is a constant feature in liver cirrhosis, and fasting plasma branchedchain amino acids are often lower, although branchedchain amino acid intolerance can be shown under loading conditions.29 Both elevations or reductions in plasma branched-chain amino acids have been associated with hyperglycemia resulting from a decreased effect of insulin on liver gluconeogenesis and glycogenolysis.28,30 Branched-chain amino acids most likely accumulate in the plasma because of either deficient peripheral insulin-dependent influx toward oxidation or because a relative lack of amino acids limits their use for protein synthesis.31 Branched10A THE AMERICAN JOURNAL OF CARDIOLOGY姞

TABLE 1 Composition of 100 g of the Proposed Formulation of Amino Acids Amino Acid Leucine Isoleucine Valine Cysteine Phenylalanine Tyrosine Lysine Threonine Histidine Methionine Tryptophan

Weight 31.25 g 15.62 g 15.62 g 3.75 g 2.5 g 0.75 g 16.25 g 8.75 g 3.75 g 1.25 g 0.50 g

chain amino acids must be considered an important part of the complex and varied cellular needs for initiating and maintaining protein synthesis. Although amino acids can modulate insulin activity in conditions of reduced insulin sensitivity,32,33 branchedchain amino acid accumulation in plasma of patients with insulin resistance could be a nitrogen-sparing, compensatory mechanism for facilitating entry of amino acids into cells (before starting synthesis, when requirements for other amino acids would be fulfilled), and insulin levels would be increased by meals.34 A very high percentage of proteins in dietary calories have been shown to worsen glucose tolerance in individuals with diabetes.8 The use of a specific alimentary protein contained in high-caloric food to evaluate nitrogen metabolic effects may be misleading because the ratio of the amino acid content exceeds that necessary to humans. In the late 1970s, nitrogen introduction and calcium urinary losses were correlated, using beef as a reference protein.35 Further studies by the same authors showed that calciuria correlated mainly with acidification of nephron proximal tubules and excretion of sulfur-containing amino acids, which were abundantly present in test meals.36 Finally, it has been established that nitrogen input is necessary for successfully treating osteoporosis, and it is indispensable for promoting collagen anabolism in bones.37 Conversely, formulations of amino acids can be tailored on demand according to metabolic requirements and for balancing food deficiencies. Many different possibilities exist for developing dietary supplementation with formulations of amino acids targeting specific aspects of metabolism.24,38 In patients with chronic heart disease, a formulation of amino acids programmed to provide balanced amounts of propionate/acetate, for example, would be suitable to release the brake on PDH that was previously applied by excess acetate from FFA oxidation. A positive regulatory effect on entry of pyruvate in the TCA cycle would be observed, thus optimizing ATP production for any mole of O2 and competitively reducing FFA oxidation– dependent oxygen drag, ultimately improving myocyte performance.10

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THE RIGHT NITROGEN SUPPLY: PROPOSING A SUITABLE FORMULATION Nutritional supplementation and qualitative modifications of dietary macronutrients targeted at improving the health of selected patients comprise a promising new frontier for research.39,40 In the exploration of new therapies, the benefits and risks certainly should be evaluated in clinical trials. Polyunsaturated FFAs appear to have a positive role on cardiac death. Polyunsaturated FFAs act to metabolically stabilize myocyte membranes, thus improving oxygen consumption and recovery of contractile function, but they are ineffective at decreasing the progression of atherosclerosis.41 FFA oxidation is a producer of oxidative intermediates, such as reactive oxygen species,42 a key factor in apoptosis and aging that is particularly detrimental for heart interfibrillar mitochondria.43 Any alimentary protein has a particular content of amino acids, and their concentrations exceed ratios required by humans. All amino acids not suitable for synthesis or not rapidly used for energy purposes as precursors of oxaloacetate must be eliminated, and their carbon skeleton deposited as lipid,44 as summarized in Figure 1. The appropriate amount and ratio of amino acids needed to promote synthesis and contribute to energy production has not yet been fully determined. A total of 75% of the body’s nitrogen requirement is supplied by 5 amino acids: leucine, isoleucine, valine, threonine, and lysine.45 A key to understanding the energy needs of all other essential amino acids has been obtained by serial analyses of amino acid consumption by training athletes (⬎4 hr/day) maintained on hypocaloric (about 0.8 kcal/kg) diets for agonistic purposes (bodybuilding) for long periods (ⱖ4 weeks). These observations have contributed to the development of an amino acid formulation with potential beneficial effects. Leucine is utilized as isoleucine or valine, and the sum of branched-chain amino acids is approximately twice that of threonine and lysine. Histidine supplies the remaining 25% of essential amino acid needs; this amino acid is limiting for hemoglobin and myofibrillar protein synthesis. Methionine, a sulfur-containing amino acid essential in methylation reactions, should be present in formulations for human use, in concentrations half that of cysteine, to prevent appearance of the toxic intermediate of methionine toward cysteine synthesis, namely, homocysteine.46 Cysteine, in turn, is also indispensable for maintaining synthesis of glutathione from glutamate. The appropriate formulation should also contain the essential amino acids phenylalanine and tyrosine. Tyrosine can be synthesized only in the liver; thus, its availability significantly ameliorates protein synthesis in peripheral tissues.41 Tryptophan is a neurotransmitter precursor, necessary to activate RNA-dependent messages; thus, its supplementation should be calculated in a small but sufficient amount to prevent free and albumin-bound pools to shrink, when syntheses are triggered by mass action of the other essential amino acids.47

Based on these assumptions, a formulation of amino acids whose reciprocal stoichiometric ratios have been calculated for matching either energetic needs of metabolism or the maintenance of protein synthesis even in extreme physiologic and pathologic conditions is shown in Table 1 (Big One; Professional Dietetics SRL, Milan, Italy).

CONCLUSIONS Amino acids can be metabolized either for energy production (the primary goal of metabolism) or for synthesis of proteins. Only those amino acids not used for maintaining energy production will be suitable, and will control, protein synthesis. Certain stoichiometric ratios of some amino acids will drive glucose toward full oxidation, whereas other ratios and formulations will promote acetate accumulation, hindering pyruvate derived from entry into the TCA cycle. Specifically tailored amino acid formulations can be a powerful tool in achieving specific metabolic targets. 1. McNulty PH, Cline GW, Whiting JM, Shulman GI. Regulation of [13C]glucose

metabolism in conscious rats. Am J Physiol Heart Circ Physiol 2000;279:H375– H381. 2. Malloy CR, Sherry AD, Jeffrey FMH. Evaluation of carbon flux and substrate selection through alternate pathways involving the citric acid cycle of the heart by 13C NMR spectroscopy. J Biol Chem 1988;263:6964 –6971. 3. Taegtmayer H. Energy metabolism of the heart: from basic concepts to clinical applications. Curr Prob Cardiol 1994;19:59 –113. 4. Randle PJ, Hales CN, Garland PB, Newsholme EA. The glucose fatty-acid cycle: its role in insulin sensitivity and the metabolic disturbances of diabetes mellitus. Lancet 1963;1:785–789. 5. Terruzzi I, Allibardi S, Bendinelli P, Maroni P, Piccoletti R, Vesco F, Samaja M, Luzi L. Amino acid- and lipid-induced insulin resistance in rat heart: molecular mechanisms. Mol Cell Endocrinol 2002;190:135–145. 6. Wolfe RR. Metabolic interactions between glucose and fatty acids in humans. Am J Clin Nutr 1998;67(suppl):519S–526S. 7. Stellingwerff T, Watt MJ, Heigenhauser GJF, Spriet LL. Effects of reduced free fatty acid availability on skeletal muscle PDH activation during aerobic exercise. Am J Physiol Endocrinol Metab 2003;284:E589 –E596. 8. Linn T, Geyer R, Prassek S, Laube H. Effect of dietary protein intake on insulin secretion and glucose metabolism in insulin-dependent diabetes mellitus. J Clin Endocrinol Metab 1996;81:3938 –3943. 9. Rodrigues B, McNeill JH. The diabetic heart: metabolic causes for the development of a cardiomyopathy. Cardiovasc Res 1992;26:913–922. 10. Ferranini E, Bevilacqua S, Lanzone L, Bonadonna R, Brandi L, Oleggini M, Boni C, Buzzigoli C, Ciociaro D, Luzi L, De Fronzo RA. Metabolic interactions of amino acids and glucose in healthy humans. Diabetes Nutr Metab 1988;1: 175–186. 11. Jefferson LS, Rannels DE, Munger BL, Morgan HE. Insulin in the regulation of protein turnover in heart and skeletal muscle. Fed Proc 1974;33:1098 –1104. 12. Daniel PM, Pratt OE, Spargo E. The metabolic homeostatic role of muscle and its function as a store of protein. Lancet 1977;310:446 – 448. 13. Swan JW, Anker SD, Walton C, Godsland IF, Clark AL, Leyva F, Stevenson JC, Coats AJ. Insulin resistance in chronic heart failure: relation to severity and etiology of heart failure. J Am Coll Cardiol 1997;30:527–532. 14. Mitch WE, Goldberg AL. Mechanism of muscle wasting. N Engl J Med 1996;335:1897–1905. 15. Booth FW. Time course of muscular atrophy during immobilization of hindlimbs in rats. J Appl Physiol 1977;43:656 –661. 16. Balady GJ. Survival of the fittest: more evidence [editorial]. N Engl J Med 2002;346:852–854. 17. Thompson PD. Additional steps for cardiovascular health [editorial]. N Engl J Med 2002;347:755–756. 18. Diabetes Prevention Program Research Group. Reduction in the incidence of type 2 diabetes with lifestyle intervention or metformin. N Engl J Med 2002; 346:393–403. 19. Bylund-Fellenius A, Ojama KM, Flaim KE, Li JB, Wassner SJ, Jefferson LS. Protein synthesis versus energy state in contracting muscles of perfused rat hindlimb. Am J Physiol 1984;246:E297–E305. 20. Obled C, Barre F, Millward DJ, Arnal M. Whole body protein synthesis: studies with different amino acids in the rat. Am J Physiol 1989;275(pt 1):E639 – E646. 21. Giordano M, Castellino P, De Fronzo A. Differential responsiveness of protein synthesis and degradation to amino acid availability in humans. Diabetes 1996;45:393–399.

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22. Streat SJ, Beddoe AH, Hill GL. Aggressive nutritional support does not

prevent protein loss despite fat gain in septic intensive care patients. J Trauma 1987;27:262–266. 23. Goldberg AL, Chang TW. Regulation and significance of amino acid metabolism in skeletal muscle. Fed Proc 1978;37:2301–2307. 24. Layman DK. The role of leucine in weight loss diets and glucose homeostasis [review]. J Nutr 2003;133:261S–267S. 25. Bennet WM, Connacher AA, Smith K, Jung RT, Rennie MJ. Inability to stimulate skeletal muscle or whole body protein synthesis in type 1 (insulindependent) diabetic patients by insulin-plus-glucose during amino acid infusion: studies of incorporation and turnover of tracer L-[1-13 C]leucine. Diabetologia 1990;33:43–51. 26. Nair SK, Ford GC, Ekberg K, Fernqvist-Forbes E, Wahren J. Protein dynamics in whole body and in splanchnic and leg tissues in type I diabetic patients. J Clin Invest 1995;95:2926 –2937. 27. Moore MC, Hsieh PS, Flakoll PJ, Neal DW, Cherrington AD. Net hepatic gluconeogenic amino acid uptake in response to peripheral versus portal amino acid infusion in conscious dog. J Nutr 1999;129:2218 –2224. 28. Forlani G, Vannini P, Marchesini G, Zoli M, Ciavaella A, Pisi E. Insulindependent metabolism of branched-chain amino acids in obesity. Metabolism 1984;33:147–150. 29. Marchesini G, Bianchi GP, Vilstrup H, Checchia GA, Patrono D, Zoli M. Plasma clearances of branched-chain amino acids in control subjects and in patients with cirrhosis. J Hepatol 1987;4:108 –117. 30. Kabadi UM. The association of hepatic glycogen depletion with hyperammonemia in cirrhosis. Hepatology 1987;7:821–824. 31. Wright PD, Holdsworth JD, Dionigi P, Clague MB, Jones OF. Effect of branched chain amino acid infusions in cirrhosis of liver. Gut 1986;27(suppl): 96 –102. 32. Patti ME, Brambilla E, Luzi L, Landaker EJ, Kahn CR. Bidirectional modulation of insulin action by amino acids. J Clin Invest 1998;101:1519 –1529. 33. Sutherland C, O’Brien RM, Granner DK. Phosphatidylinositol 3-kinase, but not p70/p85 ribosomal S6 protein kinase, is required for the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin: dissociation of signalling pathways for insulin and phorbol ester regulation of PEPCK gene expression. J Biol Chem 1995;270:15501–15506.

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