- Email: [email protected]
Amino acid control of muscle protein turnover in renal disease
Amino Acid Control of Muscle Protein Turnover in Renal Disease Arny A. Ferrando, PhD,* Dominic Raj, MD,† and Robert R. Wolfe, PhD* This review discusses the concept that skeletal muscle intracellular amino acids (ICAAs), in particular the essential amino acids, are regulated throughout a wide range of physiologic circumstances. Whether in critical illness, severe injury, or healthy states, ICAAs are closely regulated by a coordinated response in 1 or more of the processes of synthesis, breakdown, and tissue transport. For a given metabolic signal (hormonal, change in plasma amino acid concentrations), the regulation of ICAAs entails appropriate and corresponding changes in amino acid kinetics. These changes vary according to the strength of the metabolic signal and the existing requirement to maintain the ICAA pool. For the patient with end-stage renal disease (ESRD), frequent dialysis induces an abrupt removal of half of the circulating amino acids, which in turn results in a substantial efflux of amino acids from skeletal muscle. ICAAs are maintained through the increase in protein breakdown, and similar to other stress states, there is a concomitant increase in protein synthesis. Thus, the regulation of ICAAs often pushes subsequent adaptations in amino acid kinetics to maintain the existing homeostasis. This regulatory mechanism is evident in circumstances ranging from increased amino acid availability in healthy volunteers to a change in anabolic signal in severe injury. Despite the substantial evidence of ICAA regulation, its physiologic significance is not evident. However, the regulation of ICAAs represents a method by which skeletal muscle ensures its capacity for anabolism. © 2005 by the National Kidney Foundation, Inc.
I
N A HEALTHY INDIVIDUAL, muscle homeostasis is maintained by a complex interaction of hormonal and nutritional signals. When this system is perturbed by critical illness or severe injury, the accompanying neuroendocrine changes are directed toward the enhancement of survival. Skeletal muscle then serves as a primary source of both gluconeogenic precursors and precursors for synthesis of essential proteins, such as those involved in immune function and wound healing. In this mode, muscle experiences a persistent and substantial decrease in protein content and an accelerated release of amino acids. Because amino acids, in particular intracellular amino acids (ICAAs), are required for protein synthesis, it is tempting to speculate that their continued release from the precursor pool could adversely affect protein synthesis. However, in severe trauma such as burns, muscle protein synthesis is
*Department of Surgery, University of Texas Medical Branch, Galveston, TX. †University of New Mexico, Albuquerque, NM. Address reprint requests to Arny Ferrando, PhD, 815 Market St, Galveston, TX 77550. E-mail: [email protected] © 2005 by the National Kidney Foundation, Inc. 1051-2276/05/1501-0008$30.00/0 doi:10.1016/j.jrn.2004.09.014
34
elevated 45% over normal values despite a substantial efflux of amino acids from the muscle.1 Therefore, to maintain an elevated synthetic rate, the intracellular amino acid precursor pool must also be maintained. Cumulative evidence from our laboratory suggests that ICAAs, particularly the essential amino acids, are closely regulated throughout a wide range of physiologic circumstances, from critical illness and severe injury to healthy volunteers and hyperaminoacidemia. Because the essential amino acids must be present for protein synthesis to occur, their regulation is crucial to the replenishment of muscle proteins. Nonessential amino acids can be derived from other sources and from interorgan transport,2 and are therefore not as closely regulated. An alteration in ICAA availability because of a change in metabolic signal encompasses a coordinated response in 1 or more of the processes of synthesis, breakdown, and tissue transport to maintain or restore these concentrations. To illustrate this concept, we discuss the circumstances of critical injury or illness, because the accompanying and altered metabolic demands magnify the requirement to maintain ICAAs. In particular, we examine burn injury and ESRD patients undergoing dialysis, in whom the predominant effect is an outflow of amino acids from skeletal muscle. Journal of Renal Nutrition, Vol 15, No 1 ( January), 2005: pp 34-38
MUSCLE PROTEIN TURNOVER
35
Delivery In A
Inward Transport Protein Synthesis
Shunting
M
Protein Breakdown V
Outward Transport
Delivery Out
A = femoral artery Figure 1. Three-pool model V = femoral vein of leg amino acid kinetics. M = vastus lateralis muscle
Although the theoretical considerations for this hypothesis have been detailed elsewhere,3 the purpose of this article is to illustrate the tight control of ICAAs throughout a range of circumstances in which alterations in the primary hormonal or nutritional signal impact muscle protein metabolism.
trations, allows for a coordinated investigation of protein synthesis, breakdown, and amino acid transport during any physiologic alteration. In this article, the regulation of ICAAs is discussed in light of the resultant changes in each of these measured parameters.
Model of Amino Acid Kinetics
Increased Amino Acid Availability
Over the past decade, we have developed4 and used1,5-7 a 3-compartment model that quantifies amino acid kinetics across leg muscle (Fig 1). Stable isotope methodology is used in conjunction with the sampling of blood that enters the leg (femoral artery) and leaves the leg (femoral vein), as well as muscle tissue (via biopsy), to determine amino acid kinetics between compartments. With this model, the rate of exchange or the movement of any amino acid can be quantified. In particular, this methodology determines the rate of amino acids entering the intracellular pool via inward transport or protein breakdown and the rate at which they leave by outward transport or protein synthesis.4 When a tracer that is not oxidized by muscle, such as phenylalanine, is used, disappearance from the intracellular pool entails either outward transport or incorporation into protein. Conversely, appearance into the intracellular pool is derived from inward transport or as a result of protein breakdown. Thus, the use of an appropriate amino acid tracer enables the calculation of protein synthesis and breakdown. This methodology, in conjunction with the determination of intracellular amino acid concen-
Our laboratory has shown that the infusion8,9 or ingestion10,11 of amino acids produces a substantial increase in muscle protein synthesis. The resultant increase in arterial amino acid concentrations serves to signal an increase in protein synthesis. The precursor amino acids, the ICAAs, required to support an increased synthetic rate are derived by increased inward transport,8 such that there is no concomitant requirement to derive ICAAs from an increased protein breakdown.8,9,12 Protein synthesis increases steadily with the increase in arterial amino acid concentrations until the point at which synthetic capacity is maximized.13 Intracellular amino acid concentrations remain fairly constant throughout a wide range of extracellular amino acid concentrations until the point at which extracellular concentrations are large enough to move to the intracellular space by mass action. Thus, the elevation of arterial amino acid levels in healthy individuals serves as a signal to stimulate muscle protein synthesis. To maintain the precursor ICAA pool, inward transport is also stimulated. The result is maintenance of the ICAA pool by inward transport alone and the provision of ade-
36
FERRANDO ET AL
quate ICAA precursors to support elevated protein synthesis.
Decreased Amino Acid Availability—Hemodialysis Dietary protein and calorie intake decrease progressively with decline in renal function. Preliminary evidence indicates that protein turnover is adaptively decreased and protein balance is maintained in uncomplicated uremia.14 Similarly, Castellino et al15 reported that although insulinmediated suppression of proteolysis is preserved in patients with chronic renal failure, the ability to increase protein synthesis in response to amino acid infusion is impaired in chronic renal failure. The impaired response to amino acids is further exacerbated by the fact that renal disease, by its nature, severely restricts protein and amino acid intake. Among the many complications in patients with end-stage renal disease (ESRD), protein and calorie malnutrition is a major risk factor for increased mortality.16 ESRD patients are forced to accommodate a limited protein intake while subjected to chronic intermittent hemodialysis. Fasted skeletal muscle protein kinetics in ESRD patients are similar to those of healthy volunteers,6,17 especially if blood bicarbonate levels are normalized.6 However, hemodialysis dramatically alters muscle protein kinetics. Arterial amino acid concentrations are decreased by 20% to 40% during hemodialysis.6,17 Hemodialysis serves as a catabolic signal that results in an efflux of amino acids, primarily essential amino acids, from muscle.6,17 This efflux is facilitated by an increase in outward transport kinetics.6 In human beings, hemodialysis results in a more negative net protein balance, indicating net protein breakdown. Raj et al6 showed that both protein synthesis and breakdown increase during hemodialysis, with a greater increase in breakdown. The net effect is a substantial loss of amino nitrogen from skeletal muscle.6,17 The increase in protein turnover is consistent with a stress state, in which both synthesis and breakdown are increased 50% to 100% above normal values.1,5 Hemodialysis in ESRD patients has been shown to increase other catabolic indicators and signals, such as cortisol,6 and proinflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor␣.18 The increased production of cytokines is linked to several cellular markers of protein
breakdown.18 Thus, hemodialysis creates a catabolic environment for skeletal muscle. Despite the many changes in amino acid kinetics during hemodialysis, there is a remarkable consistency in ICAAs. Both the essential and the nonessential amino acid concentrations are maintained during hemodialysis.6 Because the primary metabolic signal is one of catabolism, ICAAs are maintained by a substantial increase in protein breakdown, as there is no increase in inward transport.6 The increase in protein breakdown is sufficient to maintain the ICAA pool and to support an increase in both outward transport and protein synthesis.19 Viewed from the perspective of our proposed hypothesis of ICAA regulation, to maintain the ICAA pool and provide amino acids centrally, protein breakdown must increase. The increase in protein synthesis may be the result of a push effect from increased amino acid availability, much as increased availability from amino acid infusion/ingestion stimulates protein synthesis. To further illustrate the concept of ICAA regulation, we examine the example of severe burn injury and the subsequent alterations in amino acid kinetics that result from an alteration in metabolic signal.
Enhancement of Anabolic Signal in Severe Burns As occurs with hemodialysis in the ESRD patient, protein breakdown is elevated to the point at which both increased efflux and protein synthesis are supported in the severely burned patient.1 ICAA concentrations achieve a new homeostasis in burn injury, with some amino acids present in greater concentrations (phenylalanine, leucine), whereas others are reduced (glutamine, lysine, alanine).1,20 Burn injury entails a profound insensitivity of skeletal muscle to the effects of insulin on both glucose and protein metabolism.21 In an attempt to overcome this insulin resistance, Sakurai et al administered high-dose insulin to severely burned patients for 7 days to investigate the effects on protein metabolism and amino acid kinetics.7 The resultant blood insulin concentrations (approximately 900 U/mL) presented a substantial increase in anabolic signal, such that net protein synthesis increased 2- to 4-fold. To support the increased stimulation of protein synthesis with adequate ICAA precursors, protein breakdown paradoxically increased 2.5fold, whereas inward transport increased 6-fold. An
37
MUSCLE PROTEIN TURNOVER
increase in inward transport is remarkable in these patients because net amino acid transport is uniformly outward.1 The fact that the primary transport kinetics can be affected with sufficient signal provides evidence of the regulatory role of ICAAs. These dramatic changes in amino acid kinetics enabled ICAA concentrations to remain unchanged.7 Thus, the regulation of ICAAs during stimulated protein synthesis was accomplished not only by increasing inward transport but also by increasing protein breakdown. The substantial increase in protein breakdown was capable of supporting both the ICAA precursor pool for protein synthesis and the anticipated efflux of amino acids from skeletal muscle. This response suggests a metabolic hierarchy, with the maintenance of ICAAs taking precedence over the inhibitory action of insulin on protein breakdown.22 This is also reflected in the cumulative data, which suggests that the in vivo response of muscle to insulin administration is highly dependent on the availability of amino acids.22 We subsequently investigated the effects of lowerdose insulin on protein metabolism and amino acid kinetics in severely burned patients. Exogenous insulin was administered for 3 to 4 days to achieve blood concentrations of approximately 240 U/ mL.5 Again, this resulted in a substantial anabolic signal to skeletal muscle such that net protein synthesis increased 2-fold. However, unlike the previous study with 3-fold greater insulin levels, there was no statistical increase in protein breakdown or transport kinetics.5 Apparently, the existing (high) rate of protein breakdown was sufficient to maintain the precursor pool for increased protein synthesis and the outward flux of amino acids. In this case, protein synthetic efficiency increased, meaning that for a given quantity of ICAAs derived from protein breakdown and inward transport, a greater portion was directed toward protein synthesis. In this circumstance, the regulation of ICAAs was accomplished with minimal changes in protein kinetics. Burn injury leads to a dramatic reduction of testosterone production in male patients. After severe burn injury, testosterone concentrations are far below the normal physiologic range.23,24 In an attempt to restore this important anabolic influence, we administered exogenous testosterone to male patients with severe burn injury. The intent was to normalize circulating testosterone concentrations to investigate the effects on muscle protein metabolism and amino acid kinetics. Patients were studied before and after 2 weeks of
testosterone enanthate (200 mg/week intramuscularly). Blood testosterone concentrations increased to the low-normal range after the first injection and to the upper-normal range after the second injection.24 Patients were first studied approximately 2 weeks after the severe burn, with the second study at approximately 4 weeks postburn. Protein turnover was typically elevated, with a marked net loss (negative net balance) from skeletal muscle before testosterone administration. After 2 weeks of testosterone, protein breakdown decreased 2-fold without a concomitant change in protein synthesis.24 As a result, net protein balance was restored by the concurrent enteral feeding. Muscle protein synthesis was unchanged; however, protein synthetic efficiency increased 2-fold. In addition, amino acids from protein breakdown were redirected to protein synthesis at a 2-fold greater rate. ICAAs remained unchanged after testosterone administration, with the exception of increased leucine uptake, most likely directed toward oxidation.8 Skeletal muscle ICAA was maintained by altering transport and protein breakdown. After testosterone administration, the elevated muscle protein synthesis associated with burn injury was supported by reducing the outward transport of amino acids from muscle. The reduction in protein breakdown could potentially detract from the required precursor pool for protein synthesis; however, protein breakdown was still elevated to a sufficient level. Evidence for the adequacy of ICAA precursors derived from protein breakdown is shown by a concomitant decrease in inward transport. In other words, ICAAs could be maintained and support the existing rate of protein synthesis in light of decreased protein breakdown by decreasing outward transport of amino acids. Thus, with the anabolic signal of testosterone in severe burns, ICAA regulation leads to an increase in protein synthetic efficiency and a decrease in protein breakdown and outward transport.24
Summary For a given metabolic signal, the regulation of ICAAs entails appropriate and corresponding changes in amino acid kinetics. These changes vary according to the strength of the metabolic signal and the existing requirement to maintain the ICAA pool. Throughout a wide range of physiologic changes and/or metabolic signals,
38
FERRANDO ET AL
ICAAs remain fairly constant. Extracellular signals, such as changes in hormone or nutritional concentrations, may signal or initiate changes in protein metabolism. In the ESRD patient, the sudden removal of almost half of circulating amino acids induces a substantial increase in amino acid efflux from skeletal muscle. To maintain ICAAs, protein breakdown must also increase substantially. The increase in protein synthesis may be a response to increased ICAA availability from protein breakdown. From this perspective, the regulation of ICAAs pushes the subsequent adaptations in amino acid kinetics to maintain the existing homeostasis. This form of active regulation of ICAAs enables skeletal muscle to maintain a sufficient precursor amino acid pool to support protein synthesis given an adequate anabolic signal. Despite the substantial evidence of ICAA regulation, its physiologic significance is not evident. Further, this regulation is not conducive to a teleologic argument. Evidence of the regulatory role of ICAAs is compelling and substantiates a requirement for adequate amino acid precursors for muscle anabolism to occur. This role is further substantiated by examining the interaction of amino acids and the anabolic stimuli of resistance exercise and insulin. Resistance exercise alone has a minimal effect on muscle protein balance25; however, when combined with sufficient amino acids, muscle anabolism is substantial.8 This is analogous to the effects of insulin, whereby sufficient amino acids must also be present for anabolism to occur.26 Thus, when viewed from this perspective, it is likely that the regulation of ICAAs represents a method by which muscle ensures its capacity for anabolism.
References 1. Biolo G, Fleming RY, Maggi SP, et al: Inverse regulation of protein turnover and amino acid transport in skeletal muscle of hypercatabolic patients. J Clin Endocrinol Metab 87:3378-3384, 2002 2. Abumrad NN, Williams P, Frexes-Steed M, et al: Interorgan metabolism of amino acids in vivo. Diabetes Metab Rev 5:213-226, 1989 3. Wolfe RR, Miller S: Amino acid availability controls muscle protein metabolism. Diabetes Nutr Metab 5:322-328, 1999 4. Biolo G, Fleming DY, Maggi S, et al: Transmembrane transport and intracellular kinetics of amino acids in human skeletal muscle. Am J Physiol Endocrinol Metab 268:E75-E84, 1995 5. Ferrando AA, Chinkes DL, Wolf SE, et al: A submaximal dose of insulin promotes net skeletal muscle protein synthesis in patients with severe burns. Ann Surg 229:11-18, 1999
6. Raj DS, Zager P, Shah VO, et al: Protein turnover and amino acid transport kinetics in end-stage renal disease. Am J Physiol Endocrinol Metab 286:E136-143, 2004 7. Sakurai Y, Aarsland A, Herndon DN, et al: Stimulation of muscle protein synthesis by long-term insulin infusion in severely burned patients. Ann Surg 222:283-297, 1995 8. Biolo G, Tipton KD, Klein S, et al: An abundant supply of amino acids enhances the metabolic effect of exercise on muscle protein. Am J Physiol Endocrinol Metab 273:E122-E129, 1997 9. Ferrando AA, Sheffield-Moore M, Paddon-Jones D, et al: Differential anabolic effects of testosterone and amino acid feeding in older men. J Clin Endocrinol Metab 88:358-362, 2003 10. Paddon-Jones D, Sheffield-Moore M, Creson DL, et al: Hypercortisolemia alters muscle protein anabolism following ingestion of essential amino acids. Am J Physiol Endocrinol Metab 284:E946-953, 2003 11. Volpi E, Mittendorfer B, Wolf SE, et al: Oral amino acids stimulate muscle protein anabolism in the elderly despite higher first-pass splanchnic extraction. Am J Physiol Endocrinol Metab 277:E513-E520, 1999 12. Volpi E, Ferrando AA, Yeckel CW, et al: Exogenous amino acids stimulate net muscle protein synthesis in the elderly. J Clin Invest 101:2000-2007, 1998 13. Bohe J, Low JF, Wolfe RR, et al: Latency and duration of stimulation of human muscle protein synthesis during continuous infusion of amino acids. J Physiol 532:575-579, 2001 14. Tom K, Young VR, Chapman T, et al: Long-term adaptive responses to dietary protein restriction in chronic renal failure. Am J Physiol 268:E668-677, 1995 15. Castellino P, Solini A, Luzi L, et al: Glucose and amino acid metabolism in chronic renal failure: Effect of insulin and amino acids. Am J Physiol 262:F168-176, 1992 16. Ikizler TA, Hakim RM: Nutrition in end-stage renal disease. Kidney Int 50:343-357, 1996 17. Ikizler TA, Pupim LB, Brouillette JR, et al: Hemodialysis stimulates muscle and whole body protein loss and alters substrate oxidation. Am J Physiol Endocrinol Metab 282:E107-116, 2002 18. Raj DS, Shah H, Shah VO, et al: Markers of inflammation, proteolysis, and apoptosis in ESRD. Am J Kidney Dis 42:1212-1220, 2003 19. Raj DS, Dominic EA, Wolfe R, et al: Coordinated increase in albumin, fibrinogen, and muscle protein synthesis during hemodialysis: Role of cytokines. Am J Physiol Endocrinol Metab 286:E658-664, 2004 20. Ferrando AA, Chinkes DL, Wolf SE, et al: Acute dichloroacetate administration increases skeletal muscle free glutamine concentrations after burn injury. Ann Surg 228:In Press, 1998 21. Wolfe RR: Nutrition and metabolism in burns. Crit Care 7:19-63, 1986 22. Wolfe RR, Volpi E, in Jefferson L, Cherrington A (eds): Handbook of Physiology, vol 2. New York, Oxford, 2001, pp 735-757 23. Woolf PD: Hormonal responses to trauma. Crit Care Med 20:216-226, 1992 24. Ferrando AA, Sheffield-Moore M, Wolf SE, et al: Testosterone administration in severe burns ameliorates muscle catabolism. Crit Care Med 29:1936-1942, 2001 25. Biolo G, Maggi SP, Williams BD, et al: Increased rates of muscle protein turnover and amino acid transport following resistance exercise in humans. Am J Physiol Endocrinol Metab 268:E514-E520, 1995 26. Wolfe RR: Effects of insulin on muscle tissue. Curr Opin Clin Nutr Metab Care 3:67-71, 2000
I
N A HEALTHY INDIVIDUAL, muscle homeostasis is maintained by a complex interaction of hormonal and nutritional signals. When this system is perturbed by critical illness or severe injury, the accompanying neuroendocrine changes are directed toward the enhancement of survival. Skeletal muscle then serves as a primary source of both gluconeogenic precursors and precursors for synthesis of essential proteins, such as those involved in immune function and wound healing. In this mode, muscle experiences a persistent and substantial decrease in protein content and an accelerated release of amino acids. Because amino acids, in particular intracellular amino acids (ICAAs), are required for protein synthesis, it is tempting to speculate that their continued release from the precursor pool could adversely affect protein synthesis. However, in severe trauma such as burns, muscle protein synthesis is
*Department of Surgery, University of Texas Medical Branch, Galveston, TX. †University of New Mexico, Albuquerque, NM. Address reprint requests to Arny Ferrando, PhD, 815 Market St, Galveston, TX 77550. E-mail: [email protected] © 2005 by the National Kidney Foundation, Inc. 1051-2276/05/1501-0008$30.00/0 doi:10.1016/j.jrn.2004.09.014
34
elevated 45% over normal values despite a substantial efflux of amino acids from the muscle.1 Therefore, to maintain an elevated synthetic rate, the intracellular amino acid precursor pool must also be maintained. Cumulative evidence from our laboratory suggests that ICAAs, particularly the essential amino acids, are closely regulated throughout a wide range of physiologic circumstances, from critical illness and severe injury to healthy volunteers and hyperaminoacidemia. Because the essential amino acids must be present for protein synthesis to occur, their regulation is crucial to the replenishment of muscle proteins. Nonessential amino acids can be derived from other sources and from interorgan transport,2 and are therefore not as closely regulated. An alteration in ICAA availability because of a change in metabolic signal encompasses a coordinated response in 1 or more of the processes of synthesis, breakdown, and tissue transport to maintain or restore these concentrations. To illustrate this concept, we discuss the circumstances of critical injury or illness, because the accompanying and altered metabolic demands magnify the requirement to maintain ICAAs. In particular, we examine burn injury and ESRD patients undergoing dialysis, in whom the predominant effect is an outflow of amino acids from skeletal muscle. Journal of Renal Nutrition, Vol 15, No 1 ( January), 2005: pp 34-38
MUSCLE PROTEIN TURNOVER
35
Delivery In A
Inward Transport Protein Synthesis
Shunting
M
Protein Breakdown V
Outward Transport
Delivery Out
A = femoral artery Figure 1. Three-pool model V = femoral vein of leg amino acid kinetics. M = vastus lateralis muscle
Although the theoretical considerations for this hypothesis have been detailed elsewhere,3 the purpose of this article is to illustrate the tight control of ICAAs throughout a range of circumstances in which alterations in the primary hormonal or nutritional signal impact muscle protein metabolism.
trations, allows for a coordinated investigation of protein synthesis, breakdown, and amino acid transport during any physiologic alteration. In this article, the regulation of ICAAs is discussed in light of the resultant changes in each of these measured parameters.
Model of Amino Acid Kinetics
Increased Amino Acid Availability
Over the past decade, we have developed4 and used1,5-7 a 3-compartment model that quantifies amino acid kinetics across leg muscle (Fig 1). Stable isotope methodology is used in conjunction with the sampling of blood that enters the leg (femoral artery) and leaves the leg (femoral vein), as well as muscle tissue (via biopsy), to determine amino acid kinetics between compartments. With this model, the rate of exchange or the movement of any amino acid can be quantified. In particular, this methodology determines the rate of amino acids entering the intracellular pool via inward transport or protein breakdown and the rate at which they leave by outward transport or protein synthesis.4 When a tracer that is not oxidized by muscle, such as phenylalanine, is used, disappearance from the intracellular pool entails either outward transport or incorporation into protein. Conversely, appearance into the intracellular pool is derived from inward transport or as a result of protein breakdown. Thus, the use of an appropriate amino acid tracer enables the calculation of protein synthesis and breakdown. This methodology, in conjunction with the determination of intracellular amino acid concen-
Our laboratory has shown that the infusion8,9 or ingestion10,11 of amino acids produces a substantial increase in muscle protein synthesis. The resultant increase in arterial amino acid concentrations serves to signal an increase in protein synthesis. The precursor amino acids, the ICAAs, required to support an increased synthetic rate are derived by increased inward transport,8 such that there is no concomitant requirement to derive ICAAs from an increased protein breakdown.8,9,12 Protein synthesis increases steadily with the increase in arterial amino acid concentrations until the point at which synthetic capacity is maximized.13 Intracellular amino acid concentrations remain fairly constant throughout a wide range of extracellular amino acid concentrations until the point at which extracellular concentrations are large enough to move to the intracellular space by mass action. Thus, the elevation of arterial amino acid levels in healthy individuals serves as a signal to stimulate muscle protein synthesis. To maintain the precursor ICAA pool, inward transport is also stimulated. The result is maintenance of the ICAA pool by inward transport alone and the provision of ade-
36
FERRANDO ET AL
quate ICAA precursors to support elevated protein synthesis.
Decreased Amino Acid Availability—Hemodialysis Dietary protein and calorie intake decrease progressively with decline in renal function. Preliminary evidence indicates that protein turnover is adaptively decreased and protein balance is maintained in uncomplicated uremia.14 Similarly, Castellino et al15 reported that although insulinmediated suppression of proteolysis is preserved in patients with chronic renal failure, the ability to increase protein synthesis in response to amino acid infusion is impaired in chronic renal failure. The impaired response to amino acids is further exacerbated by the fact that renal disease, by its nature, severely restricts protein and amino acid intake. Among the many complications in patients with end-stage renal disease (ESRD), protein and calorie malnutrition is a major risk factor for increased mortality.16 ESRD patients are forced to accommodate a limited protein intake while subjected to chronic intermittent hemodialysis. Fasted skeletal muscle protein kinetics in ESRD patients are similar to those of healthy volunteers,6,17 especially if blood bicarbonate levels are normalized.6 However, hemodialysis dramatically alters muscle protein kinetics. Arterial amino acid concentrations are decreased by 20% to 40% during hemodialysis.6,17 Hemodialysis serves as a catabolic signal that results in an efflux of amino acids, primarily essential amino acids, from muscle.6,17 This efflux is facilitated by an increase in outward transport kinetics.6 In human beings, hemodialysis results in a more negative net protein balance, indicating net protein breakdown. Raj et al6 showed that both protein synthesis and breakdown increase during hemodialysis, with a greater increase in breakdown. The net effect is a substantial loss of amino nitrogen from skeletal muscle.6,17 The increase in protein turnover is consistent with a stress state, in which both synthesis and breakdown are increased 50% to 100% above normal values.1,5 Hemodialysis in ESRD patients has been shown to increase other catabolic indicators and signals, such as cortisol,6 and proinflammatory cytokines, such as interleukin-1, interleukin-6, and tumor necrosis factor␣.18 The increased production of cytokines is linked to several cellular markers of protein
breakdown.18 Thus, hemodialysis creates a catabolic environment for skeletal muscle. Despite the many changes in amino acid kinetics during hemodialysis, there is a remarkable consistency in ICAAs. Both the essential and the nonessential amino acid concentrations are maintained during hemodialysis.6 Because the primary metabolic signal is one of catabolism, ICAAs are maintained by a substantial increase in protein breakdown, as there is no increase in inward transport.6 The increase in protein breakdown is sufficient to maintain the ICAA pool and to support an increase in both outward transport and protein synthesis.19 Viewed from the perspective of our proposed hypothesis of ICAA regulation, to maintain the ICAA pool and provide amino acids centrally, protein breakdown must increase. The increase in protein synthesis may be the result of a push effect from increased amino acid availability, much as increased availability from amino acid infusion/ingestion stimulates protein synthesis. To further illustrate the concept of ICAA regulation, we examine the example of severe burn injury and the subsequent alterations in amino acid kinetics that result from an alteration in metabolic signal.
Enhancement of Anabolic Signal in Severe Burns As occurs with hemodialysis in the ESRD patient, protein breakdown is elevated to the point at which both increased efflux and protein synthesis are supported in the severely burned patient.1 ICAA concentrations achieve a new homeostasis in burn injury, with some amino acids present in greater concentrations (phenylalanine, leucine), whereas others are reduced (glutamine, lysine, alanine).1,20 Burn injury entails a profound insensitivity of skeletal muscle to the effects of insulin on both glucose and protein metabolism.21 In an attempt to overcome this insulin resistance, Sakurai et al administered high-dose insulin to severely burned patients for 7 days to investigate the effects on protein metabolism and amino acid kinetics.7 The resultant blood insulin concentrations (approximately 900 U/mL) presented a substantial increase in anabolic signal, such that net protein synthesis increased 2- to 4-fold. To support the increased stimulation of protein synthesis with adequate ICAA precursors, protein breakdown paradoxically increased 2.5fold, whereas inward transport increased 6-fold. An
37
MUSCLE PROTEIN TURNOVER
increase in inward transport is remarkable in these patients because net amino acid transport is uniformly outward.1 The fact that the primary transport kinetics can be affected with sufficient signal provides evidence of the regulatory role of ICAAs. These dramatic changes in amino acid kinetics enabled ICAA concentrations to remain unchanged.7 Thus, the regulation of ICAAs during stimulated protein synthesis was accomplished not only by increasing inward transport but also by increasing protein breakdown. The substantial increase in protein breakdown was capable of supporting both the ICAA precursor pool for protein synthesis and the anticipated efflux of amino acids from skeletal muscle. This response suggests a metabolic hierarchy, with the maintenance of ICAAs taking precedence over the inhibitory action of insulin on protein breakdown.22 This is also reflected in the cumulative data, which suggests that the in vivo response of muscle to insulin administration is highly dependent on the availability of amino acids.22 We subsequently investigated the effects of lowerdose insulin on protein metabolism and amino acid kinetics in severely burned patients. Exogenous insulin was administered for 3 to 4 days to achieve blood concentrations of approximately 240 U/ mL.5 Again, this resulted in a substantial anabolic signal to skeletal muscle such that net protein synthesis increased 2-fold. However, unlike the previous study with 3-fold greater insulin levels, there was no statistical increase in protein breakdown or transport kinetics.5 Apparently, the existing (high) rate of protein breakdown was sufficient to maintain the precursor pool for increased protein synthesis and the outward flux of amino acids. In this case, protein synthetic efficiency increased, meaning that for a given quantity of ICAAs derived from protein breakdown and inward transport, a greater portion was directed toward protein synthesis. In this circumstance, the regulation of ICAAs was accomplished with minimal changes in protein kinetics. Burn injury leads to a dramatic reduction of testosterone production in male patients. After severe burn injury, testosterone concentrations are far below the normal physiologic range.23,24 In an attempt to restore this important anabolic influence, we administered exogenous testosterone to male patients with severe burn injury. The intent was to normalize circulating testosterone concentrations to investigate the effects on muscle protein metabolism and amino acid kinetics. Patients were studied before and after 2 weeks of
testosterone enanthate (200 mg/week intramuscularly). Blood testosterone concentrations increased to the low-normal range after the first injection and to the upper-normal range after the second injection.24 Patients were first studied approximately 2 weeks after the severe burn, with the second study at approximately 4 weeks postburn. Protein turnover was typically elevated, with a marked net loss (negative net balance) from skeletal muscle before testosterone administration. After 2 weeks of testosterone, protein breakdown decreased 2-fold without a concomitant change in protein synthesis.24 As a result, net protein balance was restored by the concurrent enteral feeding. Muscle protein synthesis was unchanged; however, protein synthetic efficiency increased 2-fold. In addition, amino acids from protein breakdown were redirected to protein synthesis at a 2-fold greater rate. ICAAs remained unchanged after testosterone administration, with the exception of increased leucine uptake, most likely directed toward oxidation.8 Skeletal muscle ICAA was maintained by altering transport and protein breakdown. After testosterone administration, the elevated muscle protein synthesis associated with burn injury was supported by reducing the outward transport of amino acids from muscle. The reduction in protein breakdown could potentially detract from the required precursor pool for protein synthesis; however, protein breakdown was still elevated to a sufficient level. Evidence for the adequacy of ICAA precursors derived from protein breakdown is shown by a concomitant decrease in inward transport. In other words, ICAAs could be maintained and support the existing rate of protein synthesis in light of decreased protein breakdown by decreasing outward transport of amino acids. Thus, with the anabolic signal of testosterone in severe burns, ICAA regulation leads to an increase in protein synthetic efficiency and a decrease in protein breakdown and outward transport.24
Summary For a given metabolic signal, the regulation of ICAAs entails appropriate and corresponding changes in amino acid kinetics. These changes vary according to the strength of the metabolic signal and the existing requirement to maintain the ICAA pool. Throughout a wide range of physiologic changes and/or metabolic signals,
38
FERRANDO ET AL
ICAAs remain fairly constant. Extracellular signals, such as changes in hormone or nutritional concentrations, may signal or initiate changes in protein metabolism. In the ESRD patient, the sudden removal of almost half of circulating amino acids induces a substantial increase in amino acid efflux from skeletal muscle. To maintain ICAAs, protein breakdown must also increase substantially. The increase in protein synthesis may be a response to increased ICAA availability from protein breakdown. From this perspective, the regulation of ICAAs pushes the subsequent adaptations in amino acid kinetics to maintain the existing homeostasis. This form of active regulation of ICAAs enables skeletal muscle to maintain a sufficient precursor amino acid pool to support protein synthesis given an adequate anabolic signal. Despite the substantial evidence of ICAA regulation, its physiologic significance is not evident. Further, this regulation is not conducive to a teleologic argument. Evidence of the regulatory role of ICAAs is compelling and substantiates a requirement for adequate amino acid precursors for muscle anabolism to occur. This role is further substantiated by examining the interaction of amino acids and the anabolic stimuli of resistance exercise and insulin. Resistance exercise alone has a minimal effect on muscle protein balance25; however, when combined with sufficient amino acids, muscle anabolism is substantial.8 This is analogous to the effects of insulin, whereby sufficient amino acids must also be present for anabolism to occur.26 Thus, when viewed from this perspective, it is likely that the regulation of ICAAs represents a method by which muscle ensures its capacity for anabolism.
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