Metabolism of branched-chain amino acids in altered nutrition

PROGRESS

IN ENDOCRINOLOGY

AND METABOLISM

Metabolism of Branched-Chain Amino Acids in Altered Nutrition Siamak A. Adibi Plasma concentrations of the branchedchain amino acids (leucine, isoleucine, and valine) are more prominently affected than the concentrations of other amino acids by changes in dietary--caloric, protein, fat, and carbohydrate--intake in man. For example, within a day of starvation or protein deprivation, there are increases or decreases, respectively, in concentrations of these amino acids in the plasma of healthy human volunteers. The cellular mechanisms of these changes have been investigated in rats, since the changes in the plasma branched-chain amino acid concentrations in response to the previously stated dietary alterations are similar to those found in man. Among the tissues studied (liver, skeletal muscle, heart, kidney, and intestine) only liver and the skeletal muscle exhibit changes in branchedchain amino acid concentrations in response to dietary alteration. Changes in plasma concentrations appear to reflect more intimately those of the muscle than the liver. After 8 days of starvation, there

is a 25% decrease in the muscle protein, but after 8 days of protein deprivation, there is no significant change in the muscle mass. Increases in concentrations of branched-chain amino acids in the muscle are much smaller than the amounts of these amino acids lost as protein constituents from the muscle during fasting. Changes in tissue transport, transamination, oxidation, or metabolic conversions of branched-chain amino acids are not responsible for the alteration in pool sizes of free branched-chain amino acids in tissues. It is concluded that increased muscle protein breakdown, which provides substrates for enhanced gluconeogenesis in the liver and enhanced branched-chain amino acid oxidation in the muscle, is the major mechanism of hyperbranched-chain aminoacidemia in starvation. On the other hand, the principal factors in the development of hypobranched-chain aminoacidemia during protein deprivation are absence of exogenous amino acids as well as curtailed muscle protein breakdown.

THE

three branched-chain amino acids, leucine, isoleucine, and valine, are essential nutrients for man and animals; in fact, they make up about 40% of the minimal daily requirement of indispensable amino acids of man. Branched-chain amino acids are important not only as essential substrates for protein synthesis, but also as biochemical regulators or precursors in complex metabolic reactions. Leucinc is regarded as one o f the most potent amino acids in the stimulation of insulin secretion, l as a significant precursor for sterol biosynthesis in adipose tissue and muscle, 2 and as an inhibitor of urea formation in the liver. 3 The role of branched-chain amino acids, especially that of leucine, as a regulator of protein synthesis in the skeletal muscle has been suggested by the independent studies of several investigators. 4,5 The importance of branched-chain amino acids as metabolic fuels during the stressful situations

From the Gastrointestinal and Nutrition Unit of Montefiore Hospital, University of Pittsburgh School of Medicine, Pittsburgh, Pa. Received for publication October 14, 1975. Supported by Grant AM-15855 from the National Institute of Arthritis, Metabolism, and Digestive Diseases, National Institutes of Health, United States Public Health Service. Reprint requests should be addressed to Siamak A. Adibi, M.D., Ph.D., Montefiore Hospital, 3459 Fifth Avenue, Pittsburgh, Pa. 15213. 9 1976 by Grune & Stratton, Inc.

Metabolism,Vol. 25, No. 11 (November), 1976

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of caloric deprivation (starvation) and increased caloric need (exercise) has been shown in recent observations. 6-8 During prolonged starvation, administration of a small amount of keto-analogues of branched-chain amino acids facilitates nitrogen-sparing by a mechanism other than the amination of the ketoacids and incorporation of the resulting amino acids into protein. 9 In view of the observation that the oxidation of branched-chain amino acids increases the muscle synthesis and release of alanine, it has been proposed that these amino acids may also regulate the muscle release of ammonia and gluconeogenic precursors for metabolism by the liver.l~ Various abnormalities of branched-chain amino acid metabolism caused by genetic enzymatic defects and leading to severe metabolic derangements have been well recognized. 1! Relative excess of leucine in the diet has been implicated in the etiology of pellagra ~2by showing that it adversely affects the formation of niacin from tryptophan.~3 Not only do branched-chain amino acids affect the metabolic processes in the body, but their own metabolism is profoundly altered by malnutrition. The purpose of this review is to present a brief account of recent observations on the latter aspect of branched-chain amino acid metabolism. STUDIES IN MAN

Plasma Concentration

Plasma concentrations of branched-chain amino acids are more prominently affected than those of other amino acids when human subjects are given either a protein, carbohydrate, or fat meal. The major contributing factor to hyperaminoacidemia after a protein meal ~4,15or to hypoaminoacidemia after glucose ingestion ~6 is the change in plasma concentrations of branched-chain amino acids. When a large amount of fat is ingested, the concentrations of branchedchain amino acids are uniquely increased in plasma. 17 In response to dietary deprivation in man, the most dramatic and prompt changes in plasma concentrations of amino acids are those of branched-chain amino acids and alanine. Starvation, for as brief a period as 1 day, increases the concentrations of all three branched-chain amino acids in the plasma of healthy human subjects (Fig. 1). The levels of branched-chain amino acids in these subjects reach a peak value by the second day of starvation and remain elevated during the first week. Resumption of a regular diet during the first week o f starvation (Fig. 1) or prolongation of starvation to 2 wk lowers the concentrations of branched-chain amino acids to basal levels. 18Except for a more gradual rise, the changes in concentrations of branched-chain amino acids during starvation are similar in obese and nonobese subjects. 18 21 In contrast to starvation, feeding healthy human volunteers a diet devoid of protein, but adequate in caloric content, lowers the plasma concentrations of branched-chain amino acids to below basal levels within one day (Fig. 1). This reduction is more marked for valine than for leucine and isoleucine. Reintroduction of protein into the diet promptly returns the depressed levels of branched-chain amino acids, in particular that ofvaline, to basal levels (Fig. 1). In patients with longterm protein deprivation (Kwashiorkor), plasma concentrations of branchedchain amino acids, as compared to other amino acids, are more severely decreased. 22,23

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5oo[ 450

400

350

3OO -J

250

g, IE

~k 2OO

150

Fig. 1. Postabsorptive plas-

ma concentrations of leucine ( o - o . o ) , isoleucine (A-A-A), a n d valine (o-o-e) before (day 0),

during (days 1-6), and after (days 7 and 8) each dietary deprivation in healthy human volunteers. ]9 Each value represents the mean • $EM o f concentration values in six subjects.

100

50

0 0

I

2

3

4 DAYS

5

6

7

8

Increased concentrations of branched-chain amino acids have also been observed in the plasma of patients with massive obesity.~8'24 Felig, Marliss, and Cahil124 have favored the view that the abnormalities in plasma amino acid concentrations are a secondary manifestation of the obese state, but may account for the hyperinsulinemia commonly observed in this condition. However, a decreased food intake in anticipation of weight reduction appears responsible for increased plasma concentrations of branched-chain amino acids in some obese individuals. 19Swendseid et al. 25 have not found a significant difference in branched-chain amino acid tolerance tests between obese and nonobese subjects. The direction of changes in plasma concentration of alanine during starvation or protein deprivation (Fig. 2) is opposite to those of branched-chain amino acids. The decrease in plasma alanine concentration has been interpreted as a reflection of enhanced gluconeogenesis in starvation,18-2~ and the increase in plasma alanine concentration as an indication of depressed gluconeogenesis and increased alanine synthesis in protein deprivation.J6'19 In protein deprivation, maintenance of an adequate caloric intake necessitates an increase in carbohydrate content of the diet. 19 The previous observations on plasma concentrations have provided the evidence for alteration of metabolism of branched-chain amino acids by dietary changes. Furthermore, they raise questions regarding the cellular processes

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580 560 520 480

~ 440 .~ 400

g,

3eo

28O

Fig. 2. Postabsorptive plasma concentrations of alanlne before ( d a y 0), during (days I - 6 ) , and

24O 2OO 160

0

1

2

3

4

5 6 DAYS

7

8

after (days 7 and 8) each dietary deprivation in healthy human volunteers. 19 Each value represents the mean • $EM of concentration values in six subjects.

concerned with branched-chain amino acid metabolism which are altered by changes in nutritional status. The information available on these processes will be examined in the following sections.

Renal Clearance The kidney plays an important role in homeostasis of free amino acids in the body. Although all amino acids circulating in systemic blood are regularly filtered through glomeruli, there is normally almost complete reabsorption of these amino acids by the renal tubular cells. As in the intestine, carrier systems located on the brush border membrane of the tubular cells appear to be involved in transport of free amino acids, z6 In certain hereditary disorders of transport, the levels of free amino acids in plasma are markedly changed by defective renal transport. 26 Therefore, one simple explanation of the observation of changes in plasma concentrations of branched-chain amino acids during dietary deprivations as previously discussed could be changes in renal clearance of free amino acids by these dietary manipulations. However, the renal clearances of individual amino acids, including those of the branched-chains, have been shown to remain unaffected during 1 wk of starvation or protein deprivation. 27Although renal clearances are unchanged, the amounts of daily excretion of individual amino acids are affected by diet. 27 For example, there is a greater excretion of all three branched-chain amino acids during starvation than during protein deprivation. These and other observations have suggested that the difference in the urinary excretion pattern of free amino acids exhibited during starvation and during protein-free dieting is principally a reflection of differences in plasma amino acid composition, and not renal clearance of amino acids. 27

Intestinal Absorption After a protein meal, movement of amino acids from the gut lumen into the systemic circulation could influence the concentration of amino acids in the peripheral venous blood. 14,15However, in the postabsorptive state, the intestine does not appear to play a major role in the regulation of plasma amino acid

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composition. Nevertheless, the intestinal absorption of branched-chain amino acids under conditions of optimal nutrition and dietary deprivation have been investigated in human subjects. Two functionally distinct mechanisms appear to be involved in the intestinal absorption of branched-chain amino acids; the free amino acid and the peptide carrier systems. 2s-32The peptide carrier system appears to be physiologically more important than the free amino acid carrier system in the uptake of branched-chain amino acids from protein in the gut lumen. 33'34Both starvation and protein deprivation reduce intestinal absorption of free branched-chain amino acids." However, protein-calorie malnutrition does not affect the absorption of branched-chain amino acids by the peptide carrier system. 36 These observations, therefore, do not provide evidence that alterations in intestinal transport could account for the alterations in plasma branched-chain amino acid composition during starvation and protein deprivation.

Amino A cid Exchanges A cross Organs Felig and co-workers 2~have investigated the amino acid balance across the liver of obese human subjects undergoing starvation for weight reduction. Their data show that the liver is not a source of plasma branched-chain amino acids in the postabsorptive state nor after brief (36-48 hr) or prolonged (5-6 wk) starvation. The concentrations o f these amino acids were always lower in hepatic venous blood than in arterial blood. In contrast to liver, the muscle releases branched-chain amino acids whether the subjects are in a state of postabsorption or starvation.2~ 37,a8 In a recent study, Pozefsky et al. 38 have shown that after a brief starvation (60 hr) the arterial concentrations of most amino acids are decreased, but those of branched-chain amino acids are markedly increased. The same investigators have examined the amino acid balance across deep forearm tissues, and have found that brief starvation results in a near twofold increase in the muscle release of branched-chain amino acids. 38However, due to large variations in concentrations, the changes in their release were not statistically significant. Felig et al. 2~ have shown that in prolonged fasting (4-6 wk) when there is hypo branched-chain aminoacidemia, the muscle release of these amino acids is significantly reduced. Although the above observations are suggestive of the increased muscle release of branched-chain amino acids as a major cause of the hyper branchedchain aminoacidemia of starvation, additional investigations are needed to firmly establish this hypothesis.

Metabolism of Exogenous Leucine The plasma concentration of leucine is increased considerably by intestinal infusion or oral ingestion of leucine solutions. 25,35 If the same subjects are starved for 2 wk and the same infusion studies repeated, the increases in plasma concentration of leucine are depressed twofold, a5 Furthermore, if the oral leucine tolerance test is repeated at intervals after a jejunoileostomy for massive weight reduction, the increases in plasma leucine concentration are blunted. 39 Although a decrease in the intestinal absorption rate of leucine in these individuals 35contributes to lesser increases in plasma leucine levels, it does not ap-

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pear to be the entire explanation. Starvation also decreases intestinal absorption rate of glucose, 4~ but plasma concentration of glucose during the oral glucose tolerance test is greater than before fasting. 41 The previously stated observations indicate that there is a major difference between the effects of starvation on the metabolism of leucine and that of glucose: the metabolism of leucine is accelerated by fasting, but that of glucose is depressed. STUDIES IN RATS

Dietary-induced changes in plasma concentrations of branched-chain amino acids in rats are essentially similar to those described in the preceding section in man. Starvation increases 42 and protein deprivation lowers 43 the concentrations of all three branched-chain amino acids in rat plasma.* Therefore, rats have been used as a convenient animal model for the following investigations of the effects of dietary deprivations on concentration, transport, and metabolism of branched-chain amino acids in tissues. Amino Acid Pools

Using a paper c h r o m a t o g r a p h y technique, W u in 195444 c o m p a r e d the concentrations of free amino acids in the muscle, liver, and plasma before and after 9 days of starvation in rats. He found that starvation markedly increased concentrations of branched-chain amino acids in both tissues and plasma. M o r e recently, with ion exchange c h r o m a t o g r a p h y technique, which offers more sensitivity and precision than methods previously available, concentrations of amino acids in the skeletal muscle (gastrocnemius), heart muscle, kidney cortex, intestinal mucosal cells, and the liver before and at intervals during 8 days of starvation in rats were investigated. 42 A m o n g these tissues, only the skeletal muscle and the liver exhibited increases in their concentrations of branchedchain amino acids. The increases in concentrations became evident after 4 days of starvation in the skeletal muscle, but after 8 days in the liver. Both the muscle and the liver lost weight although less by the muscle than by the liver. 42 Despite the loss of tissue mass, there was a 45~/o-95~ increase in the pool size (total a m o u n t / w h o l e organ) of each branched-chain amino acid in the skeletal muscle. However, there was a 50~o decrease in the pool size of each of these amino acids in the liver. Bloxam, 45 who has measured the portal-venous differences in the amino acid concentrations of rats starved for 1 or 3 days, has found that most amino acids flow from extrahepatic tissue to liver, but branched-chain amino acids flow in the opposite direction. However, an increased hepatic release of branched-chain amino acids is not a major factor in the development of hyperbranched-chain aminoacidemia of starvation. In rats the plasma concentrations of branched-chain amino acids do not become elevated until the sixth day of starvation when the muscle pool size and release of these amino acids have already increased. 42,46

*It should be noted that the development of hyperbranched-chain aminoacidemia is not as rapid in rat as it is in man. In rats (270-290 g) the plasma concentrations of branched-chain amino acids become elevated after 6 days of starvation, while they are increased after 1 day of fast in man.

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The effect of total starvation on the pool sizes of branched-chain amino acids is due to caloric and not protein deprivation, since feeding a protein-free diet in amounts isocaloric with a nutritionally adequate diet produces tissue changes which are opposite to those previously described. 43After l or 2 days of protein-free dieting, there are decreases in the pool size of each branched-chain amino acid in both the skeletal muscle and the liver. The changes in the liver reach a plateau within the first day, while those in the muscle continue to decrease by prolongation of protein deprivation beyond 1 day. Transport Membrane transport systems appear to be involved in mediating the cellular entry of circulating branched-chain amino acids. The characteristics of the "leucine-preferring" transport system have been studied by Christensen and his co-workers. 47 Both the muscle and liver metabolize branched-chain amino acids whether for protein and lipid synthesis or for oxidation. Therefore, the investigation of cellular transport of these amino acids into these tissues under conditions of dietary deprivation could be complicated by altered conditions inside the cells affecting their metabolism. T h e use of cycloleucine (l-amino cyclopentane-l-carboxylic acid) which is a nonmetabolizable analog of branched-chain amino acids has obviated this problem. The hepatic transport ofcycloleucine is increased within 1 day of starvation, while its muscle uptake remains unaffected after 5 days of starvation, a8 In contrast to starvation, protein deprivation for 1 or 2 days does not affect hepatic transport of cycloleucine (Adibi: unpublished observations). The increased accumulation of cycloleucine in the liver of starved rats is not unique to this amino acid, since, under the same nutritional condition, the liver transport of AIB (ot-amino-isobutyric acid), which is an amino acid model for the "alanine-preferring" transport system, is also increased. 49 The lack of alteration of cycloleucine transport by the muscle in starved rats appears specific to this substrate, since under the same nutritional condition the muscle transport of AIB and 3-0-methylglucose (a nonmetabolizable analog of glucose) is reduced. 49,5~ The preceding transport studies indicate that the increases in concentrations of branched-chain amino acids during starvation, or decreases during protein deprivation, are not due to changes in the muscle uptake of these amino acids from plasma. However, the increases in concentrations of branched-chain amino acids in the liver of starved rats may, in part, be the result of increased transport. Transamina tion Removal of the amino group is a necessary first step for the oxidation of branched-chain amino acids. Ichihara et al. 51 have described three isozymes of branched-chain amino acid transaminase in tissues. According to these investigators, all tissues contain isozyme I, but isozymes II and III are found only in the liver and brain, respectively.51 Furthermore, isozymes I and III transaminate all three amino acids, but isozyme II is specific for leucine. Taylor and Jenkins, 52,53who have purified extensively the leucine transaminase from pig

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heart muscle, have found that the major portion (85~o) of the activity of this enzyme resides in the soluble fraction of the muscle cells. Among liver, muscle, and kidney, the muscle contains the highest total transaminase activity against these amino acids. 54'55However, the specific activity of branched-chain amino acid transaminase is highest in the kidney and lowest in the liver. 54'55 The effects of dietary deprivation on the activity of branched-chain amino acid transaminase of rat tissues have also been studied in several laboratories, but some of the results are conflicting. Mimura et al. 55 found that protein deprivation for 10 days increased the activity of branched-chain amino acid transaminase in the muscle and liver but lowered that of kidney. Brown and coworkers $6reported that feeding of a low protein diet for 10 days decreased the specific activity of this enzyme in the skeletal muscle. Sketcher and associates 57 found increases in leucine transaminase activity of the muscle with both starvation and a low protein diet, while the liver activity was not affected. Wohlhueter and Harper 58 found that starvation for 1 day does not significantly alter the specific activity of leucine transaminase either in the liver or in the kidney of rats that were maintained previously on a 9~o casein diet. Adibi et al., 54 using leucine as a substrate in a recent investigation, have shown that the specific activity of branched-chain amino acid transaminase can be modulated by dietary means. When rats were starved for one full day, the activity of leucine transaminase was increased by approximately twofold in the muscle and kidney. Prolongation of fasting for 5 days resulted in an additional increase in specific activity of leucine transaminase in the muscle. 54 In contrast, protein deprivation for 1 or 5 days resulted in a significant reduction in the specific activity of leucine transaminase in the skeletal muscle. 54 Neither starvation nor protein deprivation had a remarkable effect on the activity of this enzyme in the liver. 54 Differences in experimental techniques, including the enzyme assay method, could have accounted for the varied results summarized. Nevertheless, the most consistent pattern of change emerging from the studies in the preceding paragraph is that starvation increases and protein deprivation decreases the activity of branched-chain amino acid transaminase. The effects of dietary deprivation are more pronounced on the transaminase activity in the muscle and kidney than in the liver. From the previous observations, it appears that changes in the activity of branched-chain amino acid transaminase cannot be held responsible for the expansion of pool sizes of branched-chain amino acids during starvation and their decreases during protein deprivation. The changes in transaminase activity and pool sizes are in parallel directions in each of the above nutritional conditions. Oxidation

Transamination of L-leucine, L-isoleucine, and L-valine results in formation of a-ketoisocaproic acid, a-keto-/3-methylvaleric acid, and a-keto isovaleric acid, respectively. As shown in the following equation, dehydrogenation of these ketoacids results in the loss of the carboxyl group and the formation of thioesters of co-enzyme A. The decarboxylation step is catalyzed by a high molecular weight, multi-enzyme complex involving several cofactors, H,59 such

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as nicotinamide adenine dinucleotide (NAD), flavin adenine dinucleotide (FAD), thiamine pyrophosphate, lipoic acid, and co-enzyme A. O

II

R--C--COOH

+

CoA--SH

+

N A D + --~

O R--C

SCoA

+

CO2

+

NADH

+

H+

Subcellular distribution studies in the liver have indicated that the oxidative decarboxylation activities against the three branched-chain ketoacids are located principally in mitochondria. 6~J o h n s o n and Connelly 6~ have presented evidence suggestive of the existence o f a branched-chain ketoacid dehydrogenase in the soluble fraction of bovine liver different from that found in the particulate fraction, Connelly et al. 61 have reported isolation of two branchedchain a-ketoacid dehydrogenases from bovine liver; one catalyzes the decarboxylation of a-ket0isocaproic acid and a-keto-~-methylvaleric acid, and the other that of a-keto~sovaleric acid. However, the problem of whether there are several branched-chain a-ketoacid dehydrogenases does not yet appear fully resolved. II According to established biochemical patlaways, 59 the three branched-chain acylCoA products formed as a result of decarboxylation are further oxidized through a series of reactions to either acetyl (leucine) or to acetyl and propionate fragments (isoleucine and valine). The final products o f oxidation are CO: and water. The potential of tissues to oxidize branched-chain amino acids varies. According to t h e data of Odessey and Goldberg, 62 the order of tissue potential (as defined by the rate of CO: released per gram of tissue) to oxidize leucine is as follows: kidney > brain > adipose tissue > muscle > liver. However, several lines of evidence have indicated that oxidation o f these amino acids is accomplished principally in muscles: (1) In eviscerated rats, there is marked oxidation of intravenously administered (~4C) branched-chain amino acids to 14CO2,63 while these rats hardly oxidize other amino acids; 63 (2) After hepatectomy, plasma concentrations of branched-chain amino acids are decreased, while those of others are increased; 64 (3) Rate o f CO2 production from leucine in the muscle is higher than in the liver but lower than in the kidney when expressed per gram o f tissue weight. 62,6sHowever, if the oxidative capacity is expressed per total weight of the organ, the total capacity of the muscles to oxidize branched-chain amino acids far exceeds that of kidney. The small capacity for transamination of branched-chain amino acids appears to be a major hindrance to the oxidation of these amino acids by the liver. Featherston and H o r n 66 have found in chicks that addition of kidney homogenate, which is rich in branched-chain amino acid transaminase, to the muscle and liver homogenates does not increase CO: production from leucine by the muscle, but it markedly increases CO2 from leucine by the liver. Recent studies 67 have shown that the oxidation of leucine does not proceed to completion in the muscle, and CO: production from leucine is limited to o~-decarboxylation in this tissue. In contrast, liver can degrade a-ketoisocaproic acid to acetyl-CoA and acetoacetate. 6s However, the ketone bodies are not

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utilized by the liver 69but are efficiently oxidized by muscles to C02 .70 These observations suggest a complex collaborative arrangement for total oxidation of leucine between muscles, the liver, and possibly other tissues. The oxidation of branched-chain amino acids is influenced by the state of nutrition. After intraperitonea165 or intragastric injection ~7 of (~4C) leucine, starved rats expire a greater amount of ~4CO2 than fed rats. The tissue principally responsible for the increased leucine oxidation appears to be the skeletal muscle. Muscle preparations, such as gastrocnemius muscle slices or diaphragm of starved rats, have been shown to have an increased capacity to oxidize (~4C) leucine as assessed by the rate of 14C02 production by these tissues. 6,7 The increased capacity becomes apparent within 12 hr after starvation and is reduced within 24 hr after refeeding. 6 The starvation-induced increase in CO2 production from leucine by the muscle is the result of increased a-decarboxylation of this amino acid by this tissue. 67 Independent studies from three laboratories have all shown that starvation does not affect leucine oxidation by the liver. 6,7,65 Total starvation includes caloric as well as protein deprivation. Investigations of the effect of protein deprivation on branched-chain amino acid oxidation have produced conflicting results. Adibi et al. 6 have found that protein deprivation for a period of 1 day is without effect on the oxidation of leucine by the gastrocnemius muscle slices. In contrast, several other investigators 57,7~,v2 have reported a decrease in the oxidation of branched-chain amino acids in rats subjected to low protein diets. However, more recently Neale and Waterl o w 73 have been unable to observe any reduction in the oxidation of branchedchain amino acids in rats being fed a protein-free diet for 20 days. Protein deprivation, whether lacking an effect or reducing branched-chain amino acid oxidation, does not appear to account for the increased leucine oxidation during starvation. Wohlhueter and Harper 58 have studied in detail the responses of the liver and kidney branched-chain a-keto acid dehydrogenases to increases in dietary protein and the supplementation of diet with each of the branched-chain amino acids. They found that increasing the protein content of the diet from 0 to 30~o resulted in a linear increase in the specific activity of branched-chain a-ketoacid dehydrogenases in the liver. The effect had plateaued when the dietary protein content was increased to 50~. Supplementation of the diet with leucine, isoleucine, or valine also increased the liver dehydrogenase activity against all three branched-chain a-ketoacids. The specific activity of branched-chain a-ketoacid dehydrogenase in the kidney was not changed by alteration of protein content of the diet. These studies have indicated the adaptive nature of the liver branched-chain a-ketoacid dehydrogenases to variation in dietary protein intake. The studies of branched-chain amino acid oxidation just summarized do not provide a satisfactory explanation for the alteration of plasma concentration of branched-chain amino acids during starvation and protein deprivation. The unchanged or lowered oxidation of branched-chain amino acids would not account for decreases in concentration of these amino acids in tissue and plasma during protein deprivation. The increased oxidation of branched-chain amino acids during starvation should actually lower and not increase plasma and tissue concentrations of these amino acids. Nevertheless, the increased

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oxidation of branched-chain amino acids in starvation may serve a useful and perhaps important function. In comparison to carbohydrate, the body contains a large amount of protein and fat. During starvation a large portion of body proteins, which contain considerable amounts of branched-chain amino acids, are catabolized. 42 The amounts of branched-chain amino acids in free amino acid pools as compared to those in body proteins are quite small. 42,43 Leucine is not a gluconeogenic amino acid and the other two branched-chain amino acids are poor glucose precursors. Therefore, the increased oxidation by the muscles provides an explanation for the loss of these amino acids from body protein during starvation. The increases in muscle oxidation of fatty acids 6'74 and branched-chain amino acids 6,7 and the decrease in muscle oxidation of glucose 6'75 during starvation appear to reflect an adaptive response o f the skeletal muscle to improve its efficiency to utilize available metabolic fuels. In view of the alteration o f plasma concentration of branched-chain amino acids by glucose and fat discussed in the Section on plasma concentration, it is pertinent to examine the effect of these agents on the oxidation of branchedchain amino acids by the muscle. Indeed, the effects of glucose and fatty acids on the oxidation of leucine by intact muscle preparations such as diaphragm or heart in vitro have been investigated; however, the results of some of these studies are conflicting. While Johnson et al. 76 and Manchester 77 did not find glucose to alter the oxidation of leucine, Odessey and Goldberg 62 and Buse et al. 78 observed an inhi~bition of leucine oxidation by glucose. Buse et al. 78 reported a significant stimulation of leucine oxidation by palmitate, but Odessey and Goldberg did not find this effect. 6zPossible variations due to transport and the lack of a precise knowledge of the intracellular concentration complicate the interpretation of the data regarding amino acid oxidation by intact muscle preparations. The studies of amino acid oxidation by cell-free preparations may obviate these difficulties. More recently, the effect of glucose and fatty acids on the a-decarboxylation of leucine by a homogenate of gastrocnemius muscle has been investigated. 67 Glucose and palmitic acid (C~6) when added in a wide range of concentrations to the incubation medium did not significantly alter the rate of a-decarboxylation of leucine by the muscle homogenate. However, the addition of hexanoate (C6) and octanoate (C8) to the incubation medium significantly increased the oxidation of leucine. When fatty acid chain length was elongated to C~o (decanoate) the stimulatory effect was not only abolished but this fatty acid significantly inhibited a-decarboxylation of leucine. Although these studies indicate a complex interrelationship between the metabolism of leucine and that of fatty acids, they do not provide evidence for the muscle oxidative metabolism as a basis for alteration of plasma concentrations of branched-chain amino acids by dietary glucose and fat.

Lipid Conversion The oxidative products of branched-chain amino acids could serve as precursors for fatty acid and cholesterol synthesis. 2'79-82 Among the three amino acids, lipid conversion is most efficiently achieved with leucine and is weakest with valine. 2 Like the capacity for oxidation, the capacity for conversion to lipid is also greater in the extrahepatic tissues. 2 Total lipid synthesis from

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leucine, when expressed per milligram of protein, is much greater in adipose tissue than in either the liver or in the muscle, z Glucose appears to enhance conversion of leucine to lipids in adipose tissue. 2'65'79 The effect of starvation on lipid synthesis from a variety of precursors has been extensively studied. Starvation reduces lipogenesis but enhances lipolysis. 83 Therefore, it may be anticipated that conversion of leucine to fatty acids is reduced in starvation. Experimental data, although limited at present, appear to support this thesis. 65 The impaired conversion of branched-chain amino acids to lipid could be considered as a possible mechanism for the accumulation of these amino acids in starvation. However, this could not be counted as a principal mechanism, since the increase in plasma and tissue concentration of valine, which is a poor lipid precursor, is comparable to that of leucine which is excellent in this regard. Protein Metabolism

The concentrations of essential amino acids in the tissue to a large extent are governed by a balance between protein synthesis and protein degradation. Starvation and protein deprivation affect both these processes in the liver and muscle. 84'85 During starvation, protein catabolism exceeds protein anabolism. In comparison to starvation, there is greater conservation of body proteins when adequate amounts of calories in the form of carbohydrate and fat are provided in the diet. 27 For example, after 8 days of starvation, 65~o of liver and 25~ of muscle (gastrocnemius) protein is catabolized 42 while after 8 days of protein deprivation, there is no significant change in the protein content of the muscle, and only 30~ decrease in the liver protein. 43 CONCLUSION

In both man and rats, caloric deprivation increases and protein deprivation decreases plasma concentrations of branched-chain amino acids. Mechanisms of these changes have been investigated in rat tissues. The tissue branched-chain amino acids are essentially contained in proteins, and most of these proteins are in the muscle. The concentrations of branched-chain amino acids in tissue free amino acid pools are always trace amounts as compared to their concentrations in proteins. Although there is extensive proteolysis in tissues, such as the liver and intestine, the breakdown of muscle proteins is the major source of free amino acids during starvation. The increased protein breakdown provides needed substrates for the enhanced gluconeogenesis in the liver. Since branchedchain amino acids either lack or have weak potential as glucose precursors, oxidative degradation to CO2 and water, as metabolic fuels, becomes the main pathway for their utilization in starvation. The extrahepatic tissues, such as muscle and kidney, appear as the principal sites for the initial steps, namely transamination and a-decarboxylation of branched-chain amino acids. These steps are stimulated by starvation. Despite enhanced oxidation, the pools of free branched-chain amino acids expand uniquely in the muscle. These expansions appear as the main source of hyperbranched-chain aminoacidemia of starvation. However, the increase in concentrations of these amino acids in free amino acid pools is quite small as compared to total amounts of tissue branchedchain amino acids catabolized in starvation. When protein is omitted from the diet for a few days, but adequate amounts

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o f c a l o r i e s in t h e f o r m o f c a r b o h y d r a t e a n d f a t a r e p r o v i d e d , l i v e r p r o t e i n s a r e c a t a b o l i z e d , b u t m u s c l e p r o t e i n s a r e s p a r e d . I n this s i t u a t i o n , l a c k o f a m i n o a c i d s in t h e d i e t a n d a b s e n c e o f an a c c e l e r a t e d m u s c l e p r o t e i n b r e a k d o w n c o n t r i b u t e to t h e d e p l e t i o n o f b o d y p o o l s o f f r e e e s s e n t i a l a m i n o acids, i n c l u d i n g those of branched-chain amino acids and subsequent hypobranched-chain aminoacidemia. Whether the mechanisms of hyper- and hypobranched-chain aminoacidemia i n d u c e d by d i e t as s h o w n in r a t s a r e r e l e v a n t t o t h e s a m e p h e n o m e n o n in m a n r e m a i n s to b e i n v e s t i g a t e d .

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