J ANIM SCI-1992-Lobley-3264-75

G E LobleyControl of the metabolic fate of amino acids in ruminants: a review.

1992, 70:3264-3275.J ANIM SCI

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Control of the Metabolic Fate of Amino Acids in Ruminants: A Review'

G. E. Lobley

Rowett Research Institute, Bucksburn, Aberdeen AB2 9SB, Scotland

ABSTRACT: In general, ruminants convert ingested feed protein (N) to body tissues with low efficiency (0 to 35%). Although some of this inefficiency is due to the peculiarities of ruminal action and digestion, a large proportion is associated with metabolic events in the tissues. In the fasted condition, amino acid catabolism is greater than in the maintenance-fed animal, and perhaps 40% of this loss is due to provision of carbon sources for gluconeogenesis. The contributions of other pathways to these basal losses are poorly quantified. Below maintenance intake, insulin seems to be a major determinant of the rate of protein loss, primarily through reduction of protein degradation (especially in muscle tissue) with an accompanied decrease in the rate of branched- chain amino acid (BCAAI oxidation. At intakes above maintenance, protein anabolism and amino

acid catabolism are more probably regulated by the growth hormone/insulin-like growth factor I (GH/IGF-I) axis, with the major control via alterations in protein synthesis. The actions of insulin and GH/IGF-I may provide overlapping regulatory mechanisms, which would explain the biphasic alterations in protein dynamics and amino acid catabolism observed for the ruminant between the fasted and ad libitum intake conditions. The BCAA may assume a key regulatory role in integrating the metabolism of peripheral tissues with the metabolic and oxidative functions of the liver. This integration seems well-coupled in the ruminant, for which the relationship between the extent of BCAA catabolism in peripheral and hepatic metabolism remains fairly constant under a range of nutritional and physiological conditions.

Key Words: Ruminants, Amino Acids, Protein Turnover, Insulin, IGF-I, Catabolism

J. Anim. Sci. 1992. 70:3264-3275

Introduction

Because of the symbiotic relationship with ruminal microorganisms, ruminants have success- fully colonized the grassland regions of the world and provided humans with a convenient source of meat, milk, and fiber. The ability to utilize forage materials is not accomplished without cost, however, and includes the relatively inefficient use made of both ingested energy and protein (nitrogen). This has been singled out as a focal point for those arguing against the poor usage of natural resources that occurs with current agricultural practices. The argument does not take into account that such resources are, in general, not usable by other major species, but, nonetheless, it

'Presented at a symposium titled "Amino Acids in Meat Animal Production: Current Concepts and Future Perspectives" at the ASAS 83rd Annu. Mtg., Laramie, WY.

Received October 28, 1991. Accepted February 21, 1982.

is necessary to improve the metabolic efficiency of productive animals, with consequent advantages for agricultural economics and better conservation of the ecosystem. To do this systematically requires knowledge of where and why the inefficiencies occur, and thus control points susceptible to manipulation must be identified. In terms of amino acid and protein metabolism the information available is rather incomplete; this review will attempt to clarify some of our current knowledge and indicate areas where further investigation is required.

General Considerations

The inefficiency of N metabolism in ruminants is illustrated in Table 1. These data come from a recent study (J. C. MacRae, personal communica- tion) in which lambs were offered 2 times main tenance intake of grass pellets between 25 and 40 kg, while they were maintained on continuous N balance. In a comparable set of lambs measure-

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RUMINANT AMINO ACID AND PROTEIN METABOLISM 3265

Table 1. Net N movements (g/d) in growing wether lambs maintained on twice-maintenance intake of

grass pellets between 25 and 40 kilogramsa

N source or fate

Intake 20 Absorbed as amino acid N 15 “Maintenance” N losses 7 “Anabolic” amino acid N losses 3.5 Retention 4.5

*Data of J. C. MacRae (unpublished observations).

ments were made of amino acid disappearance between the duodenum and ileum. Overall, the conversion of dietary N to body N was only 13%. Although 50% of ingested N was not available to the animal as absorbed amino acids, further inefficiencies occurred through animal tissue metabolism. Of these latter losses 30 to 50% could be ascribed to meeting basal N requirements, with between 250 and 420 mg of N/kg.75 needed to maintain N equilibrium (Hovell et al., 1983a; Inkster et al., 1989). The greater value is similar to fasting losses (Lobley et al., 1987) and includes specific requirements for amino acid carbon. Above N equilibrium, the fates of amino acids are distributed between gain and amino acid oxidation. The incremental efficiency of absorbed amino acids for protein gain seems to be variable; published values range from 40 to 80% (see Lobley, 1986; Rohr and Lebzien, 1991). It is the mechanisms and regulatory aspects of both the basal and anabolic losses that are the concern of this review.

Kinetic Approaches

The use of isotopes to monitor the metabolic fate of amino acids has been more limited with ruminants than with nonruminants, especially as applied to response situations so that the consequences of perturbations can be followed. One advantage of tracer technology is illustrated in Figure 1, in which protein oxidation, assessed from the irreversible loss rate (flux1 of radiolabeled leucine and I4CO2 production, is compared, between fasting and 1.8 times maintenance with the classical, nonruminant approach of using urinary N elimination as the protein oxidation index [from Lobley et al., 19871. Although the two methods show good agreement at fasting (after sequestration of labeled C02 has been taken into account), the tracer approach shows a decreased oxidation a t maintenance, although urinary N losses are increased. This latter loss of N results because only a portion of the urinary urea eliminated is derived from metabolic deamination of amino

acids, whereas the remainder is produced from NH3 absorbed from the rumen (the combined elimination would thus lead to an overestimation of tissue protein Oxidation). The trends above maintenance are similar but quantitatively different for the same reason. The kinetic approach thus reveals that protein (amino acid) oxidation by ruminant tissues is high in the fasted condition, then declines as N equilibrium is established, followed by an elevation as intake is sustained above maintenance. This is similar to the pattern of leucine oxidation observed across the hindlimb(s1 of sheep (Figure 2). This alteration in rates of protein oxidation (and, inversely, amino acid utilization for protein gain) during chronic nutritional adaptation will provide the theme of the ensuing discussion.

Tracer approaches have also demonstrated that protein metabolism in ruminants, as in nonruminants, is dynamic. Even in fasted sheep (Pel1 et al., 1986) or cattle (Lobley et al., 19871, daily protein synthesis amounts to 10 to 18 g/kg.75, values that increase to 19 to 26 g/kg.75 at supramaintenance intakes (Lobley et al., 1987; Harris et al., 1992). At similar intakes these rates are greater for cattle than for sheep, and this may relate to the observation that the bovine has a higher metabolic rate (expressed per kilogram of BW,75) than the ovine under comparable nutritional and physiological conditions. Thus, for well-fed, 35-kg lambs or 500-kg steers with daily protein gains of 30 and 150 g, respectively, the corresponding minimum

9 Z a

20 00 80 60 40

20

0 0 1 1.6

Intake (x maintenance)

Figure 1. Comparison of urinary N elimination and protein oxidation assessed from whole-body leucine kinetics and 14C02 production. Isotope data corrected for sequestration of .8 for 14C02 label. Data means (+ SE) from Lobley et al. (1987). Shaded bars = urinary N; open bars = protein oxidation N.




3266 LOBLEY

whole-body synthesis rates would be 280 and 2,700 g/d. Small changes in either the rate of synthesis or of breakdown can therefore cause great alterations in the rate of gain. This creates a difficulty in that a 2% change in synthesis rate would alter protein accretion by 20 to 40%. Therefore, the precision of the kinetic methods becomes critical because many of the procedures are not accurate to within 4 to 5%.

Unfortunately, many of the kinetic data available are for animals maintained under relatively low intakes, often < 1.8 times maintenance. Although these would be comparable to European summer grazing intakes, they are not representative of the feedlot situation used extensively in North America. The relative, but not absolute, contribution, of “maintenance” N losses (Table 2) would decrease for intensive production systems. Information is urgently needed on the protein dynamics of ruminants with high feed intakes.

Basal (Submaintenance) Needs

Amino acids play many metabolic roles in addition to providing the precursors for protein deposition. Several of these roles (Figure 31 will be established even in the fasted condition, but, unfortunately, even though the biochemical pathways are well-established, the rates for most of the individual reactions are poorly quantified or unknown in ruminants. For example, although methionine and lysine are often considered to be first-limiting amino acids for ruminants dependent

a C 0

a J

.- a

500

400

300

200

100

0 0 .5 1 1.5

Intake (x maintenance)

Figure 2. Total leucine oxidation by peripheral tissues of sheep. Data means (* SE) adjusted from hindlimb studies, assuming muscle and fat tissue of these to be representative for the whole body. Calcu- lated from Pell et al. (1986) and Harris and Lobley (1991).

Table 2. Changes in the fractional rates (x 100) of protein growth (k,), protein synthesis (k,), and

protein degradation (kd) in peripheral tissues of sheep at varying intakesa

Intake (x maintenance) k, ks kd

0 .5

1 .o 1.2 1.5

-2.9 3.8 -.3 2.3

.4 3.0

.8 5.7

.7 5.8

6.7 2.7 2.6 5.1 4.9

*Hindlimb peripheral tissues include muscle, skin, bone, and fat. Data calculated from Pell et al. (1986) and Harris and Lobley (1991).

exclusively on ruminal microbial protein or fed high-corn diets, surprisingly little is known of their regulatory fates. Methionine, besides acting as a precursor for cysteine (necessary for fiber production and a major component of unrecovered gastrointestinal tract secretions), also is a key intermediate in the transmethylation reactions. These provide one-carbon moieties for phos- pholipid biosynthesis (for membrane structure), creatine production (for energy storage and transfers), and methylation of DNA bases (a mechanism of transcriptional control), which represent just a few of the hundred or more known reactions involving S-adenosylmethionine (SAM). In addition, the transsulfuration pathway of methionine catabolism also provides the precursor for polya- mines, which are key elements in cellular prolifer- ation. The flux of the methyl-group of methionine exceeds that of the remainder of the methionine carbons by three- to fivefold (see Lobley, 19911, with homocysteine as the immediate product of demethylation of SAM. Homocysteine is toxic and unless remethylated to methionine is removed by the transsulfuration pathway to cysteine. Because the ruminant has a low provision of methyl groups available from the diet (due to ruminal action on choline, etc.1, inadequacies in one-carbon substrates for remethylation may increase methionine oxidation. Supply of methyl groups or equivalent one-carbon structures by dietary or other means may therefore reduce methionine catabolism. This approach, which also involves the other amino acids concerned in the reactions shown in Figure 3, may provide means of conserving amino acids for protein anabolism. Currently, however, it is difficult to decide the contribution of particular requirements related to these pathways of amino acid metabolism and to predict how these will change under different nutritional and physiological conditions.

GZuconeogenesis. The most studied of these reactions involves gluconeogenesis. Under most conditions the ruminant animal has an obligate need to





carnitine nucleotides 7- 7-

GIuNH, Ala Glu GIuNH,

Lys\ t Z t f GIuNH, General

Z f

Met His Phe GIT WOOL

S-adomet 1 1 1 dopamine etc. nucleotides dipeptides

Thr 1 1 Cys

Met (Met) Cys

Figure 3. Some of the obligatory pathways of amino acid metabolism that result in net catabolism either of the specific amino acids involved or of those that remain and that will be in excess to potential gain. Lys, lysine; Glu NHz, glutamine; Ala, alanine; Glu, glutamate; Met, methionine; His, histidine; Phe, phenylalanine; S-adomet, S- adenosylmethionine; Thr, threonine; Cys, cysteine; GIT, gastrointestinal tract.

synthesize glucose and, although in the fed animal propionate is the predominant precursor (30 to 6 O % ) , other sources such as glycerol, lactate, and amino acids become more important in the fasted or underfed condition (Bergman et al., 1968; Lomax and Baird, 1983).

The gluconeogenic role of amino acids has been most clearly shown in ruminants maintained on a protein-free diet. For example, sheep and cattle sustained by total intragastric infusion that contained a mixture of VFA (Cz, Cs, and Cd) but no protein showed a 40 to 6 0 % lower daily urinary N loss compared with the 500 mg of N/kg75 eliminated by fasted ruminants (0rskov et al., 1983). This was probably due to the presence of the glucogenic precursor, propionate, rather than to a simple energy-sparing effect, because infusion of either acetate or lipid alone failed to improve fasting N loss in sheep, whereas glucose did (Asplund et al., 1985). Systematic studies with abomasal infusion of incremental quantities of glucose into fasted steers (Ku-Vera et al., 1989) revealed that the sparing was maximal at 300 g of glucose (4.2 g of g l u ~ o s e / k g . ~ ~ , equivalent to only 12% of maintenance energy requirement). This is comparable to normal glucose oxidation rates for maintenance-fed cattle (Head et al., 1964). The protein oxidation spared was equivalent to 130 g/ d, but because the gluconeogenic potential of animal body protein is 30 to 55 g of glucose/100 g of protein (Krebs, 1964; Barry and Manley, 19851, only 40 to 70 g of glucose (Le., only 11 to 25% of probable glucose oxidation; Head et al., 1964) could be supplied from the endogenously released amino acid. Because this would be equivalent to only 13 to 23% of the amount of glucose required

for maximal sparing and because N sparing was approximately linear for the first 300-g increments of glucose, the use of exogenous glucose to reduce protein catabolism was clearly not the overriding metabolic priority in these animals. Quantitatively similar data in response to glucose additions were obtained in sheep maintained near N and energy equilibrium by total intragastric infusion that contained no propionate (Girdler et al., 1986).

Such data have led to the suggestion that the involvement of protein in gluconeogenesis in ruminants is small (0rskov and MacLeod, 19901 and might be easily overcome by provision of other gluconeogenic precursors, such as propionate, as feed is offered. This is not supported, however, by data from the classic isotope studies of Bergman and colleagues, who demonstrated that 11 to 18% of glucose flux (approximately equivalent to 50 to 100% of glucose oxidation; see Lobley, 1991) could be attributed to synthesis from the major glucogenic amino acids, glutamate, glutamine, and alanine, with lesser contributions from aspartate, serine, and glycine (Wolff and Bergman, 1972; Heitmann and Bergman, 1978). The involvement of the gluconeogenic essential amino acids, such as threonine, seems to be relatively trivial (Egan et al., 1983). These tracer-based calculations involve several problems, however. First, the overall contributions of amino acids are likely to be underestimated because intracellular isotope dilution was not taken into account. Second, and as a counterbalance, isotopic exchange occurs within the tricarboxylic acid cycle (see, for example, Katz and Rognstad, 19761, such that tracer enters glucose but without represent- ing a net flow of carbon. Similarly, glucose carbon




3268 LOBLEY

can recycle to amino acids, and the resultant net flow are lower than those calculated from the unidirectional isotopic movements. Without cor- rection for these transfers apparent gluconeogenesis from amino acid carbon will be overestimated. The difficulty is that it is not known which of these kinetic problems dominates, and to what extent.

Other evidence does suggest, however, that synthesis of glucose may be an important fate of amino acid carbon. The original studies of Berg mann and colleagues were performed with adult sheep either fasted or fed to approximately maintenance, conditions under which glucose requirements would be minimal and, for the fed animal, under conditions in which provision of other glucose precursors would be expected to reduce synthesis from amino acids. Indeed, evidence from studies on ovine hepatocytes in vitro indicates that the absolute conversion of alanine and glutamine to glucose is approximately twofold greater in the fasted condition than in the fed state and, as a percentage of total glucose synthesis, nutritional deprivation causes a threefold increase in the contribution (Demigne et al., 1991). Even so, approximately 15 to 20% of glucose synthesis was from the two amino acids in hepatocytes from the well-fed lambs. Again, these data may involve overestimates of the net contribution of amino acid due to isotope exchange.

However, glucose “requirement,” as assessed from the quantity oxidized, does increase at intakes above maintenance and in different physiological states, such as pregnancy and lactation [see Lobley, 19911. The contribution from other gluconeogenic substrates also alters with substrate supply and physiological state (see Veen- huizen et al., 1988) but, interestingly, protein supply also alters glucose flux (Barry and Manley, 1985). This latter observation may represent “reverse” tracer dilution, but it also could indicate that gluconeogenesis from amino acids continues even under supramaintenance conditions. This would explain, in part, the low efficiencies (40 to 80%) with which incremental quantities of absorbed amino acids are utilized by ruminants. Continued contribution of net amino acid carbon to glucose synthesis in the well-fed ruminant is difficult to prove and requires complex tracer kinetic approaches such as those outlined by Katz and Rognstad (19781, Goebel et al. (19821, or Steinhour and Bauman (19861, but, on a physiological basis, glucose homeostasis is so vital to the survival of the animal that complete suppression of particular anabolic pathways could be catas- trophic.

The mechanisms underlying the control of gluconeogenesis are well-defined, and the gluca- gondnsulin ratio is critical, both in vivo (Brockman

et al., 1975; Heitmann et al., 1987) and in vitro (Gill et al., 1985). Glucagon directs nutrients, including amino acids, toward the liver, and the net mechanism involves stimulation of pyruvate carboxylase, which enhances oxaloacetate production and thus gluconeogenesis. This can be overridden if insulin concentrations are sufficiently great. For instance, a t reduced or zero intakes plasma insulin is low and net amino acid release occurs from peripheral tissues, particularly muscle (Heitmann and Berg- man, 1980); this changes to a net uptake as feed is increased, with concurrent elevation of insulin concentration.

Hormonal Effects on Protein Metabolism

Insulin. The effect of intake on hindlimb protein metabolism is biphasic (Table 21. Under fasting conditions, protein breakdown is elevated and net tissue mobilization occurs. As intake is increased there seems f i s t to be a sharp reduction in protein breakdown, with synthesis barely affected. Con- versely, as supramaintenance conditions are reached protein synthesis starts to increase, accompanied by a lesser elevation in breakdown. Insulin status ’is primarily regulated by energy supply (Russell et al., 1988; Abdul-Razzaq and Bickerstaffe, 1989; Reynolds and Tyrrell, 19911, although secretion of the hormone is also sensitive to amino acid concentration (Kuhara et al., 1991). Administration of exogenous hormone has been shown to lower plasma amino acid concentrations (Ahmed et al., 1983; Grizard, 1983; Prior and Smith, 1983); this is probably a consequence of peripheral tissue net uptake because insulin does not alter hepatic removal of amino acids (Brockman et al., 1975). The lower plasma concentration could be due to increased protein synthesis or to reduced breakdown, or to a combination of both. Attempts to show that insulin alters protein synthesis by ovine hindlimb tissues in vivo have not generally been successful (Oddy et al., 1987; Early et al., 1988a,b). Indeed, the study of Oddy et al. (19871, on preruminant lambs, was only able to demonstrate an anabolic flowered catabolic) effect of insulin in the fasted condition, when the mechanism involved a reduction in protein degradation. If such a system were also applicable to the ruminant animal, then this would perhaps account for the decline in protein breakdown observed between the fasted and maintenance condition (Table 21. Similarly, when undernourished lambs were refed, Crompton and Lomax (1987a,b) observed that the net anabolism achieved across the leg was due primarily to a decrease in protein breakdown, although a statistically significant relationship with insulin could not be established.




RUMINANT AMINO ACID

These observations seem, initially, to contrast with the situation in growing nonruminants, in which insulin stimulates muscle protein synthesis in the fasted condition and is implicated in the synthesis increases observed when feed is given (Garlick et al., 1983). In adult nonruminants, however, neither feeding nor insulin infusion alters muscle or whole-body protein synthesis, and, in some circumstances, reductions in protein breakdown have been invoked to explain the anabolic responses (Baillie et al., 1988; Melville et al., 1989). An apparently analogous situation exists with mature Merino sheep, for which both hindlimb and whole-body protein synthesis are unaltered between the fed and fasted condition, and thus the changes in net anabolism have been ascribed to alterations in protein degradation (Teleni et al., 1988).

The ruminant may therefore, when intake is low, have more similarities to the adult nonruminant than to the rapidly growing rodent, especially when the generally lower systemic insulin concentrations are also considered. Fur- thermore, the apparent inability of fed sheep to respond to exogenous insulin probably indicates that the concentration range over which the hormone exerts its protein anabolic action may be limited. Indeed, even in rats, Jepson et al., 1988) have suggested that insulin regulation of both protein synthesis and degradation may be r e s tricted primarily to plasma concentrations of the hormone c 25 mU/L. Presumably, increases in circulating concentrations above this relate more to control of carbohydrate and fat metabolism.

Growth Hormone and Insulin-Like Growth Factor I. In growing ruminants, it is well established that increases in feed intake lead to increases in both whole-body and muscle protein synthesis; this seems to be true of the neonate (Patureau-Mirand et al., 1988, 19901, the preruminant (Oddy et al., 19871, and the ruminant (Pell et al., 1986; Crompton and Lomax, 1987a; Lobley et al., 1987; Harris et al., 19891. However, because insulin does not seem to be a prime effector of protein synthesis, attention has focused on the growth hormone (GH)/insulin- like growth factor I (IGF-I) axis.

Exogenous administration of GH has been demonstrated in both sheep and cattle to stimulate protein synthesis, particularly in skeletal muscle. Pell and Bates (1987) observed increases in both the fractional and absolute rates of muscle protein synthesis in lambs, under controlled intake. Although there were differences in the degree of response between “ red and “white” muscles, in both cases the improvements in synthesis were greater than needed to sustain the extra net anabolism. In consequence, there were concomitant, but smaller, increases in protein

AND PROTEIN METABOLISM 3269

Table 3. Effect of exogenous growth hormone (GH) on protein synthesis (PS) and breakdown (PD) in

biceps femoris muscle from sheep and cattle

Protein metabolism, g/d GH Species status PS PD

~ ~ ~~~-

Sheepa -GH 1.44 1.25 +GH 2.10 1.84

+GH 9.7 4.9 Cattleb -GH 7.7 4.4

~~ ~

*From Pell and Bates (1987). bFrom Eisemann et al. (1989); values for PD calculated from

estimated rate of muscle accretion.

breakdown (Table 3). Similar effects can be deduced from the studies of Eisemann et al. (1989) in that both synthesis and degradation increased when steers were treated with GH (Table 3).

That part of this effect probably involves IGF-I is seen in the study of Douglas et al. (1991). Young, fasted lambs infused intravenously with recom- binant IGF-I for 5 h had higher fractional synthesis rates in liver and various muscles than did saline- treated animals. The increases exceeded the associated decrease in urea clearance but were only seen at higher IGF-I dose rates. These findings are in apparent contrast to those of Oddy et al. (1991), who observed that close arterial infusion of recom- binant IGF-I across the ovine hindleg caused a significant decline in protein breakdown (leading to an improvement in net gain). On a pro-rata basis a much larger quantity of IGF-I was infused locally in the latter study, and interactions with the insulin receptor may have occurred.

Pell and Bates (1990) proposed that systemic concentrations of IGF-I mediate the growth response to diet, but in practice the IGF-I response itself may involve direct interactions with protein supply. Thus, the amount (and perhaps quality) of absorbed amino acids may determine IGF-I status (Nam et al., 1990; MacRae et al., 1991; Kriel et al., 1992). In conjunction with this is the finding that GH does not produce a sustained response in N retention when sheep receive a low protein input, even though both GH and IGF-I concentrations are maintained above those observed in normal physiological conditions (MacRae et al., 1991). This seems similar to the situation with pigs in which anabolic responses to exogenous GH administration require a minimum of 14 to 16% CP in the diet (Smith et al., 19891. Clearly, the correlation between IGF-I and protein supply has an effective range physiologically, but responses may not be observed outside this range; again, this has analo- gies to the insulin situation.

It is thus possible to create a scheme (Figure 41 to explain the observed nutritional responses in




3270 LOBLEY

I insulin 4 I

IGF-I +

‘in vivo‘ intake -,

Figure 4. Hypothetical scheme for the action of insulin and insulin-like growth factor I (IGF-I) on ovine muscle protein metabolism. The action of insulin is most pronounced at low concentrations with protein degradation (PD) most influenced, whereas IGF-I operates only at higher (protein) intakes with simulation of both protein synthesis (PS) and degradation. Combined effects produce the biphasic responses observed in vivo.

protein metabolism based on the separate but overlapping actions of insulin and IGF-I. At lower intakes, and regulated primarily by energy supply, insulin reduces net protein catabolism by reduction of protein degradation with only marginal effects on protein synthesis. As intake is increased, so is amino acid absorption; this triggers synthesis and release of IGF-I, which leads to a stimulation of protein synthesis (and to a lesser increase in protein degradation). This seems to be the situation in skeletal muscle, whereas the anabolic mechanisms in other ruminant tissues are obscured by high turnover rates (Eisemann et al., 1989). This is a very simple hypothesis and does not take into account the actions of other hormones such as the glucocorticoids, those thyroid- derived, the catecholamines, and the sex steroids. Because these hormones are affected primarily by factors other than intake they may act as media- tors of the main effects rather than as major regulators of nutritional responses.

Regulation of Branched-Chain Amino Acid Oxidation

Increases in the rate of protein deposition, whether controlled by changes in the synthetic or degradative pathways, must be accompanied by decreases in amino acid oxidation, provided that the absorption of amino acidk) has remained unaltered by the anabolic stimulus. One question that naturally follows from this is whether amino acid catabolism is a mechanism by which amino

acids excess to requirement are removed (i.e., a “passive” response) or whether the oxidation is actively regulated to allow specific quantities of the amino acid to be channeled toward protein deposition. Regardless of which operates, and both may for different amino acids or under varying physiological conditions, it is important to determine the controls involved. Despite this need, the regulatory aspects of amino acid metabolism have been poorly studied in ruminants and researchers have relied mainly on studies in vitro. The excep- tion to this involves the branched-chain amino acids (BCAA), especially leucine. Even here it is necessary to draw on findings from nonruminant studies to determine the overall picture.

Relative to other amino acids, a greater proportion of absorbed BCAA (approximately 50%) es- cape frst-pass removal by the liver (Heitmann and Bergman, 1980). Again, whereas other amino acids are oxidized mainly by hepatic and renal tissues, the catabolism of the BCAA is divided between hepatic (see Harper et al., 1984; Pell et al., 1986) and peripheral tissues, particularly muscle and fat (Goodwin et al., 1987; Bergen et al., 19881. The resistance to hepatic clearance and the peripheral tissue oxidation are probably linked and may form part of a complex regulatory loop. One notable feature of BCAA catabolism concerns the regulation of the enzyme catalizing the first irreversible oxidative decarboxylation step. The activity of this enzyme, the branched-chain oxo-acid dehydrogenase (BCOADH), is determined by its phosphorylation status; the dephosphorylated form is active and the phosphorylated form inac- tive (see Randle et al., 1984). Separate kinases and phosphatases determine the status. Phosphoryla- tion procedures are widely used in cellular regula-

c 0 (II ‘E 40- E g 30-

20- 0 * 10- if?

0 - - milk-fed lambs adult adult adult -SheeP - +Man+

Figure 5. Fraction of total leucine oxidation attributa- ble to peripheral tissues in sheep and humans under various nutritional conditions. Data calculated from Cheng et al. (1985, 1987), Pell et al. (19861, Teleni et al. (1986), Oddy et al. (1987), and Harris and Lobley (1991). Shaded bars - low intake (0 to .5 x maintenance); open bars - high intake ( > maintenance).





Table 4. Effect of exogenous insulin (I) on rates (Fmol. min-' 3 kg-' of tissue) of leucine deamination,

reamination, and decarboxylation across the hindleg of preruminant lambsa

Fastedb Fed Leucine transfers -I +I -I +I

Reamination 3.90 .88 1.32 1.21 Deamination 5.67 2.10 3.44 3.32

Decarboxylation 1.05 .69 1.75 1.83

*Data from Oddy et al., 1987. b+I and -I represent with and without exogenous insulin

administration, respectively.

tion (see Reeds, 1987) and offer a very rapid and sensitive means of control, because there is no need to rely either on the comparatively slow induction of new enzyme protein synthesis or on the relatively insensitive response to small changes in substrate concentration, based on classical Michaelis-Menten kinetics.

In nonruminant species many of the modulators of BCOADH activity have been established; increases in either the BCAA or the respective oxo- acids concentrations elevate the activity of the enzyme. Similarly, low intracellular concentration of ATP causes activation (presumably to potentially stimulate amino acid catabolism to produce more energy), as do exercise (perhaps for a similar reason) and excess glucocorticoids, which lead to a net catabolic state with reduced protein deposition and elevated amino acid oxidation (Block et al., 1987a,b). Inactivation is caused by low leucine concentrations, acetoacetyl-coenzyme A (a later product of the degradative pathway), high ATP, and insulin (Aftring et al., 1986; Hutson, 1986). Circumstantial evidence of similar regulation in vivo for ruminants has been obtained for insulin and leucine concentration. The close arterial infusion of insulin in the studies of Oddy et sl. (1987) resulted in a decrease in the rate of decarboxylation in the fasted condition, with associated decline in the rates of leucine deamination and reamination. No effect of additional insulin was observed in the fed condition, even though higher rates of decarboxylation were observed (Table 41. Similar observations were reported by Harris et al. (19921, who found that raising intake from .8 times maintenance to 1.8 times maintenance increased circulating leucine (118 vs 189 pill, P < .011 and both whole-body (.59 vs 1.71 mmol/h; P e .001) and hindlimb (18 vs 70 p o l / h , P < . O l ) leucine oxidation, under conditions in which circulating insulin concentrations were increased. The involvement of the hormone in suppression of the BCOADH activity therefore seems to be specific at either low insulin status or

when nutrient supply is limiting. This shows a (coordinated?) similarity to the effects on protein degradation.

The proportion of total leucine catabolism ascribed to peripheral tissue metabolism may be relatively constant in ruminants (Figure 51, because a value of 35 to 45% can be calculated from studies that encompass both fed and fasted animals in a variety of developmental states, from the preruminant to the mature adult. Interest- ingly, such constancy does not apply to adult humans (Figure 51, which indicates that the impor- tance of the regulation may differ between nonruminants and ruminants (including preruminants). Certainly the degree of control exerted in peripheral tissues is considerable. Based on the total enzyme activity present in ruminant muscle and fat (from Goodwin et al., 1987; Bergen et al., 19881 and the amount of oxidation that occurs across the hindleg (Harris et al., 19921, < 6% of maximal activity is achieved (Table 5). This should be compared with the 0 to 27% of potential activity expressed by rat muscle (Hutson, 19861. Similarly, liver activity is also tightly regulated, such that e 5% of maximal activity is expressed (Table 51; this should be compared with suggestions that in rodent liver the enzyme is permanently, and nearly completely, activated (Harper et al., 1984). With such low

Table 5. Extent of regulation in vivo of leucine metabolism (pmol . min-' . kg-' of tissue] in various

ovine tissues in comparison with catabolic enzyme activitiesa

Enzyme activitiesb

"In vivo" Tissue and statee BCAAT TotaYc "Actual"c activityd

BCOADH

Liver 2.7 F 11 390

S 9 to 20 93 4 to 91 1.5

F 8 to 12 18 .5 to 9 S 4 to 15 19 .7 to 8

8 to 290

Muscle

F .3 to .6 s .1 to .4 I Fat

F 6 N D ~ .3 S 1 to 3 ND .3

*Data calculated from Pel1 et al. (19861, Goodwin et al. (19871, Bergen et al. (19881, and Harris and Lobley (1991).

bBCAAT = branched-chain amino acid transferase; BCOADH = branched-chain oxo-acid dehydrogenase.

C"Actual" refers to enzyme activities measured under maximal conditions in vitro and representative of the amount of dephosphorylated form isolated. "Total" refers to activity in vitro when all enzyme dephosphorylated.

d'In vivo" estimates based on kinetics across hindlimb tissues, which include muscle and fat.

eF = fed; S starved. fND = not determined.




3272 LOBLEY

expression of enzyme activity it is difficult to decide from studies in vivo whether the major oxidative site is muscle or adipose tissue. Although the former has the greater amount of enzyme capacity (e.g., 75% of the ovine hindlimb), there is still more than fivefold the amount of activity needed in the fat cells alone. Based on total activity in both muscle and fat the BCAA transaminase would be limiting, theoretically, before the BCOADH (Goodwin et al., 1987; Papet et al., 19881, but, in practical circumstances, the latter acts as the major rate-limiter, although suggestions have been made that the transaminase may be more significant in controlling amino acid catabolism in the preruminant Papet et al., 19881.

Do these peculiarities of BCAA metabolism indicate that these amino acids play important roles in the regulation of overall amino acid and protein metabolism? At present the answer to that is probably a speculative “yes,” based on a series of observations. First, the BCAA, especially leucine, can act as insulin secretagogues (Lobley et al., 1990; Kuhara et al., 1991). This action may be direct, or indirect via their oxo-acids, on the pancreas (Blachier et al., 1989). As already seen, insulin has anabolic properties, especially at lower intakes, and interacts in regulation of BCAA oxidation. The BCAA may therefore form part of a self-regulated feedback loop involving partial control of insulin status. The situation may become more complex if the BCAA also act as positive modulators of insulin sensitivity, as has been observed for muscle in fasted rodents (Garlick and Grant, 1988).

Second, a direct role for leucine as a positive regulator of protein synthesis has been postulated. The evidence for this is somewhat controversial but, interestingly, the situations in which responses have been observed relate to either conditions of limited nutrition or systems in vitro (Buse and Weigand, 1977; Buse et al., 1970; Smith, 1985). Effects are not obvious in well-nourished situations (McNurlan et al., 19821, although effects on ovine muscle protein synthesis have been reported by Schaefer et al. (1988). Administration of a large amount of the oxo-acid of leucine provoked a substantial increase in lamb growth (+25%; Ostaszewski et al., 19911, and this may relate to observations that the metabolite may reduce protein breakdown (Goldberg and Tischler, 19811, although direct confirmatory evidence is still needed.

Third, the B C M may act as donors (or at least intermediate donors) for glutamine synthesis. Glutamine production in peripheral tissues not only provides a substrate for gastrointestinal tract metabolism (see Lobley, 1991), but it also has been suggested as a putative regulator of muscle protein breakdown (Rennie et al., 19901. In the

latter instance the concentration of leucine ad consequent regulation of BCOADH activity, acting as a feedback system on the transaminase, could act as signals for muscle protein breakdown via rates of synthesis and accretion of the intracellular glutamine pool. Alternatively, the glutamine concentration may simply reflect the nutritional or hormonal status of the animal (again with the BCAA as potential key intermediates). Thus, low levels of muscle glutamine may be a consequence of net catabolism of the tissue rather than a direct regulator.

Fourth, the BCAA may act as interorgan signals. Clearly, mechanisms must exist whereby integration is achieved between the protein anabolic demands, or the protein catabolic supply, of the peripheral tissues and the regulatory control exerted by the liver. The division of catabolic fates between peripheral and hepatic tissues for the BCAA make these attractive candidates, although the mechanisms remain obscure. The modulation of hormone activity and(or1 sensitivity (Garlick and Grant, 1988) by amino acids offer one such system, although others probably also operate.

The metabolism of the BCAA is an example of a tightly regulated system, potentially sensitive to the needs of the animal. Other areas of amino acid catabolism may be less controlled. Recently, for example, the extent of hepatic removal of arnmo- nia has been correlated with increased liver uptake of either a-amino N (Reynolds et al., 1991) or free amino acids (Maltby et al., 1991). The fate of the extracted amino acids is unclear, because in one study incremental urea N production exceeded ammonia N removal and therefore additional sources of N were necessary to account for the balance meynolds et al., 1991). In contrast, in the other report although the basal secretion of urea N from the liver was greater than ammonia N supply, the incremental changes in urea N and ammonia N matched, so that additional metabolism of other N sources was not required (Maltby et al., 1991). Tracer studies are needed to determine whether amino acid N contributes to ureogenesis as ammonia absorption increases. If this is the case, then manipulations at the rumen level to reduce N losses as ammonia would have the double advantage of potentially increasing productive conversion of dietary N to ruminal microbial protein and reducing the catabolic lqsses of the absorbed amino acids.

Future Directions

Although during the past two decades a variety of exogenous manipulations (anabolic steroids, p- agonists, growth hormone) have all been used to improve rates of protein deposition and thus, by definition, the efficiency of utilization of absorbed





amino acids, these have only made limited inroads into the potential for this improvement. Indeed, in the case of growth hormone, real responses may not be observed unless additional inputs of protein are available. Although it could be argued that these introductions have made commercial sense with marginal improvement in the usage of natural resources, the effects are limited, especially when coupled with increasing consumer resistance to “artificial” manipulations. Possible acceptable alternatives involve use of immuno- manipulation of hormone and growth factor status or the use of molecular biology techniques either to screen embryos for particular genetic traits (“accelerated” natural selection) or perhaps by transgenic manipulations. In all these instances, however, the target sites and metabolic consequences need to be identified. The aim, therefore, in ruminant metabolism is to identify those reactions where the major losses occur.

Because much of the overall loss is associated with achievement of N equilibrium, this should be an area of major emphasis. In terms of secretory losses the gastrointestinal tract and fiber production are major areas of investigation (see MacRae and Lobley, 1991). The question of whether there is a net loss of amino acid carbon for gluconeogenesis needs to be resolved. Similarly, losses to glutamine production and the other specific mechanisms need to be identified. Specific areas might include the role of methylation reactions in the regulation of methionine metabolism (see Lobley, 1991) and also the fates of lysine metabo- lites, because these two amino acids are often considered limiting under low nutrient inputs (Storm and 0rskov, 1984). The nature of efficient use of amino acids for net gain also should be studied with emphasis on the regulatory role of the endogenous hormones. In particular, we need to identify the concentrations over which insulin has an effect on protein metabolism and to identify whether the mechanism operates via protein degradation and how this affects other tissues in the body. Again, the role of endogenous IGF-I needs clarification, especially if this is limited by protein availability, a factor that will need to be taken into account in experimental designs. We have a range of powerful potential manipulatory techniques available to the animal scientist, but to optimize their use and at the same time allay public unease at their introduction and application a good understanding of their targets and effects is vital.

Implications

Within the next decade will come the potential to increase by 25 to 100% the efficiency with which ruminants convert feed nitrogen into animal pro-

tein. Part of this improvement will involve strate- gies to reduce “obligate” catabolism associated with maintenance of nitrogen equilibrium. Further gains will accrue from a better understanding of the hormonal and substrate regulation of protein metabolism and retention, which will allow new, but acceptable (by consumers), nutritional, en- docrinological, and genetic approaches to be adopted.

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