the metabolic fates of amino acids and the formation of ... · the only amino acids that can be...

THE JOURNAL OP Bmmrc~~ CHEMISTRY Vol. 253, No. 10, Issue of May 25, pp. 3685-3695, 1978

Prmted in U.S.A.

The Metabolic Fates of Amino Acids and the Formation of Glutamine in Skeletal Muscle*

(Received for publication, October 6, 1977)

TSE WEN CHANGS AND ALFRED L. GOLDBERG§

From the Department OfPhysiology, Harvard Medical School, Boston, Massachusetts 02115

These studies have examined whether skeletal muscle during fasting uses amino acids derived from muscle protein breakdown as an energy source or as precursors in the synthesis of glutamine and alanine. Diaphragms from fasting rats were incubated in vitro to study the fates of aspartate, asparagine, glutamate, isoleucine, and valine, the only amino acids that can be converted to tricarboxylic acid cycle intermediates in muscle.

Although these five amino acids comprise about 28% of the residues in diaphragm protein, they accounted for only about 14% of the amino acids released into the medium. The fraction (50%) of these amino acids generated by protein breakdown but not released by the tissue may be accounted for by conversion to glutamine. Although glutamine com- prises only about 6% of the amino acids in muscle protein, it accounted for about 25% of the amino acids released. This additional amount of glutamine was equivalent to the miss- ing amount of aspartic acid, asparagine, glutamic acid, isoleucine, and valine.

Diaphragms were incubated with L-[UJ4C]valine, L-[U-

“Claspartate, and 12,3-‘YJsuccinate, and the metabolites released into the medium were resolved by high voltage electrophoresis. In all cases, the radioactivity recovered in glutamine and glutamate was twice that in lactate and pyruvate, and 30 times that in alanine. The measured rate of lactate and pyruvate release was 1.5 times that of pyruvate oxidation, and 18 times that of alanine release. Thus, more than 50% of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine entering the tricarboxylic acid cycle appear to be converted to glutamine, while the remainder are converted via pyruvate to CO, for energy (less than 20%). or to lactate (less than 30%) and alanine (less than 2%). which are released from the muscle.

These experiments together indicate: 1) isoleucine and valine are not oxidized completely to CO, in skeletal muscle, 2) the carbon chain of alanine is not derived from other

* This work was supported by grants from the National Institute of Neurological Disease and Stroke, National Aeronautics and Space Administration, and Muscular Dystrophy Association of America. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

f Present address, Center for Cancer Research, Massachusetts Institute of Technology, Cambridge, Mass. 02139

P Recipient of a Research Career Development Award from the Nationai Institute of Neurological Disease and Stroke

amino acids and 3) aspartic acid, asparagine, glutamic acid, isoleucine and valine derived from muscle protein are mainly converted to glutamine prior to release from muscle. And this process may be an important step in gluconeogenesis from muscle protein.

The rate of oxidation of acetyl groups by diaphragms from fasting rats incubated only with glucose was calculated from the rate of OZ consumption. Amino acids derived from the breakdown of muscle protein contributed about .5% of the acetyl groups oxidized in the tissue, most of which were contributed by leucine. When leucine is provided exogenously, it can contribute a greater fraction of the acetyl-CoA oxidized.

It is generally believed that skeletal muscles oxidize some amino acids, such as the branched-chain amino acids, to CO, (l-6). The major evidence for this view is that W-labeled branched-chain amino acids as well as aspartate and glutamate can be converted to 14C0, in hepatectomized animals (7) and in isolated skeletal muscle (8-11). However, as pointed out in the preceding report (121, the production of “YJOy does not necessarily indicate that the substrates have undergone

net degradation to CO,, because WO, can be generated from 14C-labeled precursors during rearrangement of their carbon chains in the tricarboxylic acid cycle. Therefore, it is still unclear whether skeletal muscles oxidize isoleucine, valine, aspartate, or glutamate to CO, for metabolic energy. Unlike these amino acids, leucine is not a precursor for tricarboxylic acid cycle intermediates, but is degraded to acetyl groups, which can be oxidized directly in the tricarboxylic acid cycle to provide energy.

Skeletal muscles have been reported to release large amounts of alanine and glutamine both in uivo (13, 14) and in vitro (X-17). These two amino acids together may account for 35 to 60% of all the amino acids released by the tissue, even though they comprise only about 10 to 12% of the residues in muscle protein (15, 18). A variety of experiments have indicated that the additional alanine and glutamine are synthesized de novo in the muscle (17, 19).

The preceding paper (12) showed that both in viuo and in

vitro nearly all of the pyruvate for alanine synthesis is derived from exogenous glucose rather than from the amino acids generated by net protein breakdown. These findings are consistent with the original formulation of the “glucose-alanine cycle” (13, 20) and do not support the recent alternative suggestions that amino acids derived from muscle protein

3685

3686 Amino Acid Degradation and Glutamine Formation in Muscle

provide the carbon skeleton for alanine (16, 21, 22). The origin of the carbon skeleton used for the de nouo

synthesis of glutamine has not been determined. Ruder-man and Berger (17) suggested that aspartate, arginine, histidine, and proline generated from net protein breakdown provide the carbon skeleton for glutamine, while Garber et al. (16, 22) suggested that cysteine, leucine, valine, methionine, isoleu- tine, tyrosine, lysine, and phenylalanine play such a role. These workers reported that addition of these amino acids stimulated glutamine production, but such findings need not indicate conversion of the carbon chains of these amino acids into glutamine (12). Furthermore, skeletal muscle lacks pathways for converting most of these amino acids into tricarboxylic acid cycle intermediates and thus to glutamine (12). By contrast, Krebs (23) suggested that glucose is the major source of carbons for glutamine. He reasoned that the breakdown of muscle protein cannot supply sufficient glutamate for synthesis of glutamine, but did not consider the possible conversion of other amino acids into glutamate.

PEP ,/2pyr”vo+e~ CO2

MOIIC Acetyl CoA CO2 enzyme

PEP

‘7);

\ k/--- _

corbowykmase Oxaloocetate L

f Gtrate

/ 2 / \

The purpose of the present studies was to obtain quantitative answers to the following questions: 1) What are the metabolic fates of the carbon skeletons of aspartic acid, asparagine, glutamic acid, isoleucine, and valine that enter the tricarboxylic acid cycle in skeletal muscle? 2) What fraction of these carbon skeletons are converted to pyruvate for further metabolism? 3) Are these carbons oxidized to CO, and to what extent does this process provide energy to the muscle? 41 What is the origin of the carbon skeletons for the cte novo synthesis of glutamine?

Rationale - In order for an intermediate in the tricarboxylic acid cycle or a precursor of such intermediates (i.e. aspartic acid, asparagine, glutamic acid, isoleucine, valine) to be oxidized to CO, for energy, it must first be converted to pyruvate, which can then be oxidized to acetyl-CoA (24). This conversion to pyruvate requires the enzymes P-enolpyruvate carboxykinase, which catalyzes the conversion of oxaloacetate to P-enolpyruvate, or malic enzyme, which catalyzes the conversion of malate to pyruvate (Fig. 1). In fact, Garber et al. (16) and Goldstein and Newsholme (21) suggested that in muscle these enzymes are involved in the conversion of amino acid skeletons into alanine.

The issue of the metabolic fates of the carbon chains of these amino acids entering the tricarboxylic acid cycle is intimately related to the question of the origin of the carbons for the de nova synthesis of glutamine. This latter process must consume a-ketoglutarate and consequently other tricarboxylic acid cycle intermediates. Therefore, identifying the origin of the carbons in glutamine is essentially the same problem as defin- ing the processes which maintain the pool of tricarboxylic acid cycle intermediates in the course of continual glutamine production. Aspartic acid, asparagine, glutamic acid, isoleucine, and valine derived from net protein breakdown are obvious candidates for precursors of glutamine, because a fraction of their carbon chains are converted to tricarboxylic acid cycle intermediates. Alternatively, glucose may provide the carbons for glutamine (23). The conversion of P-enolpyruvate or pyruvate to tricarboxylic acid cycle intermediates must also occur in skeletal muscle, since in incubated rat diaphragm, ‘%O, can be incorporated into aspartate and glutamate residues of protein (25). However, this result does not necessarily indicate that the direction of the net flow of carbons is from P- enolpyruvate or pyruvate to tricarboxylic acid cycle intermediates rather than in the reverse direction.

The crucial point in resolving the metabolic fates of aspartic

Ii’ \ i

Malate TRICARBOXYLIC ACID lxmtrate

r CYCLE I

\

S”cckte 9 / co k

\ cd- Keto$utarate2 \ /

;uccmyl CoA Y ’ J

co2

FIG. 1. Pathways for the complete degradation of tricarboxylic acid cycle intermediates. Intermediates in the tricarboxylic acid cycle act as cofactors for the oxidation of acetyl groups to CO, and do not themselves undergo net degradation in the process. In order for a tricarboxylic acid cycle intermediate to be oxidized to CO,, it must leave the cycle and be converted to pyruvate, and then to acetyl CoA. PEP, phosphoenolpyruvate.

acid, asparagine, glutamic acid, isoleucine, and valine, and the origin of glutamine is to determine the direction of the net flow of carbon chains between tricarboxylic acid cycle intermediates and pyruvate and P-enolpyruvate. If the direction of the net flow of carbon chains is from the pools of tricarboxylic acid cycle intermediates to pyruvate or P-enolpyruvate (Fig. 2A), glucose does not contribute to the carbon chains of glutamine. In addition, a fraction of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine must be converted to pyruvate for further metabolism, and these five amino acids would be the major source of carbons for the de novo synthesis of glutamine. Conversely, if there is a net flow of carbons from P-enolpyruvate to the pools of tricarboxylic acid cycle intermediates (Fig. 2B), glucose must supply carbon chains for glutamine synthesis. Accord- ingly, aspartic acid, asparagine, glutamic acid, isoleucine, and valine would not be converted to pyruvate, and all the carbon chains of these amino acids entering the tricarboxylic acid cycle would be converted to glutamine. In other words, glutamine would be the primary end product of the metabolism of these five amino acids in the muscle, and the pyruvate for the de nova synthesis of alanine would be provided entirely by glucose.

Determination of the direction and the flux of carbon chains between the tricarboxylic acid cycle intermediates and the P- enolpyruvate and pyruvate pools is difficult. This problem was approached by 1) comparing the proportions of aspartic acid, asparagine, glutamic acid, isoleucine, and valine together, and of glutamine in muscle protein and in the amino acids released into medium, 2) comparing the rates of aspartic acid, asparagine, glutamic acid, isoleucine, and valine from protein breakdown, the rates of their metabolism in the muscle, and the rate of glutamine production, 3) tracing the fates of ‘“C- labeled aspartate, valine, and succinate (a tricarboxylic acid cycle intermediate), and 4) measuring the incorporation of W02 into glutamine to examine the magnitude of the conversion of P-enolpyruvate or pyruvate to tricarboxylic acid cycle intermediate (see Fig. 2B).

MATERIALS AND METHODS

Procedures and Assays - The maintenance of rats, the dissection

Amino Acid Degradation and Glutamine Formation in Muscle 3687

A PATHWAYS

0

:Kz /i, ZE h

‘I” \.~_/ 6,”

FIG. 2. Fates of amino acids entering the tricarboxylic acid cycle and the origin of glutamine in skeletal muscle. The carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine may be converted to either pyruvate for further oxidation and for the synthesis of alanine and lactate or glutamine. The fraction of the carbon chains of these five amino acids incorporated into glutamine depends on the relative rates of glutamine synthesis and of conversion of these five amino acids into tricarboxylic acid cycle intermediates. Two possibilities exist. A, the carbon chains of the five amino acids provide all the carbon chains for glutamine synthesis, and some remaining carbon chains are converted to pyruvate. B, the carbon chains of the five amino acids cannot provide sufficient carbon skeletons for glutamine synthesis. Under these conditions, glutamine must be the primary end product of the metabolism of these amino acids. Glucose presumably supplies the additional carbons for the synthesis of glutamine. PEP, P-enolpyruvate.

and incubation of the diaphragms, and the determination of glutamate, glutamine, alanine, and tyrosine were carried out as described in the preceding paper (12). Lactate and pyruvate were determined spectrophotometrically with lactic dehydrogenase (EC 1.1.1.27, Sigma Chemical Co.) (26, 27).

Determination ofRate ofPyruoate Oxidation- The rate of conversion of pyruvate to acetyl-CoA by pyruvic dehydrogenase was estimated by determining the rate of conversion of n-[3,4,‘4Clglucose to “CO, and by determining the specific radioactivity of the intracellular pyruvate. Because skeletal muscles lack hexose monophos- phate shunt activity (281, the “‘C at the 3 and 4 positions of glucose will appear at the 1 position of lactate or pyruvate as a result of glycolysis. Therefore, “C0, must be produced only from the oxidation of pyruvate to acetyl-CoA. It was thus assumed that the specific radioactivity of intramitochondrial pyruvate was equal to that of lactate released into medium. Because glycolysis, the primary source of pyruvate (121, occurs in the cytoplasm, intramitochondrial pyruvate must be derived from the cytoplasmic pool. Since cytoplasmic pyruvate is also a precursor of the lactate released into the medium, the specific radioactivity of the released lactate should be equal to that of intramitochondrial pyruvate. Lactate in the medium was measured instead of intracellular pyruvate, because the radioactivity in pyruvate recovered either in the tissue or in the medium was too low to be measured accurately.

Before use, the n-[3,4-L4C]glucose (New England Nuclear Chemi- cal Corp.) was evaporated to dryness to remove ethanol. At the end of incubation, 14C0, was collected and counted as described previously (9, 121. Radioactivity in [1-14Cllactate was determined by oxidizing the lactate to pyruvate and measuring the [l-14Clpyruvate formed by treatment with peroxide, which decarboxylates a-keto acids, including pyruvate (29). The increase in i4C0, upon treatment with lactic dehydrogenase (26) was taken as the 14C in lactate. One 0.5-ml sample of the medium was treated with lactic dehydrogenase. To this sample and a similar one not treated with lactic dehydrogenase were added 1.0 pmol of sodium pyruvate as a carrierand then sufficient 2 N HCl to reduce the pH below 2.0. The flasks were then closed with rubber stoppers and 30% hydrogen peroxide was injected through the stoppers to give a final peroxide concentration of 5%. The 14C0, produced was collected and determined (9).

Metabolic Fates of ‘YXabeled Precursors - Hemidiaphragms from rats fasted for 2 days were incubated with [2,3-14Clsuccinate, L-[U- 14Claspartate, or L-[U-“CJvaline (New England Nuclear) at 0.1 mM,

and 1.5 &i/ml or 25 Cilmol. At the end of incubations, tissues were removed, and the media were heated in a boiling water bath for 3 min to inactivate enzymes that may have leaked from the tissue. The precipitates were removed by centrifugation at 1000 x g for 10 min. To 2 ml of the supernatant was added 0.25 ml of glacial acetic acid in order to expel “CO, produced during incubation. After 1 h at room temperature in a hood, the solutions were lyophilized. The residue was dissolved in 0.2 ml of distilled water. 20-~1 samples were applied to Whatman filter paper and analyzed by high voltage electrophoresis (Savant Instruments, Inc.). Most amino acids were resolved using pH 1.9 buffer system (2% formic acid, 8% acetic acid). Lactate and pyruvate were separated from other metabolites at pH 6.4 (pyridine/acetic acid/water, v/v, 100:3:879). For experiments with [U-“Claspartate, the glutamine spot on the electrophoretogram from the pH 1.9 run was cut out and sealed to another piece of paper and electrophoresed again at pH 6.4. This step was necessary because the glutamine spot was too close to the spot of aspartate, the precursor. After the electrophoretogram was dried, individual strips were cut into 0.5-cm wide pieces to count for i4C. To identify the spots, we ran known amino acid standards adjacent to the samples. Since ninhydrin decarboxylates amino acids, this reagent was used only to locate spots. For measurements of 14C0, production from ‘V- labeled succinate, aspartate, and valine, these precursors were added at a specific radioactivity of 0.03 &i/ml or 0.3 Ciimol. “CO, was collected as described previously (91.

Incorporation of ‘CO2 into Glutamine - Hemidiaphragms were preincubated in 3 ml of Krebs-Ringer bicarbonate buffer containing 5 mM glucose as described for other experiments. After a 30.min preincubation, the tissues were transferred to flasks containing the same medium, and gassed with O,/CO, (95:5). Then 50 PCi of Na, ‘VO:, were added. At the end of incubation (2 hl, the tissues were removed. 2 ml of the medium were acidified with 0.5 ml of acetic acid, allowed to stand for 1 h to drive off ‘CO,, and then lyophilized. The 25.ml flask contained 75 pmol of CO, within the 3 ml of Krebs- Ringer bicarbonate buffer, and 45 pmol of CO, in the gas space (95% 0,:5% COL). Based on these values, the approximate specific activity of ‘VO, in the medium was calculated from these values. 14C in various metabolites were measured as described above.’

Determination of Rate of Acetyl Group Oxidation in Tricarboxylic Acid Cycle- We have described a new relationship between the rates of acetyl group oxidation in the tricarboxylic acid cycle and the 0, consumption (30). The ratio of these two rates was shown to be independent of the substrates being oxidized:

Acetyl group oxidation = 0, consumption x (0.34 + 0.01)

The rate of acetyl group oxidation in the diaphragm was estimated from 0, consumption. To meaure 0, consumption, hemidiaphragms were incubated in 5.0 ml of Krebs-Ringer bicarbonate buffer containing 5 rnM glucose in the chamber of the oxygen monitor (model 53 Biological Oxygen Monitor, Yellow Spring Instruments Co.). The incubation chamber were bubbled with O&O, (95/5) prior to record- ing 0, content in the chambers using an oxygen electrode.

RESULTS

Rates of Generation of Aspartic Acid, Asparagine, Glu- tamic Acid, Isoleucine, and Valine from Muscle Proteolysis and Formation of Glutamine - Together aspartic acid, asparagine, glutamic acid, isoleucine, and valine accounted for about 28% of the amino acids in proteins of diaphragms from rats fasted for 2 days. However, these residues comprised only 14% of the amino acids released by these muscles during in vitro incubation (Table I). In contrast, glutamine accounts for about 6% of the residues in muscle protein (181, but for approximately 25% of the amino acids released (Table I). The proportion of alanine was also higher in the released amino acids than was expected from the composition of muscle protein, but this difference (about 2-fold) was less dramatic than that for glutamine (Table I). These results are very similar to earlier findings on diaphragms from fed rats (15).

During fasting, muscle proteins, including those of myofi- brils, undergo net breakdown (31). The myofibrillar proteins comprise 60 to 80% of the total tissue protein (32) and contain similar proportions of alanine, glutamine, and aspartic acid, asparagine, glutamic acid, isoleucine, and valine as total


TABLE I TABLE II

Comparison of amino acid composition of rat diaphragm proteins and amuzo acids released into medium

Hemidiaphragms from six rats fasted for 2 days were homogenized in 5% sulfosalicylic acid. After homogenization, six of these hemidiaphragms were mixed, the supernatant solution was removed after centrifugation, and the pellet washed twice with 5% sulfosalicylic acid. A portion of the pellet was placed on filter paper and dried in a desiccator. 2.0 mg of the dried pellet was hydrolyzed with HCl and the products analyzed on a Beckman amino acid analyzer. The remaining hemidiaphragms from these animals were preincubated for 30 min in Krebs-Ringer bicarbonate buffer containing 5 rnM glucose. They were then transferred to flasks containing the same medium and incubated for 2 h. At the end of incubation, the medium was placed in boiling water for 2 min. After the precipitate was removed by centrifugation at 1000 x g, l-ml samples from all incubations (6 ml in total) were mixed and lyophilized. The residue was dissolved in 2 ml of water and subiected to amino acid analvsis.

Comparison of the rate of metabolism of aspartic acid, asparagine, glutamic acid, isoleucine, and saline with the rate ofglutamine

formation

Hemidiaphragms were preincubated as described in Table I. At

the end of preincubation, the hemidiaphragms were transferred to flasks containing the same medium. One of the two hemidiaphragms from each animal was treated immediately with 0.5 ml of perchloric acid (0.2 N). The other hemidiaphragms were incubated for 2 h and treated the same way. The tissues were homogenized in the acidified media. The pellets were removed by centrifugation. Glutamine and glutamate in the tissue extracts were assayed enzymatically, and tyrosine was determined fluorometrically. The increase in glutamine, glutamate, and tyrosine during the 2-h incubation was taken as the production of these amino acids by the tissue, and the values are means f S.E. of measurements on 12 diaphragms. The generation of amino acids from net breakdown of muscle proteins was estimated from the rate of tyrosine production, and the relative frequencies of these amino acids in muscle proteins. About 50% of the aspartic acid, asparagine, glutamic acid, isoleucine, and valine were released from the tissue (Table I and Ref. 12). The “+” indicates net production, while “-” indicates disappearance.

Amino acids Released into medium

Present in protein

% total Tyrosine 2.7 Aspartic acid 0.8 Asparagine Glutamic acid 5.7 Isoleucine 3.4 Valine 2.3 Leucine 4.5 Glutamine 25 Alanine 11 Proline 4.5 Glycine 9.1 Serine 6.0 Threonine 5.3 Arginine 3.9 Lysine 5~6 Histidine 3.5 Methionine 3.2 Phenylalanine 3.4

a Includes aspartic acid and asparagine. * Includes glutamic acid and glutamine.

2.8 12”

13b 6.4 4.1 9.0

6.2 5.1 9.8 5.2 4.8 4.2 7.7 3.7 2.6 3.0

muscle protein (cf. Table I and Ref. 17). Since the amino acid composition of diaphragm proteins did not differ after 2 days of starvation (cf. Table I and Ref. 151, while 30% of muscle protein is lost (331, the pattern of amino acid release cannot be due to the preferential breakdown of specialized proteins containing low amounts of branched-chain amino acids and aspartate and high amounts of glutamine and alanine. There- fore, the low amounts of branched-chain amino acids and aspartate released by muscle must indicate their conversion to CO, or to other products.

The rates of generation of all amino acids and aspartic acid, asparagine, glutamic acid, isoleucine, and valine as a group from net protein breakdown were estimated from the rate of tyrosine production and the relative frequencies of these amino acids in muscle protein (12). The measured glutamine release exceeded what could be accounted for by muscle protein breakdown (Table II). This additional amount of glutamine must have been synthesized de nouo and can account for about 50% of the aspartic acid, asparagine, glutamic acid, isoleucine, and valine that were generated by protein degradation. Since only approximately 50% of these amino acids generated by protein breakdown were released by the muscle (Table I), the remainder of these residues were metabolized in the tissue. Therefore, all or nearly all of the carbon skeletons of those amino acids that enter the tricarbox-

Rate of production

Amino acids Frequency in muscle Estimation

protein Experimental from pro- Differ- measurement tein break- ence

down 96 nmolimg tissue/Z h

Tyrosine 2.7 0.452 k 0.028 Total amino acids 100 17 Aspartic acid, as- 28 2.4 4.6 -2.4

paragine, glutamic acid, isoleucine, valine

Glutamine 6 3.62 2 0.34 1.0 +2.6 Glutamine and 12 5.00 t 0.18 2.1 +2.9

glutamic acid

ylic acid cycle may be converted to glutamine. Metabolic Fates of ‘4C-Labeled Precursors-To study the

fates of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine, diaphragms were incubated with L-[U-Wlaspartate, L-[U-Wlvaline, or [2,3-Wlsuccinate, which is a common intermediate in the degradative pathways of glutamate, isoleucine, and valine. The diaphragms were preincubated in media containing these W-labeled precursors for 30 or 90 min to equilibrate intracellular pools and then transferred into fresh identical media. Since in the 2-h incubation, the cellular concentrations of glutamine, glutamate, alanine, and lactate did not change significantly (data not shown and Ref. 22), the radioactivity recovered in metabolites in the medium was measured. This protocol allowed the measurement of the steady state production of W-labeled metabolites. In fact, the total amount of W recovered in the acid-soluble fraction of the muscles was similar when they were incubated with the W-labeled precursors for 30 or 90 min. Specifically, the relative recovery of 14C in glutamine and glutamate, in lactate and pyruvate, and in alanine were similar for incubation media or tissues following a 30 min (Table III) or a 90 min preincubation.’ Therefore, the relative amounts of 14C recovered in end products should be propor- tional to the fluxes of carbon skeletons.

Fig. 3 shows the distribution of l*C metabolites released into the medium upon incubation with [2,3-‘Qsuccinate. Over 99% of the radioactivity recovered in amino acids were in glutamine, glutamate, aspartate, and alanine. Although the

1 Unpublished observation.

Amino Acid Degradation and Glutamine Formation in Muscle

rate of release of alanine was approximately half that of glutamine and glutamate, the 14C recovered in alanine was only about 2 or 3% of that in glutamine and glutamate. The rate of release of lactate and pyruvate was 10 to 15 times that of glutamine and glutamate. Nevertheless, half as much 14C was recovered in lactate and pyruvate as in glutamine and glutamate. A similar distribution of labeled metabolites was obtained when the diaphragms were incubated with L-[U-

14C]aspartate or L-I U-14C]valine (Table III). The presence of radioactivity in alanine, lactate, and pyru-

vate indicates that the rat diaphragm contains some P-enolpyruvate carboxykinase and/or malic enzyme. In addition, the relatively high proportion of 14C recovered in glutamine indicates that skeletal muscles preferentially transform tricarboxylic acid cycle intermediates into glutamine. Thus, the carbon chains that are constantly flowing into the tricarboxylic acid cycle from the catabolism of aspartic acid, asparagine, glu-

DPM

3OOa

2000

1000 -

4wQ- Gin, Glu

2000 - CATHODE

0 IO 20

DISTANCE (cm)

FIG. 3. High voltage electrophoretic analysis of 14C metabolites from [2,3-%]succinate. Hemidiaphragms were incubated as described in Table I, and the electrophoretic analysis was described under “Material and Methods.”

tamic acid, isoleucine, and valine derived from protein breakdown leave this cycle via two exits: conversion to glutamine or to pyruvate (see Fig. 2).

Incorporation of ‘4c0, into Glutamine and Glutamatr- Further experiments evaluated whether the relative distribution of 14C metabolites upon the incubation with 14C-labeled succinate, aspartate, or valine (Fig. 3, Table III) actually reflected the metabolic fates of the carbon skeletons of these precursors. The major concern in evaluating such data is whether the reactions catalyzed by P-enolpyruvate carboxykinase or malic enzyme are primarily unidirectional (i.e. from oxaloacetate to P-enolpyruvate or from malate to pyruvate). Skeletal muscles have been reported to contain insignificant pyruvate carboxylase activity (34). I f the reaction catalyzed by P-enolpyruvate carboxykinase or malic enzyme is reversible, the fractions of 14C from 14C-labeled succinate, aspartate, or valine that are recovered in pyruvate will exceed the real

fractions of carbon chains from these precursors that are converted to pyruvate. Because glycolysis is the major source of P-enolpyruvate and pyruvate (12), the specific 14C activity of [ “C]P-enolpyruvate and 1 14C]pyruvate will be much smaller than that of li4C]oxaloacetate or l’4C]malate. Therefore, the conversion of P-enolpyruvate to oxalacetate or pyruvate to malate will carry a much smaller amount of 14C than the reverse process even if the reactions occur at similar rates in both directions.

Table IV shows that muscle can convert P-enolpyruvate to oxaloacetate or pyruvate to malate, since 14C was recovered in glutamine and glutamate upon incubation with 14C0, (see reaction scheme in Fig. 2B). This finding is consistent with the report of Manchester and Young (25) that 14C02 could be incorporated by the diaphragm into aspartate and glutamate residues in proteins. The rates of conversion were calculated using the specific radioactivity of exogenous CO, and succinate. Because the specific radioactivities of 14C0,, L’4C]oxalacetate, and 1’4C]malate in the cells were not known, the absolute rates of the opposing reactions and hence the direction of the net flow of carbons could not be determined. Nevertheless, the results showed that there was an apprecia-

ble conversion of P-enolpyruvate or pyruvate to glutamine

TABLE III Production of and incorporation of ‘4C-labeled succinate, aspartate, and saline into different metabolites in rat diaphragm

Hemidiaphragms were incubated as described in Table I. Hemi- metabolites were assayed enzymatically. The rate of pyruvate oxi- diaphragms from two rats fasted for 2 days were incubated with 0.1 dation was determined by measuring 14C0, production from D-[3,4- mM [2,3-14C]succinate, L-[U-Wlaspartate, or L-[U-‘*Clvaline (1.7 “C]glucose in quarter-diaphragms incubated independently. ‘%O, &i/ml, or 17 Ci/mol) to determine the relative distribution of 14C in production from complete oxidation (via the conversion of these 14C- glutamine, glutamate, alanine, lactate, and pyruvate. The values labeled precursors to pyruvate, to acetyl-CoA, and then CO,, see are means of two measurements. The other hemidiaphragms were Fig. 1) is impossible to measure directly. These numbers were incubated in the same ‘%-labeled precursors (0.1 mM, 0.03 &i/ml, calculated based on the assumption that the ratio of 14C recovered in 0.3 Ci/mol) to measure 14C0, production and the release of gluta- lactate to that released during pyruvate oxidation equals the ratio of mine, glutamate, alanine, lactate, and pyruvate. The values are the rate of lactate release to that of pyruvate oxidation. means f standard error of measurements on six diaphragms. These

End products of pyruvate

Precursor co, Glutamine Glutamic acid Lactate Pyruvate Alanine

Complete oxidation (via acetyl-

CoA)

[2,3-14ClSuccinate L-[U-“ClAspartate L-[U-14C]Valine

a ND, not determined.

ND”

7.4 9.5

12.6

Rate of production (nmol/mg tissue/Z h) 3.5 k 0.2 1.3 k 0.1 58 f 6 3.2 -f 0.5 3.2 zk 0.5 38 k 5

4.8 f 0.2 59 f 6

Relative radioactivities (glutamine + glutamic acid = 1.0) 1.0 0.60 10.02 0.39 1.0 0.58 co.02 0.38 1.0 0.54 co.02 0.35


TABLE IV

Incorporation of ‘4c from ‘CO, and [2,3-‘YTlsuccinate into different metabolites

Hemidiaphragms were incubated in the same fashion as described in Table I. The studies with [2,3-Wlsuccinate were similar to those in Fig. 3. For incubations with NaHWO,, 50 &i of this compound were added to the incubation flask immediately after saturating with O&O, (95:5). The values are means of determinations on two diaphragms.

Precursors Glutamine + glutamic acid Alanine Lactate +

ruvate py-

‘TO, 12.3-‘YXluccinate

nmol precursor”lmg tissue12 h

0.76 0.040 NDb 0.039 0.0012 0.017

u The molar amounts of incorporation were calculated using the specific radioactivities of exogenous precursors.

b ND, not determined.

and glutamate. Therefore, the proportion of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine that is converted to lactate and pyruvate would be smaller and the proportion converted to glutamine would be larger than the studies with W-labeled precursors have indicated.

Metabolic Fates of Pyruvate - As discussed above, WO, can be generated from W-labeled precursors simply by rearrangement of the carbon skeletons in the tricarboxylic acid cycle. It is impossible to determine directly the proportion of the carbons that are completely oxidized to CO, (via conversion to pyruvate and then to acetyl-CoA) by following the release of 14C0, from these W-labeled precursors. Therefore, the relative importance of various pathways of pyruvate metabolism were determined by measuring enzymatically lactate, pyruvate, and alanine production. Pyruvate oxidation was measured by determining ‘4c0, production from r+[3,4-14C]glucose. The rate of production of lactate and pyruvate together was found to be approximately 1.5 times the oxidation of pyruvate, and about 25 times that of alanine release (Table III). There- fore, if any of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine are converted to pyruvate, much more will be converted to lactate or oxidized to acetyl-CoA than would be released as alanine.

Since the rate of pyruvate oxidation was known, it was possible to quantitate the end products of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine on the assumption that the release of glutamine, glutamate, lactate, pyruvate, and alanine, and the pyruvate oxidation account for all the carbon chains entering the tricarboxylic acid cycle. Because of the reversible interconversion of tricarboxylic acid cycle intermediates and P-enolpyruvate or pyruvate (Table IV), the relative amounts of 14C recovered in these end products upon incubation with 14C-labeled succinate, aspartate, and valine (Table III) need not reflect their actual importance. However, the fraction of 14C recovered in pyruvate, lactate, and alanine must reflect the maximal percentage of tricarboxylic acid cycle intermediates possibly converted to these metabolites, and the fraction of 14C recovered in glutamine must represent the minimal fraction of precursors converted to this end product (Table V). Thus, conversion to glutamine and glutamate accounts for at least 50% of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine that enter the tricarboxylic acid cycle, to lactate and pyruvate for less than 30%, to alanine for less than 2%, and complete oxidation for less than 20% (Table V). These conclusions are in accord with the earlier finding that

glutamine synthesis can account for the fraction of these amino acids not released by the muscle (Tables I and II).

It is noteworthy that when the diaphragms were incubated with 14C-labeled succinate or aspartate, 14C recovered in CO, was 7 to 10 times that in glutamine and about 20 times that in lactate and pyruvate (Table III). Since the rate of lactate and pyruvate production was 1.5 times that of pyruvate oxidation, the 14C0, recovered in the incubation must have been about 30 times the 14C0, produced by the net degradation of the 14C- labeled precursors. Thus, approximately 97% (i .e . 29/30) of the 14C02 was produced by the repeated cycling of the tricarboxylic acid cycle intermediates rather than by their net degradation via conversion to pyruvate.

Contribution of Amino Acids to Energy Metabolism in Skeletal Muscle- In order to evaluate the contribution of amino acids derived from protein breakdown to the energy requirement of skeletal muscle, the rate of acetyl group formation from leucine and from aspartic acid, asparagine, glutamic acid, isoleucine, and valine, and the rate of acetyl group oxidation in the tricarboxylic acid cycle were compared. For diaphragms incubated in buffer containing 5 mM glucose, the rate of acetyl group formation from leucine and from the five amino acids was estimated from their rate of generation by net proteolysis as described in Tables I and II. The rate of production of leucine was approximately 1.5 nmol/mg of tissue/2 h and aspartic acid, asparagine, glutamic acid, isoleu- tine, and valine together 4.8 nmol/mg of tissue/2 h. Approxi- mately 50% of these two groups of amino acids generated by protein breakdown was released intact (Table I). Thus, leucine can provide about 2 nmol of acetyl group/mg of tissue in 2 h, because the degradation of each leucine molecule generates 3 acetyl groups. Since more than 50% of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine that enter the tricarboxylic acid cycle are converted to glutamine, these amino acids can provide at most 1.2 nmol of pyruvate, or at most 0.3 nmol of acetyl groups/mg of tissue in 2 h (Fig. 4).

The rate of acetyl group oxidation in the tricarboxylic acid cycle was estimated using a recently described relationship between the rates of acetyl group oxidation and 0, consumption (30). The ratio of these two rates is practically constant (0.34 * 0.011, independent of the substrates being oxidized by the muscle. Thus, the rate of acetyl group oxidation was estimated from the measurements of O2 consumption. The rate of 0, consumption was found to be 154 -t 20 nmol/mg of tissue/2 h, which is equivalent to 52 + 6.2 nmol of acetyl group oxidatiommg of tissue in 2 h. A comparison of this rate with the rate of acetyl group formation from amino acids (see above) indicates that the net breakdown of proteins provides

TABLE V

Metabolic fates of carbon chains aspartic acid, asparagine, glutamic

acid, isoleucine, and saline in rat diaphragm

The percentages were calculated according to the relative distribution of 14C recovered in the metabolites after the incubations with [2,3-Wlsuccinate, L-[U-‘4Claspartate, or L-W-‘4Clvaline (Table III). The limits “greater than” and “smaller than” were based on the results summarized in Tables I. II. and IV.

End products Fractions of carbon chains entering tricarboxvlic acid cvcle

Glutamine + glutamic acid Lactate + alanine I CO,

Lactate Alanine co*

150% <50% <30%

(2% <20%


EXTRACELLULAR SKELETAL MUSCLE (nmol/mg lissue/Zhr)

p-GGJ 86 ~

hxtate 5S

Gin .Glu 4.8 \ 1

m energy swrce

-=2 2 AlO 2.2

Asp. Asn, Glu LCU Val. IIICJ

2

Ammo oclds 5

eKeto acids A

17 othef muno acids MUSCLE

I PROTEIN

DIAPHRAGM FROM FASTED RAT INCUBATED IN 5mM GLUCOSE

FIG. 4. The interrelationships between protein, amino acid, and carbohydrate metabolism in incubated diaphragms from fasted rats. These rats were fasted for 42 to 48 h and weighed 60 to 85 g when killed. The media contained 5 rnM glucose. This figure is based in part on data presented in earlier tables. The rate of glycolysis was determined in the preceding paper (12) and is expressed as the rate of formation of pyruvate. The values given are the means of measurements on 12 to 18 diaphragms. TCA, tricarboxylic acid cycle.

about 5% of the acetyl groups oxidized, of which leucine provides about 4%, and aspartic acid, asparagine, glutamic acid, isoleucine, and valine together account for about 1%.

These numbers can also be used to calculate the fraction of total 14C0, that is produced from the cycling of the tricarboxylic acid cycle intermediates when the diaphragm is incubated with 14C-labeled amino acids, such as [U-‘4C]aspartate. As indicated above, less than 1 of the 52 run01 of acetyl groups oxidized in 2 h is provided by oxalacetate or malate (Fig. 4). The oxidation of 52 run01 of acetyl groups produces 104 nmol of CO,, the carbon atoms of which are derived from oxalacetate. The complete oxidation of 1 run01 of oxalacetate or malate gives 4 nmol of CO,. Thus, approximately 4% (i.e. 4/102) of ‘*CO, was produced from the net degradation of the labeled precursor and 96% of the 14C02 was produced from the repeated cycling of intermediates.

DISCUSSION

The production of 14C02 from 14C-labeled substrates has been frequently taken as evidence that the substrates undergo net degradation to CO, and used as a measure of the fraction of substrates oxidized to CO,. The present studies, however, emphasize that this approach is not valid for those substrates whose degradation involves conversion to tricarboxylic acid cycle intermediates. Thus, the release of 14C02 from such 14C- labeled substrates at most indicates their conversion to tricarboxylic acid cycle intermediates. In diaphragms from fasted rats incubated in buffer containing 5 mM glucose, more than 95% of the 14C0, released during incubation with [2,3- 14C]succinate or rJU-14Claspartate was generated from the spinning of the tricarboxylic acid cycle without net degradation of these precursors. Although 7 to 10 times more 14C was recovered in CO, than in glutamine and glutamate (Table III), complete oxidation to CO, accounted for less than 20%, but conversion to glutamine and glutamate for more than 50% of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine that enter the tricarboxylic acid cycle (Table V).

The complete degradation of tricarboxylic cycle intermediates of aspartic acid, asparagine, glutamic acid, isoleucine, and valine requires their conversion to pyruvate and then to acetyl-CoA. Either P-enolpyruvate carboxykinase or malic enzyme is essential for this conversion. P-enolpyruvate carboxykinase is generally classed as an essential enzyme for gluconeogenesis. It has often not been appreciated that P- enolpyruvate carboxykinase may also be essential for converting substrates, such as malate or succinate, to pyruvate so that they can be used for metabolic energy. Mutant bacteria lacking P-enolpyruvate carboxykinase fail to grow on succinate or malate as the sole carbon source (35, 36) (these strains do not normally contain malic enzyme). The failure of growth of these mutants may be a consequence of their inability to use these substrates to generate ATP.

The recovery of 14C in lactate, pyruvate, and alanine after incubation with 14C-labeled succinate, aspartate, or valine (Table III) indicates that rat diaphragms contain either P- enolpyruvate carboxykinase or malic enzyme. This finding is consistent with the report of Crabtree et al. (34) and Opie and Newsholme (37) that red and white leg muscles contain P- enolpyruvate carboxykinase activity. However, the present experiments do not provide a quantitative measure of these enzymatic activities or their significance in converting tricarboxylic acid cycle intermediates into pyruvate. In addition, an appreciable flux of carbon chains occurred in the opposite direction. Approximately 0.8 nmol of 14C0, was incorporated into glutamine and glutamate (Fig. 2B) per mg of tissue in 2 h (Table IV). This rate is substantial when compared to the rate at which amino acids derived from proteolysis enter the tricarboxylic acid cycle which is approximately 2.4 nmol/mg of tissue/2 h (Table II).

Fates of Amino Acids Entering Tricarboxylic Acid Cycle- Because the conversion of tricarboxylic acid cycle intermediates into P-enolpyruvate and pyruvate is reversible (Table IV), indirect methods had to be employed to determine the direction and the rate of the net flow of carbon chains between these metabolites (Fig. 2). Although the exact percentage of each end product was not obtained, the minimal rate of conversion of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine into glutamine and glutamate was calculated (Table V). This value was consistent with those suggested by the pattern of amino acids released from the muscle (Table I and II); however, it is not known whether conversion to glutamine and glutamate actually accounts for all the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine entering the tricarboxylic acid cycle as suggested by Table II.

As discussed above, the complete oxidation to CO, accounts for less than 20% of the fate of the carbon chains of the five amino acids that enter the tricarboxylic acid cycle (Table V). The conclusion differs significantly from the general view (l- 11) that isoleucine and valine are oxidized in skeletal muscle to CO,. It was suggested that branched-chain amino acids may contribute up to 30% of the energy consumed in skeletal muscle during fasting (15). This estimate was based on the assumption that all three branched-chain amino acids, origi- nating from muscle proteins and from the circulation are degraded totally to CO, after decarboxylation. This assumption is certainly not true for isoleucine and valine, although it may hold for leucine.

Unlike the other amino acids released from protein breakdown, leucine may supply a considerable fraction of the muscle’s energy. Leucine derived from protein breakdown


accounts for about 4% of the acetyl groups oxidized by the incubated diaphragms from rats fasted for 2 days (Fig. 4). When leucine is present in the incubation medium at 0.5 mM, it can provide up to 20% of the acetyl groups oxidized (38). It is noteworthy that under such conditions, leucine (but not isoleucine or valine) also inhibits pyruvate oxidation and thus spares gluconeogenic precursors (38).

The present experiments extend our earlier conclusion that the pyruvate used for alanine synthesis in muscle is derived almost exclusively from exogenous glucose (12). Conversion to alanine can account for at most 2% of the carbon chains of aspartic acid, asparagine, glutamic acid, isoleucine, and valine derived by net proteolysis (Table V). On the other hand, these five amino acids can supply at most 3% of the pyruvate consumed in the skeletal muscle (12). These results argue strongly against the suggestion (16, 21) that the carbon skeletons of alanine are derived from amino acids generated from muscle proteins. Furthermore, in rat diaphragms incubated in vitro (Table III and Ref. 22), and skeletal muscles in uiuo (19, 39), lactate is quantitatively a much more important end product of pyruvate metabolism than alanine. Thus, if any of the carbon chains of amino acids were converted to pyruvate, their major fates would be conversion to lactate and oxidation to acetyl-CoA.

Origin and Fate of Glutamine- Conversion of aspartic acid, asparagine, glutamic acid, isoleucine, and valine into glutamine may account for the carbons released by the muscle as glutamine. However, since glutamine contains two amino groups, additional sources of amino groups are essential to explain the formation of glutamine (Table II). Leucine can provide amino groups, but not the carbon skeletons, since it is strictly a ketogenic amino acid. In addition, a fraction of the branched-chain amino acids are released by muscle as their a+keto acids, after being transaminated with a-ketoglutarate (12, 29). Possibly other amino acids which do not enter the tricarboxylic acid cycle in muscle may also donate amino groups, although we have failed to observe conversion of Y!- labeled lysine, threonine, serine, and glycine (all at 0.1 mM) to cr-keto acids in rat diaphragms.’ Nucleotides may possibly also provide NH, through deamination (40, 41).

Amino acids which provide the carbon skeletons for glutamine may originate from the net breakdown of muscle proteins or from the circulation. Large amounts of both glutamine and alanine are released by muscles in the postabsorptive state when protein breakdown is accelerated (13, 14). Unlike alanine, glutamine is also released in large amounts after food intake (5, 17). Under such conditions, isoleucine and valine from the diet are taken up primarily by muscle (42, 43), where they may be used in the synthesis of glutamine.

If amino acids cannot supply enough carbon skeletons for glutamine synthesis, the remaining amount must be provided by glucose (see Fig. 2B). For example, providing leucine (0.5 mM) to the muscle stimulated glutamine production 2-fold, while it simultaneously decreased net protein breakdown. Under these conditions, aspartic acid, asparagine, glutamic acid, isoleucine, and valine derived from protein breakdown cannot supply enough carbon skeletons for glutamine synthesis, and glucose has been shown to provide the additional carbons for this process2 Whether glucose provides any carbon skeletons under the present incubation conditions is not known.

The physiological significance of the transformation of aspartic acid, asparagine, glutamic acid, isoleucine, and valine

* T. W. Chang and A. L. Goldberg, manuscript in preparation.

into glutamine in skeletal muscle seems puzzling, perhaps because of our lack of knowledge about the subsequent fate of the glutamine released into the circulation. If totally oxidized, glutamine provides more energy than oxalacetate, succinate, or aspartate (44). Therefore, skeletal muscle expends energy for the synthesis of glutamine from these substrates. In fact, one of the carbons in glutamine is derived from acetyl-CoA, which would otherwise be oxidized for energy.

One possible function of glutamine production is to help in the removal of ammonia and a-amino groups produced from the metabolism of amino acids and nucleotides (41) in skeletal muscle. Circulating glutamine is a major precursor for renal ammonia production, which is essential in acid excretion (45- 48). As a nitrogen carrier, glutamine differs from alanine in that the carbon skeleton of glutamine is provided primarily by other amino acids, while those of alanine are derived almost entirely from glucose (12). In other words, the release of glutamine by muscles also represents a net efllux of carbon skeletons.

The carbon chains of glutamine taken up by the kidney are oxidized to CO, (48, 49), or used for the synthesis of glucose (45, 50, 51) or of alanine (4, 53). Experiments with nephrecto- mized and eviscerated animals suggested that the small intestine is much more important than the kidney in the catabolism of glutamine (52). The intestine extracts large amounts of glutamine from the circulation, and appears to convert the glutamine into alanine, which is then released (52-54). Alanine, but not glutamine, is taken up efficiently by the liver and converted into glucose and urea (4, 13). Thus, during starvation muscles, intestine, and liver all seem to act in concert in gluconeogenesis and nitrogen disposal for aspartic acid, asparagine, glutamic acid, isoleucine, and valine generated from the net breakdown of muscle proteins.

Acknowledgments-We thank Ms. Niconila Fedele and Ms. Mary Beth Tabacco for their invaluable technical assistance and Dr. Henrich Taegtmeyer for carrying out amino acid analyses. The advice of Dr. Dominic Lam, Mr. Janis Lazdins, Dr. Mark Tischler, and Dr. Peter Libby are greatly appreciated.

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APPENDIX: A RELATIONSHIP BETWEEN THE RATES OF ACETYL GROUP OXIDATION AND THE OXYGEN CONSUMPTION OF CELLS*

(Received for publication, October 6, 1977)

TSE WEN CHANG AND ALFRED L. GOLDBERG*

From the Department OfPhysiology, Harvard Medical School, Boston, Massachusetts 02115

A simple relationship has been derived that may be useful in determining the energy production in the tricarboxylic acid cycle in most tissues or whole organisms during fasting or exercise. The ratio of the rate of acetyl group oxidation to the rate of 0, consumption is nearly constant (0.341, irrespective of the proportion of glucose and fatty acids oxidized in a tissue or an organism. This relationship is derived by multiplying the respiratory quotient by the ratio of acetyl group oxidation to total CO, production.

* These studies have been supported by research grants from the National Institute of Neurological Disease and Stroke, from the National Aeronautics and Space Administration, and from the Muscular Dystrophy Association of America. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ Recipient of Research Career Development Award from the Na- tional Institute of Neurological Disease and Stroke.

An easy method for measuring energy production in the tricarboxylic acid cycle would be useful for studies of the energy derived from different metabolic substrates by isolated cells or organisms under different physiological conditions. In the course of studies of amino acid metabolism in skeletal muscle, we have derived from known biochemical principles an interesting and potentially useful relationship between the rate of 0, consumption and the rate of acetyl group oxidation. This article demonstrates that the ratio of 0, consumption to acetyl group oxidation is practically constant, irrespective of the type of substrate being oxidized. This constant relationship should hold for most cells and tissues in vitro and for intact animals under most conditions, including exercise, the postabsorptive state, and starvation. (As discussed below, this relationship should not hold for liver or for animals or tissues undergoing net fatty acids synthesis.)

DERIVATION OFTHE RELATIONSHIP

In most cells, CO, is produced almost exclusively from the oxidative decarboxylation of three metabolites: from pyruvate

the metabolic fates of amino acids and the formation of ... · the only amino acids that can be...

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