THE JOURNAL or B~.OCVXL CHEMISTRY Vol. 253, No. 10, Issue of May 25, pp. 3677-3664, 1978

Prrnted ,r, Cl S.A

The Origin of Alanine Produced in Skeletal Muscle* (Received for publication, October 6, 1977)

TSE WEN CHANGS AND ALFRED L. GOLDBERG§

From the Department of Physiology, Harvard Medical School, Boston, Massachusetts 02115

These studies were undertaken to clarify the origin of alanine released in large amounts by skeletal muscle. Leu- tine, a ketogenic amino acid, as well as isoleucine and valine, which are glucogenic, increased the intracellular concentration of glutamate and stimulated the production of alanine and glutamine by isolated rat diaphragms. The extent of transamination of these amino acids (i.e. the sum of cy-keto acid decarboxylation and accumulation) equalled the total increase in alanine, glutamine, and glutamate. Thus the branched-chain amino acids may stimulate alanine and glutamine production simply by supplying amino groups for transamination with cY-ketoglutarate. Providing 0.3 mM NH,+ increased glutamine production because of greater conversion of glutamate to glutamine. The fall in cellular glutamate concentration can explain the associated decrease in alanine production.

The production of alanine and glutamine is limited by the supply of carbon chains as well as amino groups. Glucose stimulated alanine production by rat diaphragms. The stim- ulation was larger when the muscle was also provided with branched-chain amino acids. In addition, Z-deoxyglucose, which inhibited glycolysis, reduced alanine production. Io- doacetate and fluoride also decreased alanine and lactate production. However, observations with the latter inhibitors (unlike those with 2-deoxyglucose) are complicated because they also decreased transamination by inhibiting the oxida- tive decarboxylation of branched-chain amino acids and simultaneously promoted net protein breakdown.

Most of the amino acids in muscle protein have been suggested as sources of the carbons in alanine and gluta- mine. Experiments with diaphragm, soleus, and extensor digitorum longus, however, showed that only certain I%- labeled amino acids could be converted to “C02. Of these, only five amino acids, aspartate, asparagine, glutamine, isoleucine, and valine are converted to tricarboxylic acid cycle intermediates, and only these amino acids could pos- sibly supply the carbon skeleton for alanine or glutamine. The rate of generation of these five amino acids by the net

* This work was supported by grants from the National Institute of Neurological Diseases and Stroke, National Aeronautics and Space Administration, and Muscular Dystrophy Association of America. A preliminary presentation of some of these results ap- peared in Fed. Proc. (19’76) 35, 1574. 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 accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

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

5 Recipient of a Research Career Development Award from the National Institute of Neurological Diseases and Stroke.

breakdown of muscle proteins in uitro was estimated from the rate of tyrosine production and their relative propor- tions in muscle protein. The rate of glycolysis was measured by the :3HOH generation from exogenous I)-/ 5JHlglucose. Comparison of these rates indicated that over 97% of the carbons of the alanine, pyruvate, and lactate released by the muscle were derived from exogenous glucose and not from amino acids generated by protein degradation. Similar anal- ysis of published data on amino acid and glucose fluxes across human muscles in ho also suggest that blood glucose (and not amino acids) is the major source of pyru- vate used in alanine synthesis.

Skeletal muscle releases large quantities of alanine and glutamine into the circulation in viuo in the postabsorptive state and in fasting (l-4). Upon perfusion (5) or incubation (6, 7) in uitro, skeletal muscles also release these two amino acids in large amounts. Over the past few years, appreciable re- search has been carried out to determine the origin of alanine. Nevertheless, this issue remains a matter of controversy.

Mallette et al. (8) and Felig et al. (2) have suggested that the carbon chain of alanine is derived from circulating glucose and the amino group from the catabolism of amino acids in muscle. They thus hypothesized the existence of a glucose- alanine cycle in which the alanine released from muscle serves as a precursor for gluconeogenesis in the liver and helps transport amino groups from muscle to liver for disposal as urea. Odessey et al. (6) found that alanine production was linked to the metabolism of branched-chain amino acids. Addition of these amino acids, but not other plasma amino acids, stimulated alanine production. In addition, the catabo- lism of branched-chain amino acids increased in muscles from fasting rats (9-11). In such tissues, the branched-chain amino acids stimulated alanine production more than in muscles from fed rats.

Garber et al. (7, 12) and Goldstein and Newsholme (13) suggested a very different model for the origin of alanine, in which most of the alanine was derived not from glucose but from other amino acids produced by the net breakdown of muscle proteins. Thus, the glucose-alanine cycle would be of minor import. These workers reported that alanine production was stimulated by aspartate, leucine, isoleucine, valine, thre- onine, and serine (12), or by glutamate and isoleucine (131, and consequently concluded that carbon chains from these amino acids were used in alanine synthesis. In addition, they reported that iodoacetate (7) and fluoride (13) inhibited glycol- ysis without reducing alanine synthesis. Because leucine was

3677

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3678 Origin of Alanine Produced in Skeletal Muscle

one of the amino acids that stimulated alanine production, Garber et al. (7) also hypothesized a new pathway for convert- ing the carbon chain of leucine into pyruvate. Such a hypo- thetical pathway would negate the classic distinction between glucogenic and ketogenic amino acids.

The present studies were undertaken to choose between these two alternative models by (i) testing whether the stim- ulation of alanine production by branched-chain amino acids (5, 7, 13) is explicable simply by increased transamination or whether it is necessary to hypothesize the conversion of the carbon chains of these amino acids into pyruvate, (ii) deter- mining which amino acids enter the tricarboxylic acid cycle in skeletal muscle and can thus serve as potential precursors of alanine, (iii) investigating the effects of glucose and inhibitors of glycolysis on alanine release, protein balance, and amino acid metabolism, and (iv) quantitating the relative impor- tance of glycolysis, protein breakdown, and amino acid metab- olism as sources of pyruvate.

MATERIALS AND METHODS

Escherichia coli alanine dehydrogenase (EC 1.4.1.1) was obtained from Boehringer Mannheim Co., NAD+, sodium iodoacetate, sodium fluoride, E. coli glutamic dehydrogenase (EC 1.4.1.31, and E. coli glutaminase (EC 3.5.1.2) from Sigma Chemical Co., L-11-14Clleucine, L-Il-14Clisoleucine, L-[l-14Clvaline, L-[UJ4Clleucine, L-[U-L4Clvaline, L-IU-14Clasparagine, L-[U-14C1glycine, I,-lU-14C]threonine, L-[U- 14Cllysine, L-(U-‘4C]phenylalanine, L-lU-L4Cltyrosine, and scintilla- tion liquid Aquasol 2 from New England Nuclear Co., and D-[5-

3Hlglucose from AmershamlSearle Co. Male rats were purchased from Charles River Breeding Labora-

tories and were fed ad libitum Purina chow. Diaphragms without ribs, soleus, and extensor digitorum longus muscles were dissected as described previously (10). Animals were deprived of food for 42 to 48 h prior to the experiments and during this time, they lost approximately 30% of their body weight. They weighed between 60 and 85 g when killed. Quarter-diaphragms or leg muscles were preincubated for 30 min at 37°C in 3 ml of Krebs-Ringer bicarbonate buffer (pH 7.4) containing glucose (5.0 mM) and chloramphenicol (0.3 mg/liter), and saturated with OJCO, (955). The muscles were then transferred to fresh media and incubated for 2 h.

At the end of incubation, the tissues were removed and homoge- nized in 1.0 ml of cold perchloric acid (0.2 N). The homogenates were centrifuged at 1000 x g, and the supernatants were neutralized with potassium hydroxide (2.5 N). The potassium perchlorate precipitate was removed by centrifugation. The incubation media were acidified to release 14C0, by injecting 0.5 ml of perchloric acid (1.5 N) through the rubber stoppers. The released 14C0, was collected in 0.2 ml of phenylethylamine which was added to a well within the incubation flask after removal of the tissue. Radioactivity was counted as discussed previously (10). The acidified media were neutralized with potassium hydroxide and treated as described above.

Alanine, glutamate, and glutamine in the neutralized samples were determined spectrophotometrically using alanine dehydrogen- ase (14) and glutamic dehydrogenase (15). To assay glutamine, the samples were first treated with glutaminase to convert glutamine to glutamate (16) and the total glutamate in the sample was then measured. Glutamine was then calculated as the difference between the basal and glutaminase-treated samples. Tyrosine was assayed according to Waalkes and Udenfriend (17). In order to determine the radioactivity in a-keto acids derived from 1-14C-labeled branched- chain amino acids, 0.5 ml of the acidified media (from which free 14C0, had been driven off) was pipetted into 25-ml vials. Wells containing 0.2 ml of phenylethylamine were then installed to collect 14C0,, and 0.5 ml of hydrogen peroxide (15%) was injected through the rubber stoppers to decarboxylate the a-keto acids. This approach allows complete and specific decarboxylation of the a-keto acids.’

The rate of glycolysis was determined by the method of Ashcroft et al. (18). Since the conversion of each D-[5-3Hlglucose molecule to pyruvate or phosphoenolpyruvate produces one 3HOH molecule, the rate of glycolysis from exogenous glucose can be estimated from the production of 3HOH. Initially, the aqueous stock solution Of D-b

_----~ -~~__~~~ __-..

1 R. Odessey and A. L. Goldberg, submitted for publication.

3Hlglucose was evaporated to dryness using nitrogen gas to elimi- nate any 3HOH generated during storage. Quarter-diaphragms were then preincubated for 30 min and incubated for 2 h in media containing n-[5-3Hlglucose (5 mm, 0.3 &i/ml). To determine the 3HOH produced during incubation, a 0.2ml sample of the neutral- ized medium was pipetted into plastic wells suspended inside a 20- ml counting vial containing 0.3 g of anhydrous calcium chloride on the bottom. A strip of filter paper was introduced into the wells to increase the surface area for evaporation of the liquid. The vials were then incubated at 37°C for about 8 h, by which time the liquid in the wells had evaporated completely. The wells were then removed, 1.0 ml of water was added to dissolve the CaCl,, and 15 ml of Aquasol 2 were added prior to liquid scintillation counting.

Measurements on paired leg muscles from the same animal or on pieces of the same diaphragm were compared by the Student t test.

RESULTS AND DISCUSSION

Role of Transamination in Alanine and Glutamine Pro- duction - Previous studies from this laboratory showed that leucine, isoleucine, and valine (but not other plasma amino acids) together stimulated alanine production by incubated rat diaphragms and concluded that these amino acids increase alanine production by donating amino groups (6). Goldstein and Newsholme (13) and Garber et al. (7) also observed that these amino acids stimulated alanine production and proposed they supply the carbon chains for alanine synthesis. Experi- ments were, therefore, undertaken to determine whether branched-chain amino acids stimulate alanine production sim- ply by increasing the rate of transamination of pyruvate.

The first step in the degradation of branched-chain amino acids is their transamination with a-ketoglutarate’ (Fig. 1). Therefore, providing these amino acids in the incubation medium should increase the intracellular concentration of glutamate, which can then either undergo transamination with pyruvate to form alanine or incorporate NH3 to form glutamine. To test this model, rat diaphragms were incubated with 0.5 mM of branched-chain amino acids. The effects of these amino acids on cellular glutamate concentration and the extent of their transamination were measured and compared with the increase in alanine, glutamine, and glutamate.

Leucine, isoleucine, and valine at approximate plasma concentrations (0.5 mM) each increased tissue glutamate con- tent and stimulated release of alanine, glutamine, and gluta- mate (Table I). It is noteworthy that leucine (unlike isoleucine and valine) is strictly ketogenic and cannot be converted to tricarboxylic acid cycle intermediates or pyruvate. Neverthe- less, leucine has a similar effect as isoleucine and valine, both of which are glucogenic amino acids, on the production of

NH;

c+Ketwci.s

t

t

t-

NH4

CO2 GlUt-3lhM

0

R-LSSCoA

FIG. 1. Pathway for metabolism of branched-chain amino acids and synthesis of alanine and glutamine. The arrows indicate the direction of net changes when muscles are supplied with branched- chain amino acids. This transamination scheme does not indicate the origin of the pyruvate used for the synthesis of the alanine, the origin of glutamate for the synthesis of glutamine, or the subsequent fate of carbon chains of the branched-chain amino acids.

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Origin of Alanine Produced in Skeletal Muscle 3679

alanine, glutamine, and glutamate. The total extent of transamination of L-l-W-labeled branched-chain amino acids was determined by measuring the total amount of l-W-a- keto acids produced by the muscle. Most of the labeled a-keto acid underwent decarboxylation, which was determined by measuring 14C0, production. In addition, 20 to 40% of the a-

TABLE I

Effects of branched-chain amino acids on release of glutamate,

glutamine, and alanine, and tissue glutamate concentration in rat

diaphragm

Quarter-diaphragms from six rats fasted for 2 days were preincu- bated for 30 min in Krebs-Ringer bicarbonate buffer containing 5 rnM glucose and the specific branched-chain amino acid. The tissues were then transferred to flasks containing the same medium. After incubating for 2 h, alanine, glutamine, and glutamate released into medium or recovered in tissue homogenates were determined enzy- matically. Values are means +- SE. of determinations on six quarter-diaphragms. All changes upon addition of amino acids were highly significant (p < 0.01).

tk$itioM;S Glutamic acid Glutamine Alanine Tissue gluta- m mine

released nmollmg tissue12 h nmollmg tissue

None 1.08 k 0.10 3.92 t 0.08 1.64 ‘- 0.10 1.57 t 0.14 Leucine 1.70 ? 0.15 7.08 f 0.32 2.81 + 0.22 3.21 -t 0.19 Isoleucine 2.04 k 0.09 7.70 t 0.16 2.75 2 0.18 3.15 T 0.23 Valine 1.36 2 0.14 6.37 2 0.28 2.41 ? 0.14 2.48 f 0.20

TABLE II

Comparison of rates of transamination of branched-chain amino

acids an.d stimulation ofglutamine, glutamate, and alanine

production

These data and those in Table I were obtained simultaneously in the same incubation. The total increase in glutamine, glutamate, and alanine caused by the addition of branched-chain amino acids represents the sum of the increases in glutamine, glutamate, and alanine (Table I). The total extent of transamination of supplied branched-chain amino acids is the sum of the a-keto acid released from the tissue and decarboxylated (see Fig. 1). L-1-“‘C-labeled branched-chain amino acids were added at 0.1 &i (0.3 Ci/mol). The amounts of l-*4C-a-keto acids were determined by measuring “CO, released upon addition of hydrogen peroxide. Values are means f S.E. of measurements on six diaphragms.

“C recovered Total increase

in u-k&o Total transa- in glutamine,

acids mination glutamic acid, and alanine

nmollmg tissuei h

Leucine 5.19 t 0.24 0.62? 0.04 5.81 ? 0.25 4.95 +- 0.42 Isoleucine 4.71 ? 0.07 1.20 f 0.10 5.90 2 0.14 5.85 2 0.26 Valine 2.69? 0.16 l.lB? 0.10 3.87 t 0.19 3.40 t 0.34

keto acids produced were released into the medium. Table II indicates that the total extent of transamination was nearly equal to the total increase in alanine, glutamine, and gluta- mate. These observations suggest that the production of alanine and glutamine may be limited by supply of amino groups for transamination with a-keto acids. Thus, the branched-chain amino acids seem to promote the production of alanine and glutamine by increasing cellular glutamate content.

Further evidence for this conclusion came from the studies on the effects on NH,+. Glutamate can either undergo trans- amination and provide amino groups to pyruvate for alanine synthesis or can incorporate NH,+ to form glutamine (Fig. 1). As shown in Table III, the addition of 0.3 mM NH,+ to the medium, either in the absence or in the presence of branched- chain amino acids, increased the production of glutamine but reduced that of alanine and glutamate. In contrast to branched-chain amino acids (Tables I to III), the presence of NH,+ did not alter the total amount of a-amino groups recovered in glutamate, glutamine, and alanine (Table III). The decrease in tissue glutamate is probably a consequence of greater conversion of glutamate to glutamine. The fall in glutamate is consistent with earlier findings that skeletal muscle has a limited glutamate dehydrogenase activity (19). Decreased glutamate in the muscle should reduce the extent of glutamate transamination and of alanine synthesis as was observed in Table III. Similar effects of NH,+ were found in the perfused rat hindquarter by Ruderman (20). These obser- vations appear to contradict those of Garber et al. (7) who reported that NH,+ stimulated both glutamine and alanine production. The basis of this discrepancy is unclear; however, these workers used NH4+ at 5 mM concentration which is 50 to 100 times higher than normal plasma concentration.

In isolated hepatocytes, NH,+ at 5 or 10 mM inhibits proteolysis (21). However, in the incubated diaphragm, NH,+ at 0.3 mM had no effects on net protein breakdown as judged from the rate of tyrosine release (22) (Table III). Therefore, the decrease in alanine production in the presence of NH,+ is not due to changes in protein balance.

Previous studies of amino acid release in intact animals (23) or human forearm (3) have demonstrated appreciable release of glutamine and net uptake of glutamate. By contrast, most investigations with incubated (6, 7) (Table I) or perfused (5) muscles have reported appreciable release of glutamate from the muscle. The finding (Table III) that addition of NH,+ to the medium increased glutamine release at the expense of glutamate suggests that NH4+ ions may have been limiting in most in vitro studies which generally use buffers (e.g. Krebs-

TABLE III

Effects on NH,+ on tissueglutamate concentration, release ofglutamate, glutamine, and alanine, and netprotein breakdown

Muscles were incubated as described in Table I for 1 h. Tyrosine release into the medium was taken as a measure of net protein breakdown (22). Tyrosine was assayed fluorometrically. Each value is mean f SE. for measurements on six diaphragms.

Additions (0.3 nm) Tissue glutamic acid Glutamic acid Glutamine Alanine Tyrosine

nmollmg tissue nmolimg tissue/2 h

None 2.14 +- 0.31 1.45 ‘- 0.16 2.48 f 0.22 0.88 f 0.04 0.444 f 0.029 NH,+ 1.49 * 0.05” 0.77 k 0.10n 3.80 f 0.06” 0.58 f 0.03* 0.432 ? 0.038

In presence of branched-chain amino acids (0.5 mM each)

None 3.20 t 0.21 2.42 k 0.17 3.48 f 0.08 1.89 t 0.04 0.351 ? 0.018 NH,+ 2.24 k O.OBb 1.61 k 0.18* 5.34 f 0.36b 1.44 f 0.15’ 0.346 i 0.024

a Significant effects of NH,+p < 0.01. b Significant effects of NH,+p < 0.02.

( Significant effect of NH,+p < 0.05.

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3680 Origin of Alanine Produced in Skeletal Muscle

Ringer bicarbonate buffer) lacking this ion. TABLE IV

Metabolism of amino acids by rat soleus and extensor digitorum longus muscles

The incubations were performed as described in Table I. U-Y- Amino-acids were present at 0.1 mM (0.3 Ci/mol). Values are means % SE. of determinations on muscles from six animals.

Together Tables I, II, and III demonstrate that alanine and glutamine production in the diaphragm is limited primarily by the supply of amino groups via transamination, and that supply of NH, + influences relative rates of production of alanine and glutamine. Thus the stimulation of alanine pro- duction by leucine, isoleucine, or valine cannot be taken as evidence for the conversion of the carbon skeletons of these amino acids into pyruvate, as has been hypothesized recently (7, 13).

Although the carbon skeleton of leucine, like those of fatty acids, can not be converted to pyruvate in mammalian cells, leucine (0.5 mM) can increase the net production of pyruvate and lactate in skeletal muscle (24). Similar results have been obtained with high concentration of isoleucine, and this result has been interpreted as evidence for the conversion of the carbon chain of isoleucine into pyruvate (13). In an accompa- nying report, we present evidence that the stimulation of pyruvate production results from an inhibition of pyruvate oxidation by these amino acids (24).

Amino Acids Which Are Converted to Tricarboxylic Acid Cycle Intermediates in Skeletal Muscle-In addition to branched-chain amino acids and aspartate, Garber et al. (7) proposed that methionine, serine, and threonine supply car- bon chains for alanine synthesis, while phenylalanine, tyro- sine, and lysine provide carbon chains for glutamine synthe- sis. Ruderman and Berger (5) proposed that lysine, histidine, and proline provide chains for glutamine production in per- fused rat hindquarter. Conversion of an amino acid to alanine or glutamine must involve the conversion of the carbon skeleton of this amino acid to tricarboxylic acid cycle inter- mediates or pyruvate. We have tested whether such pathways actually exist in muscle with %-labeled substrates, since the conversion of ‘%-labeled amino-acids into tricarboxylic acid cycle intermediates or pyruvate must lead to the release of ‘TO,.

Manchester (25) and Goldberg and Odessey (26) showed that in rat diaphragms %-labeled aspartate, glutamate, leucine, isoleucine, valine, and alanine were converted to %O,. By contrast, 14C0, was not produced to any significant extent when the diaphragms were incubated with %-labeled methi- onine, serine, threonine, proline, histidine, phenylalanine, tyrosine, and lysine. Analogous experiments were undertaken to test whether the pattern of amino acid metabolism may be similar in other rat muscles. These studies examined the soleus, which is composed mostly of red fibers, and extensor digitorum longus, which is composed of pale fibers (27). With all the amino acids tested, these muscles behaved like the diaphragm. As shown in Table IV, they generated ‘%O, only from %-labeled leucine, valine, and asparagine. The absence of ‘%O, production from ‘%-labeled glycine, threonine, lysine, phenylalanine, and tyrosine is not due to lack of precursor transport, since comparable radioactivity was recovered in the acid-soluble pools and in muscle protein after incubation with these labeled amino acids as with branched-chain amino acids, aspartate, or glutamate (Table IV).

U-W-Amino-acids ‘&C recovered in

and muscles

lo-‘0 mollmg tissue

Leucine Soleus EDL”

Valine Soleus EDL

Glycine Soleus EDL

Threonine Soleus EDL

Lysine Soleus EDL

Phenylalanine Soleus EDL

Tyrosine Soleus EDL

Aspartic acid Soleus EDL

0.98 2 0.12 1.02 f 0.11

0.46 k 0.02 0.73 f 0.08

0.00 t 0.00 0.00 2 0.00

0.00 t 0.00 0.00 2 0.00

0.00 -c 0.00 0.00 -t 0.00

0.00 5 0.00 0.00 2 0.00

0.00 2 0.00 0.00 f 0.00

0.37 2 0.03 0.27 5 0.04

0.72 IT 0.04 0.75 5 0.06

0.85 t 0.01 1.02 f 0.02

3.10 ? 0.28 2.28 -t 0.07

2.35 e 0.07 2.28 f 0.25

2.28 k 0.15 2.63 k 0.10

0.99 k 0.05 1.18 i- 0.04

1.42 i 0.03 1.41 t 0.04

4.10 -t 0.39 2.78 ‘- 0.20

1.68 i 0.09 1.55 ? 0.10

0.79 i- 0.09 0.32 i 0.04

0.31 i 0.04 0.14 i 0.01

0.79 -t 0.12 0.38 i 0.04

0.37 i 0.02 0.18 i 0.01

0.55 k 0.05 0.19 k 0.01

0.45 2 0.06 0.22 T 0.02

0.55 k 0.05 0.41 k 0.04

a EDL, extensor digitorum longus.

TABLE V

Effects of amino acids on release ofglutamate, glutamine, and alanine from rat diaphragm

Measurements were performed as described in Table I. Values are means IT S.E. of observations on six diaphragms.

Additions (0.5 KIM) Glutamine Glutamic acid Alanine nmolimg tissue/Z h

None 1.09 f 0.05 3.37 i 0.12 2.99 k 0.17

Net change Aspartic acid t-O.70 i 0.08” +0.70 k 0.28” +0.34 i 0.15” Cysteine, methio- +O.lO f 0.03” -0.07 k 0.20 +0.38 k 0.28

nine, serine Tyrosine, lysine, -0.02 -t 0.05 -0.02 i- 0.01 +0.23 f 0.36

phenvlalanine

” p < 0.01. b p < 0.05.

novo synthesis of alanine and glutamine. Although leucine is rapidly oxidized, this amino acid may not provide carbon chains for the net synthesis of pyruvate, since it is a ketogenic amino acid, which is metabolized to acetyl-CoA.

The lack of ‘%02 production from %-labeled glycine, thre- onine, phenylalanine, tyrosine, and lysine (Table IV) (25, 26) or from methionine, serine, proline, and histidine (26) indi- cates that these amino acids in muscle are not converted to tricarboxylic acid cycle intermediates or pyruvate. Therefore, they cannot serve as precursors of alanine and glutamine. In skeletal muscle, only aspartate, asparagine, glutamate, iso- leucine, and valine can supply carbon skeletons for the de

In accord with this conclusion, aspartate at 0.5 mM stimu- lated alanine and glutamine production, whereas cysteine, methionine, and serine together, or tyrosine, lysine, and phenylalanine together, each at 0.5 mM did not promote alanine or glutamine production in the rat diaphragm (Table V). The lack of stimulation of alanine or glutamine production by the latter amino acids appears to contradict the results of Ruderman and Berger (5) and of Garber et al. (71, who reported that these amino acids at 10 mM increased either

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Origin of Alanine Produced in Skeletal Muscle 3681

alanine or glutamine release. Possibly at such high concentra- tion (approximately 100 times blood level) these amino acids may undergo some transamination with cu-ketoglutarate and thereby promote alanine and glutamine production. This suggestion was supported by the finding that infusion of high concentrations of the cr-keto analogues of methionine or phen- ylalanine in intact animal increased the release of the corre- sponding amino acids from muscle (28). However, such effects appear to be insignificant at physiological concentrations.

Effects of Glucose and Glycolytic Inhibitors on Alanine Production - Since alanine is formed from pyruvate by trans- amination with glutamate (Fig. 11, the addition of pyruvate stimulates alanine production by skeletal muscles (29, 30). Similarly, the presence of glucose in the medium should increase alanine by supplying pyruvate (provided that pyru- vate supply limits alanine synthesis). In the incubated rat diaphragm (6) and the perfused rat hindlimb (31), increasing glucose concentrations promoted alanine production. Table VI also shows that addition of glucose to the medium raised alanine production by the diaphragm and this effect was greater when the muscles were also provided with leucine (0.5 mM) or all three branched-chain amino acids (each at 0.3 mM). The simplest explanation for the larger stimulation of alanine production by glucose in the presence of the latter amino acids is that the carbon skeleton for alanine synthesis may become limiting when there is an ample supply of amino groups. In other words, the effects of glucose and leucine on stimulating alanine production are additive (Table VI).

Studies with 2-deoxyglucose provided additional evidence

TABLE VI

Effects of glucose on release of alanine

Measurements were performed as described in Table I. Experi- ment I used quarter-diaphragms from six rats. Experiment II used quarter-diaphragms from six different rats. All animals were fasted for 2 days. Amino acids were used at plasma concentrations except for alanine, glutamine, glutamate, and tyrosine.

Alanine Additions

Control +Glucose (5 mu) Increase nmollmg tissueI h

Experiment I None 1.51 f 0.17 2.19 2 0.15” 0.68 f 0.09 Leucine (0.5 mhi) 2.61 f 0.31 4.08 ? 0.17* 1.47 -t 0.18

Experiment II Amino acids 1.79 k 0.07 2.87 f 0.14* 1.08 k 0.12

(plasma concen- trations)

a Significant effect of glucosep < 0.05. * Significant effect of glucosep < 0.01.

TABLE VII

Effects of 2-deoxyglucose on alanine and lactate production and net

protein breakdown

The measurements were performed as in Table I. Values are means k S.E. of determinations of six animals.

Additions (1 IIM) Alanine Lactate Tyrosine nmollmg tissue12 h

None 2.46 YL 0.19 52.8 2 5.6 0.378 f 0.014 2-Deoxyglucose 1.17 k 0.13 14.7 -t 1.8 0.437 f 0.026

% change

-52 =k 9” -72 f 15” +14 k 76

u p i 0.01. b p < 0.05.

that supply of pyruvate limits alanine production. 2-Deoxyglu- case, which inhibits glycolysis, reduced lactate and alanine production dramatically in the rat diaphragm (Table VII). Under similar conditions, 2-deoxyglucose did not affect the transamination or the oxidative decarboxylation of the branched-chain amino acids by rat muscles. For example, when the soleus muscles from fasted rats were incubated with 1 l-Wlvaline (0.5 mM) and glucose (5 mM), the total transami- nation of valine equaled 0.55 ? 0.062 and 0.55 ? 0.059 nmoli mg of tissue/2 h in the absence and presence of 1 mM 2- deoxyglucose.

Thus, the production of alanine appears to be regulated by the supply of both amino groups and carbon skeletons. Addi- tion of glucose, 2-deoxyglucose, or other factors may affect alanine production by influencing the supply of pyruvate, of amino groups, or of both. Glucose, for example, not only increases the availability of pyruvate, but also reduces the supply of amino acids by inhibiting net protein breakdown (22) and may also reduce degradation of the branched-chain amino acids (10, 32). These opposite effects of glucose may account for the contradictory reports in the literature (6, 12, 13) and the failure of workers under certain conditions to demonstrate changes in alanine production with treatments that alter glycolytic rates (12, 13). Probably because 2-deoxy- glucose reduced glucose uptake, it enhanced net protein breakdown as judged from the rate of tyrosine release (Table VII). Since tyrosine is neither synthesized nor degraded in skeletal muscle, the rate of tyrosine accumulation can be used to determine the rate of net protein breakdown (22). (The net production of tyrosine under these conditions indicates that the rate of tyrosine generation by protein catabolism exceeds its rate of reincorporation by protein synthesis.) Increased net protein breakdown upon treatment with X-deoxyglucose gen- erates more alanine directly. Thus, the observed inhibition of alanine production by 2-deoxyglucose must underestimate the actual reduction in de nouo synthesis of alanine.

Studies on the effects of iodoacetate and fluoride further illustrated that the supply of both pyruvate and amino groups limit alanine production. Besides their recognized effect on glycolysis, however, these highly reactive compounds, unlike 2-deoxyglucose, were also found to affect two other processes which determine the supply of amino groups for alanine synthesis: 1) the rate of amino acid production by net protein breakdown, and 2) the rate of degradation of branched-chain amino acids. Iodoacetate at 0.2 mM accelerated net protein breakdown by about 90% (Table VIII) and inhibited almost completely the oxidative decarboxylation of valine (Table VIII) and leucine (10). In addition, iodoacetate decreased the transamination of valine by about 95%, and reduced the alanine release by 80%. Fluoride also increased net protein breakdown, inhibited valine oxidation, and decreased alanine release, although it was less effective than iodoacetate. How these compounds inhibit amino acid decarboxylation is un- known. Since the decarboxylation requires lipoic acid and CoA, the reaction should be sensitive to sulfhydryl inhibitors such as iodoacetate. The mechanism by which iodoacetate and fluoride increased net protein breakdown is also not clear. It is likely that they block energy production and thereby reduce protein synthesis. In addition, they may enhance protein degradation by preventing glycolysis (22).

The net inhibition of alanine production by iodoacetate and fluoride suggests that although the increased proteolysis gen- erates more alanine directly and supplies amino acids for the

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3682 Origin of Alanine Produced in Skeletal Muscle

TABLE VIII

Effects of iodoacetatr and fluoride on saline metabolism, net protein degradation, and alanine production

Hemidiaphragms were obtained from six rats fasted for 2 days. The tissues were incubated as in Tables I and II. Valine was present in the medium at 0.5 mM. Values are means i SE. of observations on six diaphragms.

“C recovered in Additions Total transamination Tyrosine Alanine

co, a-K&o acid

nmollmg tissue/2 h

Experiment I None 5.28 ? 0.41 0.899 2 0.046 6.18 t 0.61 0.164 t 0.018 4.01 2 0.41

Iodoacetate (0.2 mM) 0.064 ? 0.007” 0.290 k 0.042” 0.354 f 0.005” 0.289 -c 0.018” 0.836 f 0.050”

Experiment II None 6.17 f 0.30 0.501 f 0.071 6.67 -t 0.32 0.323 k 0.020 2.54 -t 0.24

Sodium fluoride (10 mM) 1.68 * 0.19” 0.342 t 0.093” 2.02 -t 0.22” 0.542 ? 0.037” 1.93 ? 0.196

“p < 0.01.

* p < 0.05.

synthesis of alanine, these effects are much smaller than the dramatic effects on amino acid catabolism and glycolysis. In any case, because of their multiple effects, it is probably impossible to use iodoacetate and fluoride to examine unam- biguously whether glycolysis or protein breakdown contribute to alanine production. Nevertheless, other investigators have used iodoacetate (7) and fluoride (13) to determine whether glucose uptake by muscle correlates with alanine production. Garber et al. (7) did not observe any effects of iodoacetate in rat epitrochlaris incubated with 10 mM glucose, while Gold- stein and Newsholme (13) found that fluoride did not decrease alanine production in diaphragms in glucose-free medium. On the other hand, Taegtmeyer et al. (33) found that these compounds markedly inhibited alanine production in incu- bated rabbit heart papillary muscles in accord with the present results. Although the experiments with iodoacetate and fluoride may be uninterpretable and have given divergent results (Table VIII) (6, 12, 13) for unknown reasons, the clear effects of 2-deoxyglucose, a specific glycolytic inhibitor, un- ambiguously showed that pyruvate supply from glucose af- fected alanine production.

Source of Pyruvate for Alanine Synthesis-The fundamen- tal disagreement about the origin of alanine concerns the relative importance of glucose or of amino acids generated by protein breakdown as a source of pyruvate. In order to settle this issue, the rates of pyruvate generation from exogenous glucose by glycolysis and the maximal rate of pyruvate pro- duction from amino acids derived from net protein breakdown of muscle proteins were determined.

As discussed above, aspartate, asparagine, glutamate, iso- leucine, and valine are the only amino acids which can be converted to tricarboxylic acid cycle intermediates in muscle. Together these amino acids comprise about 28% of the residues in rat muscle proteins (6, 34). When aspartate, glutamate, isoleucine, and valine are produced by the net breakdown of muscle proteins, a certain fraction (about 50%, Refs. 6 and 34) of these amino acids are released directly by the muscle (5, 6) and some are released as their a-keto acid derivatives (Table II.’ Although the amount of asparagine released into medium by the incubated muscle could not be measured in these studies, the data of Jefferson et al. (35) on perfused rat hemicorpus suggest that 50% or more of asparagine from net proteolysis is released by the tissue and this figure has been used in the present calculations. The carbon chains of the remaining fraction of these amino acids enter the tricarboxylic acid cycle and may be used for the synthesis of alanine and glutamine (34). In any case, the maximal possible rate of production of pyruvate from amino acids derived from net

protein breakdown may be obtained by multiplying the rate of generation of amino acids from muscle protein by 0.28.

The rate of generation of amino acids from protein break- down was estimated from the measured rate of tyrosine release and the frequency of tyrosine in muscle protein (Table IX). The number obtained by this approach was very similar to that measured by amino acid analysis of the total amino acids released (6). The rate of generation of aspartate, aspar- agine, glutamate, isoleucine, and valine from net protein breakdown during the 2-h incubation was calculated to be 5.3 nmol/mg of tissue (Table IX). This value represents the maximal rate of pyruvate production from net protein degra- dation.

In addition, the rate of glycolysis from exogenous glucose was estimated by incubating muscles with D- 1 5-3H]glucose. One 3HOH molecule is generated from the conversion of one D-[ 5-RH]glucose molecule to pyruvate or to phosphoenolpyru- vate molecules. Thus, the production of “HOH can be used to measure the rate of glycolysis2 (18). The mean rate of glucose utilization was 43 + 3.3 nmol/mg of tissue/2 h, which is equivalent to 86 ? 6.6 nmol of pyruvatelmg of tissue/2 h (Table IX). The number is approximately similar to or slightly less than the rate of consumption of pyruvate (see subsequent paper). Muscle glycogen may undergo significant breakdown in vitro, which may account for the small additional amount of pyruvate. These findings also indicate that amino acids from protein breakdown can supply at most 6% of the pyruvate produced. Since about 50% of this group of amino acids are released as amino acids or as their a-keto acid derivatives (Table II) (6, 34), the amino acids generated from net protein breakdown can supply at most 3% of the pyruvate for alanine synthesis.

As shown in the subsequent manuscript (34), the total consumption of pyruvate, which includes pyruvate oxidation to acetyl-CoA, and lactate, pyruvate, and alanine released into the medium, ranged between 100 and 110 nmolimg of tissue/2 h. Thus, amino acids can supply at most 3% of the pyruvate consumed by the diaphragm.

Additional evidence that glycolysis is much more important than amino acids as a source of pyruvate was obtained by similar analyses of the published data on the amino acid

2 There is possibly insignificant production of3HOH in the absence of net glycolysis due to a futile cycle between glucose and glyceral- dehvde 3-Dhosnhate and dihvdroxvacetone Dhosohate (36). because skeletal k.wcie contains very little glucose-&phosphatase (37). Therefore, the generation of one 3HOH molecule should indicate the breakdown of one o-15-3Hlglucose molecule to pyruvate or P-enolpy- ruvate.

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Origin of Alanine Produced in Skeletal Muscle 3683

TABLE IX TABLE X

Maximal amount ofpyruvate that may be derived from net protein breakdown in incubated rat muscles

Aspartic acid, asparagine, glutamic acid, is&u&e, and valine

Maximal amount ofpyruvate that may be derived from net protein breakdown in human forearms

comprise about 28% of total amino acids in diaphragm muscle, and therefore upon net protein breakdown, this fraction should represent 28% of the total amino acids produced. The amount of these amino acids that is metabolized in the muscle must represent the difference between the amount generated by protein breakdown and the amount that is released into medium. For analysis on the dia- phragm, quarter-diaphragms from rats fasted for 2 days were incubated as described in Table I. Data for epitrochlaris on glucose uptake, the release of total amino acids, and the release of aspartic acid, glutamic acid, isoleucine, and valine were based upon Figs. 2 and 5 of Garber et al. (12).

Glucose uptake, release of total or-amino nitrogen were taken from Table I of Pozefsky et al. (38). The fraction of aspartic acid, asparagine, glutamic acid, isoleucine, and valine that were released by the muscle could not be obtained. The calculation of the maximal fraction of pyruvate that could be provided by net protein breakdown was made on the assumption that all these five amino acids from net protein breakdown were converted to pyruvate.

A&&venous difference

Diaphragm Epitrochlaris nmollmg tissue/Z h nmolig tissuelmin

Total amino acids production from protein degradation

19 95

Aspartic acid, alanine, glutamic acid, isoleucine, valine

From net protein degradation Released into medium Metabolized in muscle

Pyruvate production from exog- enous glucose

5.3 27 2.7 17 2.6 10

86 2 6.6 240

Arterio-deep vein Art&o-superficial vein

nmolllitw

Total amino acids production 0.48 0.15

from protein degradation Aspartic acid, asparagine, glu- 0.11 0.03

tamic acid, isoleucine, valine Pyruvate production from ex- 0.40 0.52

ogenous glucose Maximal fraction of pyruvate

that can be derived from net c--0.11- <-0.03

0.40 + 0.11 0.52 + 0.03 protein breakdown =22% -6%

Maximal fraction of pyruvate 2.6 10 that can be derived from net <KrE <240+10 protein breakdown =3% =4%

ignored their major fate in muscle, which is utilization in the de ~LOUO synthesis of glutamine (see subsequent paper).

CONCLUSION

release from the rat epitrochlaris incubated in vitro (Table IX) and from human forearm. The data of Garber et al. (Figs. 2 and 3 from Ref. 12) showed that the epitrochlaris took up glucose and released total amino acids at about 120 and 95 nmol/g of tissue/min, respectively. Since the intracellular concentrations of amino acids remained constant in the epi- trochlaris during the l-h incubation (7), the total release of amino acids was taken as the rate of generation of amino acids from net protein breakdown which was used to calculate the endogenous production of aspartate, asparagine, glutamate, isoleucine, and valine. Aspartate, glutamate, isoleucine, and valine release into medium accounted for about 15 nmol/g of tissuelmin. Comparison of these data showed that these five amino acids from net protein breakdown can supply at most 4% of pyruvate produced in the incubated epitrochlaris (Table IX).

Both in vitro and in viuo, exogenous glucose appears to be the major carbon source for alanine synthesis (Table IX). This conclusion is in accord with the original formulation of the glucose-alanine cycle (2, 8). Supplying branched-chain amino acids exogenously produces increased amounts of glutamate, which in turn promotes alanine and glutamine production. These results make it unnecessary to hypothesize a new pathway to convert the carbon chains of leucine into pyruvate (7). Among the amino acids released by the net breakdown of muscle proteins only aspartate, asparagine, glutamate, isoleu- tine, and valine can possibly provide carbon skeletons for the de nouo synthesis of alanine or glutamine, since they are the only ones that can enter the tricarboxylic acid cycle.

Similar calculations were made using the data of Pozefsky et al. (38) on the arteriovenous difference of a-amino nitrogen and glucose across human forearm in the postabsorptive state. The release of total a-amino nitrogen was taken as the rate of generation of amino acids from net protein breakdown. As shown in Table X, the breakdown of muscle protein can provide at most 22% and 4% of the pyruvate produced in the deep and superficial muscles of the human forearm, respec- tively. These percentages appear higher in the human forearm than in the incubated rat muscles. This difference may reflect the lack of specific information on the actual release from forearm muscles of the five amino acids that can be precursors of pyruvate.

It was recently proposed by other investigators that amino acids derived from the net protein breakdown provide carbon skeletons for alanine synthesis (7, 13). The present studies, however, fail to support this conclusion and have provided alternative explanations of the published experiments by these workers (7, 12, 13). Although the largest fraction of the pyruvate and alanine are derived from exogenous glucose, it is still possible that a small fraction of pyruvate (e.g. 3% in the incubated rat diaphragms, see Table IX) can be provided by amino acids derived from protein breakdown. This impor- tant issue has been investigated in the subsequent report (34).

The above analyses are of course highly conservative esti- mates and calculate only the maximal percentage of pyruvate provided by protein breakdown. These calculations have as- sumed the complete conversion of the carbon chains of aspar- tate, asparagine, glutamate, isoleucine, and valine entering the tricarboxylic acid cycle into pyruvate and have thus

One major problem in accepting the glucose-alanine cycle is that the shuttling of pyruvate residues between glucose and alanine (like the Cori cycle) does not provide net gluconeo- genie substrates to the organism (26). Yet gluconeogenesis from amino acids derived from muscle protein is essential during fasting, and alanine is quantitatively the most impor- tant gluconeogenic amino acid in the liver (4). These consid- erations led other investigators to the hypothesis that alanine synthesized in muscle is derived from other amino acids (12, 13, 39). However, amino acids derived from muscle protein may still serve as substrates for gluconeogenesis without being converted to alanine. The amino acids not entering the tricarboxylic acid cycle in muscle appear to be released di-

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3684 Origin of Alanine Produced in Skeletal Muscle

rectly into circulation as amino acids or cr-keto acids, and thus may be used for hepatic or renal gluconeogenesis without prior conversion to alanine. In addition, as shown in the next report (341, the major fate of aspartato, asparagine, gluta-

mate, isoleucine, and valine in skeletal muscle is conversion to glutamine, which probably also serves as a gluconeogenic precursor.

15.

Acknowledgments-We thank Ms. Nicolina Fedele and Ms. Mary Beth Tabacco for their excellent technical assistance throughout this work. We are grateful to Drs. Richard Odes- sey and Mark Tischler for helpful suggestions.

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