Alanine and Glutamine Synthesis and Release from Skeletal Muscle I. GLYCOLYSIS AND AMINO ACID RELEASE*

(Received for publication, April 21, 1975)

ALAN J. GARBER, IRENE E. KARL, AND DAVID M. KIPNIS+

From the Metabolism Division, Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110

The synthesis and release of alanine and glutamine were investigated with an intact rat epitrochlaris muscle preparation. This preparation will maintain on incubation for up to 6 hours, tissue levels of phosphocreatine, ATP, ADP, lactate, and pyruvate closely approximating those values observed in gastrocnemius muscles freeze-clamped in uiuo. The epitrochlaris preparation releases amino acids in the same relative proportions and amounts as a perfused rat hindquarter preparation and human skeletal muscle. Since amino acids were released during incubation without observable changes in tissue amino acids levels, rates of alanine and glutamine release closely approximate net amino acid synthesis. Large increases in either glucose uptake or glycolysis in muscle were not accompanied by changes in either alanine or glutamine synthesis. Insulin increased muscle glucose uptake 4-fold, but was without effect on alanine and glutamine release. Inhibition of glycolysis by iodoacetate did not decrease the rate of alanine synthesis. The rates of alanine and glutamine synthesis and release from muscle decreased significantly during prolonged incubation despite a constant rate of glucose uptake and pyruvate production. Alanine synthesis and release were decreased by aminooxyacetic acid, an inhibitor of alanine aminotransferase. This inhibition was accompanied by a compensatory increase in the release of other amino acids, such as aspartate, an amino acid which was not otherwise released in appreciable quantities by muscle. The release of alanine, pyruvate, glutamate, and glutamine were observed to be interrelated events, reflecting a probable near-equilibrium state of alanine aminotransferase in skeletal muscle.

It is concluded that glucose metabolism and amino acid release are functionally independent processes in skeletal muscle. Alanine release reflects the de novo synthesis of the amino acid and does not arise from the selective proteolysis of an alanine-rich storage protein. It appears that the rate of alanine and glutamine synthesis in skeletal muscle is dependent upon the transformation and metabolism of amino acid precursors.

Recent studies on the regulation of hepatic gluconeogenesis have emphasized the role of alanine as an important and potentially limiting substrate for carbon flow in this pathway (l-4). The splanchnic uptake of this amino acid, as determined by arteriohepatic venous differences in humans, greatly ex- ceeds the uptake of all other amino acids (S-S), and substantial rates of glucose formation from alanine have been observed in isolated perfused liver preparations from a number of animal species (9, 10). Correspondingly. recent studies have indicated that skeletal muscle releases large quantities of this amino acid. For example, alanine release from the perfused hindquar- ter preparation of the rat exceeds that of any amino acid other than glutamine (ll), and similar findings have been reported in the human forearm (12, 13), and leg (7, 14). In contrast to its high rate of release, the alanine content of rat or human muscle

‘This work was supported by Grants 5 ROl AM01921 and 1 F03 AM54124 from the United States Public Health Service.

$ To whom requests for reprints should be addressed.

protein is not substantially different from that of most other amino acids (15). These data imply that the comparative enrichment of alanine release from skeletal muscle results either from the selective proteolysis of an alanine-rich storage protein (11) or that de nouo synthesis of alanine occurs in skeletal muscle.

Based on these observations, a glucose-alanine cycle has been proposed (9, 16). In this formulation, glucose enters the skeletal muscle cell, is metabolized to pyruvate, subsequently transaminated to alanine, then released from muscle and taken up by the liver where it is reconverted to glucose. Ac- cording to this hypothesis, glucose carbon is recycled to the liver in much the same manner as lactate is recycled in the Cori cycle (17). One consequence of the operation of such a glucose- alanine cycle is that it cannot account for the net flow of carbon from protein to carbohydrate which is known to occur during fasting (17, 18). I f glucose formation from lactate and alanine represents recycling of already existing glucose carbon, then

826

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Muscle Alanine and Glutamine Release 827

there is no known mechanism to account for the net appear- ance of significant amounts of new carbon from extra hepatic sources into glucose. In light of this inconsistency, the interre- lationships in skeletal muscle between glucose metabolism and amino acid release were investigated using an isolated intact rat skeletal muscle preparation. Portions of this study have been presented previously (19).

MATERIALS AND METHODS

Adenylate kinase (EC 2.7.4.3), alanine aminotransferase (EC 2.6.1.2), aspartate aminotransferase (EC Z&1.1), glucose-6-phosphate dehydrogenase (EC 1.1.1.491, glutamate dehydrogenase (EC 1.4.1.3), glutaminase (EC 3.5.1.2), hexokinase (EC 2.7.1.1), lactate dehydro- genase (EC 1.1.1.27), XAD+-malate dehydrogenase (EC 1.1.1.37), NAD+, NADH, NADP’, and phenazine methosulfate were obtained from the Sigma Chemical Co. (St. Louis). Aminooxyacetic acid was purchased from Eastman Organic Co. (Rochester, N. Y.). Tetrameth- ylphenylenediamine was obtained from K & K Laboratories (N. Y.). Acetone (histologic grade) was from Fisher Scientific Co. Glucagon- free crystalline insulin was a generous gift of Dr. J. Galloway (Eli Lill) Co., Indianapolis).

Male Sprague-Dawley rats weighing from 60 to 160 g were main- tained ad libitum on Purina LAB Chow. Animals were killed by a blow to the back of the head, and the muscle under study was isolated rapidly. The entire length of the muscle was visualized by blunt dissection and loosened from the fascial planes to which it adhered. The proximal and distal tendons were transected near their insertions and the muscles removed with further blunt dissection where necessary. Excess connective and tendon tissues were trimmed, and the muscle rinsed briefly in isotonic sodium chloride, blotted, and placed in 0.5 ml of modified Krebs-Henseleit buffer (pH 7.4). This medium contained one-half the calcium concentration as originally formulated with increased buffering capacity provided by the addition of 5 mM Hepes’ (pH 7.4). The flask was then gassed with 95% OJ5% CO*, stoppered, and incubations were carried out in a shaking water bath at 35” for periods ranging from 30 to 360 min. At the end of the incubation, the muscle was removed rapidly, rinsed in cold buffer, blotted, and frozen in liquid nitrogen. The incubation media were then heated at 100” for 2 min to destroy trace amounts of enzymatic activities released hy the muscle during incubation which might interfere with subsequent analytical determinations. Lactate dehydrogenase was the principal enzyme found in the media, but even after prolonged incubation, this release amounted to less than 0.01 units/ml of media. All muscle samples and incubation media were stored at -80”. In a room maintained at 15”, frozen muscle tissue was powdered in a percus- sion mortar chilled in liquid nitrogen. The powder was rapidly homogenized in 300 ~1 of 3 M perchloric acid, 1 ml of iced water was added, and the suspension centrifuged at 4500 x g for 15 min. The supernatant was neutralized with a solution containing 3 M potassium hydroxide, 0.4 M imidazole, and 0.7 M potassium chloride (20). and the potassium perchlorate precipitate removed by further centrifugation. For determination of in uiuo substrate levels, rats were anesthetized with intraperitoneal injections of pentobarhital (Nemhutal. 50.0 mg/ kg). The gastrocnemius muscles were visualized by blunt dissection, and then frozen rapidly in situ usin g Wollenherger tongs previously chilled in liquid nitrogen. Tissue extracts were prepared as described above.

Fluorometric microtechniques (20) were adapted or developed for determinations of enzyme activities and substrate levels in both muscles and incubation media for glucose, pyruvate, aspartate, ATP, ADP, AMP, and phosphocreatine. Lactate (211, alanine (‘21, gluta- mate, and glutamine* were all determined enzymatically. Since aminooxyacetate is an irreversible inhibitor of transaminases (23), coupled enzymatic assays for alanine and aspartate in the incubation media were performed using samples pretreated with acetone for 1 hour at 3’i” to remove the inhibitor. Unreacted acetone was volatilized at 100° for 10 min.

RESULTS

Metabolic Characteristics of Various Muscle Preparations- The suitability of a number of intact rat skeletal muscle prep-

‘The ahhreviation used is: Hepes, N-2-hydroxgethylpiperazine- N’-2-ethanesulfonic acid.

*I. E. Karl and D. M. Kipnis, manuscript in preparation.

arations for studying amino acid release in uitro was inves- tigated. Owing to the large free pools of alanine and glutamine in muscle tissue (24), only intact muscle preparations were examined. In skeletal muscle,’ the ability to store high energy phosphate in the form of phosphocreatine, and to maintain normal ATP levels are sensitive indices of tissue integrity. As shown in Table I, only the epitrochlaris preparations maintained high energy phosphate levels comparable to that seen in uiuo. Indeed, the epitrochlaris preparations maintained these levels after incubations up to 6 hours duration. In contrast, both the soleus and extensor digitorum longus, after 3 hours, failed to conserve high energy phosphate, frequently exhibited considerable rigor, and showed evidence of significant tissue hypoxia as judged by elevated lactate to pyruvate ratios. The epitrochlaris preparations used for the studies noted in Table I weighed 30 mg or less, whereas the extensor digitorium and soleus preparations weighed upward of 60 mg. The epitrochlaris was consistently less than 1.0 mm in thickness as compared to a greater than 2 mm thickness noted for the other two muscle preparations. It seems likely that the decreased viability shown in uitro by the extensor digitorum longus and soleus results from their greater thickness and a consequent decreased oxygenation of the central core of the muscle preparation. Despite these metabolic differences, rates of alanine, glutamate, and glutamine release by the three muscle preparations were generally similar (Table II). The intracellular free amino acid pools after incubation were comparable in all three muscles.

Glucose uptake by the epitrochlaris muscle preparation remained constant in the presence or absence of insulin throughout 3 hours of incubation (Fig. 1A). Pyruvate release remained constant during this period in the presence of insulin; in the absence of the hormone, pyruvate release decreased about 40% between the 2nd and 3rd hour of incubation-the lactate to pyruvate ratio, however, remained constant through- out (Fig. 1B). Insulin regularly increased glucose uptake 4- to 5-fold. Since the in vitro epitrochlaris preparation exhibited stable metabolic parameters similar to those of skeletal muscle in uiuo, this preparation was used exclusively for the remainder of this study.

The pattern of amino acid release by the epitrochlaris skeletal muscle preparation is shown in Fig. 2. Alanine and glutamine together account for more than 6O?G, of the total amino acid release. The pattern of amino acid efflux from the epitrochlaris preparation is remarkably similar to that noted in the perfused rat hindquarter preparation (11) and human skeletal muscle with the exception of a small net glutamate uptake in the latter tissue (13).

The data shown in Table I were obtained in the presence of glucose and insulin. Since this hormone influences the trans- port of both glucose and amino acids across the muscle cell membrane (25), the effects of insulin on the release of alanine, aspartate, glutamate, and glutamine were investigated (Fig. 3). Over a wide range of concentrations (10 microunits/ml to 700 milliunits/ml), insulin did not significantly affect the release rate of any of the amino acids studied.

Kinetics of Amino Acid Release-significant decreases in the rates of amino acid release were observed during the course of 3 hours of incubation (Fig. 4). In general, the rate of’ release of alanine, aspartate, glutamine, and glutamate decreased linearly with increasing time of incubation so that after :I hours had elapsed, the observed rates were about two-thirds the rates noted after 30 min of incubation. These decreases do not

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828 Muscle Alanine and Glutamine Release

TABLE I

Adenine nucleotide and lactate-pyruuate balance in intact rat skeletal muscles after incubation

Various intact rat skeletal muscle preparations were dissected, rinsed, and placed in 500 ~1 of Krebs-Henseleit media buffered with 5 mM Hepes, pH 7.4. Glucose (5 m~) and insulin (100 milliunits/ml) were included in these experiments. The muscles were incubated in an atmosphere of 95% 0,/S%, CO2 at 37” in a shaking water bath for 3 hours. At the end of the incubation, these were rapidly removed, rinsed, blotted, and frozen in liquid nitrogen. The tissues were subsequently weighed, powdered, and extracted with 10 volumes of 3 M perchloric acid. Fluorometric microtech- niques for the enzymatic determinations of substrates and cofactors were used on the neutralized perchlorate extracts as outlined under “Materials and Methods.” Values for muscle intermediates are means l S.E. in micromoles/g of muscle, wet weight, with the number of preparations studied given in parentheses.

Tissue Levels

Muscle Preparation

ATP Lactate -- P-Creatine ATP ADP AMP Lactate Pyruvate ADP Pyruvate

~moles/g

Epitrochlaris 13.11 4.52 0.68 0.05 2.31 0.12 6.78 17.29 (20) +0.37 +0.31 - - +0.05 LO.01 LO.37 - +O.Ol +0.17 +1.64 -

Extensor 8.08 2.92 0.69 0.10 4.58 0.08 4.27 61.92 Digitorum Longus +0.60 +0.35 kO.04 to.01 20.88 +0.02 +0.23 +2.71 - - - -

(24)

Soleus 5.24 2.22 0.64 0.11 4.38 0.06 3.37 73.94 (24) +0.29 +0.16 +0.05 +0.01 +0.22 +0.01 +0.51 +4.87 - - - - - - - -

Freeze-Clamped 15.61 4.68 0.61 0.06 2.08 0.13 7.72 15.76 Gastrocnemius Muscle +1.09 +0.24 +-0.06 +0.02 +0.24 +0.02 +0.38 + 1.56 - - - - - - - In Vivo

(8)

TABLE II

Free amino acid tissue leuels and rates of amino acid release by various skeletal muscle preparations

Skeletal muscle preparations from the rat were obtained and incubated for 1 hour as outlined in Table I. Tissue levels of amino acids were determined in neutralized perchloric acid extracts of musc!es frozen rapidly in liquid nitrogen after incubation. Values given are the means L S.E. in nanomoles/mg with the number of experiments in parentheses. Amino acids released were determined enzymatically on the incubation media with rates (mean L S.E.) given as nanomoles/min/g of muscle, wet weight.

Muscle Preparation

Alanine PeleaSe

Tissue Level to \Icdiur: nnoles mfz nnoles/min/p

Glutmate Glutamine Release i:elease

Tissue Level to Medium. Tissue Level to Medium l-lldC.S/~g nmoles/min/g lll?lOl~S/IIl~ nnoles/min/g

1:pitrochlaris 1.55 + (j.07 20.23 .+_ l.!il lJ.Y4 + 0.09 9.30 + 1.43 3.66 + 0.42 30.75 + 2.63 .- -. -- -.. (2(l)

Lxtensor ijioitorum Loniws “(24) '- 1.43 - + ti.19 10.55 - + 1.56 0.93 - + c.12 7.68 - + 1.28 4.11 - + 0.63 24.77 -. + 1.22

Soleus 1.36 t 0.13 lb.54 + 2.05 0.94 - + 0.16 13.81 + 2.21 3.69 + C.40 33.71 3.58 (24)

- .- 5

Freeze-Clamped Gastrocnemius Muscle 1.61 + 0.12 1.02 + 0.10 3.65 + 0.33 .- .- - In Vivo

(8)

appear to be attributable to a decrease in the viability of the remainder of the incubation (Fig. 4B). No change in the intra- preparation, since with more prolonged incubations of up to 6 cellular free pools of glutamate and aspartate was noted hours in length, phosphocreatine levels, the ratios of ATP and throughout the 3 hours of incubation (Fig. 4, C and D). Since ADP, and the rate of glucose uptake were the same as those the net release of amino acids cannot be accounted for by presented in Table I. With each amino acid studied, the initial corresponding changes in their free intracellular levels, rates tissue level found after 30 min of incubation was not sig- of amino acid release must approximate rates of formation. nificantly different from that found in muscle freeze-clamped The small decline in tissue levels of alanine observed during in situ (Table II). Tissue levels of alanine remained constant the 3rd hour of incubation accounts for less than 12% of the for 120 min of incubation, but after 3 hours, decreased approxi- alanine released during this time period, and in the case of mately 10% (Fig. 4A). Tissue glutamine remained constant glutamine, the decline in intracellular level during the 3rd hour for 90 min of incubation and then declined 25% during the of incubation accounts for less than 20% of the amino acid

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Muscle Alanine and Glutamine Release 829

I GLUTAMINE 150 A 1

-

1 0 120 180

MINUTES

34B

01 / 1 1 ‘0 0 60 120 180

MINUTES

FIG. 1. Time course of’ glucose uptake and lactate and pyruvate production by the rat epitrochlaris skeletal muscle preparation. Intact epitrochlaris preparations were incubated m Krebs-Henseleit hufl’er containing .5 mu Hepes and 5 IBM glucose for 30 to 180 min. Insulin (100 milliunits/ml) was added where indicated. Glucose uptake (A) and pyruvate production and the ratio of lactate to pyruvate release CR) are shown for each time period studied. Values shown are means 1 S.E. for at least eight experiments

Il WE

FIG. 2. Amino acids released from the rat epitrochlaris muscle. Intact rat epitrochlaris skeletal muscle preparations were incubated for 1 hour as outlined under “Materials and Methods.” The amino acid content of the media was then determined hy column chromatography using a Beckman amino acid analyzer. Values shown are the means + S.E. (indicated by the vertical line) for at least eight experiments.

released. It seems clear therefore, that the decline in rates of amino acid release with prolonged incubation is not a result

of a rapid depletion of the intracellular amino acid pool. It is possible that the re-uptake of amino acids released into

the incubation medium may produce an artifactual decline in the rate of amino acid release noted on prolonged incubation. To exclude this possibility, muscles were transferred hourly

GLUTAMATE

ASPARTATE

3 :ic a

-2 -1 0 1 2 log INSULIN] mu/ml

Flc. 3. Effect ol’ exogenous insulin on alanine, aqartate, pluta- mate, and glutamine release from rat skeletal muscle. Epitrochlaris muscles were incubated in Krehs-Henseleit hui’f’er (pH 7.4~ modified with Hepes (5 mu). Increasing amounts of insulin were added in the concentrations indicated. After incubation, the amounth (11’ amino acids in the media were determined enwmatically, with values noted as means L S.E. f’or at least eight expe&ents.

into fresh incubation media during a Shour incubation and the rate of amino acid release determined for each time period. As shown in Fig. 5, the decrease in alanine and glutamine release was still noted and was virtually identical with that observed under standard incubation conditions (Fig. 4, A and B). Furthermore, glucose uptake and pyruvate production rates in these transfer experiments were similar to those noted earlier (Fig. 1). These results also suggest that the decreased forma- tion of alanine and glutamine with prolonged incubation was caused by mechanisms unrelated to either amino acid transport or glucose uptake.

Glucose Metabolism and Amino Acid Release-The failure

to observe a correlation between the rates of glucose uptake (Fig. 1) and alanine release (Fig. 4.4) was contrary to expecta-

tions based on current concepts of the glucose-alanine cycle (9, 16). To investigate this point further, epitrochlaris muscle

preparations were incubated for 1 hour in varying concentra- tions of glucose (0 to 20 mM) in the presence and absence of insulin (Table III). No changes were observed in alanine, aspartate, glutamate, or glutamine release despite a range of glucose uptake rates of 0 to 413 nmol/min/g and pyruvate release rates of 13.8 to 29.6 nmol/min/g.

Low concentrations of artificial electron acceptors such as phenazine methosulfate and tetramethylphenylenediamine doubled the rate of glucose uptake and lactate release (Table IV). Pyruvate production was increased more than X-fold and the ratio of released lactate to pyruvate declined significantly from 16:l to 1O:l indicating a shift to a more oxidized state in the cytosolic oxidation-reduction potential. Despite increases

in the rates of glucose metabolism and pyruvate production, alanine release was markedly decreased. However, both gluta- mine and glutamate release increased under these conditions so that the total amount of alanine, glutamine, and glutamate

released was not significantly altered by these electron accep- tors. These changes cannot be attributed to alterations in the energetic state of the muscle preparations, since ATP and phosphocreatine levels were not altered during incubation for 1

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830 Muscle Alanine and Glutamine Release

60 120 180 MINUTES

60 120 It30 MINUTES

60 120 180 MINUTES MINUTES

FIG. 4. Effect of duration ol’ incubation on amino acid release and tissue amino acid levels in rat skeletal muscle. Epitrochlaris muscle preparations were incubated for varying times in Krehs-Henseleit buffer in the presence and absence of exogenous insulin (100 milliunits/ml). Alanine release rates and tissue levels (A), glutamine release rates and tissue levels (B). glutamate release rates and tissue levels (C), and aspartate release rates and tissue levels (D) were determined for each preparation. All values represent the mean * S.E. for at least eight experiments.

30

GLUTAMINE 20

IO

FIG. 5. Time course of glucose uptake and pyruvate and amino acid release from muscle during sequential changes of the Incubation media. Muscles were incubated as described in Table I and under “Materials and Methods.” At the end of each hour of incubation, muscles were transferred into fresh media and the incubation contin- ued. The uptake and release of substrates were determined hourly for each preparation. Rates are expressed as nanomoles/min/g of muscle, wet weight, and represent the means l S.E. of at least eight experi- ments.

hour with either phenazine methosulfate or tetramethyl- phenylenediamine. These agents also appear to affect amino acid efflux mechanism(s), since the intracellular levels of alanine and glutamine were increased 10 to 20% in the presence of both electron acceptors (Table V). The increase in the intracellular alanine pool, however, accounts for less than a third of the decrease observed in alanine release. Estimates of net amino acid formation indicate that despite increased glucose uptake and pyruvate formation, alanine formation was decreased 50 to 60% by these electron acceptors (Table V). In contrast, glutamine formation was increased approximately 35

to 55% by these agents (Table V). The interrelationships of glucose uptake, pyruvate forma-

tion, and alanine production and release were further studied

by inhibiting the glycolytic pathway with iodoacetate. Under these conditions, alanine, glutamate, and glutamine release were increased despite marked reductions in glucose uptake and lactate and pyruvate production (Table VI). The increased rate of release was accounted for almost exclusively by loss from the intracellular pool; iodoacetate did not significantly affect the net formation of alanine, but reduced the net formation of glutamine and glutamate -40 and - 1570, respec- tively (Table VII). At a concentration of 0.2 mM, iodoacetate did not alter the ATP level after 1 hour of incubation, although phosphocreatine was reduced -50% (Table VIII). At higher concentrations (e.g. 0.5 mM), iodoacetate depressed both ATP and phosphocreatine levels.

Since the foregoing data failed to demonstrate a direct relationship between glucose metabolism and alanine and glutamine formation in skeletal muscle, the effects of other

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Muscle Alanine and Glutamine Release 831

TABLE III

Relationship between alterations in glucose uptake, pyruuate production, and amino acid release by skeletal muscle

Intact rat epitrochlaris muscle preparations were incubated for 1 hour in Krebs-Hens&it buffer with 5 rn~ Hepes [pH 7.4), together with

variable amounts of glucose (0 to 20 mM) and insulin (100 milliunits/ml) added where indicated. Glucose uptake, pyruvate production. and amino acids released were determined enzymatically in the incubation media and values noted are means i SE. for at least eight experiments

and are given as nanomoles/min/g of muscle, wet weight. Other details are as in Table I and under “Materials and Methods.“

Glucose insulin Glucose Pyruvatc Alanine (;lutamate (;lutamine

Concentration Added Uptake Released ti: munits/ml nnoles/r:in/ fi

0 0 0

0 100 n

5 0 22.4 +10.1 --

5 100 122.9 +15.1 -

20 0 lu4.8 +22.4 -

20 100 413.4 +25.6 -

13.81 +0.55 -

14.02 +0.47

15.11 +1.30 -

l’J.4h +1.62 -

15.41 +1.65 .-

2~. 64 +4.71 -

22.20 1.26 +1.65 +o. 09 - -

21.14 l.L’i 21.19 +0.12

22.24 +1.3’.1 -

21.43 +1.70 -

21.10 +1. YU -

211.70 +2.11 -

1.10 +0.1tI -

Ij.96 +O. 06 -

1.27 +0.2(, --

1.33 f0.21 -

10.25 +1.13 -

10.49 +0.73 -

10.82 +l.OG -

11.05 +1.61 -

10.41, +1.71 -

33.20 +1.t,7

31.17 t1.72 -

30.52 +1.74 -

32.51 +2.05 --

28.64 t2.11 .-

29.93 i-2.71 --

TABLE IV

Effects of phenazine methosulfate and tetramethylphenylenediamine on glucose metabolism and amino acid release by skeletal muscle

Intact rat epitrochlaris preparations were incubated in Krebs-Henseleit buffer for 1 hour as detailed in Table I and under “Materials and Methods.” Phenzazine methosulfate and tetramethylphenylenediamine were added at the concentrations indicated. Values given are the

means + S.E. for at least six experiments.

Metabolite Uptake or Release

Control Phenazine Methosulfate

10 /!M 20 ,uM nmoles/min/g

Tetramethyl Phenylenediamine

10 PM 20 GM

Glucose Uptake

Lactate Release

117.4 -i: 20.6 202.4 + 10.3 219.8 f 10.8 252.0 i- 34.1 275.8 i 44.3

310.2 ? 22.9 449.0 I- 15.7 619.7 i 10.3 422.6 2 74.7 521.7 i 38.7

Pyruvate Release 18.6 + 2.08 48.2 ir 5.01 59.7 C 4.69 47.1 i 7.80 50.3 * 9.91

Alanine Release 20.4 i 1. 16 3.5 i 1.19 1.4 k 0.30 8.5 i- 1.13 9.2 k 1.06

Glutamate Release 9.7 i 0.59 11.5 f 1.24 12.0 i 1.31 9.5 i 1.71 9.7 f 0.95

Glutamine Release 30.1 * 1.88 35.1 + 2.74 41.0 k 2.34 34.7 i 2.32 37.1 i 2.05

intermediates on the synthesis and release of these amino acids were examined. The addition of 10 mM pyruvate to the incubation medium produced a 40“’ increase in the rate of alanine release and comparable decrease in both glutamate

and glutamine release (Table IX). Glutamate addition,(Z mM) increased alanine release and proportionately reduced pgru- vate release. In the presence of 5 mM ammonium chloride, alanine release wds increased and pJmvate efflux reciprocally decreased. Under these conditions, both glutamate and gluta- mine release were increased.

Since none of the preceding studies excluded the possibility that alanine released by muscle reflects the selective proteol- ysis of an alanine-rich storage protein, this hypothesis was

tested experimentally by blocking de nouo alanine formation in muscle with aminooxyacetate, an inhibitor of alanine amino- transferase (23). Increasing amounts of this inhibitor (Table X)

produced a progressive decrease in alanine release and a reciprocal increase in glutamine formation and the release of other amino acids such as aspartate.

DISCUSSION

An in vitro rat skeletal muscle preparation has been de- scribed which on prolonged incubation demonstrafes (a) con- stant rates ofglucose uptake and lactate and pJ.ruvate produc- tion, (6) levels of phosphocreatine, ATP, and ADP which are comparable to levels found in skeletal muscle freeze-clamped in uiuo, (c) uncut muscle fibers with minimal tendonous tissue

and grossly no observable contamination by adherent adipose or connective tissue, and (d) the release of’ amino acids in a profile similar to that seen with the perfused rat hindquarter preparation or by the human forearm muscle.

These studies with the epitrochlaris preparation, as with

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832 Muscle Alanine and Glutamine Release

TABLE V

Effects ofphenazine methosulfate and tetramethylphenylenediamine on net alanine, glutamine, and glutamate formation in skeletal muscle after incubation

Epitrochlaris muscle preparations were incubated as outlined in Table IV. Free amino acid levels were determined enzymatically on the neutralized perchloric acid extracts obtained from muscles rapidly frozen after incubation. Total amino acid released during 1 hour of incubation was determined for each muscle. Net formation of amino acids was calculated for each muscle preparation using the tissue level after incubation and the amount released to the media. Values are the means + S.E. given as nanomoles/mg of muscle, wet weight.

Amino Acid

Alanine

Control Phenazine Methosulfate Tetramethyl Phenylenediamine

10 PM 20 jrM 10 ~J.M 20 ~J.M nmolesfmg

Tissue Level Released Net Formation

Glutamine

1.55 It 0.07 1.89 i 0.17 1.97 + 0.12 1.66 i- 0.18 1.75 C 0.26 1.21 f 0.15 0.25 k 0.07 0.09 2 0.02 0.51 i- 0.07 0.56 + 0.06 1.21 + 0.15 0.59 f 0.09 0.51 i 0.04 0.66 + 0.08 0.76 2 0.09

Tissue Level 3.71 i 0.24 4.06 i 0.21 4.29 i 0.30 3.82 i 0.36 4.07 i 0.29 Released 1.83 + 0.13 2.11 i- 0.16 2.46 i 0.14 2.09 c 0.14 2.23 ? 0.12 Net Formation 1.83 i 0.13 2.46 k 0.21 3.04 2~ 0.42 2.20 5 0.18 2.59 + 0.30

Glutamate

Tissue Level 0.95 + 0.06 0.88 + 0.06 0.88 * 0.09 0.93 2 0.13 0.92 ?r 0.10 Released 0.57 i 0.04 0.69 + 0.07 0.73 2 0.08 0.57 It 0.10 0.58 2 0.06 Net Formation 0.57 ? 0.04 0.62 + 0.06 0.66 I! 0.08 0.55 f 0.09 0.55 2 0.07

TABLE VI

Effects of iodoacetate on glucose metabolism and amino acid release by skeletal muscle

Intact epitrochlaris muscle preparations were incubated for 1 hour in Krebs-Henseleit buffer (pH 7.4), containing glucose (5 mM), insulin (100 milliunits/ml), and Hepes (5 mM). Iodoacetate was added in the concentrations indicated. Other details as noted in Table I and under “Mate- rials and Methods.” Values given are the means * S.E. for at least four determinations.

Metabolite Uptake or Release

Glucose Uptake

Pyruvate Release

Lactate Release

Alanine Release

Glutamate Release

Glutamine Release

Iodoacetate Control 0.2 r&l 0.5 InM

nmolesjminfg

119.5 f 21.20 26.8 f 12.30 8.1 f 4.20

19.3 * 2.03 8.5 * 0.81 6.2 -f- 2.93

303.8 f 20.70 110.7 f 8.52 64.7 * 6.71

20.2 f 1.64 25.2 + 1.52 29.6 3~ 2.53

9.6 * 0.67 10.1 f 1.15 12.8 f 1.35

30.3 f 2.21 34.5 f 3.51 43.3 + 4.52

other muscle preparations, demonstrate that alanine, gluta- of incubation either in the presence or absence of insulin (26). mate, and glutamine account for greater than 70% of the total Oxidation of released amino acids by skeletal muscle, at the amino acids released from muscle. The results of this study concentrations found in the incubation media (40 PM), would further demonstrate that the efflux of these amino acids from account maximally for less than 10% of the amount of amino skeletal muscle closely approximates their rate of net forma- acid released based on the data obtained with the rat hemidia- tion. This conclusion is based on two types of experimental phragm preparation (27). Preliminary studies carried out in observations. First, amino acid balance studies during the first our laboratory indicate this figure is considerably less in the 90 and 120 min of incubation indicate that amino acid release epitrochlaris preparation. Alanine oxidation (using [U-“C]ala- occurred without change in the tissue amino acid pool, thereby nine) was linear over incubation medium concentrations be- demonstrating that de nouo amino acid synthesis must take tween 0.1 to 5.0 mM and ranged from 0.5 to 1.5% of the rate of place. Second, the metabolic fate of newly formed amino acids alanine formation and release reported in this study under in skeletal muscle is rather limited. Incorporation into muscle comparable experimental conditions. For example, alanine protein of released amino acids does not appear to be impor- oxidation was 0.11 * 0.03 nmol/min/g when alanine was tant quantitatively since the addition of cycloheximide at a present in the incubation medium at 0.5 mM. It therefore seems concentration known to inhibit protein synthesis did not alter reasonable to conclude that the rate of alanine and glutamine either the rates of amino acid release by the epitrochlaris release, as measured in these studies, is a good approximation preparation or the tissue amino acid pool during 60 to 120 min of their rate of formation.

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Muscle Alanine and Glutamine Release 833

TABLE VII

Effects of iodoacetate on amino acid release and net amino acid formation from skeletal muscle

Intact epitrochlaris muscle preparations were incubated for 1 hour as outlined in Table VII. Free amino acid levels were determined

enzymatically on the neutralized perchloric acid extracts obtained f’rom muscles rapidly frozen after incubation. Total amino acid released

during I hour of’ incubation was determined for each muscle. hTet formation of amino acids was calculated for each muscle preparation using

the tissue level after incubation and the amount released to the media. Values are the means + S.E. given as nanomoles/mg of’ muscle, wet weight.

Iodoacetate

Amino Acid Control 0.2 mM 0.5 mM nmolesimg

Alanine

Tissue Level Released Net Formation

Glutamine

1.55 f 0.09 1.12 f 0.11 0.79 + 0.15 1.21 + 0.10 1.55 + 0.09 1.87 + 0.15 1.21 + 0.10 1.12 + 0.16 1.11 t 0.11

Tissue Level 3.62 -r 0.21 2.62 + 0.11 1.99 -r 0.15 Released 1.82 + 0.13 2.07 + 0.21 2.60 + 0.27 Net Formation 1.82 i: 0.13 1.07 k 0.18 0.97 f 0.16

Glutamate

Tissue Level 0.92 -r 0.07 0.79 + 0.06 0.61 f 0.02 Released 0.57 k 0.04 0.64 + 0.06 0.77 + 0.08 Net Formation 0.57 + 0.04 0.51 2 0.07 0.46 -t 0.08

TABLE VIII

Effects of iodoacetate and ATPand phosphocreatine levels in skeletal

muscle

Intact epitrochlaris muscle preparations were incubated for 1 hour at 37” in Krebs-Henseleit buffer as outlined in Table VII. Increasing concentrations of iodoacetate were added where indicated. The mus- cles were rapidly frozen in liquid nitrogen after the incubations, and ATP and phosphocreatine determined on the neutralized, perchloric acid extracts. Values given are means + S.E. of four to six experi- ments.

Iodoacetate ATP Phosphocreatine

rnM wlollg

None 4.89 l 0.28 13.21 i 0.58

0.20 4.55 i 0.46 6.02 i 0.23 0.50 1.57 * 0.09 3.31 i 0.16

Glucose-Alanine Cycle in Muscle-The formulation of the glucose-alanine cycle is based on two types of experimental observations. The first is that alanine is extracted by the liver in viuo to an extent greater than that of any other amino acid and supports substantial rates of gluconeogenesis in the isolated perfused rat liver (4-11). The second is that alanine release by muscle exceeds greatly the release of every other amino acid except glutamine, despite the fact that the alanine content of muscle protein is not greatly different than that of most other amino acids (12, 15). It has been postulated that pyruvate derived from glucose is the carbon precursor for alanine and that the rate of pyruvate formation via glycolysis is the rate-limiting step for alanine synthesis. Although the present study does not define the quantitative contribution of

glucose-derived pyruvate carbon to alanine, it does show clearly that there is no discernible relationship between the rate of glucose uptake or glycolysis and the formation and release of alanine by resting skeletal muscle. For example, varying the rate of glucose uptake more than 40-fold by increasing the extracellular glucose concentration in the presence and ab-

sence of insulin did not alter the rate of alanine and glutamine formation (Table III). Furthermore, stimulating both glucose uptake and glycolysis with artificial electron acceptors actually reduced alanine formation and release (Tables IV and V). Since glutamine synthesis and release were increased recipro-

cally in the latter circumstance so that the sum of alanine, glutamine, and glutamate released was not significantly al- tered, there must exist in muscle interdependent mech- anism(s) regulating the formation of these three amino acids.

The converse of these observations also appears to be valid; namely, that a marked decrease in glycolysis (as produced by iodoacetate) does not alter alanine synthesis or release (Table VI). Taken together, these data indicate that alanine forma- tion is fundamentally unrelated to glucose uptake and glyco- lytic activity in skeletal muscle. Rather, the results obtained with the addition of ammonium chloride (Table IX) suggest that the rate-limiting determinant of alanine, glutamate, and glutamine release is the amount of nitrogen equivalents

available for the synthesis of these amino acids. In this context, it is of interest to note that even when glycolysis was markedly inhibited by iodoacetate, pyruvate, and lactate release contin- ued at low basal levels of 70 to 120 nmol/min/g (Table VI). Even if one assumes that the glucose taken up under these conditions was converted exclusively to lactate and pyruvate, this would account for only 20 to 40% of their observed rate of release. Since the tissue glycogen level remained unchanged

during this study, the implication is that approximately 55 to 65 nmol/min/g of lactate and pyruvate are derived from other sources. If these other sources are amino acids, then the nitrogen equivalents donated by these amino acids might be

expected to appear as glutamate and glutamine (2 nitrogen equivalents/molecule) and, indeed, under the condition of the studies carried out in Table VI, between 75 to 99% of such nitrogen equivalents can be accounted for in this manner.

The suggestion that pyruvate derived from glucose is the limiting carbon source for alanine formation is based on two

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a34 Muscle Alanine and Glutamine Release

TABLE IX

Effects of pyruuate, glutamate, and ammonium chloride addition on amino acid and pyruuate release from skeletal muscle

Intact rat epitrochlaris preparations were incubated for 1 hour at :1’7” in Krebs-Henseleit buffer, pH 7.4, containing glucose (5 mM), insulin (100 milliunits/ml). and Hepes (5 mM). Neutral solutions of’ ammonium chloride, glutamate, and pyruvate were added to the concentrations

indicated. After incubation, the release of amino acids and pyruvate was determined enzymatlcally in the media. Values given are means + S.E. for at least four experiments and are expressed as nanomoles/min/g of muscle, wet weight.

Pyruvate Alanine Glutamate Glutamine Addition Concentration Released

d’ nmoles/min/~

:, one 20.20 2 1.16 20.12 + 1.01 8.86 + 0.51 30.13 + 1.20 .- --

ryruvate 10 N . li . ,‘c 2C1.78 + 2.35 5.84 + 0.s3 20.35 + 1.81 - - -

Glutamate 2 13.1? + 2.06 29.51 + 2.06 ;;.D." 52.10 + 3.68 - -

Ammonium Chloride 5 11.96 + 0.87 30.20 + 2.45 15.37 + CA.82 40.10 + 2.42 - - _- -

* N . I). indicates that the intermediate was not determined.

TABLE X

Effect of aminooxyacetate on glucose uptake and amino acid release by skeletal muscle

Intact rat epitrochlaris muscle preparations were incubated f’or 1 hour in Krebs-Henseleit buffer with 5 mu glucose and 100 milliunits of

insulin/ml. Aminooxyacetate was added in the concentrations as indicated, and metabolite changes were determined in the media after incubation. Other details as in Table I and under “Materials and Methods.” Values given are means + S.E. for at least six determinations.

Aminooxy- GlUCOSe

acetate Uptake

NOlIe 121.6 + 12.7

0.2 109.3 + 16.8

1.0 112.9 + 14.7

2.0 118.8 r 10.3

Alanine Aspartate Glutamate Glutamine Released

nmoles/min/g muscle wet wt.

19.86 + 1.24 0.88 + 0.10 8.63 + 0.71 29.79 + 1.49

18.12 + 1.40 1.57 f 0.26 8.45 + 0.86 30.16 -f 2.01

10.82 + 0.45 3.60 + 0.83 7.96 -f 0.49 34.58 2 1.78

7.08 + 0.94 5.09 2 1.02 8.05 + 0.69 37.21 + 1.44 -

types of in. viva studies. Felig and Wahren (7) observed positive correlations between pyruvate and alanine with increasing muscle work in human subjects. Posefsky and Tancredi showed that the intraarterial infusion of large quantities of pyruvate in human subjects resulted in an increase of approximately 50% in alanine release (13), but this was accompanied by decreases in the release of a number of other amino acids. These findings with human muscle in vioo are similar to the data with rat muscle presented in Table IX which also shows a 50% increase in alanine formation produced by the addition of pyruvate.

However, proportionate decreases occurred in the release of other amino acids such as glutamine and glutamate. The reciprocal changes in alanine and glutamine indicate that a net

flow of amino nitrogen toward alanine and away from gluta- mate must have occurred without any change in the amount of amino acids released. The in viuo data cited above, taken together with the data of Table IX, can be interpreted as demonstrating that alanine aminotransferase in skeletal mus- cle functions at or near equilibrium. This enzyme has been shown to function in liver in a near equilibrium state (9, 28). Alanine aminotransferase activity is relatively high in skeletal muscle (8 units/g, wet weight) (28). Intermediates of this

enzymatic reaction have been assayed in extracts of freeze clamped muscles from rats under various dietary and hormo-

nal states.2 In each condition, calculation of the equilibrium constant of the enzyme in uiuo from the tissue concentration of substrates and products produced values which were consist-

ently within 20%. of the equilibrium constant (0.45) of the purified enzyme in vitro (29). Based on these data, it seems probable that alanine aminotransferase in skeletal muscle functions at or near equilibrium and that factors producing perturbations in such an equilibrium will produce both direct and reciprocal changes in each component of the reaction, provided that all other factors remain constant. For this

reason, measurements of changes in alanine alone are insuffi- cient to define the precursors of alanine formation in muscle if one or another intermediate participating in the equilibrium is uncontrolled and free to vary according to the perturbation.

Precursors of Alanine and Glutamine Formation-The dem- onstration that inhibition of alanine aminotransferase with

aminooxyacetate reduced the rate of alanine release indicates that release proceeds as a consequence of de nouo synthesis,

and specifically excludes the possibility of selective proteolysis of an alanine-rich storage protein (Table IX). Since the results of the present study indicate that glycolysis is not rate-deter- mining for alanine and glutamine formation, it seems reasona- ble to suggest that alanine and glutamine release is dependent upon the transformation and metabolism of other amino acids liberated by proteolysis in the muscle cell to provide the nitrogen source and contribute to the carbon pool from which alanine and glutamine are synthesized. If alanine is derived from other amino acids, these other amino acids should be released at rates considerably less than would be predicted on the basis of their relative preponderance in muscle protein. A

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Muscle Alanine and Glutamine Release 835

case in point is that of aspartate which has the same relative concentration in protein as alanine (15), yet an efflux rate which is extremely low. The inhibition of de novo alanine formation by aminooxyacetate decreased alanine release from muscle and resulted in a striking increase in the release of aspartate, a finding consistent with a precursor roie of aspar- tate for alanine formation. Similar changes in release rates

would also be anticipated for other amino acids which are precursors for alanine formation. Thus, it seems probable that the metabolism of amino acids in skeletal muscle provides not only the nitrogen source for alanine and glutamine but also a proportional amount of carbon into a general metabolic pool reflected in the pyruvate, lactate, alanine, and glutamate released from this tissue. This hypothesis is examined in the accompanying paper (30).

Acknowledgments-We are indebted to Paul Max, Jerry

Kropp, and Thomas Howard for their excellent technical assistance. We wish to thank Dr. Ralph Feigin for the amino acid analyses.

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A J Garber, I E Karl and D M Kipnisamino acid release.

Alanine and glutamine synthesis and release from skeletal muscle. I. Glycolysis and

1976, 251:826-835.J. Biol. Chem. 

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