PYRUVATE AND ACETATE METABOLISM IN THE ISOLATED RAT DIAPHRAGM*

BY JOHN M. FOSTERt AND CLAUDE A. VILLEE$

(From the Department of Biological Chemistry, Harvard Medical School, and the Research Laboratories of the Boston Lying-In Hospital,

Boston, Massachusetts)

(Received for publication, June 28, 1954)

Previous experiments (l-6) have indicated that, in addition to an im- pairment of glucose utilization, diabetic muscle suffers some involvement of its oxidative metabolism. Villee and coworkers (l-3) and Pearson et al. (5) reported that the utilization of pyruvate by the isolated diabetic rat diaphragm was significantly lower than normal. In addition, the oxida- tion of pyruvate-2-C4 and acetate-l-Cl4 to CO2 was depressed. The metabolism of pyruvate could be restored to normal by addition in vitro of insulin; that of acetate could not. Insulin was without effect, however, on either pyruvate or acetate metabolism in normal tissue. Charalampous and Hegsted (6) injected p-aminobenzoate into normal and diabetic rats and found that the rate of acetylation was much less in diabetic animals. Insulin restored the acetylation to normal. The rate was also restored by injection of various members of the Krebs cycle, diacetyl, acetyl phosphate, or adenosinetriphosphate, but not by pyruvate or lactate.

These observations suggest that one or more of the reactions of the citric acid cycle or of the incorporation of pyruvate and acetate into the cycle are interfered with in diabetes. They do not, however, establish the precise location of this interference. The observation that insulin added in vitro does not increase acetate metabolism in diabetic muscle raises the question of whether the changes in metabolism of diabetic diaphragm are due directly to insulin lack. The present paper reports experiments in which pyruvate-1-, 2-, and 3-Cl4 and acetate-l- and 2-Cl4 were used to study these reactions in greater detail.

Materials and Methods

Animals-Male Wistar rats, weighing between 150 and 200 gm. when fasted 24 hours, were used in all experiments. Diabetes was induced by injection of 45 mg. per kilo of alloxan into the femoral vein. Only those

* From a thesis presented by John M. Foster to the Division of Medical Sciences’ Harvard University, in partial fulfilment of the requirements for the degree of Doctor of Philosophy.

t Fellow of The National Foundation for Infantile Paralysis, 195254. 1 Aided by a grant from the Charles A. King and Marjorie King Fund.

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798 PYRUVATE AND ACET.4TE IN DIAPHRAGM

animals with a blood sugar of 200 mg. per cent or higher after a 24 hour fast were included in the diabetic series. All rats were fasted 24 hours before being used in an experiment.

Substrates-Carboxyl- and methyl-labeled acetate were obtained from Oak Ridge; carbonyl- and methyl-labeled pyruvate were synthesized from them by standard procedures (7). Carboxyl-labeled pyruvate was syn- thesized from sodium acetate and NaCY*N by similar methods.

1Medium-The medium in all experiments was identical with that em- ployed by Villee and Hastings (1) and contained 0.040 M sodium phosphate, 0.005 M MgC12, 0.08 M NaCl, and 0.01 M labeled potassium pyruvate or sodium acetate. When pyruvate was the substrate, 200 mg. per cent glucose was also added. The initial pH was 6.8. The insulin (0.5 unit per ml.) used was obtained from Eli Lilly and Company and had been treated with trypsin to remove the hyperglycemic-glycogenolytic factor.

Procedure

The rats were killed by a sharp blow on the head, and the diaphragms were quickly removed, one-half at a time, and blotted on filter paper. A small piece was taken from each, weighed on a torsion balance, and placed at once in boiling 30 per cent KOH for glycogen analysis. In the initial experiments, the remaining tissue was then weighed and placed imme- diately in the incubation vessels. In the later experiments, however, each hemidiaphragm was divided in two and placed in a large volume of ice- cold incubation medium, without substrate, for half an hour. This proce- dure was suggested by Walaas and Walaas (8) for removing endogenous lactate formed by rapid glycogen breakdown when the animal is killed. It also afforded a means of pooling tissues from several animals and re- tarded glycogenolysis; hence the diaphragms were in a more nearly uniform state when the incubation began. At the end of half an hour the dia- phragms were removed, blotted, weighed, and placed in standard 15 ml. Warburg incubation vessels. Each vessel contained 3 ml. of medium and the equivalent of one whole diaphragm (about 300 mg. of tissue). In some experiments the animals were paired, one hemidiaphragm from each animal being treated with insulin and the other remaining untreated. In other experiments the samples were selected randomly from the pooled tissues. A st,rip of filter paper and 0.2 ml. of COz-free NaOH were placed in the center wells, and the flasks were gassed with 100 per cent 02 for 7 minutes and incubated for 2 hours at 37.5”. The diaphragms were then removed for glycogen determinations (I, 9). Aliquots of the incubation media were analyzed for pyruvate (lo), lactate (II), and glucose (12). The contents of the center wells were transferred to 10 ml. volumetric flasks and made up to volume with COz-free water. Aliquots were ana-

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J. M. FOSTER AND C. A. VILLEE 799

lyzed for COZ on the Van Slyke manometric apparatus. The media containing no insulin were then combined in one sample, and those con- taining insulin in another. The protein and phosphate were removed by addition of 4 volumes of ethanol and centrifuging, and the samples were taken to dryness overnight in a stream of filtered air. Lactate and certain members of the citric acid cycle were isolated from the dried samples by silica gel chromatography (13). In this procedure, lactic, succinic, and cr-ketoglutaric acids are eluted together. The a-ketoglutaric acid was converted to its 2,4-dinitrophenylhydrazone and extracted with ether; the lactic and succinic acids were then separated by rechromatography.

The effluent fractions were collected in 18 X 150 mm. test-tubes and analyzed by titration with 0.01 N NaOH. Use of an acid-base indicator in titration was found to be undesirable because it diluted the Cl4 in the sample. The titrator was therefore equipped with a glass electrode assem- bly based on a concentric design of Cannon (14). All titrations were carried to pH 8.0. The fractions containing each acid were combined, evaporated to dryness, and analyzed for total carbon by the wet combus- tion method of Van Slyke et al. (15, 16). The resulting CO2 was converted to BaC03 (17) for radioactivity determinations. Enough lactate was produced by the amount of tissue employed so that its specific activity could be calculated from the titration, Van Slyke, and radioactivity data. The amounts of the citric acid cycle intermediates, however, were very small; hence accurate titration was impossible. Consequently, carrier fumarate, succinate, cY-ketoglutarate, malate, and citrate were added to the sample before chromatography to locate these intermediates. Only the total radioactivity in each intermediate could then be determined.

The chromatographic technique was also used as a convenient specific method for the quantitative estimation of acetate. When acetate was the substrate, it then became possible to estimate its utilization by the dia- phragm. Precautions were taken to keep the samples alkaline to prevent losses by volatilization.

Radioactivity was determined on stainless steel planchets in the propor- tional gas flow counter at the Harvard Riophysical Laboratory. Pyruvate was counted as the 2,4-dinitrophenylhydrazone (l), glycogen by hydroly- sis to glucose and conversion to the phenylosazone (l), COZ, lactate, and the citric acid cycle intermediates as BaC03. The ether extract of a-keto- glutarate was counted as such. Acetate was counted as its benzylpseudo- thiuronium salt (18).

Results

E$ects of Presoaking Technique-In the initial experiments, the condi- tions employed by Villee and Hastings (1, 2) were duplicated as nearly as

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800 PYRWATE AND ACETATE IN DIAPHRAGM

possible. Comparable results were obtained. However, presoaking the tissue had a marked effect upon pyruvate metabolism (Table I). The utilization of pyruvate and its oxidation to CO2 by normal tissue were reduced to the levels found in diabetic tissue. In addition, the stimulatory effect of insulin on pyruvate metabolism in diabetic diaphragm disap-

TABLE I

Effect of Presoaking in Chilled Buj’er on Rat Diaphragm Mefabolism

Values given as the mean f the standard error of the mean in micromoles per gm. of wet weight per hour.

Pyruvate utilization

Lactate production

Oxygen uptake

COZ produced

Pyruvate metabolized to

co2

-

-

Normal rats Diabetic rats

UIl- treated

dia- Presoaked diaphragm ?hragm

Untreated diaphragm

Insulin

Presoaked diaphragm

-I-/+I-I+I-I+

15 38 32

43.5 29.8 30.4 f2.4 f0.8 10.8

35.9 29.4* 30.3* f6.5 f0.9 f0.7

36.6 37.5 39.8 f2.3 A.6 f0.3

74.0 63.5 65.5 f4.0 f1.6 f1.5

11.2 8.0 8.4 A.0 fO.6 f0.2

No. of experiments -

6

37.9

f1.9 25.9

f8.8 35.5

A.2 75.9

zk9.0 12.0

&3.7

51.3

42.6

45.5

13.6

1.5 9

31.8 32.7 fl.O Al.4

24.6t 25.7$ f1.2 fl.O

33.8 36.1 f2.5 f0.5

62.0 65.0 f1.8 f2.6

7.8 8.0 Al.0 fl.1

* Seventeen experiments. t Twelve experiments. $ Eleven experiments.

peared. These changes cannot be attributed to a general decrease in the metabolic rate of the tissue, for there was no change in oxygen consump- tion nor in lactate production. The decrease in COZ produced was also slight compared to the changes in pyruvate utilization.

Rates of Conversion of Pyruvate Carbons to COz-The rates at which carbons 1, 2, and 3 of pyruvate are metabolized to COz are given in Table II. The total amount of substrate metabolized to CO, was calculated from the ratio of the total counts in the expired COZ to the total counts originally

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J. M. FOSTER AND C. A. VILLEE 801

present in the medium, multiplied by the initial pyruvate concentration. The percentage of CO2 derived from pyruvate is equal to the ratio of the specific activity of the COz to that of the substrate (in counts per minute per millimole of carbon) times 100. Both of these calculations assume that oxidation of the substrate molecule is complete; i.e., that each labeled carbon from the substrate which is converted to CO2 is accompanied by 2 unlabeled ones. The data in Table II show that this assumption is not

TABLE II

Rates of Conversion of Pyruvafe Carbons to CO2

Values given as the mean f the standard error of the mean.

Substrate

Pyi-uvate oxi- dized to COn, pmoles per gm. per hr..

co2 derived from pyru- vate, %. .

strictly true. The 3 carbons of pyruvate appear in CO2 at slightly differ- ent rates over the 2 hour incubation period. In normal diaphragm carbon 1 appears in carbon dioxide more rapidly than carbon 2, and carbon 2 more rapidly than carbon 3. This is in accord with what a consideration of the fate of the carbons of pyruvate in the reactions of the citric acid cycle would predict.

Normal rats Diabetic rats

Insulin

-I+l-l+l-I+ No. of experiments No. of experiments

8 8 9 9 12 12 13 13 10 10 10 10

9.2 9.2 10.0 10.0 7.9 7.9 8.4 8.4 5.7 5.7 7.c 7.c &0.5f0.6fO.6f0.2&0.4f0.4 &0.5f0.6fO.6f0.2&0.4f0.4

41.9 46.5 36.0 35.5 28.8 30.7 k2.7f2.8f2.3 fl.6fl.lfl.~

-l+l-If/-If

8 8 5 5 5 5 3 3 5 5 3 3

10.2 10.2 10.7 10.7 7.8 7.8 8.0 8.0 9.5 9.5 9.4 9.4 frl.lfl.lfl.0f1.0f0.9f0.8 frl.lfl.lfl.0f1.0f0.9f0.8

49.4 49.4 48.9 48.9 34.3 34.3 35.0 35.0 54.0 54.0 47.5 47.5 k4.2f5.0f3.3 f4.1f3.8,f2.5 k4.2f5.0f3.3 f4.1f3.8,f2.5

There was no difference between normal and diabetic diaphragm in the rate of oxidation of carbons 1 and 2. However, the oxidation of carbon 3 by diabetic muscle, instead of being less than that of carbon 2, appears to be almost as fast as the oxidation of carbon 1.

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802 PYRUVATE AND ACETATE IN DIAPHRAGM

Production of Radioactive Lactate-The specific activity (counts per minute per micromole) of the lactic acid isolated from the incubation medium was calculated from the titration, combustion, and radioactivity data by means of the following equation:

C.p.m. per mg. BaC03 X mol. wt. l&CO3 X mg. C X 1000

Atomic weight C X pmolcs lactic acid = specific activity

Since the equilibrium betmreen pyruvate and lactate is freely reversible, the

TABLE III

Specific Activity oj Lactic Acid Produced by Rat Diaphragm

Specific activity = counts per minute per millimole.

I Insulin

Experi- ment No.

-/+ -/+ -/+

Specific activity of lactate Specific activity of pyruvate

N5 1 141,000 N8 51,900 NS 276,000 N11 180 ) 000 N 12

Normal rats

173,000 103, oil0

/ 178,000 82 92 103,000 50 76

379,000 379,000 73 65

/ 184,000 70,600 / 1 217,000 223,000 95,000 83 83 74

Diabetic rats

D3 161,000 119,000 285,000 253,000 57 47 D5 359,000 438,000 82

/

D8 73,300 72,700 126,000 133,000 58 55

specific activities of these two compounds should be identical after a 2 hour incubation. This was found to be the case in the initial experiments with untreated tissue. However, when the diaphragms were subjected to the presoaking procedure, the specific activity of the lactate was always less than that of the pyruvate (Table III) and varied considerably. This indicated that under these conditions complete equilibrium is not attained and suggested a study of the changes in specific activity with time during the incubation period. For these experiments the labeled pyruvate was placed in the side arms of the incubation flasks and tipped in at zero time, after gassing and equilibration. Fig. 1 shows that the specific activity of pyruvate decreased in linear fashion during the incubation; that of lactate at first rose sharply, and then approached the pyruvate values slowly.

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J. M. FOSTER AND C. A. VILLEE 803

However, if the lactate values are corrected for the unlabeled lactate produced during the equilibration period before incubation, which will have a large diluting effect on the small amounts of radiolactate formed initially, the curve indicated by the broken line in Fig. 1 is obtained. It is clear that there is a rapid formation of labeled lactate from pyruvate in the early part of the incubation period, after which the specific activity of lactate decreases more rapidly than that of pyruvate.

LACTATE L 0

.

II I I I I I I I I I I 30 60 90 120

FIG. 1. The change of specific activity of pyruvate and lactate with time in rat di- aphragm. 0 values have been corrected for unlabeled lactate formed prior to addi- tion of labeled pyruvate.

Incorporation of Pyruvate into Citric Acid Cycle Intermediates-The total radioactivity isolated in fumarate, succinate, a-ketoglutarate, malate, and citrate was sufficiently variable that no differences between the rates of incorporation of the 3 carbons of pyruvate could be detected. (Carbon 1 of pyruvate does not enter the citric acid cycle intermediates by the usual reaction, but may enter via carbon dioxide fixation.) The values obtained from all 3 carbons have therefore been averaged and are given in Table IV. The data give some indication that the amount of radioactivity remaining in the Krebs cycle is less in diabetic than in normal muscle. The values are not affected in any consistent fashion by insulin.

Acetate Metabolism-The metabolism of acetate-l- and 2-Cl4 by normal and of acetate-l-Cl4 by diabetic muscle was studied in four experiments. The results are given in Table V. The same rate of oxygen utilization was

MINUTES

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804 PYRUVATE AND ACETATE IN DIAPHRAGM

obtained as when pyruvate was the substrate, but the omission of glucose from the medium resulted in much less CO2 and very little lactate being formed. Contrary to expectation, however, approximately 60 per cent more acetate was taken from the medium by diabetic than by normal diaphragm muscle. The fact that there was no decrease in the specific

TABLE IV

Incorporation of Pyruvate Carbon into Citric Acid Cycle Zntermediates

Values in per cent of the total pyruvate counts disappearing from the medium.

Normal rats I

Diabetic rats

Intermediate Insulin

I - I + I - I -t Fumarate. 5.54 5.47 2.53 2.56 Succinate 1.27 0.46 0.31 0.57 a-Ketoglutarate 3.12 2.33 3.40 3.63 Malate........................ 1.12 1.30 0.62 0.60 Citrate........................ 0.79 1.23 1.08 0.08

TABLE V

Metabolism of Acetate by Rat Diaphragm

Values given as the mean i the standard error of the mean in micromoles per gm. of wet weight per hour.

Acetate utilized

Lactate produced Oxygen uptake

COz produced

Normal rats (6 each)

19.4 19.8 f2.4 f2.6

4.6 4.5 40.4 37.8

f0.8 f1.5 49.8 49.2

f1.5 fO.8

Diabetic rats (2 each)

29.9 35.0

1.3 2.4 29.1 33.4

35.2 37.7

activity of the acetate in the course of the incubation indicates that the muscle does not synthesize acetate under these conditions. Since there is no acetate production, acetate disappearance, measured chemically, repre- sents actual utilization.

The amount of acetate oxidized to CO2 and the composition of the CO2 (Table VI) were calculated in the same manner as the pyruvate data. The data in Columns 1 to 4 show again that the substrate carbons are not oxi- dized at the same rate, but that the difference is small. The higher rate of oxidation of the carboxyl carbon is consistent with our knowledge of

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J. M. FOSTER AND C. A. VILLEE 805

the reactions of the citric acid cycle. A comparison of Columns 1 and 5 reveals that, even when the presoaking technique is employed, the metab- olism of acetate to CO2 is less in diabetic muscle than in normal muscle. Only four diabetic animals were used, however, and no statistical signifi-

TABLE VI Rate of Conversion of Acetate Carbons to CO*

The figures are mean values.

Normal rats

CHoC*OOH C*HaCOOH

Diabetic rats

CH8C*OOH

Substrate

Acetate oxidized to COZ, pmoles per gm. per hr.. . .

COz derived from acetate, ye. .

Insulin

+ - + - +

(1) (2) (3) (4) (5) (6) ___.

8.5 6.5 5.5 5.6 4.8 6.3 31.6 25.4 24.1 24.0 27.0 31.1

TABLE VII Calculation of Carbon Balances

Values in per cent of the total substrate counts disappearing from the medium.

coz ........................ Lactic acid ................. Glycogen ................... Citric acid cycle ............

Total.

- Pyruvate

i Acetate

Insulin

-I + Normal

21.0 23.2 62.5 65.2

1.6 1.7 11.8 10.8

~__ 96.9 100.9

-I+/-/+I-/+ Diabetic Normal Diabetic

24.3 22.5 29.7 29.0 15.9 18.1 41.6 30.8 0 0 0 0 0.9 1.0 0 0 0 0 7.9 7.4 0 0 0.7 1.0

74.7 61.7 29.7 29.0 16.6 19.1

cance can be ascribed to these differences. In contrast to previous reports (3), no difference in the percentage of the respiratory CO2 derived from acetate by normal and diabetic muscle was observed. This may be due to the difference in experimental conditions. The diabetic diaphragm has a higher rate of acetate utilization (Table V), but a smaller fraction going to carbon dioxide (Table VI).

The levels of radioactivity incorporated into citric acid cycle interme-

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806 PYRUVATE AND ACETATE IN DIAPHRAGM

diates from acetate were too low to permit detection of any possible differ- ence between normal and diabetic diaphragms.

Calculation of Carbon Balances-The percentages of the substrate radio- activity incorporated by the diaphragm which appear in the various fractions studied have been totaled in Table VII. All of the radioactive pyruvate incorporated by normal diaphragm can be accounted for, the largest proportions going to lactate and COz. In diabetic diaphragm, however, 25 and 40 per cent of the radioactivity cannot be accounted for in the absence and presence of insulin, respectively. The largest change is a marked decrease in the proportion of the pyruvate which diabetic muscle converts to lactate.

Only a small proportion of the acetate utilized by the normal rat dia- phragm can be accounted for, and even less in diabetic diaphragm. The radioactivity unaccounted for appears to be in the medium when either substrate is employed. Combustion of samples of tissue after incubation to determine their radioactive carbon content showed that only 1.5 per cent of the pyruvate activity and 11 per cent of the acetate activity taken up remained in the tissue.

DISCUSSION

The rate at which the specific activity of the lactate produced by the rat diaphragm decreased with time was greater than the rate of decrease of the pyruvate specific activity (Fig. 1). If there were complete equilibrium between the pyruvate and lactate, the specific activities should be the same and should decrease at the same rate. It must be pointed out that the measurements of specific activity were performed on substances iso- lated from the medium. Thus the cell membrane, by retarding the passage of one or the other substance, might prevent equilibrium from being established in the medium. The lactate must of necessity have come from the intracellular pyruvate pool, and its specific activity should reflect that of the pool. Since the specific activity of lactate decreases faster than that of pyruvate, it follows that the specific activity of the pyruvate pool decreases faster than that of the pyruvate in the medium by dilution with unlabeled pyruvate from other sources. The simplest assumption to account for these phenomena is that pyruvate formed in the intact dia- phragm cell cannot pass freely through the cell membrane into the medium.

The absence of any differences in the rate of incorporation of the 3 pyru- vate carbons into the Krebs cycle intermediates is of interest. There was as much carboxyl carbon incorporated as there was carbonyl or methyl carbon. According to our present knowledge of the reactions of the citric acid cycle, the only means by which carbon 1 of pyruvate can enter the cycle is by the COZ fixation reactions. These data indicate that CO2

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J. M. FOSTER AND C. A. VILLEE 807

fixation is a major phenomenon in diaphragm muscle and are in accord with the experiments of Crane and Ball (19), who found an appreciable incorporation of C14-bicarbonate into rat diaphragm.

The present experiments lend additional support to the suggestion, put forth by various investigators, that there is some interference in the metab- olism of pyruvate and acetate in the Krebs cycle in diabetes. Most of these observations could be accounted for by an interference in the con- densation of acetyl coenzyme A with oxalacetate. The fact that pyruvate metabolism by normal diaphragm can be reduced to the diabetic level by presoaking in chilled buffer suggests that the interference in diabetes is not due directly to a lack of insulin. This is further borne out by the disap- pearance of any effect of insulin on pyruvate metabolism in diabetic dia- phragm upon presoaking and by the insensitivity of acetate metabolism to insulin with or without presoaking. The observations point rather to a loss or unavailability in diabetes of some cofactor essential to these reac- tions, which can be duplicated in the normal tissue by presoaking in chilled buffer. In this connection, an involvement of insulin in the efficiency of oxidative phosphorylations has been suggested previously (20, 21).

It is possible that the pyruvate counts which cannot be accounted for in diabetic diaphragm will be found in acetoacetate and other ketone bodies. This seems especially likely in view of the large proportion of acetate that disappears into unknown compounds. While the liver is generally considered the main site of ketone body formation in diabetes, it is conceivable that under these conditions muscle may synthesize them in appreciable quantities as well.

SUMMARY

1. The metabolism of pyruvate-1-, 2-, and 3-Cl4 and acetate-l- and 2-C’* to COZ, lactate, glycogen, and to intermediates of the Krebs cycle by the normal and diabetic rat diaphragm in vitro has been studied.

2. In initial experiments, results for pyruvate metabolism comparable to those of previous workers were obtained. In later experiments pre- soaking the tissue in chilled buffer was employed to remove endogenous lactate. Under these conditions the utilization of pyruvate by normal muscle and its oxidation to CO2 were reduced to the same level as that in the diabetic. Neither type of tissue responded to insulin added in vitro.

3. The oxidation of acetate-l-Cl4 to COz by diabetic muscle remained less than normal under the new experimental conditions. It was not xffected by insulin. There was no decrease in the radioactivity of the acetate, indicating that muscle does not synthesize acetate.

4. The rates of oxidation of the 3 carbons of pyruvate and the 2 carbons of acetate were shown to be slightly different. The carboxyl carbon was

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808 PYRUVATE AND ACETATE IN DIAPHRAGM

oxidized most rapidly, the methyl carbon least rapidly, in normal tissue. In diabetic diaphragm the oxidation of carbon 3 of pyruvate was at least as rapid as the oxidation of carbon 2. The pattern is in accord with our knowledge of the reactions of the Krebs cycle.

5. The specific activity of the lactate isolated after incubation was lower than that of the pyruvate, indicating a lack of complete equilibrium between them.

6. There was as much pyruvate carboxyl carbon incorporated into the Krebs cycle intermediates as carbonyl or methyl carbon, indicating ap- preciable CO2 fixation. The incorporation of radiopyruvate into the Krebs cycle intermediates was somewhat lower in diabetic than in normal muscle. Almost no activity from labeled acetate was found in either normal or diabetic diaphragm.

7. Nearly all of the pyruvate counts incorporated by normal diaphragm could be accounted for, but only 60 to 75 per cent in diabetic diaphragm. Only a small portion of the acetate counts disappearing could be accounted for. The possibility that the missing counts will be found in ketone bodies is suggested.

BIBLIOGRAPHY

1. Villee, C. A., and Hastings, A. B., J. Biol. Chem., 1’79, 673 (1949). 2. Villee, C. A., and Hastings, A. B., J. Biol. Chem., 181,131 (1949). 3. Villee, C. A., White, V. K., and Hastings, A. B., J. Biol. Chem., 195, 287 (1952). 4. Pearson, 0. H., Hastings, A. B., and Bunting, H., Am. J. Physiol., 168,251 (1949). 5. Pearson, 0. H., Hsieh, C. K., Dutoit, C. H., and Hastings, A. B., Am. J. Physiol.,

168, 261 (1949). 6. Charalampous, F., and Hegsted, D. M., Federation Proc., 8, 379 (1949). 7. Calvin, M., Heidelberger, C., Reid, J. C., Tolbert, B. M., and Yankwich, P. E.,

Isotopic carbon, New York, 210 (1949). 8. Walaas, O., and Walaas, E., J. Biol. Chem., 187, 769 (1950). 9. Good, C. A., Kramer, H., and Somogyi, M., J. Biol. Chem., 100,485 (1933).

10. Friedemann, T. E., and Haugen, G. E., J. Biol. Chem., 147, 415 (1943). 11. Barker, S. B., and Summerson, W. H., J. Biol. Chem., 138, 535 (1941). 12. Nelson, N., J. BioZ. Chem., 153, 375 (1944). 13. Bulen, W. A., Varner, J. E., and Burrell, R. C., Anal. Chem., 24, 187 (1952). 14. Cannon, M. D., Science, 108, 597 (1947). 15. Van Slyke, D. D., and Folch, J., J. BioZ. Chem., 138, 509 (1940). 16. Van Slyke, D. D., Plazin, J., and Weisiger, J. R., J. BioZ. Chem., 191, 299 (1951). 17. Van Slyke, D. D., Steele, R., and Plazin, J., J. BioZ. Chem., 192, 769 (1951). 18. Donleavy, J. J., J. Am. Chem. SOL, 68, 1004 (1936). 19. Crane, R. K., and Ball, E. G., J. BioZ. Chem., 188, 819 (1951). 20. Sacks, J., in McElroy, W. D., and Glass, B., Phosphorus metabolism, Baltimore,

2, 653 (1952). 21. Haugaard, N., Marsh, J. B., and Stadie, W. C., J. BioZ. Chem., 189, 59 (1951).

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John M. Foster and Claude A. VilleeDIAPHRAGM

METABOLISM IN THE ISOLATED RAT PYRUVATE AND ACETATE

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