THE ACTION OF EPINEPHRINE AND INSULIN IN FROGS UNDER ANAEROBIC CONDITIONS

BY K. W. BUCHWALD AND CARL F. CORI

(From the State Institute for the Study of Malignant Disease, Buffalo)

(Received for publication, May 8, 1931)

The predominantly oxidative metabolism of a homothermal animal represents such a complex system that it is usually difficult to decide what is the primary effect of a hormone in the body and what are merely secondary changes. For instance, the decrease in muscle glycogen and increase in blood lactic acid after epinephrine injection could be ascribed to a direct accelerating effect on the glycogenolytic process, but it could also be due to vasoconstriction and hence to a diminished supply of oxygen. Since it was found that epinephrine acts on muscle glycogen when supplied at a rate which causes dilatation rather than constriction of the blood vessels of muscle, the second possibility seemed less probable (1).

A further investigation of this problem is made possible by the fact that cold blooded animals are able to survive a period of oxy- gen want of several hours’ duration. During anaerobiosis oxidative processes are eliminated or greatly restricted and the action of epi- nephrine and insulin, if any, can thus be studied under simplified conditions. Moreover, a possible curtailment of t,he oxygen supply to the tissues through vasoconstriction cannot be a factor in the action of epinephrine under anaerobic conditions. If, therefore, epinephrine should still be able to increase lactic acid formation when oxygen is excluded, this would have to be ascribed to a direct point of attack of epinephrine in the muscle cell.

Lesser (24) made numerous experiments concerning the effect of anaerobiosis on the carbohydrate content of frogs. He found that there occurred a progressive increase in sugar and lactic acid and that the former was derived from liver glycogen and the latter from muscle glycogen. Meyerhof and Meier (5) measured the rate of lactic acid formation in frogs kept anaerobically. In the

355

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Epinephrine and Insulin in Frogs

present experiments determinations of the sugar and lactic acid content of the whole animal were made in preference to glycogen determinations in liver and muscle, because the basal values of the former were less subject to individual variations than the latter. Lesser states that when glycogen is determined it is necessary to analyze at least fifteen frogs in each series in order to obtain a satis- factory average; according to our experience six to eight experi- ments are sufficient in the case of sugar and lactic acid determina- tions. Experiments on hepatectomized frogs to be described in the following paper (6) show that an increase in sugar contentafter epinephrine injection is dependent on glycogenolysis in the liver since it does not occur in the absence of the liver and that muscle glycogen is not a source of sugar but yields lactic acid. There is therefore some ground for the assumption that whenever the sugar or lactic acid content increases, this is due to a corresponding decrease in liver or muscle glycogen.

EXPERIMENTAL

Male frogs (Runa pipiens) of 20 to 35 gm. in weight were used. Since the experiments were made in the early winter months, the animals were in a good nutritional condition. They were kept in an aquarium at a temperature of about 10’. On the night previous to the experiment some frogs were placed in a cage standing in shallow water so that the animals could not submerge. For the experiments the frogs were put into large fruit jars provided with a rubber stopper, thermometer, and inlet and outlet tubes. The jars, which were kept in a water bath, had an inside temperature of 15 f 0.5”. Air or nitrogen, saturated with moisture, was passed through at a rate of about 15 liters per hour. In order to produce rapid anaerobiosis, one jar was filled with water and the nitrogen allowed to displace it. Traces of oxygen were removed from the nitrogen by passing the latter over copper gauze heated in a silica tube and then cooling it in a copper coil. When the nitrogen so prepared was bubbled for 20 minutes through 10 per cent pyro- gallic acid, only a slight yellow &or was produced on addition of an equal volume of 10 per cent sodium hydroxide; i.e., the Hopkins test for oxygen was negative.

After 3 hours the frogs were put into chamois bags containing COz snow to which a few drops of ether had been added. When

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K. W. Buchwald and C. F. Cori 357

thoroughly frozen they were cut into thin slices by means of a tobacco cutter and the slices placed in 50 cc. of ice-cold, 2 per cent sulfuric acid. The whole mass was frozen solid by addition of CO2 snow and then allowed to thaw slowly at room temperature, which required about 30 minutes. During this time the acid had thoroughly penetrated into the tissues and passing them once through a small meat grinder sufficed to reduce them to a fine pulp. The tissue residue was separated on a Buchner funnel and was sus- pended three times in succession in 40 cc. of water, being filtered each time. The combined extracts were made up to 200 cc. and proteins were precipitated with mercuric sulfate, as described by West, Scharles, and Peterson (7). To 25 cc. of the extract, 20 cc. of water and 5 cc. of 30 per cent HgS04 in 10 per cent H2S04 were added. Freshly precipitated barium carbonate was then added and the whole shaken until no more COZ was evolved and the reac- tion was faintly alkaline to neutral litmus paper. After filtration on a Buchner funnel and addition of a drop of strong sulfuric acid to precipitate the excess barium, HzS was passed through the solu- tion. The mercuric sulfide was filtered off and the H,S expelled by aeration. After neutralization with NaOH aliquot portions were analyzed for sugar, the HagedornJensen and in some later experi- ments t.he Benedict method (8) being used. Lactic acid was deter- mined according to Friedemann and Kendall (9) after removal of sugar by copper sulfate and lime.

Injections were made into the throat lymph sack. Epinephrine was given in doses of 0.005 to 0.1 mg. but in the majority of cases a dose of 0.02 mg. WM used. Insulin (2 units) was injected 18 to 42 hours previous to the experiments. Control animals were given an equivalent amount of salt solution.

Recovery of Added Glucose and Lactic Acid-Amounts of glucose and lactic acid, covering the extreme ranges encountered in the present work, were added to the frog extract before precipitation with mercuric sulfate. The results obtained are shown in Table I. On an average 99.1 per cent of added glucose and 81.6 per cent of added lactic acid were recovered. Since the recovery of lactic acid in pure solution is between 95 and 100 per cent, it may be concluded that the mercuric sulfate precipitation leads to a loss of lactic acid amounting to nearly 20 per cent. A correction for this loss was not applied. It should be noted that other protein precipitants lead

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358 Epinephrine and Insulin in Frogs

to similar losses of lactic acid and besides, most of them do not yield a filtrate which is suitable for sugar determinations owing to t.he presence of a large amount of non-sugar reducing substances. The chief advantage of the mercuric sulfate precipitation is that sugar and lactic acid can be determined in the same filtrate and that the loss of the latter, though fairly large, is reasonably con- stant.

Fermentution with Yeast.-In order to determine the nature of the reducing substances in the filtrate from the mercuric sulfate precipitation, the filtrate was treated with washed yeast for 20

TABLE I

Effect of Mercuric Sulfate Precipitation on Recovery of Added Glucose and Lactic Add

25 cc. of the same frog extract were precipitated in each case as described in the text.

-

-

Found Added

ma.

0.312 0.586 0.704 0.780 0.919 1.085

w7.

0.36 0.48 0.60 0.72 0.90

- Lactic acid

T Recovered -

-

- ml. per cent

0.274 76 0.392 81.5 0.478 80 0.607 84.5 0.773 86

Average.. . . . . . . . . . . . . . . . . -

- 81.6

-

Found

w?.

0.129 0.243 0.289 0.327 0.186* 0.215*

- .-

-

GlUCOS-3

Added 1

m7.

Recovered

mg.

0.12 0.114 0.16 0.160 0.20 0.198 0.12 0.122 0.15 0.151

per cent

95 100 99

101 100.5

99.1

* One-half of the usual amount taken for analysis.

minutes at room temperature. After centrifuging off the yeast, the supernatant fluid was used to make up a 0.01 per cent glucose standard from a 0.1 per cent glucose stock solution. At the same time 0.01 per cent glucose standards, the same pipette, stock solu- tion, and volumetric flask being used, were prepared with the supernatant fluid of yeast that had been suspended in water and of yeast that had acted on a pure glucose solution. With the Bene- dict copper method, the three glucose standards gave the same reading within the limit of error of the method. This experiment was repeated three times on different filtrates and essentially the same result was obtained. It shows that the reducing substance

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K. W. Buchwald and C. F. Cori

determined in the mercuric sulfate filtrate by means of the Benedict method is completely fermentable. When the Hagedorn-Jensen method was used, the glucose standard prepared with the fer- mented mercuric sulfate filtrate always gave higher reducing values than the other two standards. The non-fermentable residue in the case of the Hagedorn-Jensen method was found to be chiefly crea- tine which reduces the ferricyanide reagent even in the presence of glucose. Benedict’s copper reagent is not affected by creatine in the presence of glucose, though creatine alone has a weak reducing action. Apparently the Benedict method gives true sugar values when applied to the mercuric sulfate filtrate, while the values obtained with the Hagedorn-Jensen method are somewhat too high. The difference in reducing value obtained by means of the Benedict and Hagedorn-Jensen methods on unfermented mercuric sulfate filtrates corresponds closely to the non-fermentable residue found in the latter method. This is illustrated in the following example. With the Benedict method 58.6 and with the Hagedorn- Jensen method 75 mg. of sugar per 100 gm. of frog were found, the difference being 16.4 mg. The non-fermentable reducing sub- stance for the Benedict method was 0, for the HagedornJensen method 15.2 mg. per 100 gm. of frog. Generally the difference between the two methods was smaller than in the above example.

Incomplete Precipitation of Creatine-According to our present knowledge the most important reducing substances which interfere with the determination of the true sugar content of an acid extract of muscle are hexosephosphate, glutathione, and creatine. This will be discussed in a subsequent paper on muscle sugar. The former two substances are carried down by the mercuric sulfate- barium carbonate treatment of the extract. Creatine is, however, incompletely precipitated under these conditions. The concen- tration of creatine in the final filtrate used for the sugar analysis is remarkably constant, in spite of variations in its concentration before precipitation. The following figures per 2 cc. of final filtrate (this being the amount usually taken for sugar analysis) illustrate this point: 0.111,0.108, 0.107,0.098,0.092,0.105,0.094,and0.112, average 0.103 mg. of creatine’ or 60 mg. of creatine per 100 gm.

1 The values recorded are those obtained with the alkaline picrate method after conversion of creatine into creatinine. The values obtained by means of the diacetyl color reaction for creatine were approximately 15 per cent lower.

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360 Epinephrine and Insulin in Frogs

of frog. Since creatine in the presence of glucose has a reducing power for the ferricyanide reagent approximately It of that of glucose, 60 mg. of creatine increase the reducing value by about 6 mg. per 100 gm. of frog.

Comparison of the Hagedorndensen and Benedict MethodsBe- fore realizing the advantage which a specific copper reagent offers

TABLE II

Effect of Epinephrine Injections on Sugar and Lactic Acid Content of Normal and Insulinized Frogs under Aerobic and Anaerobic Conditions

The temperature was 15”. All values were calculated in mg. per 100 gm. of body weight.

Normal frogs

All frogs injected with insulin 18 to 42 hrs. previously

-

_

. _

-

Aerobic (3 hrs.)

Epinephrine- injected

Sugar Sugar

28.8 42.8 53.0 33.6 42.5 49.0 41.0 56.6 60.6 40.3 49.0 59.0 42.0 25.0 50.1 41.7 35.6 53.1 39.2 66.5 63.0 35.4 60.0 52.5

37.8 jz3.t

47.7 k1O.C

55.0 It4.i

36.0 45.3 34.6 26.9 54.1 33.9 34.8 52.0 44.1 39.5 57.0 42.5 31.6 40.1 38.2 38.2 48.1 43.8

34.5 zk3.1

49.4 f4.S

39.5 zk3.t

-

Controls

Sugar

68.5 60.4 63.0 65.0 78.5 52.9 62.0 74.0 47.0 55.0 51.6 56.5 76.9 70.0 65.0 41.0

64.1 59.4 f7.4 k8 t

48.3 54.5 56.5 46.5 70.5 40.3 57.5 35.8 53.1 50.0 55.4 44.9

56.9 45.3 h4.i *5.:

Anaerobic (3 hrs.)

Epinephrine- injected

-T

89.5 72.9 96.5 82.0 86.5 57.7 82.4 70.1

113.0 64.0 121.1 64.9 111.0 75.0

90.5 71.0 --

98.8’ 69.7 ~12.3 f5.C --

108.0 33.9 81.1 56.0 95.0 48.5 71.5 42.0 86.5 50.5

100.0 51.0 --

90.3 46.9 zt9.0 +6.(

“2% 121.5 134.0 113.5 97.5

129.0 166.0 143.0 131.0

129.4 t14.3

79.0 81.5 92.0

102.0 100.1 105.0

93.2 f7.2

for this type of work, many determinations were made with the Hagedorn-Jensen method only. Recently comparisons between the two methods were made on thirty-seven filtrates from mercuric sulfate precipitations (Table III of this and Table III of the follow-

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K. W. Buchwald and C. F. Cori 361

ing paper (6)). On an average the Hagedorn-Jensen method indi- cated 9.7 mg. more reducing substances per 100 gm. of frog than the Benedict method. In the preceding section it was shown that 6 out of the 9.7 mg. may be ascribed to the reducing action of creatine on the ferricyanide reagent. The nature of the remaining 3.7 mg. of non-sugar reducing substances was not ascertained.

Effect of Epinephrine under Aerobic and Anaerobic Conditims- In Table II two series of experiments are recorded, one aerobic and the other anaerobic. Each of these series is again subdivided into a control group and into a group injected with epinephrine. Whenever possible, experiments on four frogs, one for each of the four groups, were run on the same day. If this could not be done, experiments on one control and one injected frog of the same series were run at the same time. This is of some importance in the case of frogs, because experiments performed on the same day show much better agreement than experiments performed on different days. In order to examine individual experiments, figures on the same horizontal line should be compared. Epinephrine acting under aerobic conditions causes an increase in the sugar and lactic acid content of the frogs. This may.be observed in each of the individual experiments. On an average the sugar content in- creased by 45.5 and the lactic acid content by 34.4 per cent. The response of the cold blooded animal to epinephrine is therefore of the same nature as that of the mammal.

When the frogs were kept for 3 hours in nitrogen, there resulted a marked increase in the sugar and lactic acid content, the former rising 57 and the latter 107 per cent. It should be noted that all frogs survived a period of anaerobiosis of 3 hours’ duration, though some animals became paralyzed toward the end of the period. When placed in air,they recovered completely within a short time. Epinephrine injected at the beginning of anaerobiosis caused an increase in the rate of lactic acid formation. Each of the indi- vidual experiments was decidedly positive. The effect of epineph- rine on the sugar content under anaerobic conditions was not nearly so marked as under aerobic conditions. Apart from a good deal of overlapping of the values, there was one negative (the fourth experiment in Table II) and several weakly positive experi- ments. On an average the increase in sugar content amounted to 17.3 per cent. It is probably correct to assume bhat epinephrine

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362 Epinephrine and Insulin in Frogs

has but a slight accelerating effect on sugar formation under an- aerobic conditions.

The above experiments are open to the objection that there is oxygen left in the frogs when they are placed in nitrogen. At the rate of oxygen consumption prevailing at 15” it may roughly be calculated that 20 minutes would elapse until most of the oxygen is used up. During this period of partial anaerobiosis epinephrine might have exerted its influence on sugar and lactic acid formation. In order to test the validity of this objection experiments of the

TABLE III

Effect of Epinephrine Znjcclions on Sugar and Lactic Acid Content of Decere- braled Frogs ILnder Anaerobic Conditions

In these experiments (in contrast to those in Table II) the animals were analyzed 2s hours after the beginning of anaerobiosis and 2 hours after the injection of epinephrine. All values were calculated in mg. per 100 gm. of body weight.

Sugar T

63.4 60.3 60.6 58.5 43.8 74.5 91.5

53.5 54.0 38.8 56.0 78.4

Lactic acid

Epinephtine-injected

102.0 93.4 72.5 91.0 64.0

104.0 123.5

_

-

62.0 60.5 75.1 75.8 48.0 77.5 86.5

58.6 68.5 38.2 64.5 69.0

102.5 111.5 99.1

117.0 91.8

120.5 148.0

_ _ _ 64.6 56.1 92.9 1 69.3 1 59.7 1 112.9

Sugar - I Lactic acid

type shown in Table II were repeated 1 year later with the follow- ing modifications. The frogs were decerebrated by means of a Goltz puncture on the day preceding the experiment. As is well known, decerebrated frogs, when undisturbed, remain perfectly motionless for long periods of time. A possible error due to unequal muscular activity of the different animals was thus elimi- nated. Secondly, the animals were kept in nitrogen for a prelimi- nary period of 30 minutes before epinephrine was injected. In this manner the action of epinephrine was confined to a period of

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K. W. Buchwald and C. F. Cori

more complete anaerobiosis. Finally, the animals were kept under anaerobic conditions for a total period of only 23 hours. Owing to this fact they showed no signs of paralysis when removed from the containers. The results of this series of experiments are shown in Table III; on comparing them with the corresponding ones in Table II attention should be paid to the fact that epineph- rine was allowed to act for a different length of time in the two cases. When calculated per hour, the excess lactic acid formation as the result of the epinephrine injection was the same; i.e., an average of 10.0 mg. for the experiments in Table III and of 10.2 mg. for those in Table II. The conclusion that epinephrine is able to accelerate lactic acid formation under anaerobic conditions is therefore sustained.

The influence of epinephrine on sugar formation was even weaker in the experiments in Table III than in those in Table II. Glycogen determinations in the liver of decerebrated frogs showed that there was enough material present for sugar formation. There is the possibility that the anaerobiosis itself caused sugar formation to proceed near its maximal rate so that a further acceleration could not occur when epinephrine was injected.

E$ect of Insulin under Aerobic and Anaerobic Conditions-In contrast to epinephrine which acts rapidly in cold as well as in warm blooded animals, insulin action has a marked temperature coefficient. The effect of temperature on the production of con- vulsions in frogs by insulin administration was studied in detail by Huxley and Fulton (10). They found that at 6-8” it took 120 to 144, at 15” 60 to 70, at 20” 43 to 49, at 25” 24 to 27, and at 30” 14 hours until hypoglycemic convulsions supervened. On plotting the rate of action of insulin and the O2 consumption of frogs at different temperatures they noted a surprising coincidence between the two curves and by extrapolation they arrived at the conclusion that if a frog could exist for any length of time at 37”, convulsions would occur as rapidly as in a mammal.

In the present experiments the animals were used 18 to 42 hours after the insulin injection. Since they were kept at about lo”, they were still far removed from the convulsive state. Epineph- rine, when injected into these insulinized frogs, produced hardly any increase in the sugar content either aerobically or anaerobi- cally. But not only the glycogenolytic action of epinephrine was

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364 Epinephrine and Insulin in Frogs

antagonized by the preceding insulin treatment. The increase in sugar content which occurred when frogs were kept anaerobically was also markedly inhibited by the insulin injection (Table II). In mammals insulin prevented the decrease in liver glycogen which occurred at a certain time interval after the injection of epineph- rine (11, 12).

Aerobic lactic acid formation after epinephrine injection and lactic acid formation produced by anaerobiosis were only slightly retarded by insulin administration, the former by 11 and the latter by 9 per cent. In rabbits insulin inhibited the rise in blood lactic acid after epinephrine injection by about 14 per cent (13). In view of these results it was surprising to find that the accelerating effect of epinephrine on lactic acid formation in anaerobic frogs was almost completely inhibited by insulin (Table II).

DISCUSSION

Lesser (3) determined the amounts of glycogen which disap- peared from liver and rest of body during 3 hours of anaerobiosis at 14”. Per 100 gm. of frog an average of 112 mg. was lost from the liver and of 165 mg. from the rest of the body. In other experi- ments Lesser (4) determined sugar and lactic acid in the whole frog instead of glycogen. During 3 hours of anaerobiosis there were formed per 100 gm. of frog 102 mg. of sugar and 98 mg. of lactic acid. Meyerhof and Meier (5) found considerably less lactic acid formation during anaerobiosis, i.e. an average of 45 mg. per 100 gm. of frog in 3 hours. The value of 51 mg. found in theexperi- ments in Table II agrees much better with that found by Meyerhof than with the value recorded by Lesser.

As stated in the introduction, the fact that epinephrine is able to accelerate lactic acid formation under anaerobic conditions makes it very improbable that this effect is due to vasoconstriction in muscle. The same conclusion was arrived at in experiments on mammals in which epinephrine raised the blood lactic acid (and blood sugar) level at rates of intravenous injection far below those required to raise the blood pressure. It is of interest t,hat the amount of lactic acid formed as the result of epinephrine injection is nearly twice as large under anaerobic as aerobic conditions. The significance of this finding will be discussed in the followmg paper 03).

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K. W. Buchwald and C. F. Cori 365

The idea that epinephrine is oxidized in the tissues and that it is this oxidized form in the nascent state which produces the effects, is not contradicted by the fact that epinephrine was found to act under anaerobic conditions. Traces of oxygen will undoubtedly remain in the tissues for some time in spite of anaerobiosis.

Von Issekutz (14) found that the isolated and perfused liver of frogs which had received an insulin injection on the preceding day, formed only one-fifth as much sugar as the liver of uninjected frogs. Furthermore, addition of epinephrine to the perfusion fluid increased sugar production much less in the liver of the insulinized frogs than in those without insulin injection. The present experiments on intact frogs may be regarded as a cotirma- tion of these results; i.e., an injection of insulin, 18 to 42 hours previously, prevented the glycogenolytic action of epinephrine on the liver. Insulin was also found to inhibit the hydrolysis of liver glycogen which takes place when frogs are kept for 3 hours in an atmosphere of nitrogen, showing that this action persists under anaerobic conditions. The inhibitory action of insulin on hepatic glycogenolysis has also been demonstrated in mammals; it possibly is one of the primary effects of insulin in the body.

SUMMARY

Epinephrine injections increased the sugar and lactic acid con- tent of frogs under aerobic as well as anaerobic conditions. When epinephrine was injected into frogs which had received insulin 18 to 42 hours previously, an increase in sugar content did not take place either aerobically or anaerobically. This effect of insulin is ascribed to an inhibitory action on hepatic glycogenolysis, becom- ing especially noticeable when the latter is augmented by epineph- rine or anaerobiosis. The fact that epinephrine is able to acceler- ate lactic acid formation under anaerobic conditions argues against the idea that this effect is due to vasoconstriction and hence to asphyxia in muscle. The evidence is in favor of the assumption that epinephrine has a direct accelerating effect on the glycogeno- lytic process in muscle.

BIBLIOGRAPHY

1. Cori, C. F., Cori, G. T., and Buchwald, K. W., Am. J. Physiol., 93,273 (1929).

2. Lesser, E. J., 2. Biol., 66,467 (1911); 60,388 (1913).

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366 Epinephrine and Insulin in Frogs

3. Lesser, E. J., Biochem. Z., 146,577 (1923). 4. Lesser, E. J., Biochem. Z., 146,560 (1923). 5. Meyerhof, O., and Meier, R., Arch. ges. Physiol., 204,488 (1924). 6. Cori, C. F., and Buchwald, K. W., J. Biol. Chem., 92,367 (1931). 7. West, E. S., Scharles, F. H., and Peterson, V. L., J. Biol. Chem., 82,137

(1929). 8. Benedict, S. R., J. Biol. Chem., 76,457 (1928). 9. Friedemann, T. E., and Kendall, A. I., J. Biol. Chem., 83,23 (1929).

10. Huxley, J. S., and Fulton, J. F., Nature, 113,234 (1924). 11. Sahyun, M., and Luck, J. M., J. Biol. Chem., 86,l (192930). 12. Cori, G. T., Cori, C. F., and Buchwald, K. W., J. Biol. Chem., 86, 375

(1930). 13. Cori, C. F., and Cori, G. T., J. Biol. Chem., 84,683 (1929). 14. von Issekutz, B., Biochem. Z., 147,264 (1924).

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K. W. Buchwald and Carl F. CoriANAEROBIC CONDITIONSINSULIN IN FROGS UNDER

THE ACTION OF EPINEPHRINE AND

1931, 92:355-366.J. Biol. Chem. 

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