THE EFFECT OF INSULIN ON MUSCLE RESPIRATION*

BY F. J. STARE AND C. A. BAUMANN

(From the Cancer Research Laboratory, and the Department of Biochemistry, College of Agriculture, University of Wisconsin, Madison)

(Received for publication, October 9, 1939)

According to Krebs and Eggleston (1) insulin accelerates the respiration of muscle tissue in vitro. The effect was observed with pigeon breast muscle suspended in 0.1 M phosphate buffer, pH 6.8, supplemented with muscle juice, and citric or some dicarbox- ylic acid. In this system insulin was the limiting factor. Shorr and Barker (2) studied the effect on rabbit, cat, and dog muscle, and failed to observe any response to insulin. In pigeon muscle they observed a 20 per cent increase, in contrast to the 90 per cent increase reported by Krebs and Eggleston. Banga and co- workers (3) observed stimulation due to insulin in certain brain preparations fortified with fumarate, adenylic acid, and pyruvate.

Like Shorr and Barker we have failed to observe a marked in- crease in respiration under the experimental conditions originally outlined by Krebs and Eggleston, but when a Ringer-phosphate medium was used, insulin increased respiration, particularly in the breast muscles of depancreatized pigeons (4). A detailed study of this insulin effect has therefore been made, including the character of the respiration, the response to various di- and tri- carboxylic acids, the effect of pancreatectomy, and a correlation of insulin effects in vivo and in vitro.

Methods

The oxygen consumption of minced pigeon breast muscle was measured in the usual way in a standard Warburg respirometer (4, 5). The medium was a Ringer-phosphate buffer free of cal- cium (6), pH 7.4, containing 0.2 per cent glucose. Approximately

* Supported by the Jonathan Bowman Cancer Fund and the Wisconsin Alumni Research Foundation.

453

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454 Insulin Effect on Muscle Respiration

150 mg. of fresh tissue were suspended in 2 cc. of medium. The medium was supplemented with 0.1 or 0.2 cc. of muscle juice (Kochsaft) (6), and a di- or tricarboxylic acid. The following acids were studied: fumaric, malic, succinic, cr-ketoglutaric, citric, and glutamic. The solutions were brought to pH 7.4 before addition to the medium, and the final concentration of the ‘(acid” was 0.001 or 0.005 M. Both the muscle juice and the solutions of dicarboxylic acid were prepared at weekly intervals. oc-Keto- glutaric acid was always dissolved immediately prior to use.

Two forms of insulin were available for these studies, amorphous insulin,’ and a commercial preparation, iletin, containing 80 units per cc. In the earlier experiments up to 40 units of insulin were used per 2 cc. of medium, but these higher amounts fre- quently inhibited the respiration of normal muscle (4). The optimum concentration appeared to be 4 units per 2 cc. of me- dium, and this amount was Iater adopted as standard. The experiments were continued for 4 hours. This long experimental period raised the question whether bacterial contamination af- fected respiration results. Accordingly, bacterial plate counts were made every hour for 9 hours. Under the usual laboratory conditions, with clean glassware and instruments, the number of organisms present was too small to affect respiration until after the 5th hour.

To obtain muscle low in insulin, birds were depancreatized 1% to 4 weeks prior to use. Complete surgical removal of the pan- creas was attempted, but was probably never achieved, as small bits of pancreas containing islet tissue were observed on autopsy. However, at least 90 to 95 per cent of the pancreas had been removed.

E$ect of Insulin on Respiring Muscle-Insulin increased the respiration of pigeon breast muscle both in the presence and absence of supplements which themselves also increased respira- tion appreciably.

Either insulin, fumarate, or muscle juice alone increased the oxygen uptake about 20 per cent over that of the control. Insu- lin and fumarate or insulin and muscle juice were somewhat more effective than either alone, and the three together were more

1 We are indebted to Dr. G. H. A. Clowes of Eli Lilly and Company for this preparation.

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F. J. Stare and C. A. Baumann 455

effective than any two, causing an increase of 80 per cent over the unsupplemented control. The effect of insulin was most pro- nounced during the latter part of the experiment. During the 3rd and 4th hours respiration had practically ceased in the un- supplemented control tissues, whereas in media containing insulin, either in the presence or absence of other supplements, muscle continued to respire appreciably. Insulin, therefore, tended to preserve respiration rather than to increase it. A typical experi- ment with the various controls is given in Table I.

TABLE I*

Effect of Insulin and Other Supplements on Oxygen Consumption of Breast Muscle from Depancreatized Pigeon

02 = total c.mm. of oxygen consumed per mg. of dry weight of tissue during the time t (6).

Supplements

None, control ....................................... Insulin (lunit) ...................................... Fumarate (0.001 M) .................................. Muscle juice (0.2 cc.) ................................ Malonate (0.001 M) .................................. Insulin + fumarate ..................................

‘I + muscle juice ............................... I< + malonate ..................................

Fumarate + muscle juice ............................ L‘ + malonate ...............................

Muscle juice + malonate. ........................... Insulin + fumarate + muscle juice. .................

“ + “ + tl I‘ + malonate ......

- t

-

39 (4 lm.)

20.6 0.9 28.0 3.0 26.1 1.9 25.4 2.2 4.7 0.3

32.8 4.5 32.0 4.4 5.1 0.3

28.0 2.5 25.7 1.8 15.5 1.3 37.2 6.4 36.9 6.3

L

* To conserve space the results have been skeletonized. Detailed protocols of preliminary experiments are given elsewhere (4).

Importance of Di(Tri)-Carboxylic Acids-Apparently active di(tri)-carboxylic acids must be present in the respiring mixture for insulin to exert its effect. In addition to fumaric acid, the following were used to “sensitize” muscle to insulin : succinic, malic, citric (Fig. l), ar-ketoglutaric, and glutamic (Table II) acids. Since these substances are normally present in muscle, it is possible to observe an increase in respiration when insulin alone is added, though the effect is enhanced by the presence of

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456 Insulin Effect on Muscle Respiration

more dicarboxylic acid. When the dicarboxylic acid is effectively inactivated by malonate, respiration is strongly inhibited both in the presence and absence of insulin (Table I). The addition of fumarate restores respiration and sensitivity to insulin, even in the presence of malonate, since fumarate nullifies the action of malonate in equivalent concentrations (5).

The degree of malonate inhibition in systems containing insulin and other di(tri)-carboxylic acids varied with the kind and amount

CONTROL

INSULIN

SUCCiNATE+MUSCLE JUICE

INSULIN + SUC.+ M. JUICE

FUMARATE+M. JUICE

INS. l FUM. + M. JUICE

MALATE * M. JUICE

INS.+ MALATE + M* JUICE

CITRATE + M. JUICE

INS. l CIT. + N*JUlCE

r

r 1

1 a

I 1

I 1

FIG. 1. The effect of insulin and supplements on the oxygen consumption of breast muscle from a depancreatized pigeon, during the 3rd and 4th hours of the experiment. 0.001 M succinate, fumarate, malate, and citrate were used, 0.2 cc. of muscle juice, and 1 unit of amorphous insulin. Blood sugar before pancreatectomy was 197 mg. per cent, after 11 days 282 mg. per cent.

of acid present, in accordance with the ability of the acid to compensate for the malonate effect in the absence of insulin (5). In systems fortified with fumarate, muscle juice, and insulin, no inhibition within experimental error was observed in the pres- ence of equimolar amounts of malonate (Tables I and III). When citrate was substituted for fumarate, however, respiration was inhibited 68 per cent. In the presence of insulin only, malonate

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F. J. Stare and C. A. Baumann 457

inhibition was 88 per cent (Table III). Glutamic acid, like citric, failed to compensate for malonate (5), and insulin systems con- taining glutamic acid were also markedly sensitive to malonate (Table II). Malic and cr-ketoglutaric acids were like fumaric acid in protecting insulin respiration from malonate, whereas succinic acid was somewhat less effective.

Another factor in respiration appeared to be cocarboxylase, since it increased respiration in the presence of other supplements (Table II). Cocarboxylasez alone had no effect on the respira-

TABLE II Effect of Insulin, Glutamic and c+Ketoglutaric Acids, and Other Supplements

on Respiration of Pigeon Breast Muscle

Supplements 02 (4 lxx.) Qon (4th hr.)

Control. ............................................. Glutamic acid (0.005 M). ............................. Muscle juice (0.2 cc.). ............................... Insulin (4 units). .................................... Cocarboxylase (100-y) ................................ Malonate (0.005 M) ..................................

Glutamic acid + muscle juice ....................... ‘I “ + “ “ + insulin ............. ‘I “ + “ (‘ + “ + malonate. ‘I “ + “ “ + Ii + cocar-

boxylase ........................................... or-Ketoglutaric acid (0.005 M) ........................

“ “ + muscle juice. ................. I‘ “ + “ “ + insulin

c.mm. 15.4 24.2 17.7 14.5 13.4 4.6

38.5 44.2 12.7

50.0 11.0 18.4 2.4 24.6 2.8 40.0 8.0

1.2 2.7 1.5 0.5 0.4 0.1 5.0 9.6 0.8

tion of normal muscle, although it did increase the respiration of muscle from a depancreatized pigeon (Table III).

Effect of Pancreatectomy-A marked difference between the muscles of normal and depancreatized birds was observed, both in the oxygen uptake and in the sensitivity to insulin. To a considerable extent it was possible to correlate these differences with the time elapsed from pancreatectomy until the bird was used for the experiment (Table IV). The birds were grouped into five groups of two to five each. Group 1 consisted of five representa-

2 We are indebted to Professor C. A. Elvehjem for this preparation.

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458 Insulin Effect on Muscle Respiration

tive normal birds; Group 2, three pigeons used in the 2nd week after pancreatectomy; Group 3, three pigeons in good condition, used during the 3rd week after pancreatectomy; Group 4, two extremely emaciated birds used during the 3rd week after pan- createctomy; and Group 5, three healthy birds used during the 4th week after pancreatectomy.

TABLE III Effect of Malonate on Respiration o.f Pigeon Breast Muscle Fort&d with

In&in, Fumaiate,-and Citrate

Pigeon No. Supplements

4. Depan- creaked

7. Depan- creatized

None, control Malonate (0.001 M)

Insulin (4 units) “ + malonate

Fumarate (0.001 M)

“ + malonate “ + muscle juice

Insulin + “ “ + fumarate I‘ + “ “ + “ + mal-

onate None, control Malonate (0.005 M)

Insulin (4 units) “ + malonate

Citrate (0.005 M)

“ + malonate “ + muscle juice

Insulin + citrate + muscle juice “ -I- “ + “ “ f malonatc

Cocarboxylase (100 y) Citrate + insulin + muscle juice + cocar-

boxylase

02 4 hrs.)

C.nwn.

11.1 5.2

20.0 5.9

18.7 18.1 21.1 30.1 29.0

10.6 3.0

18.3 2.2

15.5 5.5

17.9 24.5 7.7

19.3 31.0

Per cent ohibi- on by mal- mate

53

71

3

4

72

88

64

68

During the 2nd week of recovery, control respiration was ac- tually somewhat higher than that of muscle from normal pigeons and the sensitivity to insulin had increased markedly. Insulin increased respiration 62 per cent in the unsupplemented tissues and 40 per cent in the supplemented tissues (Group 2), as com-

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F. J. Stare and C. A. Baumann 459

pared to increases averaging 20 and 16 per cent respectively in normal muscle (Group 1). Although the sensitivity to insulin had increased, the per cent of increase due to the complete system

TABLE IV

Effect of Pancreatectomy on Oxygen Uptake of Pigeon Breast Muscle and Its Sensitivity to Insulin

31ood wgar

-

1

.-

P’

.-

--

-

-

- 02 uptake of

control (unmp- plemented

timle)

Per cent change in 4 hr. 0s uptake upon addition of various supplements

.

1st hr. 1

4th hr. Insulin

to contra

’ I 1 j

,

Insulin

&, acid + muscle juice

c.nwn.

9.9 12.6

8.7 9.1

12.1

e.mm.

18.2 $36 +94 0 26.2 +51 +ss +30 12.2 +17 +195 +12 17.1 +10 +182 +I6 26.6 -5 +I8 +19

10.5 20.1

19.1 28.1 20.6

+20 +llS +I6

12.2 14.7 12.5

j-100 +100 +16 +47 +110 +s9 +40 +85 +32

13.1

7.4 5.4 7.8

+62 +98

+80 +171

+14 +llS +73 +131

+40

+43 +14

+161

6.9

6.1 5.3

5.7

22.6

11.1 7.9

10.6

9.9

7.7 6.0

6.9

+56 +140 +73

-18 +137 +57 -10 +87 +44

-14 $112 $51

10.7 19.8 -24 +13 -28 9.6 19.5 -15 +52 -14

10.8 20.2 -22 +25 -18

10.4 19.8 -

-20

--

- +30 -20

‘igeon NO.

I Group No.

_

days

7 8 9

11 13

w. 67 ccl

190 180 187 184 210

1. Normals

190 Average.....................

9 11 11

210 216 282

236

290 170

2. Depancreatized 1 2 3

Average.....................

Average..................... 230 _

205 220 __

213 Average.....................

5. Depancreatized 9 21 8 24

11 26

240 238 243 -

240 Average.....................

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460 Insulin Effect on Muscle Respiration

-di(tri)-carboxylic acid, muscle juice, insulin-had decreased from an average of 116 to 98 per cent. This meant that the sensi- tivity of the muscle to supplements other than insulin had de- creased appreciably, presumably because insulin was a limiting factor in this tissue. During the 3rd week following pancreatec- tomy, control respiration decreased markedly; the oxygen uptake for the 1st hour averaged 6.9 c.mm. as compared to 10.5 for normal muscle, but the sensitivity to insulin persisted. Insulin alone increased respiration 56 per cent; insulin in the presence of a di(tri)-carboxylic acid and muscle juice increased respiration 73 per cent. The complete system increased respiration 140 per cent. Thus when suitable supplements were added, respiration was nearly equal to that of normal tissues simi1arIy supplemented, indicating that the respiratory capabilities of the muscle had been unimpaired. Presumably the lower respiration of the unsupple- mented tissue was due to a lack of other essential factors as well as insulin.

Somewhat different results were obtained with the muscles of two extremely emaciated birds (Group 4), likewise used during the 3rd week following pancreatectomy. The oxygen uptake was approximately half that of normal muscle, averaging only 5.7 c.mm. The unsupplemented tissue did not respond to insulin, an average inhibition of 14 per cent being observed. However, in tissue supplemented with dicarboxylic acid and muscIe juice, the addition of insulin produced an increase of 51 per cent, and the fully supplemented tissue gave an increase over the unsupple- mented tissue averaging 112 per cent. It thus appeared that the muscle from the emaciated birds had been so deficient in other factors that insulin activity could not be demonstrated until they were supplied.

Pigeons which survived 4 weeks following pancreatectomy were lively and appeared to be fully recovered. The oxygen uptake of the muscle tissue also was similar to that of normal birds, but the response to insulin and the dicarboxylic acids was markedly less (TabIe IV, Group 5). Insulin alone inhibited respiration 20 per cent when added either to the unsupplemented tissues or to tissues supplemented with acid and muscle juice. The respiration of tissue fully supplemented with insulin, dicarboxylic acid, and muscle juice was only increased 30 per cent above that of the

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F. J. Stare and C. A. Baumann 461

control, an increase far lower than is usually observed. Thus it appeared that whereas the tissues from these birds had a nor- mal oxygen uptake, the dicarboxylic acids exerted very little effect, and insulin actually inhibited respiration. Since the tissue seemed to be respiring at a greater rate during the 4th week than the 3rd, it is possible that the restored respiration involved some other mechanism than that concerned with insulin and the dicarboxylic acids.

Action of Treated Insulin-The question arose whether the effectiveness of insulin preparations in muscle respiration could be correlated with their action in lowering blood sugar in rabbits. Accordingly, insulin samples were treated to destroy their effec- tiveness in the living animal, and their activity on muscle was tested as before. The treatments included boiling for 30 minutes at pH 7.0, and incubation for 3 hours at 40” in the presence of M/30 alkali. When levels equivalent to 4 or 8 units of insulin were given, the treated samples failed to lower the blood sugar of rabbits, whereas 4 units of the untreated insulin lowered blood sugar markedly and produced convulsions. The treated insulin preparations, neutralized before use, were inactive in stimulating muscle respiration (Table V). Similar results were obtained with normal muscle and muscle from depancreatized birds.

Respiratory Quotients-Since the respiration experiments ex- tended over a 4 hour period, it was desirable to know whether the oxygen uptake represented a true respiration, particularly with the highly fortified tissues. The respiratory quotient was there- fore determined during various intervals of the experimental period on muscle from both normal and depancreatized pigeons. Muscle from normal pigeons had a normal R.Q., varying from 0.89 to 0.98 during the 1st hour of the experiment, regardless of whether the tissue was unsupplemented, or supplemented with insulin, fumarate, and muscle juice (Table VI). During the 3rd and 4th hours the R.Q. of the unsupplemented control decreased to practically zero, indicating cessation of true respiration, whereas the R.Q. of tissue supplemented with insulin, fumarate, and muscle juice remained normal at 1.05. With muscle from depancreatized pigeons, the respiratory quotients were quite comparable. During the 1st hour of the experiment the R.Q.

of unsupplemented or supplemented tissues remained between

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462 Insulin Effect on Muscle Respiration

0.89 and 1.1. In the 2nd hour of the experiment there appeared to be no significant change, but in the 3rd hour of the experiment, the R.Q. of the unsupplemented control dropped to zero, whereas in the presence of insulin, or insulin plus acid plus muscle juice, no such decrease was observed. In other words, a prolonged up- take of oxygen in the presence of insulin, etc., indicated a prolon- gation of true respiration.

Insulin versus Malonate in Viva-The inhibition of insulin action by malonate in vitro (Table III) raised the question whether malonate might not also hinder the action of insulin in the living

TABLE V Effect of Treated Insulin on Respiration of Pigeon Breast Muscle

Normal pigeon Fumarate (0.005 M) + muscle juice + untreated insulin

(4 units).............................................. 35.5 +6.1 Fumarate (0.005 M) + muscle juice + insulin (boiled). 29.3 -0.1

“ (0.005 ,‘) + “ “ + “ (alkali- treated).............................................. 29.8 +0.4

Fumarate + muscle juice (no insulin). 29.4 Depancreatized pigeon

Fumarate (0.005 M) + muscle juice -I- untreated insulin (4 units)............................................. 18.2 +6.6

Fumarate (0.005 M) f muscle juice + insulin (boiled). 12.8 +1.2 ‘I (0.005 “ ) + ‘( I‘ + “ (alkali-

treated).............................................. 10.2 -1.4 Fumarate (0.005 M) + muscle juice (no insulin). . 11.6

animal. Accordingly, a 15 per cent solution of neutralized sodium malonate was injected subcutaneously into rabbits, either before or simultaneously with a solution of insulin. The sugar content of the blood was then determined at intervals, by the Shaffer- Hartmann method. Both fed and fasted rabbits were used. The exact dosages and the results of the experiments are presented in Table VII. The blood sugar of fed rabbits which received 4 units of insulin fell rapidly until the animals went into convulsions (Rabbits 1,2, and 3). Fed Rabbits 4, 5, and 6 received the same amount of insulin, but in 2 cc. of 1 M malonate solution. Their

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F. J. Stare and C. A. Baumann 463

blood sugar showed some decrease, but much less than when insu- lin was given alone. Furthermore, none of these rabbits went into convulsions. Similar results were obtained with fed Rabbit 7, which received malonate 15 minutes before the insulin. Fed Rabbits 8 and 9 received only malonate and their blood sugar increased somewhat.

TABLE VI Respiratory Quotient of Pigeon Breast Muscle

-

Normal

Depancrea- tieed

‘igeon NO.

1 2

3 14 14

4 4 4

5 5 5

Supplement

None, control Insulin + fumarate + muscle

juice None, control

‘I ‘I

Insulin + fumarate + muscle juice

None, control Insulin

‘I + fumarate + muscle juice

None, control Insulin

“ + cr-ketoglutaric acid + muscle juice

None, control Insulin

I‘ + citrate + muscle juice None, control Malonate + citrate Insulin + citrate + muscle juic

-

e -

1st hr. od hr.

0.91 0.95

0.89 0.92 0.98

0.91 0.89 0.97

1.02 0.96 0.96

0.89 0.94 0.89 1.00 1.10 1.09

0.88

0.92 1.03

- 3

I

-

rd hr.

0.15 1.05

0.0 0.92 1.90

0.0 1.30 1.02 0.0

1.3

* Insulin used = 4 units; muscle juice used = 0.2 cc.; di(tri)-carboxylic acids = 0.005 M.

Similar results were obtained with rabbits fasted for 24 hours. Malonate retarded the action of insulin in lowering blood sugar for about 1 hour (Rabbits 6 through 9, as compared with Rabbits 1 to 5), but as time went on the malonate effect wore off, and the blood sugar decreased. With the rabbits receiving malonate without added insulin (Rabbits 10 through 14) there was a tendency for the blood sugar to rise slightly. Rabbits 12 and 13,

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464 Insulin Effect on Muscle Respiration

TABLE VII E$ect of Insulin and Malonate on Blood Sugar of Rabbit

Rabbit No. Treatment

-

4 units insulin Same

‘I

4 units insulin in 2 cc. 1 M malonate Same

‘(

10 cc. 1 M malonate, 15 min. later 4 units insulin

8 10 cc. 1 M malonate at 0 hr., 10 cc. at 1 hr.

9 10 cc. 1 M malonate at 0 hr., 5 cc. at 1 hr.

-

7 8

9

10 11 12 13 14

Blood Sugar

Ohr. 1 lhr. 1 Zhrs. jdhrs.

Fed rabbits (2-2.5 kilos)

-

“:sa 104 101 120 116 142 96

103

115

117

75 85

Fasted (24 hrs.) rabbits 2-2.5 kilos in weight

4 units insulin Same

‘I “ ‘I

10 cc. 1 M malonate, 15 min. later 4 units insulin

Same 20 cc. 1 M malonate, 15 min. later 4

units insulin Same

10 cc. 1 M malonate Same 20 cc. 1 M malonate Same 10 cc. 1 M malonate at 0 hr., 10 cc. at

1 hr.

86 26’ 93 46 82 58 98 52 83 51 96 83

100 104 61 64 83 70 66 36*

77 85

93 108 96 114

106 110 84 109

110 99

56’ 61

75 90

150

148

%nPt~ 39*

40* 113 111

155

139

47 39 45 42 55

54*

39* 35* 37*

Died, no convul-

sic LS 97 96

114 115 121 123 102 135 212 107

* Convulsions.

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F. J. Stare and C. A. Baumann 465

which received twice as much malonate as Rabbits 10 and 11, showed a definite rise.

The true meaning of this antagonism between insulin and malonate in vivo is not clear, since a proper interpretation depends upon what insulin does in the living animal. If the decrease in blood sugar following administration of insulin is entirely due to withdrawal of sugar into liver storage, this phenomenon would appear to concern an aspect of insulin activity different from its effect in increasing the oxygen consumption in vitro. Nevertheless it is curious that malonate and insulin should counteract each other both in vitro and in vivo. Since the action of malonate in vitro presumably concerns the Cb acids, it is likely that these acids are also somehow involved in the action of insulin in vivo.

Insulin in Other Species-Since most of the experiments with insulin in vitro have been carried out with pigeon muscle, it should be pointed out that this animal exhibits certain peculiarities. The pigeon normally has a high blood sugar content and removal of 95 per cent of the pancreas does not materially alter this value. Furthermore the pigeon is able to tolerate tremendous doses of insulin. We have given up to 150 units within 24 hours without killing the bird, and the blood sugar was reduced only 50 per cent. The apparent recovery of depancreatized birds after 4 weeks might indicate either the presence of an auxiliary respiratory mechanism or the ability to regenerate islet tissue in some other organ. Thus results with pigeon muscle can be applied to mam- mals only with caution. Hence, our basic experiments in vitro were repeated on rabbit heart, rabbit skeletal muscle, and chicken breast muscle. With these more slowly respiring tissues, the results were qualitatively similar, though quantitatively less spectacular than with the more rapidly respiring pigeon breast muscle (4).

SUMMARY

Our results support the general conclusion that insulin is a factor in the respiration of muscle. Other necessary factors are the di(tri)-carboxylic acids, cocarboxylase, and substances in muscle juice. Insulin added to normal pigeon breast muscle in vitro increased respiration about 20 per cent both in the presence and absence of other supplements; the sensitivity to insulin was in-

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466 Insulin Effect on Muscle Respiration

creased to 60 per cent by removal of the pancreas. The optimum response was obtained 1 to 23 weeks after pancreatectomy.

Insulin prolonged respiration, the effect being most pronounced after 2 hours. In muscle from depancreatized birds, insulin also increased respiration. The R.Q. was maintained at normal levels for 4 hours by insulin alone or insulin plus other supplements. Insulin preparations inactivated by heat or alkali failed to stimu- late muscle respiration. Insulin action was inhibited in vitro by malonate, and insulin and malonate appeared to counteract each other to a certain extent in lowering blood sugar in the rabbit.

BIBLIOGRAPHY

1. Krebs, H. A., and Eggleston, L. V., Biochem. J., 32, 917 (1938). 2. Shorr, E.. and Barker, S. B., Proc. Am. Physiol. Sot., Am. J. Physiol.,

61, 213 (1939). 3. Banga, I., Ochoa, S., and Peters, R. A., Proceedings of the Biochemical

Society, 202nd meeting (1939). 4. Stare, F. J., and Baumann, C. A., in Cold Spring Harbor symposia on

quantitative biology, Cold Spring Harbor, 7 (1939). 5. Baumann, C. A., and Stare, F. J., J. Biol. Chem., 133, 183 (1940). 6. Stare, F. J., and Baumann, C. A., Proc. Roy. Sot. London, Series B,

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F. J. Stare and C. A. BaumannRESPIRATION

THE EFFECT OF INSULIN ON MUSCLE

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