Seasonal differences and the effect of insulin on pyruvate uptake, oxidation and synthesis to glycogen by frog skeletal muscle

Comp. Biochem. Physiol., 1969, VoL 29, pp. 509 to 524. PergamonPress. Printed in Great Britain

SEASONAL D I F F E R E N C E S AND T H E E F F E C T OF I N S U L I N ON PYRUVATE UPTAKE, O X I D A T I O N AND SYNTHESIS TO GLYCOGEN BY FROG S K E L E T A L MUSCLE* D. R. H. GOURLEY, TAE KYU S U H t and L. L. B R U N T O N Department of Pharmacology, University of Virginia School of Medicine, Charlottesville, Virginia 22903 (Received 30 ,September 1968)

Abstract--1. In frog sartorius muscle, insulin stimulated the uptake of pyruvate, the synthesis of pyruvate to glycogen, and in summer, hut not in winter, the oxidation of pyruvate. 2. Insulin shifted the utilization of pyruvate away from pathways not studied in this investigation. In summer muscles, the major shift was toward oxidation, while in winter muscles, the major shift was toward glycogen synthesis. INTRODUCTION ISOLATED intact frog skeletal muscle appears to be an excellent preparation in which to demonstrate the stimulation by insulin of carbohydrate oxidation. In frog sartorius muscle, insulin increases the oxidation of lactate to CO2 and the synthesis of glycogen from lactate (Gourley & Suh, 1967, 1969). In order to be oxidized or synthesized to glycogen in isolated muscle, lactate must be converted to pyruvate. In preliminary experiments, we have found no effect of insulin on the conversion of lactate to pyruvate by lactic dehydrogenase, which suggests that the effects of insulin observed with lactate as substrate are beyond the pyruvate level. There have been several experimental indications that insulin influences pyruvate utilization. Delrue & Dekeyser (1940) found that treatment of rabbits with insulin prevented the rise in blood pyruvate which normally followed the intravenous injection of pyruvate. Rice & Evans (1943) demonstrated that insulin increased the utilization of pyruvate and the uptake of oxygen by minced breast muscle of the pigeon. The amount of pyruvate metabolized to COg and the total utilization of pyruvate were found by Villee & Hastings (1949) to be less in the diaphragm muscle from diabetic rats than in normal muscle, and both could be restored to normal by the addition in vitro of insulin. In diabetic humans, the * Supported in part by U.S. Public Health Service Grant A-4718 from the National Institute of Arthritis and Metabolic Diseases. A preliminary report of this work was presented at the Fall Meeting of the American Society for Pharmacology and Experimental Therapeutics, 1966 (Pharn~cologist 8, 201). t Present address: Kyungpook National University School of Medicine, Taegu, South Korea.

509

510

D. R. H.

GOURLEY,TAE KYU SuH ANDL. L. BRUNTON

plasma concentration of pyruvate is increased during insulin-glucose tolerance tests (Fry & Butterfield, 1962). Since the ratio of lactate to pyruvate in the plasma is apparently not changed in m a n b y diabetes or b y glucose or insulin (Klein, 1942), the increased plasma pyruvate observed by F r y & Butterfield might result f r o m impaired pyruvate oxidation. T h u s , a considerable amount of evidence supports the hypothesis that pyruvate oxidation via the K r e b s cycle is stimulated by insulin. Dixit et al. (1967) found that the citrate content of skeletal muscle f r o m diabetic rats was greatly increased and citrate oxidation was greatly decreased f r o m normal. However, their data do not indicate the precise step in the K r e b s cycle which is controlled by insulin. T h i s p a p e r reports experiments in which an a t t e m p t was made to further delineate the point at which insulin stimulates pyruvate oxidation b y the use of pyruvate labeled in carbon atoms 1, 2 or 3. I t is well known that frog tissues show seasonal differences in their metabolism (Smith, 1950; F r o m m & Johnson, 1955; Mizell, 1965; H o n g et al., 1968). T h e respiratory response of the sartorius muscle of the frog to insulin also varies seasonally (Gourley & Suh, 1967). T h e experiments reported in this p a p e r were conducted with muscle f r o m b o t h s u m m e r and winter frogs and striking differences in the effect of insulin on pyruvate oxidation were observed. MATERIALSAND METHODS

The materials and methods used in this study are similar to those described previously (Gourley & Suh, 1967, 1969) and are summarized here only briefly with the addition of certain modifications of the previous methods. Intact sartorius muscles, which usually weighed in the range of 70-110 mg, were carefully dissected from healthy male and female specimens of Rana pipiens. The muscles were washed in Ringer's solution (buffered with phosphate at pH 7.4; described by Gourley, 1965) and stored overnight at 5°C to reduce their lactate content to a minimum. After equilibration at room temperature to restore the intraceUular concentrations of sodium and potassium to their original levels, each muscle was blotted, weighed and transferred to a respirometer flask. One muscle of each pair was placed in Ringer's solution while the other was placed in Ringer's solution containing insulin (50 mu/ml) for a further equilibration period of 1 hr. Weighing the muscles prior to the measurement of oxygen consumption is a modification of the earlier procedure. It resulted from the observation that during the incubation period there was a significant increase in the weight of muscles exposed to pyruvate plus insulin (about 4 per cent), which would introduce an error in all calculations of effects of insulin which were based on the wet weight of these muscles determined at the end of the incubation period (Gourley & Suh, 1966). The oxygen consumption of the muscles was measured by the Warburg manometric method at 24"0 _+0"02°C for 2 hr before the addition of pyruvate and for an additional 6 hr in the presence of pyruvate. At the end of the incubation period, each muscle was prepared for determination of total glycogen and C14-glycogen, and samples were prepared for the determination of the pyruvate remaining in the Ringer's solution and the Ct402 trapped in the NaOH of the center weU of the respirometer flask. As in the previous work, the values for C'~O2 reported in this paper have been corrected for the Cx~O~ not released from the muscle and the Ringer's solution. The appearance of C x4 in COs was taken to indicate the oxidation of pyruvate and the appearance of C x4 in glycogen was taken to indicate glycogenesis from pyruvate.

INSULIN ON PYI~UVAT1~METABOLISMIN MUSCLE

511

All of the experiments were done during two different seasons over a 2-year period. Muscles dissected from frogs in late May, June and July have been called " s u m m e r " muscles, while those dissected and used in October, November and December have been called "winter" muscles. T h e months in which each experiment was performed are given in Table 1. Experiments which have been given the number 1 in this paper (Tables 1 and 3) were performed in sequence; that is, pyruvate-l-(314 was studied first in eighteen pairs of muscles (expt. la), then pyruvate-2-C 1¢ was studied in eighteen pairs of muscles (expt. l b ) and so on. T h e frog-to-frog variation in the 02 consumption, glycogen concentration and pyruvate uptake of the muscles in the first series of experiments made comparisons of effects of the differently labeled pyruvates difficult and the experimental design was changed in the subsequent experiments, numbered 2 and 3. No further study of pyruvate-3-C 14 was undertaken since the preliminary results were not sufficiently different from those observed with pyruvate-2-C 14. Instead, with a given batch of frogs on a given day, half of the muscles received p y r u v a t e - l - C 14 and half received pyruvate-2-C 14. Thus, the data for expt. 2a were obtained at the same time with muscles from the same batch of frogs as the data for expt. 2b. T h e same procedure was followed in expts. 3a and 3b. This method greatly reduced the animal-to-animal variation in the results and permits comparisons of the data obtained with p y r u v a t e - l - C 14 and pyruvate-2-C 1. to be made with a considerable degree of confidence.

Chemicals Crystalline zinc insulin (beef) was supplied by the Lilly Laboratory for Clinical Research through the courtesy of Dr. W. R. Kirtley (lot No. 535,664; activity --- 25"6 u/mg; zinc = 0.49% ; glucagon = 0.4-0.5%). Fresh solutions were prepared on the day of use. Sodium pyruvate was supplied by Sigma Chemical Co. (Type II, dimer free, lot No. 35B2030). T h e concentration of pyruvate chosen for study was 2"5 m M in order to compare the results with those obtained earlier with 2.5 m M L-(+)-lactate. T h e actual concentration of pyruvate when first added was determined analytically and is given with the data for each experiment in Table 3. T h e C14-pyruvate, labeled in carbon atoms 1, 2 or 3, was supplied by New England Nuclear Corp. In each experiment, the labeled pyruvate was diluted with unlabeled pyruvate so that about 0.5 #c of C 1. was added to each respirometer flask. T h e specific activity of the added pyruvate was about 5 x 105 dis/min per #mole (range, 3.6-6.2 x 105 dis/min per #mole in individual experiments). T h e instability of pyruvate-C 14 in aqueous solution is well known (yon Korff, 1964; Silverstein & Boyer, 1964). Therefore, pyruvate-C 14 was used usually within a month of receipt from the supplier and was stored in the interval at - 1 5 ° C as the crystalline sodium salt. Aqueous solutions were freshly prepared on the day of use and the radiochemical purity was tested by paper chromatography using the following solvent systems: (1) 95% ethanol-sodium acetate ( 7 : 3 , v : v ) ; (2) n-butanol-waterpropionic acid (10 : 7 : 5, v : v : v). Radioactive peaks obtained with a Vanguard automatic chromatogram scanner indicated purities in the range of 97-99%.

Analytical methods Glycogen was isolated from the muscles b y a modification of the method described by Walaas & Walaas (1950) and reprecipitated twice to eliminate C 1' contamination. After hydrolysis of the glycogen, glucose was determined b y the Glucostat (Worthington Biochemical Corp.) procedure. All muscle glycogen values reported in this paper have been expressed as pyruvate equivalents. Pyruvate in the Ringer's solution was determined by the method of Friedemann & Haugen (1943). T h e pyruvate uptake b y the muscle was taken as the difference between the amount added at the end of the second hour and the amount remaining in the Ringer's solution at the end of the incubation period. N o account was taken of pyruvate produced by the muscle during the experiment.

512

D. R. H. GOURLEY,TAE KYU Sun AND L. L. BRUNTON

Radioactivity was counted at 0°C in a Packard liquid scintillation spectrometer. Pyruvate or glucose (from glycogen) samples (0.5 ml) were added to 10 ml of Bray's (1960) scintillation mixture for counting. The C x4 derived from the CO2 absorbed by NaOH in the center well of the flask was added as a 1.0-ml. aqueous sample to 10 ml of 3"5% CabO-Sil gel in Bray's solution. Throughout this paper, each value preceded by + is the S.E. of the mean. RESULTS

Glycogenesisfrom pyruvate The data in Table 1 indicate that during the 6 hr that the muscles were exposed to labeled pyruvate, significant quantities of C 14 appeared in the muscle glycogen. Under identical experimental conditions, Gourley & Suh (1967) demonstrated that when muscles were exposed to equal amounts of C 14, either as bicarbonate or as lactate-l-C 14, the C14-glycogen derived from the labeled bicarbonate was less than 1 per cent of that derived from the labeled lactate. Thus the values given in Table 1 for C 14 appearing in the glycogen are assumed to represent glycogenesis from pyruvate. Furthermore, in a given season, the number of moles of glycogen synthesized was roughly the same regardless of the location of the label in the pyruvate molecule. This suggests that pyruvate was incorporated into glycogen as an intact molecule. The glycogen content of the muscles was substantially higher in winter muscles than in summer muscles, confirming earlier observations of Hall et aL (1954) and Gourley & Suh (1967). This is shown in summary form in Table 2 in which the average values for summer muscles and winter muscles are given. Similarly, the C14-glycogen was less in summer than in winter muscles. In fact, the number of molecules of pyruvate synthesized to glycogen under the same experimental conditions but in different seasons was roughly proportional to the glycogen content, as shown by the relative constancy of the C14-glycogen expressed as a percentage of the total glycogen content (Table 1). The average ratios of C14-glycogen/total glycogen for summer and winter muscles are given in Table 2. In every experiment (Table 1), insulin significantly increased the incorporation of labeled pyruvate into glycogen by amounts ranging from 19 to 89 per cent. No seasonal trend in the effect of insulin on glycogenesis was apparent. In only two of the experiments was this effect of insulin reflected in a significant increase in the total glycogen content of the muscles. As has been suggested previously (Gourley & Suh, 1967), reliance on mean glycogen contents of paired frog muscles as an indication of net glycogen synthesis can often be misleading because the large S.E. of the means of the glycogen contents precludes consistent establishment of small changes in glycogen content with statistical confidence. The effect of insulin on glycogenesis is reflected in the ratios of CX4-glycogen/total glycogen content which were invariably higher in insulin-treated muscles.

Effect of pyruvate on 02 consumption The rates of 0 2 consumption of the muscles during the second hour and the average rates during the 6 hr after pyruvate was added, in the absence and presence

INSULIN O N PYRUVATE METABOLISM IN MUSCLE

~13

of insulin, are given for all experiments in Table 3. In agreement with our previous experience (Gourley & Suh, 1967, 1969), there was a wide range in the resting rate of O 2 consumption (second-hour rate without insulin) which apparently is not correlated with the glycogen content of the muscles and therefore with season (Gourley & Suh, 1967). The addition of pyruvate (in a final concentration of about 2.5 raM) to control muscles stimulated the O 2 consumption by an amount that varied from 0"38 to 0.82/~mole/g in different experiments. The upper limit of the absolute increases in 02 consumption produced by pyruvate is reasonably close to the corresponding increases produced by 2.5 mM L-( + )-lactate which were observed in two earlier experiments to be 0.85 and 0.89/~mole/g (Gourley & Suh, 1967, 1969). Effect of insulin and imulin + pymvate on 02 consumption Insulin consistently stimulated the 0 2 consumption during the second hour when no substrate was present in the Ringer's solution (Table 3). This effect, which is now well established in frog muscle (e.g. Gourley & Suh, 1967), indicates that insulin stimulates the oxidation of substrates which are endogenous to muscle tissue. The data confirm the earlier conclusion (Gourley & Suh, 1967) that insulin is a more effective stimulant of the oxidation of endogenous substrates in summer than in winter. In the presence of pyruvate, insulin also stimulated the 02 consumption (second last column, Table 3). In every experiment the absolute increase due to insulin after the addition of pyruvate was greater than the corresponding increase before pyruvate was added. Furthermore, the increase in O 2 consumption in the insulintreated muscles after pyruvate was added paralleled the increase in the 02 consumption in the same muscles without pyruvate in that it was greater in summer than in winter muscles. The absolute increase in 02 consumption produced by pyruvate in insulintreated muscles was also greater than that produced by pyruvate in control muscles (last column, Table 3). In the control muscles, the increase produced by pyruvate was generally less in summer than in winter muscles, but there was no consistent seasonal trend in the corresponding increases in insulin-treated muscles. The extra 02 consumption, i.e. the O 3 consumed by the control and insulintreated muscles in the presence of pyruvate over and above the assumed rate of 02 consumption in the same muscles without pyruvate, is recorded for each experiment in Table 1 and average values for each season are given in Table 2. In general, the extra 02 consumption was lower in summer than in winter muscles in both the control and insulin-treated muscles. Although the average extra 02 consumption values in Table 2 indicate a substantial increase produced by insulin in both seasons, the data in Table 1 for the individual experiments reveal that the differences were not statistically significant in two of the seven experiments. Oxidation of pyruvate to CO 2 The data in Table 1 for the pyruvate equivalents which appeared as C~402 indicate that in a given season more of carbon 1 than of carbons 2 and 3 appeared as

514

Kvu SuH A~D L. L. BRUNTON

D. R. H. GOURLEY, T ~

TABLE I--SYNTH~SIS TO GLYCOGEN~OXIDATIONAND UPTAKEOF PYRUVATELABELEDIN THE 17 2 OR 3

Pyruvate carbon labeled

Expt. No. and months

No. of muscle pairs

la C-1

May, June

47"13 4,6"31

6-5

+

5"77 4-0'62

51'21 4-6'66

11"3

(+89%)*

(+9%)t

0

5"33 4- 0-44

123'0 q- 8.2

4"5

+

8.21 4- 0"47 (+49%)* 2-49 =1=0.08

133"2 ± 7"9 (+8%)* 66.24 4-2.51

6'2

+

2'97 4- 0-20 (4.19%)§

64"60 4- 3"09

4"6

0

3.27 4-0.17

51.66 -4-2.48

6.3

+

4"47 •-t-0.25 (+37%)* 5.30 4-0.38

53.28 4-1.90

8'4

118.1 4-5'6

4.5

7-40 5:0"34 (+40%)* 2.36 =t=0.16

123 "6 + 6"3

6 '0

72.33 4.4'41

3.3

+

3'37 4,0"17 (4.43%)t

73'14 4-5"37

4'6

0

2"91 4- 0"20

40"0 4-1-9

7"3

+

3"83 4-0"23 (4-32%)*

41 "6 4-2'2

9"2

0

June, July

June

18

2b

0

October, November, December

18 +

3b

0

June, July

9

lc July

3-8

9

lb

C-3

3"05 4-0"39

Total glycogen (%)

18

3a

C-2

0

Insulin

Cl~-glycogen

18

2a October, November, December

CX4-glycogen (/zmoles/g)

Total glycogen content (/zmoles/g)

18

Insulin concentration, 50 mu/ml. All figures in parentheses represent percentage increases in the experimental

* P
't"P
,'I:P
§ P
C1402 (for pyruvate-3-C x4, data are available for summer muscles only). In contrast to the seasonal differences in extra 02 consumption, the C1402 produced from pyruvate-l-C 14 and pyruvate-2-C14 was greater in summer than in winter muscles. This is clearly shown by the average values in Table 2.

INSULIN

ON PYRUVATE METABOLISM

IN

515

MUSCLE

CARBON ATOMS IN FROG SARTORIUS MUSCLE DURING SUMMER AND WINTERj AND THE EFFECTS OF INSULIN

Extra Os consumption

C1401

C140,

Extra Ot (#moles pyruvate equivalent/g) consumption 1'48 4.0'15 2"53 4-0.29 (%71%)t 1"64 +0"19

3"91 -4-0"41 7"91 4-0"84 (+102%)* 1"50 4-0"21

Pyruvate uptake (#moles/g)

Pyruvate uptake accounted for (%)

CltOs

Cl4-glycogen

Pyruvate uptake (%)

Pyruvate uptake (%)

CX~-glycogen CI~0,

2"6

10"92 +0"63

64

36

28

0"8

3'1

17"32 4-0'70 (+59%)* 15"24 4-0'71

79

46

33

0"7

46

10

36

3"7

18"20 +0"77 (+19%)* 11.89 +0.49

54

9

45

4"9

56

35

21

0.6

0"9

2"00 4-0'20 (+22%)§ 0.79 +0.14

1"66 +0"17

0"8

4.17 4-0.54

5.3

1.29 -I-0.09 (+64%):[:

6.76 4-0"54 (+62%)*

5.2

15.52 4-0.47 (+31%)*

63

44

19

0.4

1"5

10"48 +0"31

45

14

31

2"2

14"45 +0.50 (+38%)* 14.79 +0.63

51

20

31

1"6

42

6

36

5.6

17.58 +0.52 (+19%)* 10-63 4-0.59

48

6

42

7.0

43

21

22

1.1

1"02

1'50

+0'10

4-0"18

1"10 +0.13

2"86 4-0.37 (+91%)* 0.94 +0.15

2"6

2.14 +0.18 (+37%)t 0.76 4-0.14

1.06 +0.16

0.5

2.20 +0.26

2.9

1.07 +0"21

3.96 4-0"54 ( + 80%)t

3.7

14.73 +0"62 ( + 39%)t

50

27

23

0.9

1.57 +0.18

0.6

1"48 +0-18

0"90 +0"13

0'6

10"46 +0"34

36

9

28

3"2

1"92 +0-71

2"20 +0"23

1"2

15"16 4-0"87

39

14

25

1"7

(+30%)§

(+144%)*

(+45%)*

values for paired muscles due to insulin which are statistically significant.

The most striking seasonal difference in the oxidation of labeled pyruvate to C140~ is seen in the effect of insulin (Table 1). In summer muscles, insulin always had a marked stimulating effect on pyruvate oxidation to Ct4Os, regardless of the position of the label. However, in winter muscles insulin had no significant effect on pyruvate oxidation.

516

D . R . H . GOURLmr,TAE KYu SUH AND L. L. BRUNTON

T A B L E 2 - - S U M M A R Y OF SOME OF THE DATA I N T A B L E

1

SHOWING MEAN VALUES FOR ALl-

SUMMER AND ALL WINTER MUSCLES, AND THE EFFECTS OF INSULIN

Summer

Winter

Type of data

Control

Insulin

Control

C14-glycogen (~moles/g) Total glycogen content Ozmoles/g) CX4-glycogen/(total glycogen) (%) Extra O3 consumption (/zmoles pyruvate equivalent/g) CI~O2 (/~moles pyruvate equivalent/g): From pyruvate-l-C 14 From pyruvate-2-C 14 CX402/(extra 02 consumption) (for both pyruvate-l-C 1~ and pyruvate-2-Cx~) Pyruvate uptake (#moles/g) Ct4-glycogen/Ci40 ~: For pyruvate-l-C 14 For pyruvate-2-C 14

2"91 (72) 52.0 (72) 5"9 (72) 1"19 (72)

4-31 (72) 53"7 (72) 8"4 (72) 1"68 (72)

5"42 (36) 7-81 (36) 121 (36) 128 (36) 4"5 (36) 6"1 (36) 1"61 (36) 2"07(36)

4.00 (27) 1.73 (27) >1

7"35 (27) 3"23 (27) >1

1"50 (18) 1"66(18) 0"94 (18) 1"06(18) <1 <1

10"8 (72)

15"5 (72)

15"0 (36) 17'9 (36)

0.7 (27) 1.8 (27)

0"6 (27) 1"4 (27)

3-7 (18) 5"6 (18)

Insulin

4"9 (18) 7"0 (18)

All figures in parentheses are the numbers of muscles represented by the adjacent mean value. The pronounced seasonal differences in oxidative metabolism are further emphasized by the ratio, C1402/(extra 02 consumption). If the extra 02 consumption following addition of labeled pyruvate were entirely accounted for by the Ct402, the ratio would be 1. As shown in Table 1, the ratio was generally much greater than 1 in summer muscles (expt. lc is an exception) and less than 1 in winter muscles. This is owing to the fact that in summer muscles the extra 02 consumption was lower while the C1~O2 released was higher than in winter muscles. It will also be seen in Table 1 that the ratio, C1402/(extra 02 consumption), was generally greater in insulin-treated muscles than in control muscles during the summer (expt. 3a is an exception), but there was essentially no difference in the ratios in the winter.

Uptake of pyruvate The uptake of pyruvate by control muscles was approximately the same in all summer experiments and considerably lower than the pyruvate uptake by control muscles in winter experiments (Table 1). Insulin always significantly increased pyruvate uptake by the muscles, but the effect of insulin in summer muscles was about double that in winter muscles (Tables 1 and 2). A considerable proportion of the pyruvate taken up by the muscles was accounted for as Cl~-glycogen and C1402 (Table 1). Since the amount of released C1402 depended upon the position of the label in pyruvate, the proportion of pyruvate uptake accounted for by oxidation and glycogenesis together likewise

517

I N S U L I N ON PYRUVATE METABOLISM I N MUSCLE

T A B L E 3 - - E F F E C T OF I N S U L I N ON THE CONSUMPTION OF OXYGEN BY FROG SARTORIUS MUSCLE I N THE PRESENCE OF SODIUM PYRUVATE

Oxygen consumption Pyruvate Experiment Months labeling Final 6 hr Increase and No. of expt. and cone. Second hr* (average)* over second hr muscle pairs performed (mi) Insulin (#moles/g) (#moles/g) (#moles/g) la

(18)

May, June

C-1 (2"56)

0 +

lb

(18)

June

C-2 (2.48)

0 +

lc

(18)

July

C-3 (2.50)

0 +

2a

(18)

C-1 (2-59)

0 +

2b

(18)

C-2 (2"59)

0 +

3a

(9)

October, November, December October, November, December June, July

C-1 (2.58)

0 +

3b

(9)

June, July

C-2 (2.58)

0 +

1.24+0.08 2"09 +0"13 ( + 0"85) 1"96 + 0"11 2.89 +0.16 (+0.93)t 1.53 +0.07 2.33_+0.07

1.98 +0.12 3"36+0"16 ( + 1"38) 2"47 + 0"08 3.50 +0.09 (+1.03) 2.27+0.10 3.29+0.11

(+0-80)

(+1.o2)

1"77_+0"10 2.47 +_0.12 ( + 0"70) 1"79_+0"16 2.28_+0"10 ( + 0"49) ++ 1.94_+0"12 2.68-+ 0.12 (+0.74)++ 2.00-+0.18 2"87 _+0.14 ( + 0.87)

2.59_+0.12 3.47 -+0.13 ( + 0"88) 2"46_+0"11 3.31 _+0.13 ( + 0"85) 2.33 +0.09 3.32-+ 0.11 (+0.99) 2.38_+0.16 3.40 _+0.09 ( + 1.02)

0"74 1"27 0"51 0.61 0.74 0.96 0"82 1.00 0"67 1'03 0"39 0.64 0"38 0.53

The insulin-treated muscles were continuously exposed to the hormone (50 mu/ml) beginning 1 hr prior to the experiment. * Figures in parentheses in this column represent the average absolute difference in the oxygen consumption of the paired muscle treated with insulin compared with the control muscle. All differences here and in the last column are statistically significant at P < 0.001 unless a different probability is noted. t P<0"05. + P<0"01. varied with the position of the label, being greatest for p y r u v a t e - l - C t4 and somewhat less for p y r u v a t e - 2 - C x4 and p y r u v a t e - 3 - C 14 respectively. F o r a given labeling, the a m o u n t of pyruvate uptake accounted for was generally slightly less in winter muscles than in s u m m e r muscles. I n every experiment, m o r e of the pyruvate u p take was accounted for in the insulin-treated muscles than in the corresponding control muscles. T h e proportion of the pyruvate accounted for as C1402 and as C14-glycogen separately has also been entered in T a b l e 1. W i t h p y r u v a t e - l - C t4' m o r e of the pyruvate taken up was oxidized to CO2 than was synthesized to glycogen in s u m m e r muscles. T h e reverse occurred in winter muscles in which the difference

518

D. R. H. GOURLEY,TAE KYu SUH ANDL. L. BRUNTON

was quite marked. The proportion of the pyruvate-2-C t4 uptake synthesized to glycogen and the seasonal reversal were essentially the same as was observed with pyruvate-l-C 14. The proportion of the pyruvate-2-C 14 uptake oxidized to COs was much less than was observed with pyruvate-l-C 14 in both seasons, but again the proportion oxidized to CO2 was considerably greater in summer than in winter muscles.

Relationship between pyruvate synthesized to glycogen and oxidized to C02 The ratio, Ct4-glycogen/CX40~, varied markedly with the season (Table 1). In winter muscles, the usual ratio of 4-5 moles of pyruvate-l-C 14 synthesized to glycogen for each mole oxidized was observed (summary, Table 2). However, in summer muscles, less than 1 mole of pyruvate-l-C a4 was synthesized for each mole oxidized. The corresponding ratios for pyruvate-2-C 14 and pyruvate-3-C 14 were larger because smaller amounts of C 14 from these substrates were recovered as C~40~. In winter muscles, insulin increased the ratio of pyruvate synthesized to pyruvate oxidized; in summer muscles, insulin consistently decreased the ratio. DISCUSSION

Glycogenesis from pyruvate The appearance of C t4 in the glycogen of frog skeletal muscle incubated with labeled pyruvate confirms the observations of others in the diaphragm muscle of the rat (Villee et al., 1952; Hiatt et al., 1958). The possibility that a significant proportion of the incorporation of C 14 in glycogen of frog muscle might result from exchange reactions with C140~ was eliminated previously by comparing the C 14 activity in the glycogen of muscles exposed to equivalent amounts of C 14 as bicarbonate and as lactate-l-C 14 (Gourley & Suh, 1967). The radioactivity which was incorporated into glycogen from labeled bicarbonate was less than 1 per cent of the incorporation from labeled lactate. The fact that in the present experiments the amount of C 14 incorporated into glycogen in a given season was essentially the same regardless of the position of the labeled carbon atom in the pyruvate molecule further supports the conclusion that pyruvate is incorporated into muscle glycogen as an intact molecule. Two features of the synthesis of glycogen from pyruvate in frog skeletal muscle differ from the corresponding process in rat diaphragm muscle. First, one-quarter to one-third of the pyruvate uptake was synthesized to glycogen in frog muscle compared to about 13 per cent in rat muscle (Villee et al., 1952). Secondly, in frog muscle, insulin always increased glycogenesis from pyruvate whereas, in rat muscle, insulin had no detectable effect on the synthesis of glycogen from pyruvate. However, it is likely that these differences are only quantitative rather than qualitative. As expected, the amount of pyruvate synthesized to glycogen in summer frog muscles was very similar to the amount which was previously found to be synthesized from L-(+)-lactate in the same season (Gourley & Suh, 1969).

I N S U L I N O N PYRUVATE METABOLISM I N MUSCLE

519

C1402 recovery from pyruvate labeled in different carbon atoms For reasons that are not clear, the amount of C1409. derived from pyruvate-l-C 14 in summer muscles was more than double the amount previously reported to be derived from L-(+)-lactate-l-C x4 in comparable experiments (Gourley & Suh, 1969). The ratios of the Cx409. derived from pyruvate labeled in the 1, 2 or 3 carbon atoms were 1 : 0.4 : 0.2 in the control summer muscles and 1 : 0"4 : 0.3 in the corresponding insulin-treated muscles (averages in Table 2 plus values for pyruvate3-C 14 in Table 1). Similar ratios for the C1~O9. derived from pyruvate-l-C 14, pyruvate-2-C 14 and pyruvate-3-C 14 have been observed in rat adipose tissue (White et aL, 1968). The first step in the complete oxidation of pyruvate is decarboxylation to COs (from carbon 1) and acetyl CoA (from carbons 2 and 3). This accounts for the greater yield of C1409. from pyruvate-l-C 14 than from pyruvate-2-C 1~ or pyruvate-3-C 1~. Carbon atoms 2 and 3 that enter the Krebs cycle as acetyl CoA are eventually converted to COs but some of the acetate-C 14, derived from carbons 2 and 3 of pyruvate, may be incorporated into fatty acids. Hence, delayed cycling in the Krebs cycle and disappearance into fatty acids account for the reduced recoveries of C1409. from pyruvate-2-C 14 and pyruvate-3-C 1~. Insulin increased C14Os production from L-(+)-lactate-l-C 14 by 68 per cent (Gourley & Suh, 1969). In summer frog muscles, insulin also stimulated the production of C14Os from pyruvate when the C 14 was in any one of the three carbon atoms. These results can be rationalized regardless of the step or steps in the complete oxidation of pyruvate to CO9. that are stimulated by insulin. The data could be explained by the hypothesis of Randle (1964) that insulin maintains the pyruvate dehydrogenase reaction indirectly by preventing the build-up of acetyl CoA from fatty acids. However, the data are also consistent with the observations of Dixit et aL (1967), who found a marked decrease in citrate oxidation in rat skeletal muscle when the animals were made diabetic with alloxan, thus pointing to control by insulin of some reaction or reactions in the Krebs cycle. Unfortunately, the present observations in frog muscle shed no light on the precise reaction or reactions in the Krebs cycle which are controlled by insulin. Relative oxidation of pyruvate and endogenous substrates The extra 02 consumption is an estimated value calculated on the assumption that the muscle will continue to oxidize endogenous substrates at the second-hour rate during the succeeding 6 hr of the experiment if an exogenous substrate such as pyruvate is not added (Gourley & Suh, 1967). When the extra 02 consumption and the C14Os derived from labeled pyruvate are expressed in the same units, a ratio of Cx*Og./(extra 09. consumption) of 1 means that all of the extra Oz consumption is accounted for as pyruvate oxidation. A ratio less than 1 means that only part of the extra 09. consumption is a result of pyruvate oxidation; the remainder must be a result of additional oxidation of endogenous substrates. A ratio more than 1 means that pyruvate is oxidized in preference to the endogenous substrates which would have been oxidized had no pyruvate been added.

520

D.R.H.

GOURLEY, TAE

K Y u SUH AND L. L. BRUNTON

Thus, in winter muscles, in which the ratio C140~/(extra Oz consumption) is less than 1, the addition of pyruvate stimulated the oxidation of endogenous substrates. Insulin appears to have increased slightly the proportion of endogenous substrates oxidized, although the effect is of dubious quantitative significance. On the other hand, in summer muscles, in which the ratio C140~/(extra O3 consumption) is more than 1, the addition of pyruvate spared the oxidation of endogenous substrates. Insulin, which stimulates the oxidation of endogenous substrates throughout the year but more during the summer than during the winter (Gourley & Suh, 1967), appears to have increased the oxidation of pyruvate relative to that of endogenous substrates in summer muscles. In fact, it was only in summer muscles that insulin stimulated pyruvate oxidation. The nature of the endogenous substrates which are oxidized by frog skeletal muscle is unknown, but there is general agreement that resting muscle derives a large proportion of its energy from the oxidation of fatty acids and acetoacetate.

Effect of season on pyruvate metabolism The differences in the metabolism of pyruvate in summer and winter muscles are summarized in Fig. 1. The values shown in Fig. 1 are the averages obtained

I suMME I C5) glycogen

[ W,.TE. 1 (5)*

(5) glycogen (8__)* 36%t45%

26°/0128%

(I I) pyru'vote (16)*

(15) pyruvate (18)~ ~

,0"/. ~_9*/.

3 8"/,1z__7"~ ~*/.1~ */.

lactate, (4) amino acids

COt (8)~

(2)

COe

o

54"/.~46_'/. (--2) lactate, amino acids

FIG. 1. A diagram comparing the fate of pyruvate-l-C TM in the sartorius muscle of summer and winter frogs. Pyruvate not accounted for is labeled "lactate, amino acids". In each pair of average values, the value on the left represents control muscles and the value on the right (underlined) represents the corresponding insulin-treated muscles. An asterisk denotes a significant difference between control and insulin-treated muscles. Figures in parentheses are the /zmoles/g of each substance expressed in pyruvate equivalents. Figures beside the arrows are the percentages of the utilized pyruvate which go in the directions indicated (the proportions are indicated roughly by the thickness of the arrows). Original data are given in Table 1, expt. la, 2a and 3a. with pyruvate-l-C 1~, but the results obtained with pyruvate-2-C 14 are qualitatively the same. When compared to winter muscles, the pyruvate uptake by summer muscles is considerably less and a smaller proportion of it is synthesized to glycogen

INSULIN ON PYRUVATE METABOLISM IN MUSCLE

521

than is oxidized or converted to other forms. Seasonal variations in the blood glucose level, the liver glycogen concentration and the relative weight of the fat body of Rana pipiens have been reported by Mizell (1965). Gourley & Sub (1967) reported corresponding variations in frog muscle glycogen concentration which have been confirmed in Rana temporaria by Hong et al. (1968). During the summer months corresponding to these experiments, the muscle and liver glycogen contents in a frog are at a low ebb, and the blood glucose level and the weight of the fat body are starting to increase. Thus, during the period that fatty acids are being synthesized to lipid, a large proportion of the energy required by the muscle can be provided by pyruvate oxidation which also spares the oxidation of endogenous substrates (fatty acids ?). Although the glycogen content of the summer muscles is low, the amount of pyruvate synthesized to glycogen is less than in winter muscles. This seems somewhat paradoxical, but it may be that the mechanism which accelerates glycogen synthesis seasonally does not become effective until late summer. In winter muscles, when the demand for energy was not as great, relatively little of the added pyruvate was oxidized and the largest proportions were converted to glycogen and other forms. During the winter months corresponding to these experiments, the glycogen content of both muscle and liver in the frog is at a peak, while the blood glucose level and fat body weight are falling. During this period of the year, possibly the oxidative mechanism of muscle is geared more toward fatty acid oxidation while synthesis of glycogen and protein from carbohydrate intermediates is favoured. The greater uptake of pyruvate by winter muscles supports the suggestion that synthetic processes are favoured during the early winter months. It is also clear from Fig. 1 that a major effect of insulin on pyruvate metabolism is to shift it away from pathways not studied in this investigation toward the synthesis of glycogen and, in summer only, toward oxidative pathways.

Relationship between pyruvate synthesized to glycogen and oxidized to COs From his studies of the aerobic utilization of lactate and pyruvate by frog muscle in the 1920's, Meyerhof (1941) concluded that for each mole of lactate or pyruvate oxidized, 2-4 moles were synthesized to glycogen. This estimate was based on the assumption that all of the lactate or pyruvate that disappeared and could not be accounted for as extra O3 consumption was synthesized to glycogen. Recent data from experiments in which Ct4-1abeled substrates were used, such as those presented in this paper, show that this assumption is incorrect. Nevertheless, the ratios of Ct4-glycogen/Cx4Oz obtained with lactate-l-C x4 (Gourley & Suh, 1967) and with pyruvate-l-C x4 (Table 2) during the winter months are in good agreement with Meyerhof's early conclusion. However, in summer muscles in which glycogenesis decreased and oxidation was greatly increased, less than 1 mole of pyruvate was synthesized to glycogen for every mole oxidized to COs. The seasonal variations in the insulin effects are also reflected in the ratio C14-glycogen/Ca4Oa. In winter muscles, the ratio was increased by insulin because insulin stimulated glycogenesis without affecting pyruvate oxidation; in summer

522

D . R . H . GOURLEY,TAE KYU SUH AND L. L. BRUNTON

muscles, the ratio was decreased slightly by insulin because insulin stimulated pyruvate oxidation relatively more than it stimulated glycogenesis. SUMMARY 1. When pyruvate-C la was added to intact sartorius muscles of the frog in vitro, significant quantities of C 14 appeared in the glycogen. The number of moles of glycogen synthesized was roughly the same regardless of which of the three carbon atoms of pyruvate was labeled, indicating that intact pyruvate molecules were incorporated into glycogen. 2. Insulin significantly increased the incorporation of labeled pyruvate into glycogen by amounts ranging from 19 to 89 per cent, but the total glycogen content of the muscles was increased significantly in only two of seven experiments. 3. The 02 consumption of normal muscles was significantly increased by the addition of pyruvate and further increased significantly when insulin was also present. 4. More of pyruvate carbon atom 1 appeared as C1402 than of carbon atoms 2 or 3. Insulin stimulated the oxidation of all three carbon atoms of pyruvate to C14Oz in summer but not in winter muscles. 5. Insulin increased the uptake of pyruvate by muscles and a considerable proportion of the pyruvate taken up was accounted for as C~4-glycogen and C1402. More of the pyruvate uptake was accounted for as these two products in muscles treated with insulin than in control muscles. 6. The uptake of pyruvate, synthesis to glycogen and oxidation to CO2 varied seasonally. When compared to winter muscles, the uptake of pyruvate by summer muscles was lower, the synthesis of pyruvate to glycogen was lower, the extra 02 consumption in the presence of pyruvate was lower, but oxidation of pyruvate to C~4Oz was higher. In winter muscles, 4-5 moles of pyruvate-l-C 1~ were synthesized to glycogen for each mole oxidized; in summer muscles, less than 1 mole of pyruvate-l-C 14 was synthesized for each mole oxidized. 7. The effects of insulin on pyruvate utilization by muscle also varied seasonally. When compared to winter muscles, insulin stimulation of the oxidation of endogenous substrates in summer muscles was greater, the stimulation of pyruvate uptake was greater, the stimulation of 02 consumption after the addition of pyruvate was greater and the oxidation of labeled pyruvate was significantly increased. In winter muscles, insulin had no effect on pyruvate oxidation. 8. It was concluded that in summer frog muscles, most of the pyruvate taken up is oxidized or converted to other uses not studied in this investigation; in winter frog muscles, the major pathways of pyruvate utilization are synthetic. In both seasons, insulin shifts pyruvate metabolism away from pathways not studied in this investigation. In summer muscles, the major shift is toward oxidation, while in winter muscles, the major shift is toward glycogen synthesis.

Acknowledgements--The competent technical assistance of Mrs. Chuen Chu Lung in some of the experiments is greatly appreciated. The authors are indebted to Dr. R. M.

INSULIN ON PYRUVATEMETABOLISMIN MUSCLE

523

MacLeod for the use of the scintillation spectrometer and to Dr. W. A. Volk for the use of the strip scanner and chart recorder used in this investigation. REFERENCES BRAY G. A. (1960) A simple efficient liquid scintillator for counting aqueous solutions in a liquid scintillation counter. Analyt. Biochem. 1, 279-285. DELRUE G. & DEKEYSER J. (1940) Action de l'insuline sur le m4tabolisme de l'acide pyruvique. C. r. Sdanc. Soc. Biol. 133, 709-710. DIXlT P. K., DEVILLIERS D. C., JR. & LAZAROWA. (1967) Citrate metabolism in d i a b e t e s - II. Tissue citrate content, citrate synthase and oxidation in alloxan-diabetic rat. Metabolism 16, 285-293, FRIEDEMANN T. E. & HAUGEN G. E. (1943) Pyruvic a c i d - - I I . T h e determination of keto acids in blood and urine. ~. biol. Chem. 147, 415-442. FROMM P. O. & JOHNSON R. E. (1955) T h e respiratory metabolism of frogs as related to season. 3t. cell. cutup. Physiol. 45, 343-359. FRY I. K. & BUTTEI~IELD W. J. H. (1962) Carbohydrate metabolism in diabetics. A possible intracellular block. Lancet 2, 66-68. GOURLEY D. R. H. (1965) Effect of insulin on potassium exchange in normal and ouabaintreated skeletal muscle. J . Pharmac. exp. Ther. 148, 339-347. GOURLEY n . R. H. & SoB m. K. (1966) An apparent increase in water content of frog sartorious muscle exposed to insulin and carbohydrate intermediates. Can. ~. Physiol. Pharmac. 44, 871-873. GOURLEY n . R. H. & SUH T. K. (1967) Insulin stimulation of lactate oxidation and incorporation into glycogen in frog skeletal muscle. 3t. Pharmac. exp. Ther. 157, 371-380. GOURLEY D. R. H. & SUH T. K. (1969) Effects of insulin on oxidation and glycogenesis from glucose and glucose plus lactate in frog skeletal muscle. Comp. Biochem. Physiol. 29, 137-148. HALL J. C., FISHER K. C. & STERN J. R. (1954) Stimulation of oxygen consumption by insulin in intact isolated frog muscle. A m . ~ . Physiol. 179, 29-35. HIATT H. H., GOLDSTEIN M., LAREAUJ. & HORECKER B. L. (1958) T h e pathway of hexose synthesis from pyruvate in muscle. )t. biol. Chem. 231, 303-307. HONC S. K., PARK C. S., PARK Y. S. & KIM J. K. (1968) Seasonal changes of antidiuretic hormone action on sodium transport across frog skin. Am. ft. Physiol. 215, 439-443. KLEIN D. (1942) T h e effects of administration of glucose and insulin on blood pyruvate and lactate in diabetes mellitus, ft. biol. Chem. 145, 35-43. YON Koam~ R. W. (1964) Pyruvate-C x*, purity and stability. Analyt. Biochem. 8, 171-178. MEYERHOF O. (1941) T h e significance of oxidations for muscle contraction. Biol. Syrup. 3, 239-258. MIZELL S. (1965) Seasonal changes in energy, reserves in the common frog, Rana pipiens. 3t. cell. comp. Physiol. 66, 251-258. RANDLE P. J. (1964) T h e interrelationships of hormones, fatty acid and glucose in the provision of energy. Post-grad. reed. ft. 40, 457-463. RICE L. & EVANS E. A., JR. (1943) T h e in vitro effect of insulin in pigeon breast muscle. Science 97, 470-471. SILVERSTEIN E. & BOXER P. D. (1964) Instability of pyruvate-C 1. in aqueous solution as detected by enzymic assay. Analyt. Biochem. 8, 4 7 0 4 7 6 . SMITH C. L. (1950) Seasonal changes in blood sugar, fat body, liver glycogen, and gonads in the common frog, Rana temporaria, ft. exp. Biol. 26, 412-429. VILLEE C. A. & HASTINGS A. B. (1949) T h e utilization in vitro of Cl*-labeled acetate and pyruvate by diaphragm muscle of rat. ft. blol. Chem. 181, 131-139. VILLEE C. A., WHITE V. K. & HASTINGS A. B. (1952) Metabolism of C14-1abeled glucose and pyruvate b y rat diaphragm muscle in vitro, ft. blol. Chem. 195, 287-297.

524

D. R. H. GOURLEY,T ~ KYu SUH AND L. L. BRUNTON

WALAKq O. & WALAKq E. (1950) Effect of epinephrine on rat diaphragm. ~t. biol. Chem. 187, 769-776. WHiam L. W., WILLIAMS H. R. & LANDAU B. R. (1968) Metabolism of pyruvate by rat adipose tissue in vitro. Archs Biochem. Biophys. 126, 552-557.

Key Word Index--Cl~09; frog; glycogen; glycogenesis from pyruvate; insulin; muscle; oxygen consumption; pyruvate oxidation; pyruvate uptake; seasonal differences.