pyruvate ratio in muscle of thermally acclimated goldfish

Comp. Biochem. Physiol. Vol. 74B, No. 4, pp. 775 to 780, 1983

0305-0491/83/040775-06503.00/0 © 1983 Pergamon Press Ltd

Printed in Great Britain

LACTATE D E H Y D R O G E N A S E ACTIVITY A N D L A C T A T E / P Y R U V A T E RATIO IN M U S C L E OF T H E R M A L L Y A C C L I M A T E D G O L D F I S H HIROSHI YAMAWAKI Department of Biology, Faculty of Science, Osaka City University, Sugimoto 3-chome, Sumiyoshi-ku, Osaka 558, Japan (Received 23 August 1982)

Abstract 1. Some glycolytic metabolites were determined for red muscle (RM) and white muscle (WM) of goldfish acclimated to 10 and 25°C. 2. Lactate/pyruvate (L/P) was higher in RM than in WM. 3. Lactate dehydrogenase (LDH) of RM was slightly more susceptible to inhibition by lactate than WM-LDH. At L/P found in vivo under resting conditions, RM-LDH was about three times as inhibited as WM LDH. 4. When 25°C-fish were forced to swim at 25°C, the increase of L/P in WM was mainly due to increased lactate level and that in RM was ascribed to decreased pyruvate level. These changes were obscure in 10°C-fish.

INTRODUCTION Some fish can adapt to environmental temperature in their swimming capacity (Beamish, 1978). Fry & H a r t (1948) described that goldfish show a m a x i m u m crusing speed at a temperature which depends on their thermal acclimation. This may be attributed to changes in the exercise system (Johnston, 1979; Penn e y & Goldspink, 1980), general integrated function such as nervous system and h o r m o n a l control and/or metabolic control during thermal acclimation. Although there are many data concerning metabolic changes induced by exercise in fish tissues (see Driedzic & Hochachka, 1978: Love, 1970), little information has been available on metabolic changes with thermal acclimation. In trout muscle, the E m b d e n Meyerhof glycolysis was strongly activated with cold adaptation, while the Kregs cycle activity was essentially unchanged (Hochachka, 1967). On the other hand, Dean (1969) found using lake trout that the muscle and liver of cold acclimated fish were capable of catabolizing acetate and palmitate at higher rate than those of warm acclimated fish. Another aspect of swimming capacity is related to the metabolic cooperation between the red and white muscles of fish (Wittenberger, 1972; Wittenberger & Diaciuc, 1965). It has been indicated that biochemical properties of fish red muscle may be like those of the liver. And as well known, the red muscle has higher aerobic activity than the white muscle (Love, 1970). O n the basis of the experiments with isolated carp muscle, Wittenberger et al. (1975) suggested that lactic acid may be transferred from the white muscle to the red muscle and that lactate contained in the white muscle may be formed from glucose coming from the red muscle. W h a t effects on two different muscles of fish does thermal acclimation have? Goldfish can swim for a long period without oxygen debt (Smit et al., 1971). W h e n this species was subjected to strenuous swimming (3.64 body length/ 775

sec), the fish showed more than 2-fold increase in oxygen c o n s u m p t i o n and the c o n c o m i t a n t reduction of the respiratory quotient from 1.10 to 0.86 (Kutty, 1968). These, at least in whole-body level, indicate that during exercise the anaerobic carbohydrate metabolism may not be essential for this species even though the glycolysis may be also activated. In the present experiments, changes in levels of glycolytic fuels and their anaerobic product induced by forced exercise were examined for evaluation of the contribution of glycolysis to muscle metabolism of thermally acclimated goldfish.

MATERIALS AND METHODS

Experimental animals

Goldfish, Carassius auratus, obtained from a commercial hatchery were divided into two groups and kept at 10 and 25°C for more than 4 weeks. The fish were fed once a day. Aquaria were continuously aerated and illuminated. Again, both 10- and 25°C-acclimated fish were divided into three; group I kept at their acclimated temperature allowing them to swim spontaneously, groups II and III forced to exercise for 10 min at 10 and 25°C respectively. Table 1 gives the body size of the fish. Forced exercise system

A pyrex glass tube (10 x 40cm, ca. 1900ml of volume) was used as a swimming chamber, which was equipped with a grid made of copper wire, connected to a pump and immersed in a constant temperature water bath (Fig. 1). Water was sucked from one side of the chamber by the pump and returned into the bath. The fish was kept in the chamber at its acclimated temperature for 10-14 hr before exercise. As for group I, another glass tube (10 x 30cm) was used as a chamber without a grid. In the case that a fish was forced to exercise at the temperature different from its acclimated temperature, the temperature of the water bath was quickly altered by additton of hot or ice-water into the bath. Water current was maintained at ca. 3.8 cm/sec by adjusting output of pump. The grid was

776

HIROSHI YAMAWAKI Table 1. Body size of experimental goldfish body length

(mm)

body weight

(g)

Group mean

(range)

mean

(range)

lO°-acclimation I. rest at 10°C

74.0 (68.0-85.0)

20.6

(17.3-30.3)

IL exercise at 10°C

73.6

(60.0-93.1)

20.2

(15.3-26.3)

6

III. exercise at 25°C

74.5

(67.0-81.5

19.0

(14.6-26.2)

6

I. rest at 25°C

75.3 (67.0-81.7

18.6

(15.8-20.6)

7

Z. exercise at 10°C

74.6

(70.5-80.7

18.3

(15.8-20.6)

6

Ill. exercise at 25°C

75.0 (67.5-84.5

20.5

(18.7-27.0)

6

7

25°-acclimation

carrying a 2.6 V (a.c.) charge to prevent the fish from resting during the forced exercise. During most experiments, however, the electric stimulation seemed to be unnecessary. Determination of metabolites Immediately after removal of the fish from the chamber, it was sacrificed by cutting its spinal cord. The skin beneath the first dorsal fin to tail was removed. After dissection from the dorsal muscle, the red and white muscles (0.2 1 g) were separated and immersed in liquid nitrogen. These procedures were carried out within 2 3 min. The frozen tissue was weighed and homogenized with 1 ml of cold 10~o trichloroacetic acid (TCA) by use of a glass mortar and pestle on ice. The homogenate was adjusted to final 10-20-fold volume of the tissue by addition of TCA and stood on ice for at least 20 min. Then, it was centrifuged at 10,000 O for 10 min at 5°C. The supernatant was treated with three ether-extractions. This protein-free solution was used as a source for determination of glucose, pyruvic acid and lactic acid. Pyruvate and lactate were determined with bovine heart LDH (sigma) by the methods of Czok & Lamprecht (1974) and Gutmann & Wahlefeld (1974) respectively. After determination of pyruvate and lactate, the solution was stored at - 20°C until the determination of glucose. Glucose was determined by use of glucose oxidase (Miles Laboratories) and peroxidase-like activity of Cu ion-histamine system (Asato et al., 1967). For determination of glycogen, about 100 mg of another frozen tissue was used. After the digestion of the tissue weighed in boiled 30~o KOH and precipitation of the glycogen with ethanol, the glycogen content was determined with the phenol sulfuric acid method of DuBois et al. (1956) using glucose as standard. Determination of LDH activity The muscle LDH activity was assayed by the method described in the previous paper (Tsukuda & Yamawaki, 1980). The red and white muscles were homogenized with 30mM phosphate buffer (pH 7.0) and centrifuged at 10,000 O for 20 rain at 5°C. The supernatant was dialyzed against 5 mM phosphate (pH 7.0) overnight and used as an enzymatic source. The activity was determined in the presence of 0.12 mM NADH, 0.1 mM sodium pyruvate and various concentration of L-lactate at pH 6.7, 10 and 25°C. L-lactic acid was neutralized with 0.1 N NaOH before use.

forced to swim at 10°C. Table 2 gives the results of determination of glycogen, glucose, pyruvate and lactate. The paired t-test was carried out for comparison of the metabolite levels between the red and white muscles of the same fish. For comparison between two different acclimation groups and a m o n g groups I, II and III, the M a n n - W h i t n e y U-test was carried out. U n d e r conditions of spontaneous activity (group I), all metabolites examined showed different levels between the red and white muscles in both 10- and 25°C-fish; the contents of glycogen, glucose and lactate were significantly higher in the red muscle, while the pyruvate content was higher in the white muscle. Consequently, the molar ratio of pyruvate and lactate (L/P) in the red muscle was significantly higher than that in the white muscle. The pyruvate contents of both the red and white muscles were likely to decrease with cold acclimation, although it is not statistically significant. During 10-min exercise, b o t h the red and white muscles tended to raise their glucose content and to decrease their pyruvate content, irrespective of acclimation- and experimental temperature. Both decreased pyruvate and increased lactate levels caused some increase of the L / P ratio with exercise. On the basis of the mean values given in Table 2, Fig. 2 shows relative differences in pyruvate and lactate levels between the muscles of fish allowed to rest and those of fish forced to swim at the acclimation temperature. The increased lactate level was responsible for the increase in L / P ratio of white muscle of 25°C-fish, while the change in the red muscle L/P ratio depends mainly on the pyruvate level. In

wb RESULTS

In visual inspection, all fish could exercise for imposed 10 min, although 25°C-acclimated fish appeared to be somewhat fatigued when they were

Fig. 1. Experimental swimming chamber. Arrows indicate the direction of water flow. (p) is connected to a pump. (g) a grid stimulating a fish, (wb) a temperature constant water bath.

RM

I0

WM

RM

i0

I0

RM

WM

RM

25

10

i0

at

at

25°C)

10°C)

7.6

8.2

9.1

139.3±31.6

37.3±

114.1±21.2

50.8±15.5

87.5±29.2

54.2±14.5

86.8±16.6

40.0±

158.0±26.5

81.3±21.5

126.1±29.5

49.7±

tissue

1.94±0.41 §

0.48±0.10

3.17±0.47 T

0.53±0.09

1.95±0.51

0.60±0.13

1.94±0.54

0.58±0.27

1.03±0.39

0.55±0.06

1.73±0.50

0.36±0.09

glucose

wet

0.069±0.011

0.082±0.016 %

0.074±0.025

0.195±0.062

0.057±0.007

8.19±0.91

5.84±1.61

9.45±0.92

7.23±1.51

10.68±2.07

9.41±1.22

0.091±0.022

t§

11.10±2.03

8.96±2.27

7.87±1.08

5.57±1.24

i0.20±0.94

0.085±0.017

0.185±0.033

0.084±0.012

0.169i0.032

0.094±0.018

0.213±0.048

lactate

5.34±0.77

(mean±S.E.)

pyruvate

weight

*,** Significant difference at the 5 and 1~o level between W M and RM (t-test). § Significant difference at the 5~o level with respect to 25°C-fish of the same group (U-test). t , t t Significant difference at the 5 and 1~o level with respect to group I (U-test). The glycogen content is given as glucose equivalents.

WM

25

]]I(exercise

RM

25

GrouD

WM

25

Z (exercise

WM

i0

Group

RM

25

~mol/g glycogen

at a c c l i . t e m p . )

WM

I (rest

Tissue

25

Group

Acclimation Temp. (°C)

t

8.0

141.8±31.1

98.0±44.6

210.9±75.2

77.6±37.9

202.3±43.8

165.1±62.5

167.0±46.3

51.5±12.1

120.2±32.6

40.6±11.2

120.3±15.2

32.7±

L/P r a t i o

~ §

6

6

6

6

6

6

6

6

7

7

7

7

Table 2. The white muscle (WM) and red muscle (RM) contents of glycogen, glucose, pyruvate and lactate in warm- and cold-acclimated goldfish

m-

o

O

778

HIROSHI YAMAWAKI 25° fish WM

10° fish

RM

WM

P

/

I.E

I.C

RM

/

6

0.5

~

I

i

I

I

III I

~I

I

I I II I

I II

Experimental group

Fig. 2. Relative level of pyruvate (0) and lactate (O) in white muscle (WM) and red muscle (RM) from goldfish allowed to rest (I) and those forced to exercise at an acclimation temperature (25°C-acclimated fish corresponds to group IlI and 10°C-fish to group II).

10°C-fish, such a relation was not remarkable as compared with the case of 25°C-fish. However, contribution of the change in pyruvate level to the change in the ratio was larger in the red muscle than in the white muscle. Pyruvate reduction by muscle L D H was examined in the presence of various concentration of lactate (Fig. 3). The red muscle L D H was slightly more susceptible to the product inhibition by lactate than the white muscle LDH.

DISCUSSION

During exercise, both the decrease of glycogen and accumulation of lactate were not statistically signifi-

~ 25 °

100

X~

acclimation

cant. Unfortunately, the present experiments can not display the difference in swimming capacity among individual fish, though it must be closely related to the change in metabolite levels. Furthermore, a question to arise is whether more extended periods of exercise may enhance the decrease of glycogen and/or the increase of lactate level. When Wittenberger & Diaciuc (1965) stimulated carp until the fish was exhausted, the change in glycogen content was not significant in both the red and white muscles. Concomitantly, the lactate level increased from 18.3 to 32.8#mol/g in the white muscle and from 9.0 to 27.3 #mol/g in the red muscle. According to Driedzic & Hochachka (1975), however, such an increase in the carp white muscle was not observed under hypoxic conditions. Similarly, when goldfish were exposed to hypoxia or anoxia, the decrease of glycogen and increase of lactate were not significant in the white muscle. The red muscle contents were also not affected remarkably by 1 hr-hypoxia (Thillart et al., 1980). The accumulation of lactate in the muscle may not be enhanced when goldfish is forced to exercise for more extended period. Because of relatively low pyruvate and high lactate contents, the L/P ratio in the red muscle was significantly higher than that in the red muscle (Table 2). This is important if goldfish LDH is mainly regulated by the L/P ratio. The red and white muscles of goldfish contain a different composition of LDH isozymes (Tsukuda & Yamawaki, 1980). As Fig. 3 shows, the red muscle L D H was more sensitive to lactate inhibition than the white muscle LDH. The red muscle L D H was about 30~o-inhibited at 120-125 of the L/P ratio, the value found in the tissue of the resting state. The white muscle L D H was only about 100,;,- nhibited at 30-40 of the ratio found in the tissue under the same conditions. As for the conversion of pyruvate to lactate, therefore, the degree of inhibition in the red muscle may be approx three times as high as that in the white muscle.

~

10 ° acclimation

t 25 °

25 °

50 ®

I

I

I

I

I

I

1

~. mo

u

~ 51?

® I1.

I

I

i~o ~oo 300 ~o

5~o o

,~o

~oo 300 ~o

~o

L o c t o t e / p y r u v a t e ratio

Fig. 3. Pyruvate reduction by L D H of goldfish red muscle (0) and white muscle (O) in the presence of

various concentration of lactate. The reaction mixture contained 91 mM phosphate (pH6.7), 0.12 mM NADH, 0.1 mM pyruvate and 0-50 mM lactate. The activity was assayed at 10 and 25°C. Each value indicates the mean and SD. (n = 3).

779

Lactate/pyruvate ratio in goldfish muscle With exercise, changes in b o t h pyruvate and lactate levels cause some increase of the L / P ratio. In the view of the L D H kinetics (Fig. 3), the red muscle enzyme may come to be a b o u t 40%-inhibited as the ratio increases in the tissue. In the red muscle, the increase of the ratio during excercise at the acclimation temperature is due to decreased pyruvate level rather than increased lactate level (Fig. 2). If the trend of decrease of pyruvate level is not attributed to decrease of the rate of glycolysis, alternatively it suggests that the conversion of pyruvate to any other metabolites besides lactate is activated under the conditions of exercise. Similarly, some decrease of pyruvate content seems to occur in both the red and white muscles with cold acclimation. Since 10°C-fish showed lower pyruvate level than 25°C-fish when being forced to exercise at 25°C, the decrease of pyruvate may be a result of cold acclimation but not due to a temporary effect of cold environmental temperature. Freed (1971) determined glycolytic intermediates of trunk muscle of goldfish and described that glucose-6-phosphate, fructose-6phosphate, triose phosphate and pyruvate levels decreased with cold acclimation. This may indicate that the lower pyruvate level in the cold acclimated fish reflects a reduction of the glycolytic activity. According to his results, the pyruvate contents of 5- and 25°C-acclimated fish were 0.046 and 0.298 #mol/g respectively. Is the decrease of pyruvate level with excercise also due to the reduction of glycolytic activity? To know why pyruvate decrease under those conditions, we should examine two i m p o r t a n t problems, that is (1) whether or not high aerobic activity of the tissue is observed under conditions of exercise and/or cold adaptation, and (2) whether or not other fuels such as amino acids and fatty acids (Driedzic & Hochachka, 1978) are being used rather than glycogen and glucose. If pyruvate is being aerobically catabolized in the red muscle during exercise, lactate produced in the white muscle may also be oxidized to pyruvate in the red muscle, as suggested by Wittenberger et al. (1975). As for LDH, the red muscle enzyme seems to be relatively easy to convert lactate to pyruvate as compared with the white muscle enzyme at nearly neutral pHs (Tsukuda & Yamawaki, 1980). The red muscle of goldfish was characterized by high lactate and low pyruvate contents as compared with the white muscle in the present experiments. Higher lactate content was also found in the red muscle of rainbow trout ( W a k o m a & Johnston, 1981). On the contrary, lactic acid contained in carp white muscle was twice as high as that in the red muscle (Wittenberger & Diaciuc, 1965). In goldfish investigated by Thillart et al. (1980), lactate level was slightly higher in the white muscle; 9.15 in the white muscle and 8.70 #mol/g in the red muscle. The discrepancy between their values and the present results may be attributed not only to difference in the procedure employed but to the size of the fish used. They examined the fish more than 5 times as heavy as those of the present experiments. Another characteristic of the red muscle is higher content of glycolytic fuels. Higher glucose content in the red muscle may be partly due to c o n t a m i n a t i o n of

blood because of a b u n d a n c e of vascular system in the tissue. Acknowledgements--The author wishes to thank Dr. H. Tsukuda of Osaka City University for her encouragement during the present work and Dr. K. Tsugawa of Osaka Women's University for his comments on the manuscript.

REFERENCES

ASATO R., KOMANO T., OKUYAMA T. & SATAKE K. (1967) Do ion-histamine kei no peroxidase sayo no kyoeki niyoru glucose oyobi glucose-oxidase no teiryoho. Nihon Kagaku Zasshi 88, 560-565. BEAMISH F. W. H. (1978) Swimming capacity. In Fish Physiology (Edited by HOAR W. S. & RANDALL D. J.), Vol. 7, pp. 101-187. Academic Press, New York. CzoK R. & LAMPRECHT W. (1974) Pyruvate, phosphoenolpyruvate and D-glycerate-2-phosphate. In Methods of Enzymatic Analysis (Edited by BERGMEVER H. U.), pp. 1446-1451. Academic Press, New York. DEAN J. M. (1969) The metabolism of tissues of thermally acclimated trout (Salmo gairdneri). Comp, Biochem. Physiol. 29, 185 196. DRIEDZ1C W. R. & HOCHACHKA P. W. (1975) The unanswered question of high anaerobic capabilities of carp white muscle. Can. J. Zool. 53, 706-712. DmEDZtC W. R. & HOCHACHKA P. W. (1978) Metabolism in fish during exercise. In Fish Physioloyy (Edited by HOAR W. S. & RANDALL D. J.), Vol. 7. pp. 503 543. Academic Press, New York. DuBols M., GILLES K. A., HAMmTON J. K., REBERS P. A. & SMITH F. (1956) Colorimetric method for the determination of sugars and related substances. Analyt. Chem. 28, 350-356. FREED J. M. (1971) Properties of muscle phosphofructokinase of cold- and warm-acclimated Carassius auratus. Comp. Biochem. Physiol. 39B, 747-764. FRY F. E. J. & HART J. S. (1948) Cruising speed of goldfish in relation to water temperature. J. Fish. Res. Bd Can. 7, 169-175. GUTMANN I. & WAHLEFELD A. W. (1974) L-(+)-lactate determination with lactate dehydrogenase and NAD. In Methods of Enzymatic Analysis (Edited by BERGMEYERH. U.), pp. 1464-1468. Academic Press, New York. HOCHACHKA P. W. (1967) Organization of metabolism during temperature conpensation. In Molecular Mechanisms of Temperature Adaptation (Edited by PROSSERC. L.), pp. 177-203. Horn-Sharer, Baltimore. JOHNSTON I. A. (1979) Calcium regulatory protein and temperature acclimation of actomyosin ATPase from a eurythermal teleost (Carassius auratus L.) J. comp. Physiol. 129, 163-167. KurTV M. N. (1968) Respiratory quotients in goldfish and rainbow trout. J. Fish. Res. Bd Can. 25, 1689-1728. LOVE R. M. (1970) The Chemical Biology of Fishes, pp. 17-35. Academic Press, New York. PENNEY R. K. & GOLDSPINK G. (1980) Temperature adaptation of sarcoplasmic reticulum of fish muscle. J. therm. Biol. 5, 63-68. SMIT H., AMELINK-KOUSTAALJ. M., VIJVERBERGJ. and VON VAUPEL-KLE1N (1971) Oxygen consumption and efficiently of swimming goldfish. Comp. Biochem. Physiol. 39A, 1 28. THILLART G. VAN DEN, KESBEKE F. 8~ WAARDE A. VAN (1980) Anaerobic energy-metabolism of goldfish, Carassius auratus (L.). Influence of bypoxia and anoxia on phospholylated compounds and glycogen. J. comp. Physiol. 136B, 45-52. TSUKUDA H. & YAMAWAKI H. (1980) Lactate dehydrogenase of goldfish red and white muscle in relation to

780

H1ROSHI YAMAWAKI

thermal acclimation. Comp. Biochem. Physiol. 67B, 289-295. WITTENaERGER C. (1972) The glycogen turnover rate in mackerel muscles. Mar. Biol. 16, 279-280. WITTENBERGER C. & DIACIUC I. V. (1965) Effort metabolism of lateral muscles in carp. J. Fish. Res. Bd Can. 22, 1397-1406.

WITTENBERGER C., COPREAN D. & MORAR L. (1975) Studies on the carbohydrate metabolism of the lateral muscles in carp. (Influence of phloridzin, insulin and adrenaline). J. comp. Physiol. 101, 161 172. WOKOMA A. & JOHNSTON I. A. (1981) Lactate production at high sustainable cruising speeds in rainbow trout (Salmo gairdneri Richardson). J. exp. Biol. 90, 361-364.