Dichloroacetate: Effects on exercise endurance in untrained rats

Dichloroacetate:

Effects

S. H. Schneider,

on Exercise

P. M. Komanicky,

Endurance

M. N. Goodman,

in Untrained

Rats

and N. 8. Ruderman

The effect of dichloroacetate (DCA), an activator of pyruvate dehydrogenase, on the performance of fed, untrained rats was evaluated while swimming for different durations. DCA-treated rats were able to swim almost 40% longer than controls (354 + 18 versus 255 * 18 sec. p < 901). This was associated with lower levels of blood and muscle lactate at rest and after 210 and 240 set of swimming. At exhaustion, blood lactate was the same in the two groups even though the DCA rats had worked for an additional 99 set (18.9 f 1.2 versus 15.8 + 1.2 mM/L NS). Pretreatment with DCA did not alter the usual exercise-induced decreases in muscle ATP and creatine phosphate or liver glycogen. After 210 set of exercise, plasma FFA and blood glucose and acetoacetate were also the same in the two groups; however, @hydroxybutyrate was somewhat higher, and there was a small but significant sparing of muscle glycogen in the DCA group. The data indicate that DCA enhances the ability of rats to exercise at near maximal work loads. They are consistent with the notion that improved endurance is a consequence of a decreased rate of lactate accumulation; however. the possibility that it is secondary to some other action of DCA cannot be excluded.

T

HE METABOLIC BASIS for exhaustion during exercise has been the object of increasing interest in recent years. Two types of exhaustion have been identified. During submaximal endurance exercise such as long-distance running, exhaustion generally occurs after several hours and it is related to depletion of muscle glycogen.‘*2.334*536In contrast, exhaustion during exercise at near maximal workloads occurs rapidly and is independent of glycogen depletion. Efforts to identify a factor or group of factors which limit this type of exercise have centered around the accumulation of lactate and a close correlation between blood and tissue lactate and exhaustion during intense exercise has been demonstrated.7*8~9*‘o*” Dichloroacetate (DCA) has been shown to lower blood glucose in starved and diabetic rats, and because of this, it has been evaluated as a oral hypoglycemic agent.12 DCA appears to act, at least in part, by activating the pyruvate dehydrogenase complex (PDH) in muscle and other tissues,‘3,‘4 thereby enhancing the oxidation of pyruvate and secondarily diminishing the plasma levels of the principal gluconeogenic precursors, lactate, pyruvate and alaPresumably by the same mechanism, nine. ‘5*‘6~‘7*‘2 DCA has been shown to diminish the accumulation of lactate after epinephrine and biguanide administration, during intense exercise in animals,‘8~‘9~20~2’~22~23 and in clinical states associated with lactic acidosis in

From the Department of Medicine, College of Medicine and Dentistry of New Jersey-Rutgers Medical School, Piscaraway. New Jersey and Department of Medicine, Boston University School of Medicine, Boston, Massachusetts. Received for publication October 15. 1980. Supported in part by USPHS Grants AM 19514, AM 19469. and NIH Grant DM 00625. Address reprint requests to Stephen H. Schneider, M.D., Department of Medicine, CMDNJ-Rutgers Medical School, P.O. Box 101, Piscataway, New Jersey 08854. 8 1981 by Grune & Stratton, Inc. 0026~495/81/3006-0012$01.00/0

590

man.24 This report describes the effect of DCA on the ability of the rat to perform exercise at a near maximal workload. The results indicate that DCA both retards the accumulation of lactate and prolongs the period until exhaustion.

MATERIALS

AND

METHODS

Animals Untrained male Holtzman rats weighing 19&210 grams were used in all experiments. Animals were housed in climate controlled quarters with a 12 hr light-dark cycle (8-8) and were allowed free access to Purina Laboratory Chow and water until the time of the experiment.

Materials Sodium dichloroacetate, obtained from Eastman Kodak (Roehester, N.Y.), was redistilled and neutralized to pH 7.0 with NaOH on the morning of the experiment. Enzymes for metabolite analyses were obtained from Boehringer (Indianapolis, Indiana) or Sigma (St. Louis, Missouri).

Study

Design

All studies were initiated at 8:00 a.m. Rats were weighed and then randomly assigned to groups administered DCA or saline (control group). They received either 1 ml of 0.4 M Na DCA or I ml of 0.9% NaCl intraperitoneally 60 and 20 min before the initiation of exercise. Sixty minutes after the initial injection, a weight of 20 gm was attached to the rat’s trunk, posterior to its forepaws and around the chest wall, with a loose fitting rubber band. The rat was then placed in a large vat of tap water maintained at a temperature of 280p300 C and permitted to swim either to a predetermined time (210 or 240 set), or to the point of exhaustion. Exhaustion was taken as the time when the rat was unable to break the surface of the water for 5 set on three occassions. One of us (P.K.), who was unaware whether the rat had received DCA or saline, determined when exhaustion occurred in all studies. Two of the 60 rats used in the study drowned and were excluded from the calculations. To obtain samples, the rats were stunned and decapitated, and blood was collected from the severed vessels in the neck. The right gastrocnemius and the right lobe of the liver were then removed as rapidly as possible and frozen in clamps cooled in liquid nitrogen. The tissue was stored in liquid nitrogen until analysis for glycogen and metabolites was performed.

Metabolism. Vol. 30, No. 6 tJune),

198 1

DICHLOROACETATE

591

Analyses Blood was deproteinized ized with ZM KOH liver

were

in ice cold 10% w/v HCIO,

- OSM triethanolamine.

powdered

under

liquid

and neutral-

Frozen muscle and

nitrogen

in a stainless

steel

A weighed portion of the powder was homogenized in 10 volumes of ice cold 10% w/v HCIO, and neutralized with 2M KOH - OSM triethanolamine to pH 7.0. Metabolites and tissue glycogen were assayed by standard enzymatic procedures as described previously.25~*6~*7 percussion mortar.

SALINE

Statistics Significance

groups

of differences

was determined

using

between

control

an unpaired

treated students’ t

_1:, ,,,.,,,‘I,kj:

and DCA

two-tailed

f

test.

‘.‘..‘_ __*

,

RESULTS

EndurancfJ

30

Animals pretreated with DCA showed a substantial increase in their ability to swim at near maximal work levels (Fig. 1). The duration of swimming until exhaustion was 255 t 18 seconds (mean SEM) for the control rats and 354 k 18 seconds for the DCA group. The difference was significant at the p < 0.001 level.

120

180

240

300

360

420

480

540

600

660

710

Duration of Exercise (seconds)

Fig. 1. Effect of DCA on Endurance. Display oft percent of animals able to continue swimming at various time points. Experiments which were carried out for less than 240 set utilized 30 animals in each group, those carried out for greater than 240 set utilized 15 animals per group.

Lactate and Pyruvate Blood lactate in the exhausted controls (at 240 set) was 16.8 f 1.3 mM, a value significantly greater than that of the unexhausted DCA group (Fig. 2). At exhaustion, blood lactate was not different in the control and DCA groups, even though the latter had worked for an additional 100 set (Fig. 3). The mean blood lactate at exhaustion ranged from 14.8 - 23 mM for all groups. Changes in the concentration of lactate in muscle closely followed the changes in blood.

The concentrations of lactate in blood and skeletal muscle after swimming for various lengths of time are shown in Table 1. Blood lactate was significantly lower in the DCA group at rest and it increased to a lesser extent (8.4 versus 11.2 mM) after 210 set of exercise. At 240 sec. lactate was still lower in the DCA group; however, the difference between the groups was no longer significant. At this time, six control rats were exhausted versus only two rats in the DCA group.

Table 1. Effect of DCA on Blood and Muscle Lactate and Pyruvate

--

Duration of Metabolltes Blood

EXWCKX

lactate

(mM)

Muscle

1 .B + 0.2

15.4

*

1.2

13.2

f

0.9

10

15.8

+

1.2

16.9

+ 1.2

15

3.7

t

0.5

2.8

+ 0.4

15

210

13.1

*

1.6

9.6

t

1.2*

20

240

21.7

t

1.4

17.2

”

l.lt

10

wt)

-

15

0

0.12

+ 0.008

0.09

* 0.004t

15

210

0.21

+ 0.01

0.15

i- O.OOS$

20

240

0.17

* 0.01

0.15

2 0.01

10

Exhaustion

0.29

2 0.02

0.20

+ 0.02$

15

0.10

+ 0.01

0.09

ZL 0.01

0.058

pyruvate wet

20

240

(mI4l)

lumole/g

? 0.82

Exhaustion

pyruvate

Muscle

15

9.6

+ 0.8

Exhaustion Blood

2 0.2*

13.0

0

wet

(N)

1.2

210

lactate

(urn&/g

DCA

Control

0

0 wt)

210

0.064

? 0.01

240

0.13

* 0.02

*

0.11

0.01

15 20

l

* 0.01

10

Exhaustion Results were

are means

obtained

significantly

+ SEM.

by intracardiac different

from

Exhaustion puncture.

those

occurred All other

of the control

15

at a mean samples

group

time

were

of 255

obtained

at the p < 0.05,

set from

in controls neck

p .Z 0.025,

and 354

vessels

following

and p < 0.001

set

in the DCA

treated

decapitation. levels

respectively.

group.

The symbols*,

At time t.

0, all samples

$, indicate

values

SCHNEIDER

592

Blood

lactate levels in different groups animals after various swim times (mM/L)

ET AL.

Muscle Creatine Phosphate and ATP

of

The concentration of ATP in muscle was the same in the two groups prior to swimming, however, creatine-phosphate tended to be higher in the DCA treated rats (Table 3). After 210 set of swimming, there were no significant differences between the groups, although both ATP and creatine-phosphate tended to be slightly less depleted in the DCA treated rats.

181614-

Muscle and liver glycogen Treatment with DCA had no effect either on liver or skeletal muscle glycogen in the resting state (Table 3). After 210 set of swimming, liver glycogen was

tzIO-

Blood lactate levels at exhaustion in saline and DCA treated animals

8-

(mM/L)

6-

18 16 basal

210 sec. Duration

mexhaus,ed 0

240 sec.

14 -

of exercise a

;o,;;&t;;;;d

DCA treated, unexhausted

Fig. 2. Blood lactate levels after 0. 210, and 240 sac of exertion. Hatched bar represents saline treated rats which were unable to complete a 240 set swim. Results ere means + SEM. See text and legend to Table 1 for further details.

I2 IO 86-

In keeping with previously reported findings, blood pyruvate was diminished at rest in DCA treated rats. As shown in Table 1, it also remained lower during exercise and in contrast to lactate, it was lower than in the control group at exhaustion. Interesting@, muscle pyruvate did not differ in the two groups either at rest or after 240 set of exercise although pyruvate levels were somewhat lower at 210 set in the DCA treated animals. Blood Glucose Treatment with DCA did not alter the concentration of glucose in blood either at rest or when the rats were exhausted (Table 2). In no instance was exhaustion associated with hypoglycemia.

42-

‘

255 sec.

354 sec.

Mean time to exhaustion Fig. 3. Blood lactate levels of DCA-treated and control rats at exhaustion. No difference in lactate at exhaustion was found despite the fact that DCA-treated animals worked an average of 99 set longer.

DICHLOROACETATE

593

Table 2.

Effect of DCA on Blood Glucose. Ketone Bodies, and Plasma FFA Duration of Exercise

Metabolites

lS?C.)

GlWXse (mM)

DCA

Control

(N)

0

7.1 2 0.01

7.2 + 0.2

(15)

Exhaustion

7.2 + 0.06

8.0 f 0.7

(151

303 + 10

(10)

FFA

210

(mEq/L) Acetoacetate

210

0.13

k 0.01

0.13

+ 0.02

(20)

(mMI B-hydroxybutyrate

210

0.03

k 0.02

0.08

* 0.02$

(20)

293 t 6

(mM) All values expressed as means +- SEM. (See legend to Table 1 and Methods for details).

decreased to the same extent in the two groups. Muscle glycogen was slightly but significantly less diminished in the DCA group. Free Fatty Acids and Ketone Bodies Plasma free fatty acids (FFA) and acetoacetate were similar in control and DCA treated rats after 210 set of exercise; however, B-hydroxybutyrate was higher in the DCA group (Table 2). DISCUSSION

DCA activates pyruvate dehydrogenase and stimulates the oxidation of pyruvate and its precursors, lactate, glucose and alanine in tissues of normal and diabetic animals.‘4~‘5~23~28At the same time it may diminish the oxidation of FFA.‘2,29 By decreasing the supply of gluconeogenic precursors to the liver and possibly by other mechanisms, DCA also decreases blood glucose in fasting animals3’ and man, This has prompted its evaluation as a hypoglycemic agent;““’ however, because of neurotoxic effects with prolonged therapy,3’ its use for this purpose has been discontinued. The results of the present study indicate that pretreatment of untrained fed rats with DCA allows them to engage in strenuous swimming for almost 40% longer than control animals. The precise mechanism by which this occurs is uncertain; however, the assoTable 3. Effect of DCA on the Concentrations

ciation of a decreased rate of lactate accumulation with improved performance in the DCA group is of interest. The relationship between lactate metabolism and muscle fatigue has been studied by many investigators. With the onset of work at greater than 60% of an individual’s maximal aerobic capacity, lactate accumulates exponentially in muscle and blood.32333,34The lactate results from the breakdown of circulating glucose and glycogen in the exercising muscle and the subsequent generation of pyruvate and NADH.35 Pyruvate is reduced to lactate in the cytoplasm by NADH through the action of lactic acid dehydrogenase, or it may enter the mitochondria along with its associated reducing equivalents, to be oxidized to CO, and H20.36*37When the rate of production of pyruvate and NADH exceed the capacity of the mitochondria to oxidize them, lactate and hydrogen ion accumulate in the cytosol. This, in turn, results in a decrease in intracellular pH. It has been suggested by Nakamura et al.3a that this increase in hydrogen ion concentration might impair contractile force by interfering with the binding of Ca+ + to troponin in the myocyte. The possibility that the accumulation of lactate and its associated reducing equivalents might be a limiting factor in intense exercise was first raised by A.V. Hill (in 1929) when he noted that the accumulation of lactic acid impaired contractile force in the frog

of Creatine-Phosphate

and ATP in Muscle, and Glycogen in Muscle and Liver

Exercise Metabohtes

Duratton

Control

DCA

1N)

Muscle

ATP Lumole/g wet wt) Creatine-phosphate (urn&/g

wet wt)

Glycogen (urn&

0

5.8 2 0.3

5.9 i 0.3

(151

210

4.0 ? 0.2

4.4 f 0.3

120)

0

11.5 !I 0.3

13.5 + 0.6$

(15)

210

3.2 k 0.5

4.0 + 0.7

(20)

0 glucose/g wet wt)

210

(omole glucose/g wet wt)

210

32.3

2 2.3

34.2

+ 2.3

(15)

10.2 ? 0.9

14.1 + 1.2t

(20)

367

t 36

326 k 49

(15)

161 z 17

159 i 20

120)

Liver G&Ogefl

(See legend to Table 1 and Methods for details)

0

594

SCHNEIDER

Later studies by Tesch”,” and others diaphragm.39 confirmed a reproducible relationship between exhaustion at high workloads and levels of lactic acid in the range of 8-17 mM/L in blood and approximately 25 umole/g in fast-twitch glycolytic muscle. 7.8,32.40.41,42. Several findings of the present study are consistent with the hypothesis that lactate accumulation and exhaustion are closely linked. The most noteworthy is the association of improved performance of the DCAtreated rats with a decreased accumulation of lactate. Also, at the time of exhaustion, blood lactate was similar in the two groups even though the DCA treated rats had worked an average of 99 set longer. Finally, the rats unable to complete a 240 set swim tended to have a higher blood lactate concentration than their more successful cagemates. Precisely how DCA diminishes the accumulation of lactate during exercise remains to be determined. Pyruvate dehydrogenase is substantially activated during intense exercise43 and it is not known whether it can be furthered activated by DCA in this circumstance. Alternately, DCA could act by altering blood flow and/or the transport of pyruvate and other ions in and out of the mitochondria.44 It might also diminish lactate accumulation by altering the redox state of mitochondria so as to make them more oxidized. DCA does not appear to act by inhibiting glycolysis, although this has only been studied in resting muscle.‘5 The concentrations of lactate in muscle and blood of the rats at rest were somewhat higher than those reported in several other studies.‘5,37,45 This probably is due in part to the fact that the rats had had two injections and were not fasted prior to sampling. The method used to obtain muscle may also have contributed as similar lactate levels have been reported in other studies in which muscle was excised prior to 32,46 Parenthetically, the rats were freeze-clamping. sacrificed by a blow on the head but neither tetany nor convulsions were observed. DCA did not cause hypoglycemia. While it lowers blood glucose levels in fasting and diabetic rats, DCA is not a hypoglycemic agent in the fed state.” DCA

ET AL.

probably lowers blood glucose by impairing the delivery of gluconeogenic precursors” and possibly by a direct effect on hepatic gluconeogenesis.47,4*,4’ However, gluconeogenesis is not an important determinant of blood glucose maintenance in exercise of short duration and high intensity if adequate glycogen stores are available. Therefore, the lack of a glucoselowering effect of DCA under these circumstances is not surprising. On the other hand, DCA theoretically would be hypoglycemic during prolonged submaximal exercise or during exercise performed in the fasted state, since gluconeogenesis is the principal source of glucose in these situations. Pretreatment with DCA led to a small but significant sparing of muscle glycogen. The extent of glycogen sparing was not sufficient to account for the lesser accumulation of lactate nor the greater endurance of the DCA group. The reason it occurs is unclear. In conclusion, we have shown that pretreatment of fed rats with DCA increases endurance during near maximal exercise. Although the data are consistent with the notion that this is a consequence of a decreased rate of lactate accumulation, the possibility that this was due to some other action of the drug cannot be excluded. DCA has many metabolic effects other than the activation of pyruvate dehydrogenase. These include enhanced oxidation of glucose, decreased oxidation of FFA, inhibition of hepatic gluconeogenesis and VLDL synthesis, elevation of plasma FFA and ketone bodies and alterations in the redox state. Also, it may have direct hemodynamic effects. The relationship of these and other effects of DCA to enhanced endurance will require clarification. ‘2,‘6.48.50It will also be of interest to study the effects of DCA on exercise in the fasted state and with sub-maximal workloads when the metabolic events associated with exercise may be quite different. Whatever the basis for its effects, DCA-like agents with less toxicity may provide a novel means of enhancing athletic performance in man and animals. In addition, it may prove a useful tool for studying the metabolic correlates of the exhausted state.

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DICHLOROACETATE

595

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in in

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