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The effect of exercise on the rate of oxygen uptake by rat-liver mitochondria
165
SHORT COMMUNICATIONS
sc 2369
The effect of exercise on the rate of oxygen uptake by rat-liver mitochondria Recent experiments by GLICK AND COHEN1 have demonstrated that the rate of succinate oxidation b y rat-liver mitochondria prepared at night was 41% higher t h a n the rate of succinate oxidation b y mitochondria prepared during the day. Since rats normally exercise at night and rest during the day, the experiments reported in this communication were designed to determine whether physical activity could control oxidative capacity in a similar manner. The results show that although exercise increased the rate of succinate oxidation, the most pronounced effects were obtained in the presence of those citric acid-cycle substrates which were oxidized most slowly. This phenomenon appears to be due to changes in dehydrogenase activity, and suggests that the oxidative capacity of the citric acid cycle is increased in physically active animals. Male Wistar rats were kept in pairs and were permitted food and water ad libitum for at least a week prior to sacrifice. On the day in which they were used they weighed between 18o and 25 ° g. In each pair one rat served as a control and remained in his cage without food or water for 3 h. The other animal was transferred to an activity wheel which was rotated at a speed of about 6 rev./min by an electric motor. At the end of the 3-h period both rats were sacrificed, and liver mitochondria were prepared by the method of KIELLEY AND KIELLEY2. 0 2 uptake was measured by the Clark oxygen electrode 3. P / 0 ratios were obtained by the technique of CHANCE AND W1LLIAMS 4.
The results in Table I show that the rate of 02 uptake in mitochondria from active animals was significantly increased over that in mitochondria from resting animals with each substrate tested. The .greatest percentage of stimulation was observed with malate or pyruvate which were oxidized most slowly, a-Ketoglutarate and fl-hydroxybutyrate which show relatively fast rates of oxidation were less TABLE I O2-UPTAKE RATES IN MITOCHONDRIA FROM EXERCISED RATS E a c h incubation vessel contained: i # m o l e of ADP, io #moles of MgCI~, 2o #moles of p h o s p h a t e (pH 7.o), 2 o o # m o l e s of sucrose. A m o u n t of succinate, 5/~moles; a-ketoglutarate, I o # m o l e s ; fl-hydroxybutyrate, 2 o # m o l e s ; citrate, I o # m o l e s ; malate, i o # m o l e s ; pyruvate, Io/*moles; m a l a t e - p y r u v a t e m i x t u r e : i o # m o l e s each of malate and p y r u v a t e ; s u b s t r a t e pool: 3 #moles each of all t h e above substrates except succinate. Total volume, 2.o ml; t e m p e r a t u r e , 3o°; d r y weight of mitochondria per incubation, 2.73-4.13 mg. The values reported are means 4- S.E. of the mean. The n u m b e r of animals is indicated b y the n u m b e r in parentheses. Oxygen uptake rate (l~atomlminper rag dry wt.)
Substrate
Control
Succinate 0¢-Ketoglutarate ~-Hydroxybutyrate Citrate Malate Pyruvate Malate--pyruvate Substrate pool Endogenous substrate
0.248 o.o78 o.o9 o 0.043 o.o21 0.022 o.o73 o.iio o.o13
4- O.Ol 3 (6) 4- o.oo 5 (6) 4- o.oo 5 (6) 4- o.oo2 (6) 4- 0.003 (6) 4- 0.002 (6) 4- o.o12 (4) 4- o.oo 3 (6) 4- o.oo2 (4)
Exercised
0.305 o.o96 O.lO9 o.o59 0.o35 0.039 o.o98 o.122 °.°15
4- o.o18 (6) 4- 0.007 (6) 4- 0.o07 (6) 4- o.oo 5 (6) + o.0o 4 (6) 4- 0.004 (6) 4- o.o12 (4) 4- 0.006 (6) 4- o.oo4 (4)
% Control
123 123 121 137 167 177 134 iii 115
(P (P (P (P (P (P (P (P (P
< 0.05) • o.o5) ,< o.oi) < o.oi) < o.ooi) < 0.02) < o.oi) < 0.05) < 0.4)
Biochim. Biophys. Me/a, 82 (1964) 165-167
166
SHORT COMMUNICATIONS
strikingly affected. Citrate, which has an intermediate rate of oxidation in control mitochondria, was oxidized considerably more rapidly in the experimental mitochondria. The rate of O2 uptake in the presence of a pool of substrates with DPNlinked dehydrogenases was increased only slightly in the mitochondria from the active animals. In this case the rate of oxidation was higher than that with any single substrate except succinate. The rate of oxidation for succinate was significantly increased by exercise to the same extent as that of a-ketoglutarate. The rate of endogenous substrate oxidation was found to be only slightly greater for the experimental animals than for the control animals, and this difference was not statistically significant. It is thus unlikely that this change was responsible for the effects of exercise in the presence of exogenous sub~trate. If the endogenous values are subtracted from the exogenous values, higher percentage differences result in all cases, except for the presence of the substrate pool. In contrast to the linear rate of pyruvate oxidation noted with the experimental mitochondria, pyruvate oxidation with the control mitochondria was increasingly inhibited during the course of the incubation. In the presence of a mixture of pyruvate and malate the inhibition was released, and the maximum rate of oxidation was increased. The rate of O~ uptake of the pyruvate-malate mixture was still considerably higher after exercise, although the response was not as great as with pyruvate or malate alone. Perhaps part of the effect of exercise on pyruvate oxidation is due to an increase in endogenous oxaloacetate. P/O ratios have been obtained in the presence of each of the substrates as well as in the presence of the substrate pool, and in all cases the experimental mitochondria show P/O ratios similar to those of the control mitochondria. Moreover, respiratory control does not appear to differ between the two types of mitochondria. Rates of 0 2 uptake in the absence of ADP have also been measured. When succinate is utilized there is a 33 % increase in O 2 uptake in the experimental mitochondria. Studies with the other substrates indicate that the experimental mitochondria have higher O~-uptake rates than the control mitochondria, but there were large variations in the oxidation rate for any particular substrate. In control mitochondria the wide range of rates of coupled oxidation among the substrates with DPN-linked dehydrogenases is obviously due to differences in dehydrogenase activity. In this study approximately optimal amounts of substrate and ADP were used. The fact that the greatest response to exercise was noted in the presence of either malate or pyruvate alone, when the dehydrogenase activity was low, and that the weakest response was observed in the presence of the substrate pool when the electron transport chain was presumably operating close to its maximum capacity indicated that the dehydrogenase step and not the electron transport chain was activated in tile experimental mitochondria. The significant response in the presence of succinate, the most reactive substrate, does not detract from this hypothesis, since succinic dehydrogenase is not linked to the electron transport chain by way of DPN. These effects of exercise on oxidative capacity are in marked contrast with the effects of changes in thyroid-hormone levels, as thyroid hormones increase oxidative capacity primarily by stimulating electron transport and have little effect on the slow dehydrogenases ~. NIELSON AND KLITGAARD~ have suggested the possibility of a biological r h y t h m as one explanation for their findings that maximum and minimum values of succinate Biochim. Biophys. dcta, 82 (1964) t65-I67
SHORT COMMUNICATIONS
167
oxidation in tissue homogenates reoccur over the course of a starvation period of 2 days. In contrast to our results, they have reported further that exercise tends to depress slightly succinate oxidation ~. Their results do not necessarily conflict with ours, since their experimental conditions differed considerably from ours. However, our data clearly emphasize the importance of measuring oxidation rates with a variety of substrates in any study on the control of metabolic rate. In conclusion, these results show that exercise increased the rate of oxidation of a variety of substrates by rat-liver mitochondria. The largest percentage effects were observed with the substrates oxidized most slowly, and the data indicated an approximate doubling of the effective capacity of the citric acid cycle. This investigation was supported by a grant (3TI-GM-57 o) from the U.S. Public Health Service.
Department of Zoology, Columbia University, New York, N.Y. (U.S.A.)
J.
L E S L I E GLICK
J.
RAMSEY B R O N K
1 j . L. GLICK AND W. D. COHEN, u n p u b l i s h e d . W. W. KIELLEY AND R. K. KIELLEY, J. Biol. Chem., 191 (1951) 485 . W. W. KIELLEY AND J. R. BRONK, J. Biol. Chem., 23o (1958) 521. 4 B. CHANCE AND G. R. WILLIAMS, ]. Biol. Chem., 217 (1955) 383. s j . R. BRONK, S6ience, 141 (1963) 816. * R. R. NIELSON AND H. M. KLITGAARD, Am. J. Physiol., 2Ol (1961) 37. R. R. NIELSON AND H. M. KLITGAARD,Federation Proc., 2o (1961) 316.
Received June I2th, 1963 Revised manuscript received September I6th, 1963 Biochim. Biophys. Acta, 82 (1964) 1 6 5 - I 6 7
sc 2334
Effect of 6-amino N A D and other N A D analogues on the formation of N A D H ~ P and its transphosphorylation to ATP* 6-AN produces severe toxic symptoms when given to experimental animals 1-5. Investigations of the mechanism of action have shown that under these conditions the 6-AN can replace the nicotinamide moiety of NAD giving rise to the formation of an abnormal NAD molecule *, 3 which may, at least partly, explain the defect observed. Little information is available on the effect of 6-ANAD on enzymic processes. We have been able to prepare 6-ANAD in reasonably good yield6 and are at present studying the interference of this analogue on enzymes connected with ATP synthesis, since the toxic symptoms may suggest a lesion of a major energy-generating system of the cell. This investigation describes the effect of 6-ANAD on the NAD-phosphorylating system reported recently by GRIFFITHS AND CHAPLAIN7. The sheep-heart mitochondrial system used converts appreciable amounts of NAD to the phosphorylated form in the presence of succinate. Spectrophotometric characteristics of the compound A b b r e v i a t i o n s : 6-AN, 6 - a m i n o n i c o t i n a m i d e ; 3-AP, 4-AP, 3/4-acetylpyridine; 6 - A N A D , 3 - A P A D , 4 - A P A D , a n a l o g u e s of N A D ; N A D H ,,~ P, p h o s p h o r y l a t e d N A D , * T h i s work w a s s u p p o r t e d b y a g r a n t f r o m t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t .
Biochim. Biophys. Acta, 82 (1964) i 6 7 - i 7 o
SHORT COMMUNICATIONS
sc 2369
The effect of exercise on the rate of oxygen uptake by rat-liver mitochondria Recent experiments by GLICK AND COHEN1 have demonstrated that the rate of succinate oxidation b y rat-liver mitochondria prepared at night was 41% higher t h a n the rate of succinate oxidation b y mitochondria prepared during the day. Since rats normally exercise at night and rest during the day, the experiments reported in this communication were designed to determine whether physical activity could control oxidative capacity in a similar manner. The results show that although exercise increased the rate of succinate oxidation, the most pronounced effects were obtained in the presence of those citric acid-cycle substrates which were oxidized most slowly. This phenomenon appears to be due to changes in dehydrogenase activity, and suggests that the oxidative capacity of the citric acid cycle is increased in physically active animals. Male Wistar rats were kept in pairs and were permitted food and water ad libitum for at least a week prior to sacrifice. On the day in which they were used they weighed between 18o and 25 ° g. In each pair one rat served as a control and remained in his cage without food or water for 3 h. The other animal was transferred to an activity wheel which was rotated at a speed of about 6 rev./min by an electric motor. At the end of the 3-h period both rats were sacrificed, and liver mitochondria were prepared by the method of KIELLEY AND KIELLEY2. 0 2 uptake was measured by the Clark oxygen electrode 3. P / 0 ratios were obtained by the technique of CHANCE AND W1LLIAMS 4.
The results in Table I show that the rate of 02 uptake in mitochondria from active animals was significantly increased over that in mitochondria from resting animals with each substrate tested. The .greatest percentage of stimulation was observed with malate or pyruvate which were oxidized most slowly, a-Ketoglutarate and fl-hydroxybutyrate which show relatively fast rates of oxidation were less TABLE I O2-UPTAKE RATES IN MITOCHONDRIA FROM EXERCISED RATS E a c h incubation vessel contained: i # m o l e of ADP, io #moles of MgCI~, 2o #moles of p h o s p h a t e (pH 7.o), 2 o o # m o l e s of sucrose. A m o u n t of succinate, 5/~moles; a-ketoglutarate, I o # m o l e s ; fl-hydroxybutyrate, 2 o # m o l e s ; citrate, I o # m o l e s ; malate, i o # m o l e s ; pyruvate, Io/*moles; m a l a t e - p y r u v a t e m i x t u r e : i o # m o l e s each of malate and p y r u v a t e ; s u b s t r a t e pool: 3 #moles each of all t h e above substrates except succinate. Total volume, 2.o ml; t e m p e r a t u r e , 3o°; d r y weight of mitochondria per incubation, 2.73-4.13 mg. The values reported are means 4- S.E. of the mean. The n u m b e r of animals is indicated b y the n u m b e r in parentheses. Oxygen uptake rate (l~atomlminper rag dry wt.)
Substrate
Control
Succinate 0¢-Ketoglutarate ~-Hydroxybutyrate Citrate Malate Pyruvate Malate--pyruvate Substrate pool Endogenous substrate
0.248 o.o78 o.o9 o 0.043 o.o21 0.022 o.o73 o.iio o.o13
4- O.Ol 3 (6) 4- o.oo 5 (6) 4- o.oo 5 (6) 4- o.oo2 (6) 4- 0.003 (6) 4- 0.002 (6) 4- o.o12 (4) 4- o.oo 3 (6) 4- o.oo2 (4)
Exercised
0.305 o.o96 O.lO9 o.o59 0.o35 0.039 o.o98 o.122 °.°15
4- o.o18 (6) 4- 0.007 (6) 4- 0.o07 (6) 4- o.oo 5 (6) + o.0o 4 (6) 4- 0.004 (6) 4- o.o12 (4) 4- 0.006 (6) 4- o.oo4 (4)
% Control
123 123 121 137 167 177 134 iii 115
(P (P (P (P (P (P (P (P (P
< 0.05) • o.o5) ,< o.oi) < o.oi) < o.ooi) < 0.02) < o.oi) < 0.05) < 0.4)
Biochim. Biophys. Me/a, 82 (1964) 165-167
166
SHORT COMMUNICATIONS
strikingly affected. Citrate, which has an intermediate rate of oxidation in control mitochondria, was oxidized considerably more rapidly in the experimental mitochondria. The rate of O2 uptake in the presence of a pool of substrates with DPNlinked dehydrogenases was increased only slightly in the mitochondria from the active animals. In this case the rate of oxidation was higher than that with any single substrate except succinate. The rate of oxidation for succinate was significantly increased by exercise to the same extent as that of a-ketoglutarate. The rate of endogenous substrate oxidation was found to be only slightly greater for the experimental animals than for the control animals, and this difference was not statistically significant. It is thus unlikely that this change was responsible for the effects of exercise in the presence of exogenous sub~trate. If the endogenous values are subtracted from the exogenous values, higher percentage differences result in all cases, except for the presence of the substrate pool. In contrast to the linear rate of pyruvate oxidation noted with the experimental mitochondria, pyruvate oxidation with the control mitochondria was increasingly inhibited during the course of the incubation. In the presence of a mixture of pyruvate and malate the inhibition was released, and the maximum rate of oxidation was increased. The rate of O~ uptake of the pyruvate-malate mixture was still considerably higher after exercise, although the response was not as great as with pyruvate or malate alone. Perhaps part of the effect of exercise on pyruvate oxidation is due to an increase in endogenous oxaloacetate. P/O ratios have been obtained in the presence of each of the substrates as well as in the presence of the substrate pool, and in all cases the experimental mitochondria show P/O ratios similar to those of the control mitochondria. Moreover, respiratory control does not appear to differ between the two types of mitochondria. Rates of 0 2 uptake in the absence of ADP have also been measured. When succinate is utilized there is a 33 % increase in O 2 uptake in the experimental mitochondria. Studies with the other substrates indicate that the experimental mitochondria have higher O~-uptake rates than the control mitochondria, but there were large variations in the oxidation rate for any particular substrate. In control mitochondria the wide range of rates of coupled oxidation among the substrates with DPN-linked dehydrogenases is obviously due to differences in dehydrogenase activity. In this study approximately optimal amounts of substrate and ADP were used. The fact that the greatest response to exercise was noted in the presence of either malate or pyruvate alone, when the dehydrogenase activity was low, and that the weakest response was observed in the presence of the substrate pool when the electron transport chain was presumably operating close to its maximum capacity indicated that the dehydrogenase step and not the electron transport chain was activated in tile experimental mitochondria. The significant response in the presence of succinate, the most reactive substrate, does not detract from this hypothesis, since succinic dehydrogenase is not linked to the electron transport chain by way of DPN. These effects of exercise on oxidative capacity are in marked contrast with the effects of changes in thyroid-hormone levels, as thyroid hormones increase oxidative capacity primarily by stimulating electron transport and have little effect on the slow dehydrogenases ~. NIELSON AND KLITGAARD~ have suggested the possibility of a biological r h y t h m as one explanation for their findings that maximum and minimum values of succinate Biochim. Biophys. dcta, 82 (1964) t65-I67
SHORT COMMUNICATIONS
167
oxidation in tissue homogenates reoccur over the course of a starvation period of 2 days. In contrast to our results, they have reported further that exercise tends to depress slightly succinate oxidation ~. Their results do not necessarily conflict with ours, since their experimental conditions differed considerably from ours. However, our data clearly emphasize the importance of measuring oxidation rates with a variety of substrates in any study on the control of metabolic rate. In conclusion, these results show that exercise increased the rate of oxidation of a variety of substrates by rat-liver mitochondria. The largest percentage effects were observed with the substrates oxidized most slowly, and the data indicated an approximate doubling of the effective capacity of the citric acid cycle. This investigation was supported by a grant (3TI-GM-57 o) from the U.S. Public Health Service.
Department of Zoology, Columbia University, New York, N.Y. (U.S.A.)
J.
L E S L I E GLICK
J.
RAMSEY B R O N K
1 j . L. GLICK AND W. D. COHEN, u n p u b l i s h e d . W. W. KIELLEY AND R. K. KIELLEY, J. Biol. Chem., 191 (1951) 485 . W. W. KIELLEY AND J. R. BRONK, J. Biol. Chem., 23o (1958) 521. 4 B. CHANCE AND G. R. WILLIAMS, ]. Biol. Chem., 217 (1955) 383. s j . R. BRONK, S6ience, 141 (1963) 816. * R. R. NIELSON AND H. M. KLITGAARD, Am. J. Physiol., 2Ol (1961) 37. R. R. NIELSON AND H. M. KLITGAARD,Federation Proc., 2o (1961) 316.
Received June I2th, 1963 Revised manuscript received September I6th, 1963 Biochim. Biophys. Acta, 82 (1964) 1 6 5 - I 6 7
sc 2334
Effect of 6-amino N A D and other N A D analogues on the formation of N A D H ~ P and its transphosphorylation to ATP* 6-AN produces severe toxic symptoms when given to experimental animals 1-5. Investigations of the mechanism of action have shown that under these conditions the 6-AN can replace the nicotinamide moiety of NAD giving rise to the formation of an abnormal NAD molecule *, 3 which may, at least partly, explain the defect observed. Little information is available on the effect of 6-ANAD on enzymic processes. We have been able to prepare 6-ANAD in reasonably good yield6 and are at present studying the interference of this analogue on enzymes connected with ATP synthesis, since the toxic symptoms may suggest a lesion of a major energy-generating system of the cell. This investigation describes the effect of 6-ANAD on the NAD-phosphorylating system reported recently by GRIFFITHS AND CHAPLAIN7. The sheep-heart mitochondrial system used converts appreciable amounts of NAD to the phosphorylated form in the presence of succinate. Spectrophotometric characteristics of the compound A b b r e v i a t i o n s : 6-AN, 6 - a m i n o n i c o t i n a m i d e ; 3-AP, 4-AP, 3/4-acetylpyridine; 6 - A N A D , 3 - A P A D , 4 - A P A D , a n a l o g u e s of N A D ; N A D H ,,~ P, p h o s p h o r y l a t e d N A D , * T h i s work w a s s u p p o r t e d b y a g r a n t f r o m t h e D e u t s c h e F o r s c h u n g s g e m e i n s c h a f t .
Biochim. Biophys. Acta, 82 (1964) i 6 7 - i 7 o