562 Bioeh~mica et ~iophysica Acta, 943 (1988) 562-565 Elsevier

BBA 50247 BBA Report

Effects of endurance exercise on carnitine pdmitoyltransferase I from rat heart,

skeletal muscle and liver mitochondria

Manuel Guzmhn and Jo& Castro

~ep~t~ent of Biockemjst~, Faculty of ~k~rn~st~, Universidad ~ornp~ute~.~e, Madrid (Spain)

(Received 20 July 1988)

Key words: Carnitine pabnitoyltransferase I; pH effect; (Rat heart): (Rat skeletal muscle)

Prolonged physical exercise increased the activity of camitine palmitoyl~ansfer~e I in rat heart and skeletal muscle mitochondria, whereas enzyme sensitivity to inhibition by malonyl-CoA remained unchanged. Nevertheless, inhibition of carnitine palmitoyltransferase I activity by small decreases in pH was attenuated in heart and skeletal muscle mitochondria from exercised animals. Liver enzyme did not suffer any alteration by endurance exercise.

Camitine palmitoyltransferase I, the overt form of camitine palmitoyltransferase (EC 2.3.1.21), is considered to have a regulatory role in the trans- port of long-chain fatty acids into the ~tochondrial matrix (reviewed in Refs. 1 and 2). The specific activity of rat liver carnitine palmitoyltransferase I is modulated both on the short [3] and on the long term [4-81. Long-term changes in specific activity of the hepatic enzyme are also accompanied by alterations in enzyme sensitivity to malonyl-CoA, a physiological in- tracellular inhibitor of carnitine palmitoyltransfer- ase 1[9]. In muscle, the supply of fatty acids to the mitochondrial matrix is probably a rate-control- ling factor for fatty acid oxidation (reviewed in Refs. 10 and 11). Thus, the exercise-induced adap- tive response of mitochondrial enzymes includes a significant increase in camitine palmitoyltrans- ferase activity in rat skeletal muscle [lo].

The aim of the present work was to study the effects of prolonged physical exercise on the activ-

Correspondence: J. Castro, Department of Biochemistry, Fa- culty of Chemistry, Universidad Complutense, 28040 Madrid,

Spain.

ity and regulation of carnitine palmitoyltransferase I in rat heart, skeletal muscle and liver mitochondria.

Male Wistar rats (115 + 15 g) were trained to run on a motor-driven treadmill. After 4 weeks of adaptation, animals were running continuously for 45 min at 25 m/min during 8 weeks, 5 days per week [12]. Animals were not exercised for 24 h prior to killing. At the end of the experimental period, exercised animals showed a significant 15 f 4% decrease in body weight as compared to the sedentary controls (P -z 0.01 by the paired t-test). Animals were killed by decapitation and their livers, hearts and hind-limb skeletal muscle were excised. Mitochondria were isolated according to Saggerson and Carpenter [13]. Carnitine palmito- yltransferase I activity was determined in isolated mitochondria by measuring the malonyl-CoA-sen- sitive incorporation of L-[ rneth~$‘~ Clcarnitine into n-butanol-soluble product exactly as described in Ref. 8, except that the pH of the incubation medium was varied between 4.8 and 7.6. Briefly, mitochondria (150-250 f*g of protein) were prein- cubated at 25” C for 4 mm in 1.0 ml containing 150 mM sucrose, 60 mM KCI, 25 mM Tris-HCl (pH 7.4 in standard assay), 1 mM EDTA, 1 mM

~5-2760/88/$03.50 0 1988 Elsevier Science Publishers B.V. (Biomedical Division)

dithiothreitol, 40 PM palmitoyl-CoA and 1.3 mg/ml albumin (fatty acid-free). Reactions were started by addition of 25 ~1 containing 0.25 PCi (0.4 pmol) of L-[ methyl-‘4C]carnitine, carried out for up to 4 min and stopped with 1.0 ml ice-cold 1 M HCl. The values of the concentrations of malonyl-CoA causing a 50% of the maximal inhibition (IC,,) of camitine pahnitoyltransferase I activity were directly calculated from the plots of activity versus logarithm of malonyl-CoA con- centration. These were corrected for malonyl- CoA-insensitive camitine palmitoyltransferase ac- tivity. Results shown represent the means + SD. of six animals in each group, with incubations carried out in triplicate. Statistical analysis was performed using the paired t-test.

The effects of endurance exercise on carnitine pahnitoyltransferase I activity and sensitivity to inhibition by malonyl-CoA are presented in Table I. Since both parameters have been shown to exhibit a marked dependence on the pH of the assay mixture [14,15], measurements were per- formed at pH 6.8 and 7.4. Prolonged physical exercise greatly increased carnitine palmitoyltrans- ferase I activity in heart and skeletal muscle, in- cluding both slow-twitch (soleus) and fast-twitch (extensor) muscle fibers. However, the activity of liver enzyme was not significantly altered by long-term physical training. This makes prolonged

563

physical exercise a physiological state of particular interest, since adaptive changes in camitine palm- itoyltransferase I activity occur in extrahepatic tissues, but not in the liver. On the contrary, in the different physiological states previously studied, adaptive changes in carnitine palmitoyltransferase I activity seem to be confined to the liver enzyme [4-S]. The exercise-induced increase of carnitine palmitoyltransferase I activity would sustain enhanced rates of fatty acid oxidation which ap- pear after long-term physical exercise in heart and skeletal muscle mitochondria [lO,ll].

Sensitivity of carnitine palmitoyltransferase I to inhibition by malonyl-CoA, as measured by the IC,, value, was not changed in any of these three tissues after endurance training (Table I). In tis- sues with low rates of fatty acid synthesis, such as rat heart and skeletal muscle [2,13,15] as well as sheep liver [16], carnitine palmitoyltransferase I retains a marked sensitivity to inhibition by malonyl-CoA. Nevertheless, no changes in enzyme sensitivity to malonyl-CoA have been found in extrahepatic tissues under different physiological and pathological conditions [2,6,17]. Our results on the effects of prolonged physical exercise are in agreement with this general pattern. On the other hand, it has been shown that short-term physical exercise increases blood ketone body levels and the rate of hepatic ketogenesis [18,19], but this

TABLE I

EFFECTS OF PROLONGED PHYSICAL EXERCISE ON THE ACTIVITY AND SENSITIVITY TO MALONYL-CoA OF CARNITINE PALMITOYLTRANSFERASE I IN RAT HEART, SKELETAL MUSCLE AND LIVER MITOCHONDRIA

Enzyme activity was determined at pH 6.8 and 7.4 by measuring the malonyl-CoA-sensitive incorporation of L-[ methyl-t4C]camitine

into palmitoylcamitine. The concentrations of malonyl-CoA used to calculate the values of IC,, were 0, 0.01, 0.02, 0.03, 0.05, 0.1, 0.2,

0.5, 1.0, 10 and 100 FM for mitochondria from heart, soleus and extensor muscle, and 0, 0.25, 0.5, 0.75, 1.0, 2.0, 5.0, 10, 20 and 100 pM for liver mitochondria. * P < 0.01 vs. control.

Tissue Group of animals

Camitine palmitoyltransferase I

activity (nmol/min per mg protein) IC,, for malonyl-CoA (p M)

pH 6.8

Heart

Soleus

Extensor

Liver

control

exercise control exercise control exercise control exercise

5.60 f 0.36 15.54+ 1.09 *

2.96+0.18 9.69k 1.17 * 1.3850.12 4.88 + 0.40 * 2.94 f 0.70 3.64kO.92

pH 7.4 pH 6.8

9.66 f 2.07

19.62 + 2.41 * 10.96 f 1.02 20.61 f 1.63 *

5.12k0.84 10.39& 1.42 *

2.99 f 0.63 3.61 kO.74

0.03 f 0.01

0.03 f 0.01 0.03 f 0.01 0.03 * 0.01 0.03 + 0.01 0.03 + 0.01 0.95 kO.18 1.09kO.16

pH 7.4

0.11+0.02

0.09 + 0.02 0.07 + 0.01

0.08 + 0.02 0.09 + 0.02 0.11+0.02 4.05 f 0.97 4.32 _+ 0.91

Fig. 1. Effect of pH on camitine pahnitoyltransferase I activity in rat heart (A), soleus muscle (B) and liver (C). Enzyme activity was

determined at different values of pH in exercised animals (0) and sedentary controls (o), and expressed as percentage of enzyme activity at pH 7.4. The profile obtained for extensor muscle was analogous to that presented here for soleus muscle. * P c 0.01 vs.

control.

post-exercise ketosis is attenuated in endurance- trained rats [18]. However, the concentration of malonyl-CoA in liver has been shown to be not significantly altered by either short- or long-term physical exercise [18]. In the case of adult rat liver, a positive relationship has been suggested to exist between the sensitivity of carnitine palmitoyl- transferase I to inhibitor malonyl-CoA and the concentrations of malonyl-CoA in the livers from which the mitochondria are isolated [20]. This may provide a possible explanation for the lack of effect of long-term physical training on enzyme sensitivity to malonyl-CoA in rat liver.

It may be inferred from Table I that the ratio of camitine palmitoyltransferase I activity at pH 6.8 to activity at pH 7.4 is elevated in heart and skeletal muscle mitochondria isolated from exercised animals, as compared to the sedentary controls. Hence, we determined the profile of en- zyme activity as a function of pH over the range of pH 6.8-7.6, in mitochondria from heart, skeletal muscle and liver. Camitine palmitoyltransferase I activity in heart and skeletal muscle mitochondria increased when pH was raised from 6.8 to 7.6, whereas liver enzyme activity was fairly constant over this pH range (Fig. I), in agreement with

previous reports [14,15]. More important, a strik- ing adaptive change in the sensitivity of camitine palmitoyltransferase I to variations of pH ap- peared after long-term physical exercise. Figs. 1A and 1B clearly show that in heart and skeletal muscle mitochondria, the inhibition of enzyme activity by small decreases in pH was attenuated in exercised animals as compared to the sedentary controls. This effect was absent in liver enzyme activity (Fig. 1C). Furthermore, similar profiles were obtained when the activity of camitine palm- itoyltransferase I was determined in the presence of malonyl-CoA concentrations approaching the IC,, value for this inhibitor, namely, 0.1 PM for heart and skeletal muscle and 3 PM for liver enzyme (data not shown). In heart and skeletal muscle, differences in enzyme activity between exercised and sedentary animals were maximal when measured at pH 6.8, both in the absence (Fig. 1) or in the presence (data not shown) of malonyl-CoA. Nevertheless, prolonged physical exercise had no effect on the sensitivity of the enzyme to malonyl-CoA at pH 6.8, as measured by the IC,, value for this inhibitor (Table I). Therefore, adaptive changes to small decreases in pH, occurring in heart and skeletal muscle cami-

565

tine palmitoyltransferase I after prolonged physi- cal exercise, are exclusively reflected on the specific activity of the enzyme, but not on enzyme sensitiv- ity to inhibition by malonyl-CoA. Stephens et al. [14] proposed that a decrease in intracellular pH, such as might occur in ketoacidosis, may provide a physiological mechanism to attenuate hepatic fatty acid oxidation by increasing sensitivity to malonyl-CoA of camitine palmitoyltransferase I. Now, our data suggest that long-term exercised animals might sustain enhanced carnitine palmito- yltransferase activity in heart and skeletal muscle despite exercise-induced acidosis.

In conclusion, our results suggest that camitine palmitoyltransferase I could play a central role in controlling the flux of fatty acids into the oxida- tion pathway in heart and skeletal muscle mitochondria after prolonged physical exercise. This is also the first report describing a physio- logical state associated with adaptive changes in the sensitivity of carnitine palmitoyltransferase I to physiological decreases in pH.

This study was supported by a grant from the Consejo Superior de Deportes, Spain.

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