Increased muscle carnitine palmitoyltransferase II mRNA after increased contractile activity

ZHEN YAN, STANLEY SALMONS, JONATHAN JARVIS, AND FRANK W. BOOTH Department of Physiology and Cell Biology, University of Texas-Houston Health Science Center, Houston, Texas 77030; and Department of Human Anatomy and Cell Biology, The University of Liverpool, Liverpool L69 3BX, United Kingdom

Yan, Zhen, Stanley Salmons, Jonathan Jarvis, and Frank W. Booth. Increased muscle carnitine palmitoyltrans- ferase II mRNA after increased contractile activity. Am. J. Physiol. 268 (Endocrinol. Metab. 31): E277-E281, 1995.- The capacity of skeletal muscle to oxidize fatty acids increases with endurance training. The oxidation of long-chain fatty acids occurs in mitochondria and is initiated by a carnitine- dependent transport step in which three enzymes help fatty acyl groups enter the matrix compartment. The purpose of this study was to determine whether pretranslational regula- tion of one of these three enzymes, carnitine palmitoyltransfer- ase II (CPT II), as estimated from the level of CPT II mRNA, plays a role in the doubling of CPT activity in skeletal muscle of rats subjected to daily 2-h bouts of running on treadmills (P. A. Mole, L. B. Oscai, and J. 0. Holloszy. J. Clin. Invest. 50: 2323-2330, 1971). After 100 min/day of running on motor- driven treadmills for 2 wk, CPT II mRNA in the plantaris muscle was unchanged when normalized per unit of extracted RNA but was 50% higher (P < 0.05) over sedentary controls when normalized per unit of muscle wet weight. To test whether additional contractile activity would make CPT II mRNA even higher, continuous indirect electrical stimulation was imposed on the tibialis anterior muscles. After 9 days of chronic stimulation, CPT II mRNA was 63, 221, and 137% greater than control (P < 0.001) when normalized to extracted RNA, muscle wet weight, and whole muscle, respectively, compared with the muscle in the control rats. These data indicate that pretranslational regulation of CPT II occurs in response to increased contractile activity in skeletal muscle.

free fatty acid oxidation; skeletal muscle; messenger ribo- nucleic acid; pretranslational regulation; mitochondria; tread- mill running; chronic stimulation

FAT BECOMES A MORE important source of energy for submaximal exercise after endurance training (4). For example, a two-legged cycling test revealed a more pronounced metabolism of fatty acids in the trained than in the nontrained leg during submaximal exercise in subjects who had trained with a one-leg bicycle ergometer (7). Henriksson (7) suggested that, during submaximal exercise, the trained leg oxidized more fatty acid because of an increased muscle oxidative capacity, since blood flow was similar in the two legs. Saltin and Astrand (24) indicated the complexity of pinpointing the rate-limiting step that produces increased fatty oxida- tion as a result of endurance training. They suggested that any or all of the following reported adaptations, which occur in trained skeletal muscle, could account for an enhanced oxidation of fatty acids by the trained skeletal muscle: 1) an increase in the activities of enzymes of the citric acid cycle, fatty acid oxidation, and electron transport; 2) an elevation of carnitine and of

carnitine transporters for fatty acids; 3) an increase in the concentration of fatty acid-binding proteins that transfer fatty acids through the cytoplasm; and 4) a decrease in the fiber volume supplied by capillaries. Some of the processes related to these steps do not show changes in mRNA. For example, 7 days of treadmill running did not alter the level of acyl-CoA synthetase mRNA and lipoprotein lipase mRNA in the gastrocne- mius muscle of rats (27). On the other hand, lipoprotein lipase activity was increased in the white and red portions of the vastus lateralis muscle immediately after an acute bout of swimming (13). To our knowledge, little other information is available on the response of mRNAs for proteins involved in fatty oxidation in skeletal muscle after training.

The mitochondrial oxidation of long-chain fatty acids is initiated by a carnitine-dependent transport step whereby fatty acyl groups gain entry to the matrix compartment by the actions of three enzymes: carnitine palmitoyltransferase I and II (CPT I and CPT II, respectively) and carnitine-acylcarnitine translocase (18). CPT II is located in the inner membrane of mitochon- dria and splits acylcarnitine into acyl-CoA and carnitine. Chronic electrical stimulation has been found to result in a threefold increase in the consumption of fatty acids by contracting muscles (10) and increase the levels of mRNA encoding several enzymes in the citric acid cycle and electron transport chain (22), but little information is available on the response of mRNAs of proteins involved in fatty acid oxidation. The model of chronic stimulation can provide information as to the extent of muscle plasticity (22). The aim of this study was to determine whether increased contractile activity pro- duced by chronic stimulation would increase CPT II mRNA and to compare the magnitude of such response to that induced by treadmill training.

MATERIALS AND METHODS

Animals. Female Sprague-Dawley rats (Harlan, 100-120 g and 125-149 g body wt for the treadmill running and chronic stimulation studies, respectively) were housed in temperature- controlled quarters (21°C) with a 12:12-h light-dark cycle. Animals were provided with water and chow (Harlan Teklab) ad libitum. Experimental protocols were approved by the University of Texas Health Science Center at Houston Institu- tional Animal Welfare Committee. Where indicated, anesthe- sia [a mixture of ketamine (54 mg/ml), xylazine (2.2 mg/ml), and acepromazine (3.5 mg/ml)] was injected into the right gluteus muscle (1.4 ml/kg).

Treadmill running. Seventeen rats in this study were preacclimatized to treadmill running by performing lo-min daily runs for 3 wk on an 8” inclined motor-driven treadmill. The treadmill speed was gradually increased from 0.07 m/s at

0193~1849/95 $3.00 Copyright o 1995 the American Physiological Society E277

E278 TRAINING INCREASES CPT II MRNA

the start of preacclimatization to 0.45 m/s at the end of preacclimatization. Nine rats that avoided the rear of the running compartment the most were selected for endurance training, which consisted of running 100 min/day (in 3 separate sessions of 33.3 min) at a speed of 0.36-0.45 m/s on an 8” incline for 14 days. The remaining eight rats were assigned to the control group and remained in their cages during the endurance training. At the end of the 14-day training period, plantaris muscles were taken from anesthe- tized rats.

Chronic electrical stimulation. Electrodes and devices were implanted into five rats for chronic stimulation as described by Mayne et al. (17). The deep peroneal nerve of the left hindlimb was exposed from the lateral side in anesthetized rats under aseptic conditions. A battery-powered implantable stimulator (23) was implanted in the abdominal cavity, and two stainless steel electrodes were secured N 0.5 cm apart with sutures under the deep peroneal nerve, which supplies motor fibers to the muscle in the anterior compartment of the hindlimb including the tibialis anterior muscle. The same procedure was performed for the right hindlimb except that the electrodes were not connected to a stimulator. Stimulation was com- menced 2 days later. Rectangular pulses (0.2 ms) were applied continuously at 10 Hz for 24 h/day. Tibialis anterior muscles were dissected from anesthetized rats at the end of 9 days of stimulation.

RNA isolation. Designated muscles were dissected and quickly frozen with liquid-nitrogen-cooled tongs and stored at -80°C. Before being homogenized, N 100 mg (wet wt) muscle was powdered in a mortar containing enough liquid nitrogen to prevent the sample from thawing. RNA was extracted (extracted RNA) by the RNAzol (Biotecx Laboratories, Hous- ton, TX) protocol (3). After homogenization in 3.5 ml RNAzol B, ice-cold chloroform (3.5 ml) was added to the homogenate, and the mixture was kept on ice for 15 min. After centrifuga- tion at 12,000 g for 15 min at 4”C, the extracted RNA was precipitated with isopropanol, and the RNA concentrations were determined spectrophotometrically at 260 nm wave- length.

Riboprobe generation. The CPT II cDNA cloned in pB- SIIKS+ (Stratagene) was generously supplied by Dr. J. D. McGarry (31). It was digested with Nde I and used as a template to synthesize antisense RNA. A 448nucleotide anti- sense RNA was synthesized with an in vitro transcription kit (Promega) labeled with [32P]CTP (>400 Ci/mmol; Amer-

sham). The specific radioactivity of the riboprobe was 108-log counts . min l (cpm) l Fg DNA?

Northern blot anaZysis. Extracted RNA (20 kg) was electro- phoresed in formaldehyde-agarose gels and transferred to nylon membranes by capillary blotting (25). Ethidium bromide staining of gels indicated equivalent amounts of 18s and 28s rRNA in each lane. RNA was immobilized by ultraviolet cross-linking (Stratolinker), after which the membranes were prehybridized at 60°C for 15 min in hybridization buffer containing 6~ SSPE (pH 7.0), 50% formamide, 5 x Denhart’s solution, 0.5% sodium dodecyl sulfate, and 0.1 mg/ml dena- tured salmon sperm DNA. Hybridizations were performed at 60°C for 12-14 h in the hybridization buffer with the addition of lo6 cpm/ml [32P]CPT II riboprobe. A specific band appeared at the 2580-nucleotide position upon autoradiography, estab- lished with 18s and 28s rRNA markers. Esser et al. (6) have reported that CPT II mRNA contains - 2,500 bases.

Total RNA quantification. To estimate CPT II mRNA concentration per gram of wet weight in muscles, total RNA was determined according to Munro and Fleck (2 1). The concentration of CPT II mRNA per gram of wet weight was calculated by multiplying CPT II mRNA per microgram extracted RNA by total RNA per milligram wet weight (2). CPT II mRNA per whole muscle was calculated by multiplying CPT II mRNA per milligram wet weight by muscle wet weight.

Statistics. An unpaired Student’s t-test was employed for comparisons between treadmill trained and control plantaris muscles. A one-way analysis of variance was performed for comparisons among control, contralateral control, and stimu- lated tibialis anterior muscles in the chronic stimulation study. When significant differences among groups were de- tected, a Newman-Keuls test was performed to determine differences among experimental groups. P < 0.05 was ac- cepted as significant for all statistical analyses.

RESULTS

After 14 days of treadmill running, CPT II mRNA per unit of wet weight in the plantaris muscles was in- creased 50% (P < 0.05; Table 1; Fig. 1). No significant difference in the percentage of CPT II mRNA per unit of extracted RNA was noted.

In comparisons between the tibialis anterior muscle of the stimulated leg and a separate control rat, CPT II

Table 1. CPT II mRNA levels in skeletal muscle of rats that have undergone either 14 days of treadmill running or 9 days of chronic stimulation

Treadmill Running (Plantaris) Chronic Stimulation (Tibialis Anterior)

Control (n = 15)

Trained (n = 18)

%Change vs. control

Control (72 = 7)

Contralateral control (n = 4)

Stimulated (n = 5)

%Change vs. control

CPT II mRNA IOD/ kg RNA 0.33 + 0.03 0.41+ 0.03 +25 0.40 k 0.02 0.19 + 0.04-j- 0.65 t 0.057$ +63 IOD/mg wet wt 0.32 + 0.03 0.48 + 0.05’” +50 0.43 + 0.02 0.26 + 0.07 1.38 iz O.l4”f$ +221 IOD/muscle ND ND ND 190 + 14 110 + 32’” 451 rf: 27”f$ +137

Total RNA, pg/mg wet wt 1.00 t 0.05 1.15 + 0.06’” +15 1.08 + 0.03 1.27 + 0.07* 2.112 0.117 +95 Muscle wt, g ND ND ND 0.44 + 0.02 0.42 k 0.02 0.33 * 0.03’“§ -25

Values are means + SE; IZ, no. of rats. ND, not determined. Control, set of animals separate from treatment group; contralateral control, sham-operated nonstimulated limb of treatment group. Integrated optical density (IOD) units were obtained from image analysis (BioImage; Millipore) of -2580-nucleotide band on autoradiographs of Northern blot analysis of carnitine palmitoyltransferase (CPT) II mRNA. Normalization of CPT II mRNA per unit of muscle wet weight was done by multiplying CPT II mRNA/pg extracted RNA by total RNA/mg muscle wet weight (2). Normalization of CPT II mRNA per whole muscle was calculated by multiplying CPT II mRNA/mg wet weight by muscle wet weight. See MATERIALS AND METHODS for procedures to extract RNA and total RNA. *P < 0.05 from control rats. -f-P < 0.001 from control rats. $P < 0.001 from contralateral control muscles. $P < 0.05 from contralateral control muscles.

TRAINING INCREASES CPT II MRNA E279

Trained Control

f------m i

28s - / i

I

18s - :

Fig. 1. Northern blot analysis of carnitine palmitoyltransferase (CPT) II mRNA in rat plan&is muscle after 14 days of treadmill running training (100 miniday). 32P-labeled antisense RNA was synthesized in vitro from CPT II cDNA and hybridized to 20 pg extracted RNA (see MATERIALS AND METHODS) from plantaris muscles of trained and control rats. Positions of 28s and 18s rRNA are shown. Relative integrated optical density of specific bands was determined ( - 2580 nucleotides indicated by arrow; see Table 1 for results).

mRNA was 63% (P < 0.001) greater per unit of ex- tracted RNA in the stimulated muscle (Table 1; Fig. 2). Because total RNA per gram of wet weight was 51% higher (P < 0.01) in the stimulated muscle, CPT II mRNA per gram of muscle wet weight and per whole

18s-

Fig. 2. Northern blot analysis of CPT II mRNA in rat tibialis anterior muscle after 9 days of chronic nerve stimulation (10 Hz, 24 h/day). 32P-labeled antisense RNA was synthesized in vitro from CPT II cDNA and hybridized to 20 pg extracted RNA (see MATERIALS AND METHODS) from chronic stimulated and sham-operated contralateral control (contra control) muscle and from unoperated control rat tibialis anterior muscle. Relative integrated optical density of specific bands was determined ( - 2580 nucleotides indicated by arrow; see Table 1 for results).

muscle were 221 and 137% greater, respectively, than in the sedentary control rat (P < 0.001).

Interestingly, significant changes occurred in muscles of the nonstimulated contralateral leg. Comparisons of these muscles with muscles from the unoperated un- stimulated control group revealed decreases of 52% (P < O.OOl), 40% (not significant), and 42% (P < 0.05) in CPT II mRNA per unit of extracted RNA, in CPT II mRNA per unit of muscle wet weight, and in CPT II mRNA per whole muscle, respectively (Table 1). Contra- lateral effects from chronic stimulation have previously been observed for alterations in the compositions of myosin protein isoforms and Ca2+-adenosinetriphospha- tase (ATPase) isoform mRNAs (see Ref. 15 and refer- ences therein). Leberer et al. (15) indicate that the reasons for these contralateral effects during chronic stimulation are unknown, but they speculate that the effect on the contralateral side could be caused by reflex activity from the stimulated side or by unusual long- term positions of the animal with altered loads on the contralateral dorsiflexors.

DISCUSSION

The novel observation in these studies is that in- creased contractile activity was associated with greater level of CPT II mRNA. CPT II is one of the three enzymes involved in the transfer of long-chain fatty acids from the cytoplasm to the mitochondrial matrix. A pretranslational response of these enzymes to increased contractile activity in skeletal muscle has not been reported previously. The observation of higher CPT II mRNA suggests an increase in CPT II transcription, an increase in CPT II mRNA processing, an increase in CPT II mRNA stability, or any combination of these three possibilities.

Previously, Mole et al. (19) reported that the capacity of gastrocnemius and quadriceps muscles for oxidizing palmitate, oleate, linoleate, palmityl CoA, and palmityl carnitine doubled in rats that had undergone a 12-wk program of treadmill running in which the rats were running for 2 h/day at the end of training. Two impor- tant consequences of a training-induced increase in the capacity for oxidizing fatty acids may be improved submaximal exercise endurance and body fat reduction. Aerobic training increases mitochondrial density in skeletal muscles, which then oxidize more fatty acids during submaximal low-intensity exercise; as a result, the duration of work to exhaustion is prolonged during exercise (see Refs. 8 and 24 and references therein). Endurance training affects fatty acid mobilization and oxidation independently. Issekutz et al. (11) have sug- gested that supply of fatty acids from adipose tissue is rate-limiting for fatty acid utilization. However, Kiens et al. (12) later reported that the trained leg of a subject has a greater uptake of free fatty acid (FFA) at a given delivery of FFA than that observed in the contralateral nontrained leg, which implies that endurance training can enhance the process of extraction of the supplied fatty acids by the muscles. Thus, as Saltin and Astrand (24) have stated, it is not yet possible to identify the relative role of the various adaptations that could

E280 TRAINING INCREASES CPT II MRNA

explain the shift toward greater fatty acid oxidation after training. Nonetheless, the increase in fatty utiliza- tion that results from endurance exercise training has health benefits. Despres et al. (5) have shown a greater loss of abdominal fat than midthigh adipose tissue in obese women after 14 mo of training that consisted of aerobic exercise for 90 min each day, 4-5 times/wk. Shimomura et al. (27) have reported a decrease in mesenteric, but not subcutaneous, fat in rats that run 1 h/day for 7 days. Preferential loss of abdominal fat is beneficial because a high accumulation of abdominal fat is associated with an increased risk of coronary heart disease (14).

The higher level of CPT II mRNA indicates that contractile activity exerts pretranslational control for CPT II synthesis. The absolute increase in CPT II mRNA concentration was about six times higher in chronically stimulated tibialis anterior muscles than in run-trained plantaris muscles. Results from various laboratories have similarly shown that nuclear-encoded mRNAs undergo a greater percentage increase for mito- chondrial proteins in chronic stimulation than run training. Previous reports have indicated that chronic stimulation leads to increased levels of mRNAs for nuclear encoded mitochondrial proteins: P-subunit of FIATPase mRNA increases by 165% (29), citrate syn- thase mRNA increases by 240-600% (1, 26), cyto- chrome b mRNA increases by 564% (29, 30), and subunits III and VIc of cytochrome oxidase mRNA increase by lOO-200% (9,29). The present results show a 221% higher CPT II mRNA concentration in chroni- cally stimulated muscle. In contrast to chronic electrical stimulation, most studies based on endurance training by treadmill running have been unable to demonstrate an increase in the mRNA of the nuclear-encoded mito- chondrial proteins in rat skeletal muscles. Acyl-CoA synthetase mRNA (27), 5’-aminolevulinate synthase mRNA (28), and subunits 111 and W of cytochrome oxidase mRNA (28) have been found not to increase after training by running on treadmills. However, a number of studies do report increases in mRNAs for mitochondrial proteins with treadmill running. CPT II mRNA does increase by 50% (Table l), cytochrome c mRNA increases by 18-56% (20), and cytochrome oxi- dase subunit III and WmRNAs increase 100% (16) after treadmill training.

The present study demonstrates a pretranslational regulation of CPT II mRNA in skeletal muscle by contractile activity. This mitochondrial protein, which is encoded by the nuclear genome, has an increased tran- scription, an increased mRNA processing, or an en- hanced stability of mRNA as a result of the increase in muscle contractile activity induced by treadmill running and chronic electrical stimulation. Fiber recruitment in treadmill running is often not of sufficient duration or intensity to invoke a demonstrable alteration in all mRNA concentrations for nuclear-encoded mitochon- drial proteins. On the basis of the present results, it is likely that the higher CPT II mRNA plays some role in the previously reported adaptive increases in fatty acid oxidation in endurance-trained skeletal muscle (12. 19).

We thank Drs. Dennis McGarry and Jeanie McMillin for helpful discussions and for the carnitine palmitoyltransferase II cDNA. We thank Mei-Hua Liu for technical assistance.

This research was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR-19393.

Present address for Z. Yan and S. Salmons: Dept. of Human Anatomy and Cell Biology, The University of Liverpool, PO Box 147, Liverpool L69 3BX, United Kingdom.

Address for reprint requests: F. Booth, Dept. of Physiology and Cell Biology, University of Texas-Houston Health Science Center, 6431 Fannin St., Rm. 4.100 MSB, Houston, TX 77030.

Received 28 June 1994; accepted in final form 20 September 1994.

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