The Journal of Nutrition Vol. 128 No. 5 May 1998, pp. 886-893

Carnitine Palmitoyltransferase I (CPT I) Activity and Its Regulation by Malonyl-CoA Are Modulated by Age and Cold Exposure in Skeletal Muscle Mitochondria from Newborn Pigs^1,2

Isabelle Schmidt and Patrick Herpin³

INRA, Station de Recherches Porcines, 35590 Saint Gilles, France

	ABSTRACT

Abstract Introduction Methods Results Discussion References

Whole-body lipid utilization is progressively enhanced during the first postnatal day in pigs, especially during cold exposure and muscular shivering thermogenesis. This study was designed to examine early postnatal changes in fatty acid oxidation potential, carnitine palmitoyltransferase I activity and regulation by malonyl-CoA in skeletal muscle mitochondria isolated from newborn and 5-d-old piglets. At 5 d of life, pigs were maintained for a 4-h period in thermoneutral (30°C) or cold (20°C) conditions. Intermyofibrillar and subsarcolemmal mitochondria were isolated from longissimus dorsi and rhomboïdeus muscles. In subsarcolemmal mitochondria, carnitine palmitoyltransferase I activity increased with age (P < 0.01) and was 80% lower (P < 0.001) than in intermyofibrillar mitochondria. Intermyofibrillar mitochondria had high enzyme activities and fatty acid oxidation potential from birth. The fatty acids 16:0, 18:1(n-9) and 18:2(n-6) were oxidized at a higher rate than 18:0 (37%) and 8:0 (55%). Sensitivity of carnitine palmitoyltransferase I to malonyl-CoA inhibition and malonyl-CoA levels decreased by 47% (P < 0.05) and 33% (P < 0.01) with age, respectively. After 4 h of cold exposure, sensitivity of carnitine palmitoyltransferase I to malonyl-CoA was unaffected in the rhomboideus and tended to be greater (P < 0.06) in longissimus dorsi muscle. Malonyl-CoA levels were lower (P < 0.05) in the rhomboideus and were unaffected in longissimus dorsi muscle. These results demonstrate that fatty acid oxidation is effective from birth in isolated intermyofibrillar mitochondria. The postnatal enhancement of fatty acid utilization observed in vivo can be explained, at least in part, by a rise in carnitine palmitoyltransferase I activity in subsarcolemmal mitochondria and a modulation of its activity by malonyl-CoA in intermyofibrillar mitochondria.

	INTRODUCTION

Abstract Introduction Methods Results Discussion References

The perinatal period is accompanied by dramatic modifications of the climatic and nutritional environments in pigs. First, the rapid exchange of heat across the placenta provides the fetus with a thermostable environment, whereas the newborn pig is naturally exposed to cold immediately after birth. In the absence of brown fat, newborn pigs maintain their body temperature almost exclusively by shivering (Berthon et al. 1994), and skeletal muscles therefore play a key role in preserving homeothermy. Second, the fetus is continuously supplied with carbohydrate, whereas at birth, the newborn has to withstand a brief period of starvation before being fed at intervals with colostrum and milk that constitute a high fat, low carbohydrate diet. Indeed, body and colostral carbohydrate are the predominant sources of energy utilized by newborn pigs during the first postnatal hours, but the progressive decline of the respiratory quotient during the first postnatal day provides evidence for the early and increasing importance of lipids as an energy source during this period (Berthon et al. 1993, Noblet and Le Dividich 1981). The decline of the respiratory quotient with age is even more pronounced in the cold, that is, during intense shivering thermogenesis. This suggests that the contribution of fatty acids to the energy needs of the muscle cell is progressively enhanced. The low utilization of fatty acids as an energy source during the early neonatal period in fed piglets could be due to either a limited fatty acid oxidation potential or a preferential utilization of other substrates, i.e., carbohydrate (Duée et al. 1988). In the liver, quantitative and functional changes in isolated mitochondria during the first week of life were shown by Mersman et al. (1972). Duée et al. (1994) showed that the limited capacity for hepatic fatty acid oxidation in suckling newborn pigs is due to a very low amount and activity of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-⁴CoA synthase, EC 4.1.3.5). In skeletal muscle, we showed recently that subsarcolemmal (SS) and intermyofibrillar (IM) mitochondria are functional from birth and change quantitatively with age (Schmidt and Herpin 1997). There is no available information on fatty acid oxidation by skeletal muscle mitochondria in newborn pigs and its modulation by cold exposure, but it is generally accepted that fatty acid oxidation develops rapidly after birth in the heart (Ascuitto et al. 1989). In addition, in the liver, the entry of long-chain fatty acids into the mitochondria is ensured by the carnitine palmitoyltransferase system; carnitine palmitoyltransferase I (CPT I, EC 2.3.1.21) and its regulation by malonyl-CoA play a pivotal role in the regulation of fatty acid oxidation (McGarry and Forster 1980). In nonlipogenic tissues, such as skeletal muscle, some observations suggest that malonyl-CoA/CPT I interactions may be important in the regulation of fatty acid oxidation during muscle contraction (Winder and Hardie 1996). The possibility that such a regulatory mechanism is involved in the modulation of fatty acid utilization with increasing age or during muscular shivering thermogenesis has not been investigated in pigs.

Therefore, as part of an ongoing program on the effect of age and cold exposure on skeletal muscle energy metabolism in piglets, this study was designed to examine fatty acid oxidation and CPT I activity and regulation by malonyl-CoA in skeletal muscle mitochondria, at birth and 5 d of age, in pigs exposed to thermal neutrality or to the cold. Postnatal changes were followed separately on IM and SS mitochondria isolated from a slow-oxidative, rhomboïdeus (RH) and a fast-glycolytic, longissimus dorsi (LD) muscle.

	MATERIALS AND METHODS

Abstract Introduction Methods Results Discussion References

Animals.  Forty-eight Piétrain × Large White crossbred piglets from the INRA (Institut National de la Recherche Agronomique) herd were used in the experiment. Parturition was induced with an intramuscular injection of a prostaglandin analog on d 112 of gestation, to ensure farrowing on d 113. Sows and piglets were kept under normal farrowing house conditions until the beginning of the experiment.

At birth, piglets of average body weight were killed immediately (1.6 kg, n = 10). At 5 d of life, piglets were weighed and only those exhibiting an average growth rate of 0.12 kg/d between birth and 5 d of life were selected for the experiment. They were removed from the sow immediately after suckling and maintained for a 4-h period in a respiratory chamber either in thermoneutral (TN, 30°C, n = 10, mean body weight, 2.21 kg) or in cold (C, 20°C, n = 10, mean body weight, 2.18 kg) conditions. Piglets were anesthetized by halothane inhalation and then killed by exsanguination. LD and RH muscles were immediately removed for subsequent determination of the changes in IM and SS mitochondria respiration, rate of ATP synthesis, fatty acid oxidation, CPT I activity, sensitivity to malonyl-CoA inhibition and -hydroxyacyl-CoA dehydrogenase (-OHD, EC 1.1.1.35) activity. Data related to the integrity and purity of isolated mitochondria and to the postnatal changes in mitochondrial protein mass and respiration in skeletal muscles from those piglets were presented in our preceding paper (Schmidt and Herpin 1997). Mitochondria were also isolated from the liver of piglets maintained in thermoneutral conditions at 5 d of life to measure CPT I activity, sensitivity to malonyl-CoA inhibition and fatty acid oxidation. Because of the large number of measurements performed on freshly isolated mitochondria, only one piglet was killed each day; therefore, all of the piglets were removed from different sows.

Eighteen additional piglets from a different group of sows were used at birth (n = 6) and at 5 d of life in TN (n = 6) and C (n = 6) conditions. LD and RH muscles were sampled under anesthesia and frozen in liquid nitrogen (within 30 s) to determine muscle content of malonyl-CoA and ATP, as well as acetyl-CoA carboxylase activity (ACC, EC 6.4.1.2).

Isolation of muscle and liver mitochondria.  Muscle IM and SS mitochondria exhibit differences in respiration intensity, coupling state and possibly biological function, and were therefore isolated separately by differential centrifugation as previously described (Herpin et al. 1996). Liver mitochondria were isolated as described by Mersman et al. (1972). Briefly, livers were rinsed in a medium containing 220 mmol/L mannitol, 70 mmol/L sucrose, 2 mmol/L HEPES and 0.1 mmol/L EDTA (pH 7.4). All processing steps were conducted at 4°C. After mincing, liver slices were homogenized in an ice-cold Teflon-pestle-glass Potter-Elvehjem homogenizer and centrifuged successively at 700 × g and twice at 10,000 × g for 10 min. The final pellet of mitochondria was resuspended in the isolation medium at a concentration of 20 g protein/L.

Mitochondrial oxidation of fatty acids.  Fatty acid oxidation in isolated mitochondria (0.5 g protein/L for IM and liver mitochondria and 1 g protein/L for SS mitochondria) was measured polarographically at 25°C using a Clark O₂ electrode (Oxygraph Hansatech, London, UK) in 1 mL of respiratory medium containing 75 mmol/L sucrose, 30 mmol/L KCl, 20 mmol/L KH₂PO₄, 1 mmol/L EDTA, 6 mmol/L MgCl₂, 0.1 mmol/L CoASH, 0.5 mmol/L ATP, 0.5 mmol/L GDP and 5 mmol/L malate (pH 7.4). These cofactors, i.e., CoASH, ATP, GDP and malate, are necessary to optimize the successive steps of fatty acid utilization (translocation into the mitochondrial matrix, -oxidation and Krebs cycle) (Novak et al. 1975).

Measurements were performed under both coupled and uncoupled conditions. Under uncoupled conditions [100 mmol/L 2,4-dinitrophenol, i.e., when the control exerted by the respiration chain upon the overall oxidative process has been removed (Escriva et al. 1986)], the maximal oxidation rate of the following fatty acids was compared: 40 µmol/L octanoyl-CoA (8:0), 20 µmol/L palmitoyl-CoA (16:0), 20 µmol/L stearyl-CoA (18:0), 20 µmol/L oleyl-CoA [18:1(n-9)], 20 µmol/L linoleyl-CoA [18:2(n-6)], 20 µmol/L palmitoyl-carnitine, in the presence of 2 mmol/L DL-carnitine for octanoyl-CoA and 1 mmol/L DL-carnitine for the other CoA esters. Concentration of octanoyl-CoA was two times higher than that of palmitoyl-CoA to provide the same amount of carbon atoms in the medium. Because of the limited amount of isolated SS mitochondria, these measurements were performed only on IM mitochondria. This oxidative potential was measured in natoms O/(min·mg mitochondrial protein), and was then expressed as a relative percentage of palmitoyl-CoA oxidation at birth.

Coupled conditions [10 g/L bovine serum albumin (BSA)], which reflect more closely what is actually occurring in vivo, were used to compare oxidation of 20 µmol/L palmitoyl-CoA + 1 mmol/L carnitine and 20 µmol/L palmitoyl-carnitine. The measurements give an indirect estimation of the metabolic control exerted by CPT I on fatty acid oxidation. Preliminary experiments have shown that under these conditions, oxygen consumption is directly related to the intensity of fatty acid oxidation and not to the uncoupling effect of those fatty acids because response to ADP addition was still effective at the end of the assay. Results are expressed as natoms O/(min·mg mitochondrial protein).

Mitochondrial ATP production rate.  Mitochondrial ATP production rate was determined by bioluminometry as described by Wibom and Hultman (1990). The method is based on the reaction of ATP with firefly luciferase, providing a light signal proportional to the concentration of ATP in the solution. Luminescence is measured using a bioluminometer (Luminometer 1250, Bio-Orbit, Turku, Finland) and an ATP Monitoring Reagent kit (Bio-Orbit). ATP synthesis was measured at 25°C in a reaction medium containing 80 µL of ATP Monitoring Reagent kit and 320 µL of medium containing 200 mmol/L sucrose, 5 mmol/L KH₂PO₄ and 20 mmol/L Tris acid. Mitochondrial suspensions (20-40 µL) diluted 1:500 were added to the medium as well as 500 µmol/L GDP, 5 µmol/L Malate and 100 µmol/L CoASH. Mitochondria were preincubated for 2 min with the following substrates: 20 µmol/L palmitoyl-CoA + 1 mmol/L carnitine or 20 µmol/L palmitoyl-Carnitine. After the addition of 5 µL of ADP (10 mmol/L), ATP production rate was monitored for 4 min. An internal ATP standard (20 µL, 200 pmol) was added to each assay at the end of the monitoring period. Measurements were also performed with sonicated mitochondria incubated under similar conditions to estimate ATP synthesis through adenylate kinase activity and unspecific reactions. This value was subtracted from the corresponding ATP synthesis rate and results were expressed as nmol ATP/(min·mg of mitochondrial protein).

Enzyme activities.  CPT I activity and sensibility to malonyl-CoA inhibition. CPT I activity from freshly isolated mitochondria was assayed at 30°C as the formation of [³H]-palmitoyl-L-carnitine from [methyl-³H]-carnitine and palmitoyl-CoA according to Bremer (1981) and Herbin et al. (1987). The sensitivity of CPT I to malonyl-CoA inhibition was estimated by measuring the concentration of malonyl-CoA required for 50% inhibition of the enzyme activity (IC₅₀).

Mitochondria (0.5-1 mg mitochondrial protein) were preincubated for 3 min alone or in the presence of malonyl-CoA (0.01-150 µmol/L) in 0.45 mL of a reaction medium containing 75 mmol/L KCl, 50 mmol/L mannitol, 25 mmol/L HEPES, 2 mmol/L KCN, 0.2 mmol/L EGTA, 1 mmol/L dithiothreitol, 10 g/L fat-free BSA, 80 µmol/L palmitoyl-CoA (pH 7.3) and then incubated for 6 min with 1 mmol/L L-carnitine and 37 kBq L-[methyl-³H]carnitine. The reaction was stopped by the addition of 1 mL butanol. After the addition of 1 mL saturated (NH₄)₂SO₄, extraction of [³H]-palmitoylcarnitine was performed by vortexing every 10 min for 1 h. Each sample was then centrifuged at 2500 × g (15°C) for 10 min. Subsequently, 0.8 mL of the butanol phase was transferred to a tube containing 1 mL saturated (NH₄)₂SO₄. The tubes were then vortexed and centrifuged at 2500 × g (15°C) for 10 min; 0.4 mL of the butanol phase was transferred to a scintillation vial, mixed with 5 mL of Ultima Gold scintillator fluid (Packard, Groningen, The Netherlands) and counted in a liquid scintillation counter (Tri-Carb 1600, Packard). Values of CPT I activity and IC₅₀ are expressed as nmol palmitoylcarnitine produced/(min·mg mitochondrial protein) and µmol/L, respectively. Maximum inhibition with 150 µmol/L of malonyl-CoA was 94.9 ± 1.0 and 95.2 ± 1.7% for IM and liver mitochondria, respectively.

ACC activity. The assay measured the incorporation of [¹⁴C]-bicarbonate into malonyl-CoA as described by Charkrabarty and Leveille (1969). Values are expressed as nmol bicarbonate formed/(min·g wet muscle).

-OHD activity. -OHD activity was determined by the procedure of Bass et al. (1969). Frozen isolated mitochondria (7.5-15 µg of mitochondrial protein) were placed in 190 µL of reaction medium containing 100 mmol/L triethanolamine, 5 mmol/L EDTA, 0.45 mmol/L NADH and 0.1 mmol/L acetoacetyl CoA (pH 7). The disappearance of NADH was measured at 340 nm and 30°C by spectrophotometry. Results are expressed as nmol/(min·mg mitochondrial protein).

Muscle content of ATP.  ATP level was determined by bioluminometry and a method adapted from Lundin et al. (1986). Frozen muscle powder (0.5 g) was vortexed with 4 mL of cold 153 mmol/L trichloroacetate (TCA) and 2 mmol/L EDTA buffer for 1 min, then incubated for 15 min at room temperature. After a 10-min centrifugation at 3500 × g (4°C), supernatant samples were diluted 1:100 in 100 mmol/L Tris-acetate and 2 mmol/L EDTA buffer (pH 7.75). ATP concentration was measured at 25°C in a reaction medium containing 150 µL of ATP monitoring Reagent kit (Bio-Orbit), 600 µL of Tris-acetate EDTA buffer and 20 µL of sample, before and after the addition of an internal ATP standard (25 µL, 250 pmol). Values are expressed as nmol/(min·mg of muscle).

Malonyl-CoA concentration.  Malonyl-CoA concentration was determined by reversed-phase high performance liquid chromatography (HPLC) as described by King et al. (1988) and modified by Saddick et al. (1993). An aliquot (1 g) of the frozen tissue was powered under liquid nitrogen and placed into a centrifugation tube. Two milliliters of 1.4 mol/L perchloric acid containing 200 mmol/L isobutyryl-CoA as internal standard were added to the powder. The mixture was homogenized for 2 min on ice and centrifuged at 12,000 × g (4°C) for 10 min. The pH of the resulting supernatant was adjusted to ~3 with 356 mmol/L KOH, centrifuged at 1000 × g (4°C) for 10 min and frozen at 80°C until further analysis. Finally, the pH of the samples was adjusted to ~6 just before the identification of the CoA esters by HPLC.

Statistical analysis.  Data were analyzed by the General Linear Models procedure of SAS (1989). Three-way ANOVA was used to test the effect of age, muscle type and cold stress on enzyme activities, ATP level and malonyl-CoA concentration, and the effect of age, muscle type and type of ester on fatty acid oxidation under coupled conditions. Fatty acid oxidation potential under uncoupled conditions was analyzed as follows: ANOVA was performed to test the effect of age, muscle type and cold stress on each fatty acid, and oxidation rates of different fatty acids were compared by using Duncan's means separation test (SAS 1989) at an  value of 0.05.

	RESULTS

Abstract Introduction Methods Results Discussion References

Mitochondrial respiration, amount of mitochondrial proteins, and cytochrome oxidase (EC 1.9.3.1) and creatine kinase (EC 2.7.3.2) activities in skeletal muscle from these pigs were presented in a preceding paper (Schmidt and Herpin 1997).

Fatty acid oxidation rate in IM mitochondria.  Mitochondrial oxidation of various fatty acids with different carbon chain length and saturation was obtained by measuring oxygen consumption under uncoupled conditions. As shown in Figure 1, 18:1(n-9), 16:0 and 18:2(n-6) were oxidized more rapidly (P < 0.05) than 18:0 and 8:0 in LD muscle at birth. At 5 d, 16:0 and 18:2(n-6) were 18% (P < 0.05) and 26% (P < 0.001) less oxidized than at birth, respectively. In RH muscle at birth, the highest rate of oxidation was obtained when 18:1(n-9), 16:0 and 18:2(n-6) were added to the medium. In addition, 8:0 was the most poorly oxidized of the five fatty acids studied. Its oxidation rate represented only 34% of that of 16:0; intermediary values were found for the oxidation of 18:0. At 5 d of life, 18:2(n-6) was less oxidized than at birth (P < 0.01). The same rates of fatty acid oxidation were obtained after 4 h of cold stress and after exposure to a thermoneutral ambient temperature (Fig. 1).

View larger version (34K): [in this window] [in a new window]   Fig 1. Fatty acid oxidation rate in intermyofibrillar (IM) mitochondria from longissimus dorsi (LD) and rhomboideus (RH) muscle of newborn pigs at birth and 5 d of age. Values are means ± SEM, n = 6. Piglets (5 d old) were maintained for a 4-h period in thermoneutral (5 d TN) or in cold (5 d C) conditions. There were no differences between LD and RH muscles; the absolute oxidation rate of 16:0 at birth was 41.2 ± 3.8 and 40.5 ± 2.0 natoms O/(min·mg mitochondrial protein) in LD and RH muscle, respectively. There was no effect of cold stress. a, b, c: Effect of fatty acid chain length at birth; bars with different letters are significantly different (P < 0.05). A: oxidation rates of 16:0 (P < 0.05) and 18:2(n-6) (P < 0.001) were lower in muscles from 5-d-old piglets than from newborn piglets.

CPT I activity and sensitivity to malonyl-CoA inhibition (IC₅₀ ).  Activity of CPT I was 91 and 62% lower (P < 0.001) in SS than in IM mitochondria in both muscles at birth and at 5 d of age, respectively (Table 1). There was no effect of age or muscle type on CPT I activity in IM mitochondria. On the contrary, in SS mitochondria, CPT I activity was always lower in LD than in RH muscle (P < 0.001) and increased by about 600 and 200% in LD and RH muscles, respectively, within 5 d. Because of the limited amount of available biological material in the SS mitochondria subpopulation, IC₅₀ determination was performed only on IM mitochondria. As shown in Table 1, IC₅₀ increased by about 90% (P < 0.05) in both muscles with age. These measurements were also performed on liver mitochondria. CPT I activity and IC₅₀ in the liver of 5-d-old piglets were 1.72 ± 0.08 nmol/(min·mg mitochondrial protein) and 0.103 ± 0.015 µmol/L, respectively. Activity of the enzyme in the liver did not differ from that of IM mitochondria, whereas its sensitivity to malonyl CoA was 50 times greater. Cold stress had no effect on CPT I activity but tended to decrease IC₅₀ in LD muscle (40%, P < 0.06).

ACC activity and malonyl-CoA level.  ACC, the enzyme that synthesizes malonyl-CoA, was assayed in skeletal muscle homogenates (Table 2). ACC activity did not differ in the muscles at birth, whereas it increased by 166 and 373% in LD and RH muscle, respectively, within the first 5 d of life (P < 0.001). Consequently, ACC activity was ~70% higher (P < 0.01) in RH than in LD muscle at 5 d of life. There was no effect of cold stress on ACC activity.

The highest level of malonyl-CoA was observed at birth, averaging 11.6 and 13.1 nmol/g in LD and RH muscle, respectively (Table 2). It decreased by 27% (P < 0.01) and 42% (P < 0.001) between birth and 5 d in LD and RH muscle, respectively. Further, cold stress led to a 27% (P < 0.05) reduction in malonyl-CoA concentration in RH muscle, whereas no changes were observed in LD muscle. Consequently, malonyl-CoA levels were 30% lower in RH than in LD muscle at 5 d of life in cold-stressed piglets (P < 0.05).

ATP level.  Muscle concentration of ATP did not differ in the two muscles at either age (Table 2). No changes were observed with increasing age or cold stress in RH muscle, whereas ATP concentration was 20% higher in LD muscle from cold-stressed piglets.

-OHD activity.  At birth, -OHD activity was much higher in IM than in SS mitochondria (~ +220%, P < 0.05) (Table 3). At 5 d of life, this difference was not significant, because in SS mitochondria, -OHD activity tended to increase with age in LD muscle (+59%, P < 0.07) and was unchanged in RH muscle, whereas in IM mitochondria, -OHD activity decreased by about 35% in both muscles with age (P < 0.05). Cold stress had no effect on LD muscle but tended (P < 0.08) to increase the activity of the enzyme in IM mitochondria from RH muscle; this increase was significant in SS mitochondria (P < 0.05). Finally, -OHD activity was always higher in RH than in LD muscle (P < 0.01).

View this table: [in this window] [in a new window]   Table 3. Effect of age and cold stress on -hydroxyacyl-CoA deshydrogenase (-OHD) activity in subsarcolemmal (SS) and intermyofibrillar (IM) mitochondria from longissimus dorsi (LD) and rhomboideus (RH) muscles of piglets1

Oxygen consumption rate and ATP synthesis from palmitoyl-CoA and palmitoyl-carnitine.  Oxidation of palmitoyl-CoA, which has to be transferred into the mitochondria by the CPT I system, was compared with that of palmitoyl-carnitine (Table 4). Oxidation of both substrates, which was assessed from mitochondrial oxygen consumption and ATP synthesis rate, was at least 50% lower in SS than in IM mitochondria (P < 0.001) at both ages. Oxidation of the CoA ester represented 48 and 70% of that of the carnitine ester in SS and IM mitochondria, respectively, at birth. Corresponding values of 55% (P < 0.05) and 50% (P < 0.01) are obtained when the rate of ATP synthesis is taken into account. By contrast, in the liver, oxidation of the CoA ester represented 98% of that of the carnitine ester (data not shown). In SS mitochondria from LD muscle, oxygen consumption and ATP synthesis rates increased by 30% with age, whereas they remained constant in SS mitochondria from RH muscle. Different results were obtained on IM mitochondria, with a decrease in the oxygen consumption rate in LD muscle and in ATP synthesis rate in RH muscle between birth and 5 d of life (P < 0.05). However, as a whole, there were no significant differences between LD and RH muscle. Cold stress had no effect on oxygen consumption rate or ATP synthesis.

	DISCUSSION

Abstract Introduction Methods Results Discussion References

These results clearly demonstrate that fatty acid oxidation is effective from birth in IM mitochondria from piglet skeletal muscle. The postnatal enhancement of fatty acid utilization previously observed at the whole-body level can be explained, at least in part, by a rise in CPT I activity in SS mitochondria and a modulation of its activity by malonyl-CoA in IM mitochondria. During cold stress, the muscle-specific decrease in malonyl-CoA observed in RH muscle could partly relieve CPT I inhibition.

CPT I activity in skeletal muscle and liver of newborn pigs.  CPT I activity and sensitivity to malonyl-CoA has been measured in different tissues (Mills et al. 1983) and several species (McGarry et al. 1983, Saggerson and Carpenter 1981), but no study concerned pig skeletal muscle. Duée et al. (1994) measured CPT I activity and regulation by malonyl-CoA in mitochondria isolated from the liver of 2-d old starved piglets and adult rats by the method of Bremer (1981). We used the same procedure on skeletal muscle mitochondria and also performed these measurements on liver mitochondria from 5-d old piglets to allow comparison between the studies. Results obtained on liver mitochondria are consistent with those of Duée et al. (1994) and confirmed that the sensitivity of liver CPT I to malonyl-CoA inhibition is 10-30 times higher in pigs than in other species, except guinea pigs (McGarry et al. 1983). This higher sensitivity of liver CPT I in pigs is effective in both starved (Duée et al. 1994) and fed (this study) conditions. There were no major differences in CPT I activity between IM and liver mitochondria from newborn pigs and adult rats (Duée et al. 1994), whereas activity was much lower at birth in SS mitochondria. In addition, we have compared the oxidation rates of palmitoyl-CoA and palmitoylcarnitine, which use or bypass, respectively, the step catalyzed by CPT I, and found no difference between both substrates in the liver, as shown by Duée et al. (1994). However, the oxidation rate of the CoA ester represented 70% of that of the carnitine ester in IM mitochondria and only 48% in SS mitochondria, in agreement with the difference in CPT I activity between populations. This suggests that the step catalyzed by CPT I is more rate limiting for long-chain fatty acid oxidation in SS than in IM mitochondria.

Marked differences were also noted with regard to the sensitivity of the enzyme to malonyl-CoA. In IM mitochondria, IC₅₀ was 30-50 times higher than in the liver of piglets (Duée et al. 1994, present results) and in the muscle of other species (McGarry et al. 1983). In other words, sensitivity of CPT I to malonyl-CoA was much higher in the liver than in skeletal muscle in pigs, with the opposite observed in rats. We have no explanation for this difference between species. However, in rats, two immunologically distinct isoforms of CPT I, which exhibit markedly different sensitivity to malonyl-CoA, are present in skeletal muscle and liver (Weis et al. 1994b), and both isoforms are expressed simultaneously in the heart during the neonatal period (Weis et al. 1994a). Therefore, one can postulate that in newborn pigs, the low sensitivity of muscle CPT I to malonyl-CoA could also be due to the presence of other isoforms of CPT I with different kinetic characteristics with respect to malonyl-CoA inhibition.

Effect of age and cold stress on skeletal muscle CPT I activity and regulation.  IM mitochondria. There was no change in CPT I activity with age, but there was a rise in IC₅₀ between birth and 5 d of life in LD and RH muscle. Interestingly enough, this was associated with a decrease of the tissue levels of malonyl-CoA. Similarly, in rat liver, an increase of IC₅₀ has been observed during late fetal life (Saggerson and Carpenter 1982). Taking into account the fact that the intracellular water space in skeletal muscle is ~0.8 mL/g wet weight (McMeekan 1940), it follows that the concentration of malonyl-CoA in skeletal muscle ranges between ~8 and 16 µmol/L and exceeds the IC₅₀ of IM mitochondria in pig skeletal muscle. If the response of CPT I to malonyl-CoA in the intact muscle is similar to the response obtained in isolated mitochondria, it would be strongly inhibited by malonyl-CoA as previously suggested by Winder and Hardie (1996) for rat skeletal muscle and Awan and Saggerson (1993) for myocytes. However, as suggested by Dungan et al. (1987) it is unclear at this time if muscle CPT I is exposed in vivo to the concentrations of malonyl-CoA calculated from the above-mentioned tissue content. In addition, CPT I activity in vivo may also be different due to its recently demonstrated dependence on the microenvironment of the cytosolic matrix (Guzman et al. 1994, Velasco et al. 1997). Moreover, to assess the biological importance of this regulatory system, the origin of malonyl-CoA should be established. Our results clearly show the presence of the enzyme devoted to malonyl-CoA synthesis, i.e., acetyl-CoA carboxylase, in piglet skeletal muscle. This result is consistent with the observation of Thampy (1989), demonstrating the existence of a specific isoform of acetyl-CoA carboxylase in rat heart. Surprisingly, the activity of acetyl-CoA carboxylase increased, whereas malonyl-CoA levels decreased with age, suggesting that postnatal changes in malonyl-CoA levels in skeletal muscle are related to eventual changes in its rate of utilization. Skeletal muscle is a nonlipogenic tissue, but malonyl-CoA might be involved in fatty acid elongation (Awan and Saggerson 1993) or be decarboxylated to regenerate acetyl-CoA (Kudo et al. 1995). However, our results suggest that malonyl-CoA is synthesized in skeletal muscle from the newborn pig and that its inhibitory effect on CPT I activity in IM mitochondria is weakened during the early neonatal period. Together with the enhancement of the total mitochondrial protein mass in LD (+49%) and RH (+93%) muscles with age (Schmidt and Herpin 1997), this mechanism likely contributes to the postnatal enhancement of in vivo lipid utilization in newborn pigs (Berthon et al. 1993, Noblet and Le Dividich 1981).

During cold stress and active shivering thermogenesis, i.e., muscle contraction, a muscle-specific regulatory mechanism of CPT I activity has also been identified in IM mitochondria. In the RH muscle, the sensitivity of CPT I to malonyl-CoA remained constant, whereas malonyl-CoA levels fell in the cold. Simultaneously, sensitivity of CPT I to malonyl-CoA rose and malonyl-CoA levels remained constant in LD muscle. Potentially, these changes could favor fatty acid oxidation in the most oxidative muscles during shivering thermogenesis. Interestingly, in rat red quadriceps and gastrocnemius muscles, a reduction in malonyl-CoA concentration and an enhanced rate of fatty acid oxidation (Winder and Hardie 1996) have also been observed in response to muscle contraction. However, we were not able to show an effect of cold stress on fatty acid oxidation potential in isolated mitochondria, probably because this maximal potential was measured in a malonyl-CoA-free medium.

SS mitochondria. CPT I activity was very low at birth and increased dramatically with age. In fact, CPT I behaves similarly to most other enzymes studied in our laboratory (cytochrome oxidase, creatine kinase) with regard to the differential response of SS and IM mitochondria to the effect of age (Herpin et al. 1996, Schmidt and Herpin 1997). Again, this would favor fatty acid oxidation and is likely to contribute to the postnatal enhancement of lipid utilization observed in vivo in newborn pigs. In addition, activity of CPT I was always lower in LD than in RH muscle, which is consistent with the putative difference in fatty acid oxidation between these fast glycolytic and slow oxidative muscles.

Fatty acid oxidation potential in skeletal muscle mitochondria.  The hierarchy observed in the oxidation rate of long-chain fatty acids is fairly consistent with the results obtained in vivo by Boyd et al. (1982). Oleic, linoleic and palmitic acids, which represent about 80% of the fatty acids found in sow colostrum (Herpin and Le Dividich 1995), are readily oxidized by the newborn pig and its skeletal muscle mitochondria, whereas in vivo and in vitro studies show that stearic acid is less oxidized (Boyd et al. 1982). Our results also show that octanoic acid is much less oxidized than palmitic acid in isolated mitochondria from both muscles, whereas no differences between those two fatty acids are evident in liver mitochondria (data not shown). Indeed, medium-chain fatty acids are readily oxidized in the liver, but contradictory results have been published previously with regard to octanoate oxidation in skeletal muscle. According to Groot and Hultmann (1973), rat skeletal muscle mitochondria can oxidize octanoate as well as palmitate, and this oxidation is carnitine dependent; however, Aas (1971) found no appreciable octanoate oxidation in skeletal muscle. Finally, we observed no enhancement of fatty acid oxidation potential on isolated mitochondria during cold stress and even a slight (20-30%) reduction of this potential with age for 16:0 and 18:2(n-6). As indicated earlier, this is probably because the modulation of CPT I activity by malonyl-CoA was not effective in a malonyl-CoA-free medium. However, if we take into account the postnatal increase in mitochondrial protein mass in LD (+49%) and RH (+90%) muscles from the same piglets (Schmidt and Herpin 1997), we can calculate that fatty acid oxidation potential increased by 20-40% within 5 d in piglet skeletal muscles. This result is fairly consistent with the rise of whole-body fatty acid oxidation during early postnatal development.

	ACKNOWLEDGMENTS

We gratefully acknowledge M. Fillaut and J. C. Hulin for their technical assistance during the experiment and J. P. Pégorier for the review of the manuscript.

	FOOTNOTES

Manuscript received 25 August 1997. Initial reviews completed 29 September 1997. Revision accepted 16 January 1998.

	LITERATURE CITED

Abstract Introduction Methods Results Discussion References

• Aas M. Organ and subcellular distribution of fatty acid activating enzymes in the rat. Biochim. Biophys. Acta 1971; 231:32-47 [Medline]
• Ascuitto R. J., Ross-Ascuitto N. T., Chen V., Downing S. E. Ventricular function and fatty acid metabolism in neonatal piglet heart. Am. J. Physiol. 1989; 256:H9-H15 [Abstract/Free Full Text]
• Awan M. M., Saggerson E. D. Malonyl-CoA metabolism in cardiac myocytes and its relevance to the control of fatty acid oxidation. Biochem. J. 1993; 295:61-66
• Bass A., Brdiczka D., Eyer P., Hofer S., Pette D. Metabolic differentiation of distinct muscle types at the level of enzymatic organization. Eur. J. Biochem. 1969; 10:198-206 [Medline]
• Berthon D., Herpin P., Duchamp C., Dauncey M. J., Le Dividich J. Modification of thermogenic capacity in neonatal pigs by changes in thyroid status during late gestation. J. Dev. Physiol. 1993; 19:775-781
• Berthon D., Herpin P., Le Dividich J. Shivering thermogenesis in the neonatal pig. J. Therm. Biol. 1994; 19:413-418
• Boyd R. D., Britton R. A., Knoche H., Moser B. D., Peo E. R., Johnson R. K. Oxidation rates of major fatty acids in fasting neonatal pigs. J. Anim. Sci. 1982; 55:95-100
• Bremer J. The effect of fasting on the activity of liver carnitine palmitoyltransferase and its inhibition by malonyl-CoA. Biochim. Biophys. Acta 1981; 665:628-631 [Medline]
• Charkrabarty K., Leveille G. A. Acetyl-CoA carboxylase and fatty acid synthetase activities in liver and adipose tissue of meal-fed rats. Proc. Soc. Exp. Biol. Med. 1969; 131:1051-1054 ^[Medline]
• Duée P. H., Pegorier J. P., Le Dividich J., Girard J. Metabolic and hormonal response to acute cold exposure in newborn pig. J. Dev. Physiol. 1988; 10:371-381 [Medline]
• Duée P. H., Pégorier J. P., Quant P. A., Herbin C., Kohl C., Girard J. Hepatic ketogenesis in newborn pigs is limited by low mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase activity. Biochem. J. 1994; 298:207-212
• Dungan R. E., Osterlund B. R., Drong R. F., Swenson T. L. A malonyl-CoA-binding protein from liver. Biochem. Biophys. Res. Commun. 1987; 174:234-241
• Escriva F., Ferre P., Rabin D., Rabin P., Decaux J. F., Girard J. Evidence that the development of hepatic fatty acid oxidation at birth in the rat is concomitant with an increased intramitochondrial-CoA concentration. Eur. J. Biochem. 1986; 156:603-607 [Medline]
• Groot P.H.E., Hultmann W. C. The activation and oxidation of octanoate and palmitate by rat skeletal muscle mitochondria. Biochim. Biophys. Acta 1973; 316:124-135 ^[Medline]
• Guzman M., Kolodziej M. P., Caldwell A., Corstorphine C. G., Zammit V. A. Evidence against direct involvement of phosphorylation in the activation of carnitine palmitoyltransferase by okadaic acid in rat hepatocytes. Biochem. J. 1994; 300:693-699
• Herbin C., Pégorier J. P., Duée P. H., Kohl C., Girard J. Regulation of fatty acid oxidation in isolated hepatocytes and liver mitochondria from newborn rabbits. Eur. J. Biochem. 1987; 165:201-207 [Medline]
• Herpin P., Berthon D., Duchamp C., Dauncey M. J., Le Dividich J. Effect of thyroid status in the perinatal period on oxidative capacities and mitochondrial respiration in porcine liver and skeletal muscle. Reprod. Fertil. Dev. 1996; 8:147-155 [Medline]
• Herpin, P. & Le Dividich, J. (1995) Thermoregulation and the environment. In: The Neonatal Pig. Development and Survival (Varley, M. A., ed.), pp. 57-95. CAB International, Wallington, UK.
• King M. T., Reiss P. D., Cornell N. W. Determination of short-chain coenzyme A compounds by reversed phase high performance liquid chromatography. Methods Enzymol. 1988; 166:70-79 [Medline]
• Kudo N., Barr A. J., Barr R. L., Desai S., Lopaschuk G. D. High rates of fatty acid oxidation during reperfusion of ischemic heart are associated with decrease in malonyl-CoA levels due to an increase in 5-AMP-activated protein kinase inhibition of acetyl-CoA carboxylase. J. Biol. Chem. 1995; 270:17513-17520 [Abstract/Free Full Text]
• Lundin A., Hasenson M., Persson J., Pousette A. Estimation of biomass in growing cell lines by adenosine triphosphate assay. Methods Enzymol. 1986; 133:27-42 [Medline]
• McGarry J. D., Forster D. W. Regulation of hepatic fatty acid oxidation and ketone body production. Annu. Rev. Biochem. 1980; 49:395-420 [Medline]
• McGarry J. D., Mills S. E., Long C. S., Foster D. W. Observation on the affinity for carnitine, and malonyl-CoA sensitivity, of carnitine palmitoyltransferase I in animal and human tissues. Biochem. J. 1983; 214:21-28 [Medline]
• McMeekan C. P. Growth and development in the pig, with special reference to carcass quality characters. J. Agric. Sci. Camb. 1940; 30:276-348
• Mersman H. J., Goodman J., Houk J. M., Anderson S. Studies on the biochemistry of mitochondria and cell morphology in the neonatal swine hepatocyte. J. Cell Biol. 1972; 53:335-347 ^{[Abstract/Free Full Text]}
• Mills S. E., Forster D. W., McGarry J. D. Interaction of malonyl-CoA and related compounds with mitochondria from different rat tissues. Biochem. J. 1983; 214:83-91 [Medline]
• Noblet J., Le Dividich J. Energy metabolism of the newborn pig during the first 24 h after birth. Biol. Neonate 1981; 40:175-182 [Medline]
• Novak M., Penn-Walker D., Monkus E. F. Oxidation of fatty acids by mitochondria obtained from newborn subcutaneous (white) adipose tissue. Biol. Neonate 1975; 25:95-107
• Saddick M., Gamble J., Witters L. A., Lopaschuk G. D. Acetyl-CoA carboxylase regulation of fatty acid oxidation in the heart. J. Biol. Chem. 1993; 268:25836-25845 ^{[Abstract/Free Full Text]}
• Saggerson E. D., Cappenter C. A. CPT and COT activities in liver, kidney cortex, adipocyte, lacting mammary gland, skeletal muscle and heart. FEBS Lett. 1981; 129:229-232 [Medline]
• Saggerson E. D., Carpenter C. A. Regulation of hepatic carnitine palmitoyltransferase activity during the foetal-neonatal transition. FEBS Lett. 1982; 150:177-180 [Medline]
• SAS Institute Inc. (1989) SAS User's Guide: Statistics. SAS Institute Cary, NC.
• Schmidt, I. & Herpin, P. (1997) Postnatal changes in mitochondrial protein mass and respiration in skeletal muscle from the newborn pig. Comp. Biochem. Physiol. 118B: 639-647.
• Thampy K. G. Formation of malonyl coenzyme A in rat heart. J. Biol. Chem. 1989; 264:17631-17634 [Abstract/Free Full Text]
• Velasco G., Geelen M.J.H., Guzman M. Control of hepatic fatty acid oxidation by 5-AMP-activated protein kinase involves a malonyl-CoA-dependent and a malonyl-CoA-independent mechanism. Arch. Biochem. Biophys. 1997; 337:169-175 [Medline]
• Weis B. C., Cowan A. T., Brown N., Foster D. W., McGarry J. D. Use of a selective inhibitor of liver carnitine palmitoyltransferase I (CPT I) allows quantification of its contribution to total CPT I activity in rat heart. J. Biol. Chem. 1994a; 269:26443-26448 [Abstract/Free Full Text]
• Weis B. C., Esser V., Foster D. W., McGarry J. P. Rat heart expresses two forms of mitochondrial carnitine palmitoyltransferase I. J. Biol. Chem. 1994b; 269:18712-18715 [Abstract/Free Full Text]
• Wibom R., Hultman E. ATP production rate in mitochondria isolated from microsample of human muscle. Am. J. Physiol. 1990; 259:E204-E209 [Abstract/Free Full Text]
• Winder W. W., Hardie D. G. Inactivation of acetyl-CoA carboxylase and activation of AMP activated protein kinase in muscle during exercise. Am. J. Physiol. 1996; 270:E299-E304 [Abstract/Free Full Text]

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