Stimulation of polyunsaturated fatty acid oxidation in myocytes by regulating its cellular uptake

Life Sciences, Vol. 49, pp. 185-192 Printed in the U.S.A.

Pergamon Press

STIMULATION OF POLYUNSATURATED FAq'q'Y ACID OXIDATION IN MYOCYTES BY REGULATING ITS CELLULAR UPTAKE ON THE RATE LIMITING STEP OF POLYUNSATURATED FATTY ACID OXIDATION IN HEART

Salah Abdel-aleem 1, Mostafa Badr 2, and Crist Frangalds 1 1Glaxo Inc., Department of Pharmacology, Five Moore Drive, Research Triangle Park, NC 27709 2University of Missouri-Kansas City, Department of Pharmacology Kansas City, MO 64108 (Received in final form May 14, 1991)

Summary In order to investigate the regulation of polyunsaturated fatty acid oxidation in the heart, the effect of the phosphodiesterase inhibitor enoximone on the oxidation of [1-14C] arachidonic acid, and [1-14C] arachidonyl-CoA, were studied in adult rat myoctyes, and isolated rat heart mitochondria. Enoximone stimulated arachidonate oxidation by 94%, at a concentration of 0.25 mM. The apparent Vmax value of archidonate oxidation in the presence of enoximone (6.98 nmol/mg protein/30 min), was approximately 75% higher than the value observed with the control (4.0 nmol/mg protein/30 rain) in isolated myocytes. Also, enoximone stimulated arachidonate uptake by 27% at a concentration of 0.25 mM. On the other hand, enoximone had no effect on the oxidation of [1-14C] arachidonyl-CoA in isolated rat heart mitochondria. These results suggest that the oxidation of polyunsaturated fatty acids in myocytes is regulated by the rate of uptake of these acids across sarcolemmal membranes. Although fatty acids represent the major source of energy in myocardial tissues (1), the regulation of polyunsaturated fatty acid metabolism in the heart is not fully understood. The degradation of polyunsaturated fatty acids into acetyl-CoA via the [3-oxidation pathway, requires two auxiliary enzymes in addition to the enzymes necessary for 13-oxidation of saturated fatty acids. These additional enzymes are A3-Cis-A2-trans-enoyl-CoA isomerase (EC 5.3.3.8) and 2,4-dienoyl-CoA reductase (EC 1.3.1.34) (2,3,4). However, it is not clear whether the oxidation of polyunsaturated fatty acids in the heart, is regulated by its uptake across sarcolemmal membranes, or by its mitochondrial degradation into acetyl-CoA via the 13-oxidation pathway. It was recently reported that growth hormone increases the activity of 2,4-dienoyl-CoA reductase by threefold in the isolated rat liver mitochondria (5). Furthermore, growth hormone stimulated rates of hepatic mitochondrial respiration supported by polyunsaturated fatty acylcarnitines. These findings suggest that 2,4-dienoyl-CoA reductase regulates the oxidation of polyunsaturated fatty acids in the liver. Correspondence: Dr. Salah Abdel-aleem, Glaxo Inc., Five Moore Drive, Research Triangle Park, NC 27709 0024-3205/91 $3.00 + .00 Copyright (c) 1991 Pergamon Press plc

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Recent studies at this laboratory have shown that enoximone, an inhibitor of the high affinity cyclic AMP phosphodiesterase (6,7), stimulates palmitate oxidation in myocytes by increasing its uptake rate across cellular membranes (8). The present study was undertaken to investigate the effect of enoximone on the oxidation of polyunsaturated fatty acids in myocytes and in the isolated rat heart mitochondria, which may shed light on the regulation of the oxidation of polyunsaturated fatty acids in the heart. Materials and Methods Animals: Male Sprague-Dawley rats, weighing 220-250 g, were obtained from Charles River Breeding Laboratory. Materials: [1-14C] arachidonic acid, and [1-14C] arachidonyl-CoA were purchased from New England Nuclear. Collagenase (type II) was obtained from Worthington. Sigma was the source of bovine serum albumin (essentially fatty acid free), L-carnitine, and hyamine hydroxide. Enoximone was kindly provided by Merrell-Dow Research Institute (Cincinnati, OH). Joklik minimum essential medium was purchased from Gibco Laboratories. Isolation of myocytes: Adult rat myocytes were isolated by the method of Frangakis et al. (9). Myocytes were isolated with Joklik essential medium, containing 25 mM NaHCO 3. The viability of myocytes isolated by this procedure was 80-85%, as judged by trypan blue exclusion. The same cell viability was maintained in the presence of 0.25 mM enoximone during the metabolic studies. Metabolic Studies with Myocytes: Myocytes (2 mg cell protein) suspended in 0.9 ml of Joklik essential medium, containing 25 mM NaHCO 3, 0.1 mM CaC12, and 10 mM HEPES (pH 7.4), were placed in a 25 ml Erlenmeyer flask. To this cell suspension was added 20 ~tl of the enoximone solution to give the desired concentration. After incubating the myocytes with enoximone for 5 min at 37°C, 0.1 ml of 0.2 mM [1-14C] arachidonic acid (2.7 x 105 dpm), was added to the cell suspension. Stock solution of arachidonate was prepared by dissolving arachidonic acid in a solution of defatted bovine serum albumin in the cell suspension buffer. The molar ratio of arachidonate to albumin was 4:1. The Erlenmeyer flask was then closed with a rubber septum to which a plastic center well was attached. The incubation was continued under shaking at 37°C for the indicated time periods. 0.4 ml of 1M hyamine hydroxide was injected through the septum into the center well to absorb the released CO2, and the reaction was terminated by injecting 1 ml of 10% perchloric acid through the septum into the incubation medium. Subsequently the flasks were shaken continuously for 2 hr at 37°C, at which time the plastic center well was removed, placed into a scintillation vial containing 10 ml of Scinti Verse BD, and counted in a liquid scintillation counter. Preparation of Rat Heart Mitochondria: Rat heart mitochondria were isolated by the procedure of Chapell and Hansford (10). The isolation buffer contained 0.21 M mannitol, 0.07 M sucrose, 5 mM Tris-HC1 (pH 7.4), and 1 mM EGTA. In vitro B-oxidation in rat heart mitochondria was performed according to published procedures (11, 12). The oxidation of [ 1-14C] arachidonyl-CoA was performed in a final volume of 1.0 ml, which contained 250 mM sucrose, 50 mM Tris-HC1, 120 mM KC1, 8 mM MgC12, 0.5 mM EDTA-K2, 0.1 mg/ml bovine serum albumin, 0.05 mM malate, 2 mM ADP, 2 mM L-Carnitine, 2 mM ATP and 100 IJ.M [1-14C] arachidonyl-CoA (2.8 x 104 dpm). Substrate oxidation was initiated by the addition of rat heart mitochondria (0.5-1 mg),

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which had been preincubated with or without enoximone for 5 min at 37°C, and the rate of [1-14C] arachidonate oxidation was measured by using the same procedure mentioned before for measuring the release of CO 2 with the myocytes. Proteins were measured by the Bio-Rad Protein Assay. Assay of Arachidonate Uptake: Arachidonate transport was determined by measuring the initial rate of uptake as a function of arachidonate concentration. Arachidonate was bound to BSA in a molar ratio of 4:1. At the end of the desired time, cells (1.5 mg protein preinbucated with or without 250 IxM of enoximone were separated from medium on 25 mm Nucleopore membranes using Hoefer Cell Harvester. The filters were washed twice with ice cold Joklik medium and transferred to scintillation vials containing 10 ml of Scinti-Verse BD for counting, Results Oxidation of arachidonate was monitored by the production of 14CO2 from [1-14C] arachidonic acid by isolated myocytes and trapped in 1M hyamine hydroxide. The utilization of exogenous arachidonic acid is time and concentration dependent, demonstrating linear kinetics during the first 30 min of incubation. Plateau levels of oxidation are reached after 60 minutes of incubation (data are not shown).

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Concentration of Enoximone (IxM) Fig. 1. The effect of enoximone on the oxidation of arachidonate as a function of e n o x i m o n e c o n c e n t r a t i o n . M y o c y t e s were p r e i n c u b a t e d with v a r i o u s concentrations of enoximonel(40 p ) for 5 min at 37°C. The reaction was started by the addition of 0.2 mM [1- C] arachidonate and continued for 30 min. The control value for the rate of arachidonate oxidation was 3.36 + 0.16 nmol/mg protein/30 min. Values are the mean + SD of three experiments. The phosphodiesterase inhibitor enoximone stimulates the oxidation of arachidonate in isolated myocytes in a dose and time dependent manner. Maximal stimulation of oxidation is seen at 100 ~tM, with an apparent ED50 of about 50 ~tM (Fig. 1).

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Time (min) Fig. 2. The time course of the effect of enoximone on arachidonate oxidation in myocytes. Myocytes were preincubated without ( • ) or with ( • ) 250 ~tM of enoximone for various time periods. Values are the mean of three different experiments. Fig. 2 shows that maximal stimulation of arachidonate oxidation rate is seen after 30 rain of incubation, with 75% increase. No additional increase in oxidation rates was seen with subsequent incubation times. Fig. 3 shows that the apparent Km values for arachidonate oxidation by myocytes was not significantly altered by enoximone (32 IIM for the control versus 26 tI.M in the presence of enoximone). However, the maximal rate of arachidonate oxidation was 75% higher in the presence of 250 gM enoximone. The apparent Km values for arachidonate uptake by myocytes was not significantly altered by enoximone (25 BM for the control versus 27 ~tM in the presence of enoximone). However, the apparent Vmax value for arachidonate uptake was significantly higher in the presence of 250 I.tM enoximone (3.2 nmol/mg protein/2 min in the presence of enoximone versus 2.4 nmol/mg protein/2 min for the control).

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The effect o f enoximone on the oxidation of arachidonate as a function o f arachidonate concentration in myocytes. Myocytes were preincubated without ( O ) or with ( A ) 250 I.tM of enoximone for 5 min at 37°C. At the end of this time period, various concentrations of arachidonate were added and incubations continued for 30 min at 37°C. Values are the mean of three different experiments.

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Fig. 4. The effect of e n o x i m o n e on arachidonate transport as a function o f arachidonate concentration. M y o c y t e s were preincubated with ( • ) or without ( • ) 250 ~tM of e n o x i m o n e for 15 min at 37°C. At the end o f this time, various concentrations of arachidonate w e r e added to measure arachidonate transport for 2 rain at 37°C. Values are the m e a n o f four experiments. *A result o f P < 0.05 was regarded as significant.

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Fig 5. The effect of enoximone on the oxidation of [ 1-14C] arachidonyl-CoA in isolated rat heart mitochondria. Rat heart mitochondria were preincubated without ( O ) or with ( A ) 250 p.M of enoximone for 5 min at 37°C. Reactions were started by the addition of 100 I.tM of [1-14C] arachidonyl-CoA and were continued for various time periods (10-40 min). Values are the mean of three experiments. Although we have seen in the present study a substantial increase in arachidonate oxidation rates with enoximone in intact rat myocytes, no such stimulation was observed in isolated rat heart mitochondria. The activity of the ~-oxidation pathway of polyunsaturated fatty acids by isolated rat heart mitochondria was monitored by the oxidation of [1-14C] arachidonyl-CoA. Results indicate that enoximone at a concentration up to 250 I.tM had no effect on the rate of oxidation of arachidonyl-CoA, indicating the lack of any stimulatory effect of enoximone on polyunsaturated fatty acid oxidation in mitochondria.

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Discussion The purpose of this study was to delineate the regulatory mechanisms involved in the oxidation of polyunsaturated fatty acids in the heart. In order to investigate this problem, the effect of enoximone, an inhibitor of the high affinity cyclic AMP, phosphodiesterase (6,7), on the oxidation of arachidonate was studied in isolated rat myocytes. The stimulation of palmitate oxidation by enoximone has been reported (8). Enoximone increases palmitate oxidation in myocytes by stimulating its uptake across sarcolemmal membranes. It is not known, however, whether the oxidation of polyunsaturated fatty acids in the heart is regulated by the same mechanism. In the present study, enoximone stimulated arachidonate oxidation significantly in a time and concentration-dependent manner. Recently, it was reported that the stimulation of 2,4-dienoyl-CoA reductase by growth hormone was accompanied by a parallel increase in the oxidation of polyunsaturated fatty acids in isolated rat liver mitochondria (5). In contrast, stimulation of 2,4-dienoyl-CoA reductase by growth hormone in isolated rat heart mitochondria had no effect on the oxidation of polyunsaturated fatty acids 1. These results demonstrate a tissues variation between the heart and the liver with respect to the regulation of polyunsaturated fatty acid oxidation. In the present study, enoximone had no effect on the oxidation of [1-14C] arachidonyl-CoA in isolated rat heart mitochondria (Fig. 4), supporting the contention that enoximone does not stimulate the 13-oxidation enzymes, A3-Cis-A2-trans-enoyl-CoA isomerase, or 2,4-dienoyl-CoA reductase. Furthermore, enoximone strongly inhibits the long-chain acyl-CoA synthetase in rat heart homogenates (13), which suggests that the site of stimulation of fatty acid oxidation by enoximone in myocytes is before the step of activation of long-chain fatty acids, which occurs in the cytosol. Evidence has been presented for both simple diffusion and a carrier-mediated uptake of longchain fatty acids across plasma membranes (14,15). However, in the present study, the Vmax value of arachidonate oxidation, in the presence of enoximone, was significantly higher than the value observed with the control (Fig. 3). Also, enoximone stimulated arachidonate uptake significantly at a concentration of 0.25 mM. These data suggest that enoximone increases the rate of arachidonate oxidation by increasing its uptake across the sarcolemmal membranes. The same conclusion was postulated earlier by Oram and Neely (16) for the regulation of oxidation of palmitate in the heart. They reported that palmitate oxidation in the perfused rat heart depends upon the concentration of free fatty acids and the energy demand of the heart. In conclusion, the present study shows that the oxidation of polyunsaturated fatty acids in myocytes is regulated by its rate of uptake across the sarcolemmal membranes. Also, results are in support of the findings of Nada et al. (17) that the reaction catalyzed by 2,4-dienoyl-CoA reductase is not rate-limiting in the oxidation of polyunsaturated fatty acids in the heart cells.

1Salah Abdel-aleem and Horst Schulz (unpublished data)

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References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

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