Cellular energy metabolism during fetal development

DEVELOPMENTAL

BIOLOGY

52,

161-166

(1976)

Cellular Energy Metabolism

during

VI. Fatty Acid Oxidation JOSEPH Yale

University

School

B. WARSHAW

of Medicine, Division and Gynecology,

by Developing AND MARY

of Perinatal New Haven,

Accepted

Fetal Development

March

Brain

L. TERRY

Medicine, Departments Connecticut 06510

of Pediatrics,

Obstetrics

26, 1976

We have investigated fatty acid oxidation and development profiles of palmitoyl-CoA synthetase and carnitine palmitoyltransferase in homogenates of developing rat brain. Palmitate showed a peak rate of oxidation between 10 days and the time of weaning, after which activity declined to adult levels. Acetate oxidation increased until Day 10, plateaued until Day 18 when it increased sharply and remained elevated through Day 25 before declining to the adult level. Leucine oxidation also showed a late peak as compared with palmitate.PalmitoylCoA synthetase activity was highest in late fetal development and in the newborn after which activity declined gradually to adult levels. Carnitine palmitoyltransferase activity peaked at lo-15 days of age similar to the profile obtained for long chain fatty acid oxidation. During the period of peak fatty acid oxidation, cytochrome oxidase activity increased twofold but the developmental increase in fatty acid oxidation and enzyme levels was much greater than the increase in mitochondrial number. These data suggest that during periods of high fat intake in the suckling rat the brain has an increased capacity for long chain fatty acid oxidation and that in addition to ketone bodies and leucine, fatty acids may be utilized as an alternative substrate in developing brain. INTRODUCTION

During gestation, fetuses of mammalian species are highly dependent on transplacentally derived glucose to provide energy needs. Following birth, major changes occur in nutrition and environment which are associated with an increased dependence on oxidative metabolism and an increased utilization of substrates other than glucose. In previous studies, we demonstrated that fatty acid oxidation is limited in the fetal rat and bovin heart but develops rapidly in the postnatal period (24, 26). In the rat, the postnatal emergence of myocardial fatty acid oxidation is associated with increased activities of palmitoyl-CoA synthetase and carnitine palmitoyltransferase (24). Similar data have been obtained in the developing intestine (25). It is also widely accepted that the postnatal brain has the capacity to oxidize substrates other than glucose, such as ketone bodies and branched-chain amino acids (7, 12). Previous reports by Beattie and Bas-

ford (3) and Vignais et al. (22) indicated that isolated brain mitochondria had the capacity to oxidize medium and long chain fatty acids. Since adaptation to alternate substrate oxidation by brain and other tissues is important in early metabolism, we have investigated the capacity of the developing brain in the rat (brain development in this animal being primarily postnatal) to oxidize fatty acids. We have also determined the developmental profiles of palmitoyl-CoA synthetase (EC 6.2.1.3) and carnitine palmitoyltransferase (EC 2.3.1.21, enzymes important for fatty acid oxidation. METHODS

Sprague-Dawley rats from 1 day of age to adult were killed by decapitation, and the brains were removed as rapidly as possible and placed in a cold solution of 0.25 M sucrose containing 0.5 mM EDTA and 10 mM T&-Cl, pH 7.5. Seventeen- to twenty-day fetal animals 161

Copyright 0 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.

162

DEVELOPMENTAL

BIOLOGY

were obtained by decapitation of the pregnant females, removal of the uteri to ice, and then rapid removal of the fetal brains to the cold sucrose buffer. The fetuses were alive just prior to decapitation. Brains were weighed, washed, and resuspended in 4 volumes of the cold sucrose solution, minced, and homogenized with a Brinkman PT 10 homogenizer at a rheostat setting of 4 for 30 sec. The material was kept at ice temperature at all times. The number of brains used for a preparation varied from 3 to 10 depending on age. The medium for the oxidation experiments was a modification of that used by Hulsmann et al. (14) and contained 130 n-&f sucrose, 33 mM Tris-Cl, pH 7.5,15 n&f PO, buffer, pH 7.5, 0.2 mM EDTA, 1.5 mM MgC&, 20 mM KCl, 2 mM malate, 0.1 mM carnitine, 2.7 mil4 ATP, 1.0 mM NAD (grade III-yeast), 0.03 mM cytochrome c (Type III-horse heart), and 4.8 mg of bovine serum albumin (BSA). Final concentration of acetyl-CoA was 0.2 mM containing [l-‘“Clacetyl-CoA, sp act 0.274 mCi/ mmol, and of palmitoyl-CoA was 0.1 mM containing [1-‘“Clpalmitoyl-CoA, sp act 3.22 mCi/mmol. Leucine concentration was 0.2 mM containing 1.0 mCi/mmol. When free fatty acid substrates were used, an ATP generating system containing 0.2 mM Coenzyme A (free acid), 5 mM phosphoenolpyruvate, 20 units of myokinase (Grade III-rabbit muscle), and 1.56 units of pyruvate kinase (Grade I-rabbit muscle) was added to the assay medium. Decanoate and palmitate were added as an albumin complex prepared by the method of Spector and Hoak (19). Decanoate and palmitate concentrations were 0.1 mM conand [ltaining [l-14Cldecanoate 14Clpalmitate, sp act 0.8 mCi/mmol. Acetate as Na acetate concentration was 0.35 mM containing [1-‘Qacetate, sp act 0.5 mCi/mmol. The reaction was initiated by addition of 1 mg of brain protein homogenate and the flasks were agitated gently for 90 min at 37°C in a shaking water bath. The reaction

VOLUME

52, 1976

was terminated by injecting 0.5 ml of 20% TCA through the serum caps. Then 0.3 ml of hyamine hydroxide was injected through the cap into the center well to trap the evolved ‘“C02, and the flask returned to the water bath for 45 min. The center well was removed carefully and the hyamine was transferred to a counting vial containing 10 ml of Econofluor (New England Nuclear) and counted in a Beckman scintillation spectrometer. An aliquot of the incubation medium was counted in 10 ml of Aquasol (NEN) at the same time. Blanks containing fresh distilled water in place of homogenate were incubated and treated as above to correct for any breakdown of substrate and release of 14C0, during the incubation. Palmitoyl-CoA synthetase activity was assayed by the method of Farstad et al. (9), and carnitine palmitoyltransferase by the method of Bremer and Norum (4). Cytochrome oxidase was determined polarographically as previously described (24). Protein was measured by the biuret method (15). Radioactive substrates were obtained from New England Nuclear Co. L-Carnitine was a gift from the Otsuka Pharmaceutical Factory, Osaka, Japan. The free acid of coenzyme A was obtained from Boehringer Mannheim. Other reagents was obtained from commercial sources. RESULTS

Fatty Acid nates

Oxidation

by Brain

Homoge-

The oxidation of [l-14Clpalmitate and [l“Clpalmitate-CoA to 14C0, by homogenates of developing rat brain is shown in Fig. 1. Oxidation of both the fatty acid and CoA ester showed a progressive increase and remained elevated between Days lo25 after which activity declined to adult levels. Activity in adult brain homogenates was always less than peak levels obtained during earlier development. Decanoate oxidation showed a similar developmental pattern.

163

BRIEF NOTES

I

Figure 2 shows the developmental pat20 tern of acetate and acetyl-CoA oxidation to CO,. The rate of oxidation’of acetyl-CoA was five times greater than that of acetate; however, their profiles were identical. After an initial increase observed by 10 days of age, activity plateaued until about Day 17 when it sharply increased, and later declined to adult levels after Day 254. I / ,AI 10 20 AOULT As with palmitate and decanoate, oxidaAGE Iday%) tion of these substrates by adult brain was FIG. 3. Developmental profile of leucine oxidamarkedly less than the peak activities ob- tion. Conditions are as described in the text. served in the younger rat. Synthetase and Carnitine The branched chain amino acid leucine PalmitoylXoA Palmitoyltransferase Activities also showed peak activity at about the time of weaning (Fig. 3) but in adult brain The developmental profile of palmitoylhomogenates leucine oxidation to CO, was CoA synthetase is shown in Fig. 4. Palmivirtually zero. toyl-CoA synthetase showed highest activity during late fetal development; following birth activity than gradually declined to the adult level which was approximately 25% of the peak activity. The pattern of development of carnitine palmitoyltransferase (Fig. 5) showed an increase until Days lo-15 of postnatal development after which activity declined; activity in adult brain homogenates was at a similar level to that of B-day fetuses. The developmental pattern of carnitine palmitoyltransferase mirrored that of long chain, FIG. 1. Oxidation of [lJ4Clpalmitate and Ll- fatty acid oxidation. Peak activities of pal“C]palmitoyl-CoA to TO2 by homogenates of demitoyl-CoA synthetase and carnitine palveloping rat brain. l - - -0, palmitate, 0- - -0, in developing brain palmityl-CoA. Conditions are as described in the mitoyltransferase were 4 and 10 times less, respectively, then text. found in adult liver homogenates. The activity of palmitoyl-CoA synthetase in developing brain was lo-fold greater than that of the transferase. Table 1 shows the increase in cytochrome oxidase activity of developing brain homogenates. Cytochrome oxid.ase is used as an index of mitochondrial number. The twofold increase observed by 14 days of age suggests a doubling of the number of mitochondria relative to total brain protein. Activity during early development was higher than found in the adult, sugFIG. 2. Developmental profile of acetate and acegesting further changes in mitochondrial tyl-CoA oxidation by developing rat brain. l - - -0, number relative to total brain protein. Acetate; 0- - -0, acetyl-CoA. .

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E

.

IlO-

/\

.

P c

.

.

.-*

\

164

DEVELOPMENTAL BIOLOGY

FIG. 4. Palmitoyl-CoA synthetase activity in developing rat brain. Each point represents the mean of at least two experiments carried out in triplicate. When three or more experiments were performed, the mean 2 SE is shown.

02 i

FIG. 5. Carnitine palmitoyltransferase activity in developing rat brain. Each point represents the mean of at least two experiments carried out in triplicate. When three or more experiments were performed, the mean r SE is shown.

Also shown in Table 1 are palmitoyl-CoA synthetase and carnitine palmitoyltransferase activities and oxidation of palmitic acid to CO, based on cytochrome oxidase. Dividing enzyme activities and fatty acid oxidation by ctyochrome oxidase activity expresses activity as nmol/p atom 0 which normalizes the data for the changes in mitochondrial number relative to total brain protein. The large increases observed indicate that the developmental increases are independent of the postnatal increase in brain mitochondria. DISCUSSION

During development the mammalian brain undergoes a number of major metabolic transitions which can be related to the stage of brain growth and maturation,

VOLUME 52, 1976

development of enzyme systems, and nutritional influences such as those imposed by suckling and weaning. The data available in the literature suggest that during fetal life, brain primarily metabolizes glucose anaerobically to pyruvate or lactate via the glycolytic pathway (Embden-Meyerhoff) which explains the relative resistance of the fetal and newborn brain to hypoxic insult (8). Postnatally, the brain becomes more dependent on aerobic metabolism and glucose is oxidized primarily to C02, this change being associated with an increase in the activities of the enzymes of the citric acid cycle (20). There is also evidence that the pentose phosphate pathway has higher activity in developing brain than in the matur brain (161, thus providing reducing equivalents for fatty acid synthesis as well as the ribose phosphate precursors important for synthesis of nucleotides for incorporation into RNA and DNA. The fetal rat brain is immature and its TABLE CYTOCHROME

Age

OXIDASE

Wochrome oxidasc (patom oxygen/ mg/min)

1

ACTIVITY

BRAIN n Palmitoyl-CoA synthetasc

OF DEVELOPING

Carnitine palmitoyltransferase

Palmit;;cziyi-

(nmol/ @atom oxygen) 17 Fetal days Birth 2 Days 5 Days 10 Days 15 Days 21 Days 25 Days Adult

0.019

105.3

2.9

0.082

0.021 0.024 0.028 0.032 0.034 0.032 0.029 0.024

78.6 57.1 41.1 26.6 25.0 26.6 8.6 18.8

3.9 3.5 3.4 4.7 3.5 2.5 1.4 1.3

0.079 0.074 0.087 0.226 0.294 0.264 0.146 0.167

o Cytochrome oxidase activity was determined polarographically as described in text. Activity of palmitoyl-CoA synthetase, carnitine palmitoyltransferase, and palmitate oxidation was calculated as nanomoles per milligram per minute. These values were then divided by activity of cytochrome oxidase to normalize enzyme activity and fatty acid oxidation for the observed changes in cytochrome oxidase during development.

BRIEF NOTES

growth and maturation take place primarily during the suckling period. During this period, the animal is receiving a high fat intake which should provide a source of precursors for synthesis of lipids required by the developing nervous system and also provides a source for alternate substrates in the form of ketones which can be used by the developing brain. Hawkins et al. (12) showed that ketone bodies can be oxidized by developing brain, and Tilden and Sevdalian (21) and Page et al. (17) have shown that the enzymes involved in ketone body utilization have their highest activities during the suckling period. These activities decrease after weaning the animals when the rats shift from the high fat milk diet to one higher in carbohydrate. Chaplin and Diamond (7) have further shown that branched-chain amino acids can be oxidized by developing brain probably via the ketone body pathway. This activity is lost after weaning. A number of investigators have found evidence for a low rate of fatty acid oxidation in the brain of adult rats (2, 3, 5, 10, 22, 23). In the present work, we have demonstrated developmental changes in brain fatty acid oxidation and in enzymes important for long chain fatty acid oxidation, e.g., palmitoyl-CoA synthetase and carnitine palmitoyltransferase. These changes probably reflect both changing energy requirements of the developing brain as well as changes in substrate availability imposed by birth, suckling, and weaning. The early increase in palmitoyl-CoA synthetase activity may reflect the need for activated fatty acids during the initial period of the brain growth spurt in the rat. Cantrill and Carey (6) reported a relatively constant pattern of palmitoyl-CoA synthetase activity in homogenates of developing rabbit brain. However, activity in a mitochondrial fraction increased from 2 to approximately 5 nmol/mg/min between birth and 20 days of age. Differences in the pattern of palmitoyl-CoA synthe-

165

tase development in the homogenates of rabbit brain and our own developmental data may relate to the timing of the brain growth spurt which begins prenatally in the rabbit as compared with postnatal development of the rat brain. At birth, there is a major shift in nutrition from glucose provided via the placenta to the high fat diet provided by milk. Carbohydrate intake begins to increase at about 16 days of age when the rat pups begin to nibble on standard laboratory chow; weaning is complete at 21 days of age. The activity of palmitoyl-CoA synthetase, carnitine palmitoyltransferase, and of brain fatty acid oxidation itself declines after weaning intake increases when carbohydrate sharply. Oxidation of palmitoyl-CoA closely paralleled developmental changes in carnitine palmitoyltransferase activity. Carnitine palmitoyltransferase catalyzes the reversable formation of the mitochondrial transport intermediate, palmitoylcarnitine, from palmitoyl-CoA plus carnitine. Once palmitoylcarnitine gains entry to the mitochondria, it is reconverted to palmitoyl-CoA by an intramitochondrial compartment of the transferase. In addition to its role in palmitate oxidation, carnitine palmitoylstransferase supports mitochondrial fatty acid elongation (27). Aeberhard et al. (1) demonstrated that the peak activity of fatty acid elongation by developing brain mitochondria occurs around the fifteenth postnatal day also similar to the developmental pattern of the transferase described here. The developmental increase in brain mitochondrial number reported here accounts for only part of the postnatal increase in brain carnitine palmitoyltransferase and fatty acid oxidation during the preweaning period. Fatty acid oxidation and enzyme levels showed significant increases when activity was normalized for increases in mitochondrial number by basing data on cytochrome oxidase activity. We have also confirmed previous reports

166

DEVELOPMENTAL BIOLOGY

concerning leucine oxidation by developing brain (7). Schepartz (18) reported that leucine was oxidized to CO, more readily in brain homogenates of newborn mice than in the adult. Mouse brain development closely approximates that of the rat. Since leucine degradation produces acetylCoA and acetoacetate, and the enzymes for their oxidation have been shown to be high in the suckling rat and mouse, these results are not surprising. It is of interest that peak oxidative activity of acetate was observed from Days 18-25, later than seen with long chain fatty acids. This may relate to energy needs during the period of brain myelination, and the availability of acetyl-CoA from substrates other than fatty acids after weaning. It is difficult to extrapolate in vitro data such as these to what is happening in the intact animal. However, there does appear to be increased potential for brain fatty acid oxidation during the period of early brain development in the suckling rat, and the data suggest that fatty acids in addition to ketone bodies and leucine may be utilized as alternate substrates by the developing brain. This work was supported by United States Public Health Service Grant No. HD-8293. The technical assistance of L. Barrett is greatly appreciated. REFERENCES 1. AEBERHARD, E., GRIPPO, J., and MENKES, J. H. (1969). Pediat. Res. 3, 590-596. 2. AHMED, K., and SCHOLEFIELD, P. G. (1961). Biothem. J. 81, 45-53. 3. BEATTIE, D. S., and BASFORD, R. E. (1965). J. Neurochem. 12, 103-111. 4. BREMER, J., and NORUM, K. (1967). J. Biol. Chem. 242, 1749-1755. 5. BRODY, T. M., and BAIN, J. A. (1952). J. Biol.

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Chem. 195, 685-696. 6. CANTRILL, R. C., and CAREY, E. M. (1975). Biothem. Biophys. Actu 380, 165-175. 7. CHAPLIN, E. R., and DIAMOND, I. (1974). Pediat. Res. 8, 462. 8. DAVISON, A. N., and DOBBING, J. (1968). In “Applied Neurochemistry” (A. N. Davison and J. Dobbing, eds.). Davis, Philadelphia. 9. FARSTAD, N. BREMER, J., and NORUM, K. R. (1967). Biochim. Biophys. Actu 132,492-502. 10. GEYER, R. P., MATTHEWS, L. W., and STARE, F. J. (1949). J. Biol. Chem. 180, 1037-1045. 11. GROSS, I., and WARSHAW, J. B. (1974). Biol. of the Neonate 25, 365-375. 12. HAWKINS, R. A., WILLIAMSON, D. H., and KREBS, H. A. (1971). Biochem. J. 122,13-18. 13, HIMWICH, W., ed. (1973). “Biochemistry of the Developing Brain” Vol. I. Marcel Dekker, New York. H~LSMANN, W. C., IEMHOFF, W. G. J., VAN DEN l4 BERG, J. W. O., and DEPIJPER, A. M. (1970). Biochim. Biophys. Actu 215, 5533555. 15. JACOBS, E. E., JACOB, M., SANADI, D. R., and BRADLEY, L. (1956). J. Biol. Chem. 223, 147156. 16. O’NEILL, J. J., and DUFFY, T. E. (1966). Life Sci. 5, 1849-1857. 17, PAGE, M. A., KREBS, H. A., and WILLIAMSON, D. H. (1971). B&hem. J. 121, 49-53. 18. SCHEPARTZ, B. (1963). J. Neurochem. 10, 825829. SPECTOR, A. A., and HOAK, J. C. (1969). Anal. lg. B&hem. 32, 297-302. 20. SWAIMAN, R. F. (1970). In “Developmental Neurobiology” (W. A. Himwich, ed.). C. C Thomas, Springfield, Ill. 21, TILDON, J. T., and SEVDALIAN, D. A. (1972). Arch. Biochem. Biophys. 148, 382-390. 22. VIGNAIS, P. M., GALLAGHER, C. H., and ZABIN, I. (1958). J. Neurochem. 2, 283-287. 23. VOLK, M. E., MILLINGTON, R. H., and WEINHOUSE, S. (1952). J. Biol. Chem. 195,493-501. 24. WARSHAW, J. B. (1972). Develop. Biol. 28, 537544. 25. WARSHAW, J. B., and KIMURA, R. E. (1974). Pediat. Res. 8, 361. 26. WARSHAW, J. B., and TERRY, M. L. (19701. J. Cell. Biol. 44, 354-360. 27. WARSHAW, J. B., and KIMURA, R. L. (1970). Biochem. Biophys. Res. Commun. 38,58-64.