Fatty Acid and Glucose Oxidation by Cultured Rat Heart Cells MIRIAM D. ROSENTHAL ' AND JOSEPH B. WARSHAW Division of Perinatal Medicine, Departments of Pediatrics and Obstetrics and Gynecology, Y a k University School of Medicine, New Haven, Connecticut 06510

ABSTRACT Monolayer cultures of fetal rat myocardial cells can be utilized to examine substrate preferences and interactions. The specific activity of glucose oxidation by myocardial cell cultures was high in sparse cultures but decreased with increased cell density. In contrast, palmitate oxidation was independent of initial cell density. Palmitate inhibited glucose oxidation by 50%in rat heart cultures. Glucose had only a slight sparing effect on palmitate oxidation. This suggests that fetal and newborn r a t myocardial cells in culture preferentially oxidize palmitate similar to adult heart. The sparing effect of palmitate on glucose oxidation is accounted for by inhibition of the glycolytic-aerobic pathway and not by inhibition of the pentose phosphate pathway. Data on oxidation of 14C-pyruvatespecifically labelled suggest that palmitate or a product of its oxidation such as acetyl-CoA may be acting directly to inhibit the pyruvate dehydrogenase complex. Palmitate oxidation per mg of cell protein was constant from 15 days gestational age to 2 days postnatal age. The observed differences between cultured cells and the intact heart may relate to decreased aerobic metabolism in monolayer cell culture and suggest that the increase in fatty acid oxidation observed in vivo is controlled by the oxygen environment of the cell. These studies show that heart cells in monolayer culture can be utilized to obtain metabolic information similar to an adult organ perfusion model. Cell culture provides a relatively homogeneous source of tissue which can be utilized to investigate the metabolic processes of intact cells (Warshaw and Rosenthal, '72). We have shown previously t h a t monolayer cultures of embryonic chick myocardial cells actively oxidize both glucose and fatty acids (Rosenthal and Warshaw, '73). Substrate oxidations by cultured embryonic chick heart cells were similar to those in developing chick embryo heart homogenates which exhibit active fatty acid oxidation (Warshaw, '721, although also highly dependent upon glucose (Needham and Lehmann, '37). The adult mammalian heart utilizes fatty acids preferentially to glucose (Shipp e t al., '61; Shipp, '64; Opie, '68), whereas the mammalian fetal heart appears to have a limited capacity to oxidize long chain fatty acids (Lockwood and Bailey, '70; Augenfeldt and Fritz, '70; Breuer e t al., '68; Wittels and Bressler, '63). Warshaw ('72) has reported t h a t the limited capacity of the newborn rat J. CELL. PHYSIOL., 93: 31-40.

heart to oxidize palmitate is associated with decreased activities of carnitine palmitoyltransferase and palmitoyl-CoA synthetase as compared with the activities of these enzymes in the adult tissue. In the present study, we have investigated the metabolism of fetal r a t heart cells in culture, in order to compare their utilization of fatty acids with that of the intact heart and with cultures of chick heart cells. We have also investigated whether heart cells obtained from fetuses of different ages and from neonates show the developmental differences observed in homogenates of fresh tissue. We have used the monolayers of rat heart cells to investigate further the control mechanisms involved in the interactions of fatty acid and glucose oxidation. Received July 29, '76. Accepted Mar. 3, '77. 1 Present Address: Department of Biochemistry, Eastern Virginia Medical School, Norfolk, Virginia. Please address reprint requests to: Dr. Joseph B. Warshaw, Department of Pediatrics, Yale University School of Medicine, New Haven, Connecticut 06510.

31

32

MIRIAM D. ROSENTHAL AND JOSEPH B. WARSHAW MATERIALS AND METHODS

Myocardial cells were isolated from fetal r a t hearts by procedures similar to those described previously for embryonic chick heart cell culture (Warshaw and Rosenthal, '72). Timed pregnant Sprague-Dawley rats were obtained from Charles River Breeding Laboratory (Wilmington, Massachusetts). The mothers were sacrificed by decapitation a t 19 to 21 days of gestation, and their uteri removed aseptically. Fetal hearts were rapidly removed to cold Tyrodes solution, dissected and minced. Twenty-one-day fetal hearts usually required five to six incubations of eight minutes each in 0.05% trypsin (General Biochemical, Chagrin Falls, Ohio, 1:300) in calcium and magnesium-free Tyrodes solution for complete dissociation. Cells were generally plated a t a density of 2.0 X lo6 per small Falcon flask (Falcon Plastics, Oxnard, California). Each flask contained 4 ml of F-12 medium (Grand Island Biological Co., Grand Island, New York) supplemented with 10% heat-inactivated fetal calf serum and 5% chick embryo extract. Cultures were incubated at 37" under 5% CO,; culture media were changed daily. Under these culture conditions, rat heart cultures showed a high percentage of beating cells (6040%). Both "M' and "F" cells (DeHaan, '67) were observed as well as islands of tightly packed cells. Spontaneous contractions were far more frequent than in chick heart cells cultured under similar conditions. Often entire microscopic fields were observed to pulse in synchrony. Adaptation of tissue culture flasks for measuring substrate oxidations by intact cells has been described previously (Warshaw and Rosenthal, '72). All metabolic assays were performed in Krebs-Ringer phosphate buffer which had been oxygenated just prior to use. Substrates were added at the following concentrations: glucose, 1 mM; pyruvate, 1-2 mM; palmitate, 0.16 mM, complexed to fatfree bovine serum albumin present at a final concentration of 0.025 mM (Rosenthal and Warshaw, '73). Palmitate-l-'*C and pyruvate2-14Cwere used at 0.5 mCi/mmole, and radiolabeled glucose a t 1.0 mCi/mmole. Spectrophotometric determination of lactate released during the incubation was made following the method of Hohorst ('63) and pyruvate according to Bucher et al. ('63). Palmitate oxidation by homogenates was determined using a modification of t h e

method of Beatty e t al. ('72). Adult and fetal rat hearts were removed into cold 150 mM KC1, washed, and minced with scissors. The pieces were washed twice with homogenizing medium: 100 mM KCl, 100 mM TES (N-tris(hydroxy-methyl)-methyl-2-aminoethane sulfonic acid), pH 7.4, 1 mM ATP, 5 mM MgS04 and 1mM EDTA. The tissue was then homogenized with a Brinkman P-20 high shear homogenizer for 15-20 seconds at a rheostat setting of 2; the resulting homogenate was filtered through cheesecloth. To prepare homogenates of cultures, cells were washed with cold KC1, scraped from the flask with a rubber policeman, and transferred to a test tube. They were gently centrifuged for ten minutes, resuspended in homogenizing medium, and homogenized with a small glass hand homogenizer with a loose pestle. Protein concentrations in homogenates were determined by a modification of t h e Biuret method (Jacobs e t al., '56). Homogenate assays were performed in a total volume of 1 ml in 10 ml Erlenmeyer flasks fitted with serum caps and center wells. The final incubation medium contained 70 mM KCL; 100 mM TES (pH 7.4), 1mM ATP, 1mM MgS04, 0.5 mM EDTA, 60 mM sucrose, 1mM ADP, 8 mM K2HP04,0.2 mM NADP, 0.5 mM NAD, 40 p M CoA, 1%albumin, 0.16 mM palmitate-l-14Cand 1.25 mM l-carnitine. After incubation for 40 minutes at 37O, the reaction was stopped with 20%TCA. Evolved 14C02was trapped with hyamine hydroxide and counted i n a liquid s c i n t i l l a t i o n spectrometer (Warshaw and Rosenthal, '72; Rosenthal and Warshaw, '73). Cytochrome oxidase activity was determined polarographically with a Clark oxygen electrode a s described by Warshaw and Terry ('70). Homogenates of fresh tissue and cultured cells were prepared as above except that they were washed and homogenized in buffer containing 0.23 M mannitol, 0.75 M sucrose, 0.1 mM EDTA and 10 mM Tris CL, pH 7.5. Reactions were carried out a t 30" with 0.5 to 2.0 mG of protein in a total volume of 1 ml. The reaction medium contained 160 mM sucrose, 15 mM KCL, 1mM EDTA, 50 mM Tris CL, 15 mM Na-P04, 5 mM MgC12, 20 mM ascorbate, 200 p M cytochrome C, and 300 p M ADP, at a pH of 7.5. RESULTS

Oxidation of glucose-14C (UL) and palmitate-l-14C to "CO, by cultures of fetal rat heart cells is shown in figure 1. Cells from

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33

METABOLISM OF CULTURED HEART CELLS

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PLATING DENSITY x Fig. 1 Oxidation of glu~ose-'~C (UL) and palmitate-l-I4Cto I4CO2by cells of fetal rat hearts plated at varyoxidation of gluco~e-'~C (UL); 0-0, oxidation of palmitate.1-"C in the presence ingdensities. -0, of 1.25 mM carnitine; X-X ,protein per flask. Assays were carried out after three days growth of culture as described in the test. Assay time was 60 minutes.

hearts of fetal rats of 21 days gestation were plated a t initial densities of 0.8-5.0 x lo6 cells per flask, and assays were carried out after three days in culture. The rate of oxidation of glu~ose-'~C (UL) per mg cell protein decreased with increased cell density. This decrease also observed with chick heart cells, is related to decreased activity of the pentose phosphate pathway with slower growth of more dense cultures (Warshaw and Rosenthal, '72). Similar to that observed for embryonic chick heart cells (Rosenthal and Warshaw, '731, the rate of palmitate-l-14Coxidation remained relatively constant a t all plating densities. Palmitate oxidation was also independent of length of time in culture or of total protein concentration per flask. Oxidation of palmitate was enhanced by 15-20%by addition of 1.25 mM carnitine to the assay medium. This was, therefore, included in all experiments. Palmitate oxidation by cultured heart cells was independent of developmental age (table 1). Heart cells obtained from 15 to 16 days of gestation through two days postnatal age showed similar oxidation of palmitate-l-I4Cto l4C0,.This was somewhat unexpected in view of the reported developmental changes in myocardial fatty acid oxidation (Warshaw, '73).

TABLE 1

Palmitate oxidation by heart cell obtained at different developmental ages Age (days)

Fetus - 15 days 18 days 20 days 21 days Newborn - 2 days

Palmitate oxidized cpmlrnglhr x los

1.42 1.20 1.36 1.38 1.30

All cells were plated at 1.5 X loE per flask and assayed after three days in culture. Substrate was 0.16 mM palmitate-1-"C plus 1.25 m M carnitine. Assay time was 60 minutes under conditions described in the test.

The rate of fatty acid oxidation in homogenates obtained from cells of 21-day fetal hearts after three days in culture was lower than that of the corresponding fresh fetal tissue and markedly less than seen in adult heart (fig. 2). The rates for homogenates of cell cultures assayed in modified ChappellPerry medium are similar to those observed with intact monolayers of cells. Furthermore, the cytochrome oxidase activity per mg protein was 22 natoms oxygen per minute for homogenates of cells after culture and 37 natoms oxygen per minute for homogenates of fresh fetal heart; by this index, the cells grown in vitro show about 60% of the mitochondrial number of intact fetal hearts.

34

MIRIAM D. ROSENTHAL AND JOSEPH B. WARSHAW

120

$ 100 c

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Fig. 2 Oxidation of palmitate-l-'4C by rat heart homogenates. 0-0, adult r a t heart; 0-0, 21X , 21-day fetal rat heart cells after three days in culture. The assay medium conday fetal r a t heart; Xtained 70 mM KC1.100 mM TES (pH 7.4), 1mM ATP, 1mM ADP, 1mM MgSO,, 0.5 mM EDTA, 60 mM sucrose, 8 mM K,PO,, 0.2 mM NADP, 0.5 mM NAD, 40 I.LMCoA, 1%albumin, 0.16 mM palmitate-l-'"C, and 1.25 mM I-carnitine in a total volume of 1.0 ml. Assay time was 40 minutes. TABLE 2

Interaction of glucose and palmitate oxidation in rat heart cell cultures Rat heart cells

Protein (mg/flask) Oxidation of palmitate-l-lT (cpmimg protein X 109 Control + glucose %inhibition Oxidation of glucose-"C (cpm/mg protein X lo3) Control + palmitate % inhibition

0.2210.04 13.61 1.2 12.2iz 1.4 ll.Ort4.4

9.0iz0.80 4 . 4 1 1.10 50.5iz8.60

Cells from 20 to 21-day fetal rat hearts were plated at initial densities of 1.25 to 3.0 X lo6 and assayed after three days in cul. ture. Assay time was 60 minutes. For measurement of palmitate-1-"C oxidation, the assay medium contained 0.16 mM palmitate1 - ' C and 1.25 mM carnitine. Assays were performed with palmitate-1-'Calone (control) and in the presence of 1 mM glucose. For measurement of glucose-"C oxidation, the assay medium contained 1.0 mM glucose-'C (UL). Palmitate was added at 0.16 mM; SEM of four experiments. Each experiment is the mean of 1.25 mM carnitine was used throughout. Values shown are means triplicate determinations.

*

The effects of fatty acids on glucose oxidation and of glucose on fatty acid oxidation by cultures of fetal rat heart cells are compared in table 2. The addition of palmitate to the assay medium decreased the oxidation of glu~ose-'~C (UL) to 14C02by 45-50%. Addition of glucose to the assay media resulted in inhibition of 14C02production from palmitatel-14C oxidation by only 11%.These results indicate a preference for fatty acids over glucose substrates in rat heart cell cultures.

To further define the inhibitory influence of fatty acids on glucose oxidation, we examined the effect of palmitate on the oxidation of gl~cose-l-'~C, g1ucose-6-l4C,and gluc~se-'~C (UL) by rat heart cell cultures. As is shown in table 3, the inhibitory effect of palmitate was greatest on oxidation of glucose-6-14C(52%) and least on glu~ose-l-'~C (25%). Production of 14C02 from g l ~ c o s e - l - ' ~can C be divided approximately into two components; part from the glycolytic-aerobic pathway (equal to 14C

35

METABOLISM OF CULTURED HEART CELLS TABLE 3

The effectofpalmitate on the oxidation of specifically-labeled glucose-14Cby rat heart cells Substrate

Glucose-l-'4C Control + palmitate Glu~ose-6-~~C Control + palmitate Glucose-I% (UL) Control + palmitate

Glucose oxidized

%: inhibition

13.8'1.1 10.4t0.8

25

5.2'0.22 2.5t0.12

52

7.8'0.6 4.620.6

39

Cells of 21 day fetal rat hearts were plated at an intial density of 2 0 X 10' per flask and assayed after three days in culture Control flasks contained 1 mM glucose "C (sp act 1 mCiimmole) When pres ent, palmitate was 0 16 mM 1 2 5 mM 1 carnitine was present throughout Assay time was 60 minutes Values represent means -t S E for determinations on four replicate flasks

production from glu~ose-6-'~C) and part from t h e pentose phosphate pathway (above the O, (Katz and level of g l u ~ o s e - 6 - ~ ~ Coxidation) Wood, '63, Wood e t al., '63). Inhibition of 14C0, production from g l u ~ o s e - l - ' ~(and C from glucose14C)(UL) can be accounted for entirely by inhibition of the glycolytic-aerobic pathway. Thus palmitate inhibition of glucose oxidation does not appear to affect the preferential release of the carbon-1 of glucose by the pentose phosphate pathway but inhibits flow through the glycolytic-aerobic pathway. To investigate further the effect of palmitate on glycolysis, metabolic interactions between pyruvate and glucose were studied.

-

Figure 3 shows the time course of 14C0, production from both pyruvate-l-14C and pyruvate-2-I4Cin rat heart cells. There is an initial lag in I4CO, production from pyruvate-2-14C but not pyruvate-l-14C. After the lag, 14C0, production from both substrates is linear with time up to two hours. The rate of 14C0, production from p y r ~ v a t e - l - ' ~was C higher than from pyr~vate-2-'~C. The lag with pyruvate2-14C probably relates to the different steps involved in release of different pyruvate carbons as CO,. Pyruvate carbon-1 goes directly to CO, through the action of pyruvate dehydrogenase. At the same time, carbons-2 and -3 are incorporated into acetyl-CoA. Production of 14C02from the carbon-2 of pyruvate thus involves the entire citric acid cycle and equilibration with several substrate pools. As would and be expected, oxidation of glu~ose-'~C-UL palmitate show lags similar to pyr~vate-2-'~C. Addition of palmitate to the assay medium inhibited the oxidation of pyruvate labeled in either the 1 or 2 position (table 4). Although 14C02 production from p y r ~ v a t e - l - ~was ~c higher than t h a t from p ~ r u v a t e - 2 - ~the ~ Cper, centage of inhibition of oxidation of the two labeled substrates by added palmitate was the same in any given experiment. Neither addition of carnitine nor fat-free bovine serum albumin alone had any effect on pyruvate oxidation. The proportional inhibition of 14C0, production from pyruvate-l-14Cand pyruvate2-14Cis evidence that palmitate, or a product

m

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TIME ( m i n ) Fig. 3 Time course of oxidation of pyruvate-"C to 14COz.0-0, pyruvate-l-"C; X-X , pyruvate2-14C.Cells from 21-day fetal rat hearts were plated at an initial density of 2.0 X lo6 and assayed after three days in culture. The assay medium contained 2 mM pyruvate-"C (sp. act. 0.5 mCi/mmole).

36

MIRIAM D. ROSENTHAL AND JOSEPH B. WARSHAW TABLE 4

The effect of palmitate on pyruvate oxidation by heart cell cultures Rat heart

Protein mglflask Oxidation of pyruvate-l-"C (cpmimg protein Control + palmitate %,inhibition Oxidation of pyruvate-2-'4C (cpm/mg protein Control + palmitate '%,inhibition

X

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lo3)

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Cells from 21-day fetal rat heart were plated at initial densities of 1.25 to 2.0 X 10'. All assays were carried out after three days in culture, Assays were performed with 2.0 mM pyruvate-'T alone (control) and in the presence of 0.16 mM palmitate. 1.25 mM carnitine was used throughout. Assay time was 60 minutes. Values are means It SEM of three experiments. Each experiment is the mean of triplicate determinations

-.C

T

T

Buffer

Glucose

T

Glucose Pyruvate Pyruvate a-Keto+ Glutorate + Palmitate Palmitate

Fig. 4 The effect of exogenous substrates on lactate production by cells of 21-day fetal r a t hearts. Cells were plated a t a n initial density of 2.0 X lo6per flask and assayed after three days in culture in Krebs-Ringer phosphate buffer. Substrate concentrations were: a-ketoglutarate, 1 mM; pyruvate, 2 mM; glucose, 1 mM, palmitate, 0.16 mM. 1.25 mM carnitine was added when palmitate was present. Assay time was 60 minutes. Means +. SEM of four experiments.

of its oxidation such as acetyl-CoA may be in all experiments but the increases were not acting directly to inhibit the pyruvate dehy- statistically significant. Similar data were observed when pyruvate release was measured. drogenase complex. Figure 4 compares the production of lactate DISCUSSION by rat heart cell incubated in Krebs-RingerWe have demonstrated that monolayer culphosphate buffer alone with that of cells incubated with various exogenous substrates. The tures of fetal rat heart metabolize palmitate addition of palmitate does not significantly in- to COz as well as glucose and pyruvate. The crease the endogenous rate of lactate produc- capacity of cultured heart cells to oxidize tion. Lactate production in the presence of palmitate was independent of time in culture either glucose or pyruvate was higher than in or cell density and was independent of the age buffer alone; added palmitate increased lac- of the fetal or neonatal r a t hearts cultured. tate production from these substrates slightly The latter result was somewhat unexpected

METABOLISM OF CULTURED HEART CELLS

since previous studies utilizing fetal rat heart homogenates indicated that fatty acid oxidation was limited in the fetal rat heart and became more active after birth (Warshaw, '73). However, oxidation of palmitate by cultured cells of all ages, although markedly less than in adult heart, did not show developmental variation. Cells in culture appear to revert to a more anaerobic pattern of metabolism (Paul, '65). McLimans et al. ('68) have suggested that oxygen avaliability to the cells in monolayer cultures is limited by slow diffusion rates. There is a shift in LDH isozyme pattern from predominantly H4 to M, or muscle-type when chick heart cells are grown in culture (Cahn, '64). Pious ('70) has shown that cytochrome levels of cultured human fibroblasts decrease as the oxygen tension of the cultures is further lowered below that obtained with growth under ambient air. Both HeLa cells (Cribbs and Kline, '71) and human fibroblasts (Pious et al., '72) show a preferential synthesis of M subunits of LDH when grown under decreased oxygen tensions. Our observations that cytochrome oxidase activity of rat heart cultures is lower than in the corresponding fresh tissues is consistent with these observations. In the adult rat heart, a major rate controlling step in long-chain fatty acid oxidation involves the P-oxidation process (Pande, '71). It is possible that adaptation to cell culture involves decreased mitochondrial function, so t h a t oxidative phosphorylation rather than p oxidation becomes rate limiting for fatty acid oxidation. This may explain similarities in palmitate oxidation between cultured rat hearts from different stages of development, and between cells from rat and chick hearts grown in the same culture environment. The increase in palmitate oxidation in the immediate postnatal period reported by Warshaw ('72) may, therefore, be a response to a n oxygen environment more favorable to fatty acid oxidation. Our results indicate that r a t heart cell cultures utilize palmitate preferentially to glucose or pyruvate. Whereas palmitate inhibits oxidation of glucose or pyruvate up to 50%, added glucose or pyruvate has little sparing effect on palmitate oxidation. By contrast in chick heart cultures, glucose and palmitate each spare oxidation of the other by up to 50% (Rosenthal and Warshaw, '73). The substrate preferences of fetal rat heart cell cultures also distinguish them from some of the established

37

cell lines. Significant sparing effects of glucose on palmitate-l-'* oxidation have been reported for HeLa and L cells (Geyer, '67). Adult rat hearts, however, have been shown to utilize free fatty acids preferentially to glucose (Shipp et al., '61; Shipp, '64; Opie, '68) and Wildenthal has found that fetal mouse hearts in organ culture use octanoate preferentially to glucose. We have shown that palmitate inhibits glucose oxidation to COZ via the glycolyticaerobic pathway in heart cell cultures, but does not inhibit the pentose phosphate pathway (table 3). Palmitate does not significantly affect lactate production from glucose, pyruvate, or endogenous substrates. However, palmitate inhibits CO, production from pyruvate-l-14Cand pyruvate-2-14Cto an equal extent. The data showing decrease of glucose and pyruvate oxidation to 14C0, in the presence of palmitate are consistent with inhibition of the pyruvate dehydrogenase complex as suggested by others (Evans et al., '63; Patzelt et al., '73; Taylor et al., '75). The rate of glycolysis in well-oxygenated heart muscle has also been shown to be regulated a t the level of glucose transport, hexokinase, and phosphofructokinase (Neely and Morgan, '74). Randle et al. ('66) found that in spite of a marked diminution in rates of glycolysis with fatty acid oxidation, the output of lactate plus pyruvate by rat heart was undiminished. This suggested that control of glycolysis and pyruv a t e oxidation a r e linked, presumably through coordinated regulation of phosphofructokinase and pyruvate dehydrogenase. We find that lactate plus pyruvate output by cultured rat heart cells is not significantly affected by added palmitate. Thus, coordinated control mechanisms of glycolysis would appear to be operating in these cells similar to the intact heart. Fatty acid oxidation has been shown to increase the mitochondrial and cytoplasmic ratios of NADHINAD in the heart and result in increased ratios of lactate to pyruvate in the perfusate (Williamson et al., '68).Unlike the perfused heart, fatty acid oxidation by cultured heart cells does not appear to significantly alter the release of lactate or pyruvate, nor the ratio between the two. Heart cells in culture have decreased levels of cytochrome oxidase and overall oxidative metabolism relative to anaerobic glycolysis. In these cells, fatty acid oxidation, while sufficient to produce an increase in mitochondrial levels of

38

MIRIAM D. ROSENTHAL AND JOSEPH B. WARSHAW

acetyl-CoA and a resultant decrease in glucose oxidation to COz, may not contribute as significantly to total NADH production as in the intact heart. These studies show that heart cells in monolayer culture can be utilized to obtain metabolic information in many ways similar to an organ perfusion model. Further support is provided for the hypothesis that fatty acids inhibit glucose oxidation by regulating the activity of pyruvate dehydrogenase. While cells in culture retain some of the cellular metabolic regulatory mechanisms of the intact heart, there are also adaptations to a new mode of growth in vitro, with its shift toward a more anerobic environment. Thus fatty acid oxidation in rat heart cultures reflects the environment of the cell culture rather than the developmental age of the cells. ACKNOWLEDGMENTS

This work was supported by U.S.Public Health Grant HD 08293. The authors gratefully acknowledge technical assistance of Ms. Lynn Barrett. LITERATURE CITED Augenfeldt, J., and I. Fritz 1970 Carnitine Palmityl-transferase activity in fatty acid oxidation by livers from fetal and neonatal rats. Can. J. Biochem., 48: 228-234. Beatty, C. H., M. K. Young and R. M. Bocek 1972 Respiration and metabolism by homogenates of various types of muscle. Am. J. Physiol., 223: 1232-1236. Breuer, E., E. Barta, E. Pappova and L. Zlatos 1968 Developmental changes of myocardial metabolism. 11. Myocardial metabolism of fatty acids in the early postnatal period in dogs. Biol. Neonat., 11: 367-377. Bucher, T., W. Czok, E. Lamprecbt and E. Latzko 1963 Pyruvate. In: Methods of Enzymatic analysis. H. H. Bergmeyer, ed. Academic Press, Inc. N. Y., pp. 253-258. Cahn, R. D. 1964 Developmental changes in embryonic enzyme patterns: The effect of oxidative substrates on lactic dehydrogenase in beating chick embryonic heart cell cultures. Devel. Biol., 9: 327-346. Cribbs, R. M., and E. S. Kline 1961 The effects of environment on lactate dehydrogenase isozymes of cultured somatic cells. J. Cell. Physiol., 78: 59-64. DeHaan, R. L. 1967 Regulation of spontaneous activity and growth of embryonic chick heart cells in tissue culture. Dev. Biol., 16: 216-249. Geyer, R. P. 1967 Uptake and Retention of Fatty acids by tissue culture cells. Lipid metab. Tissue cult. Cells (Wistar Symp. Monograph 6),pp. 33-47. Hohorst, H. H. 1963 L 1+1 Lactate determination with lactic dehydrogenase and DPN. In: Methods of Enzymatic Analysis. H. H.Bergmeyer, ed. Academic Press, Inc. N. Y., pp. 266-270. Jacobs, E. E., M. Jacob, D. R. Sanadi and L. Bradley 1956 Uncoupling of oxidative phosphorylation by cadmium ion. J. Biol. Chem., 223: 147-156. Katz, J., and H. G. Wood 1963 The use of l’COa yields from

gl~cose-l-and-6-’~C for the evaluation of pathways of glucose metabolism. J. Biol. Chem., 238: 517-523. Linn, T. C., F. H. Pettie, F. Hucho and L. J. Reed 1969 -Cketo acid dehydrogenase complexes. XI. Comparative studies of regulatory properties of the pyruvate dehydrogenase complexes from kidney, heart, and liver mitochondria. Proc. Nat’l. Acad. Sci., 64: 227-234. Lockwood, E. A., and E. Bailey 1970 Fatty acid utilization during development of the rat. Biochem. J., 120: 49-54. McLimans, W. F., E. J. Crouse, K. V. Tunnah and G. E. Moore 1968 Kinetics of gas diffusion in mammalian cell culture systems. I. Experimental Biotechnol. Bioeng., 10:

725-740. Needham, J., and H. Lehmann 1937 Intermediary carbohydrate metabolism in embryonic life. Biochem. J., 31:

1165-1254. Neely, J. R., and H. E. Morgan 1974 Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Ann. Rev. Physiol., 36: 413-459. Opie, L. H. 1968 Metabolism of the heart in health and disease. Am. Heart J., 76: 685-698;77: 100-122,383-410. Pande, S. B. 1971 On rate-controlling factors of long chain fatty acid oxidation. J. Biol. Chem., 245: 5384-

5390. Patzelt, C., G. Loffler, 0. H. Wieland 1973 Interconversion of pyruvate dehydrogenase in the isolated perfused r a t liver. Eur. J. Biochem., 33: 117-122. Paul, J. 1965 Carbohydrate and energy metabolism. In: Cells and Tissues in culture, Vol. I. E. N. Willmer, ed. Academic Press, N. Y., pp. 239-276. Pious, D. A. 1970 Induction of cytochromes in human cells by oxygen. Proc. Nat’l. Acad. Sci., 65: 1001-1008. Pious, D. A,, W. A. Susor, R. W. Benson and W. J. Rutter 1972 Independent regulation of cytwhruiiies and g l y colytic enzymes in human fibroblasts. J. Cell Physiol., 79:

423-428. Randle, P. S., P. B. Garland, C. N. Hales, E. A. Newsholme, R. M. Denton and C. I. Pogson 1966 Interactions of metabolism and the physiological role of insulin. Recent Prog. Horm. Res., 22: 1-44. Rosenthal, M. D., and J. B. Warshaw 1973 Interaction of fatty acid and glucose oxidation by cultured heart cells. J. Cell Biol., 58: 332-339. Rosenthal, M. D., and J. B. Warshaw Unpublished observation. Shipp, J. C. 1964 Interrelation between carbohydrate and fatty acid metabolism of isolated perfused r a t heart. Metabolism, 13: 852-867. Shipp, J. C., L. H. Opie and D. R. Challoner 1961 Fatty acid and glucose metabolism in the perfused heart. Nature,

189: 1018-1019. Taylor, S. I., Z. Mukherjee and R. L. Jungas 1975 Regulation of pyruvate dehydrogenase in isolated rat liver mitochondria. J. Biol. Chem., 250: 2028-2035. Warshaw, J. B. 1972 Cellular energy metabolism during fetal development. IV. Fatty acid activation, Acyl transfer and fatty acid oxidation during development of t h e chick and rat. Devel. Biol., 28: 537-542. Warshaw, J. B., B. Coyne and L. Barrett 1976 Effect of pH alteration on growth and glucose oxidation in myoblast cultures. Exp’t Cell Biol., 52: 283-291. Warshaw, J. B., and M. L. Terry 1970 Cellular energy metabolism during fetal development. 11. Fatty acid oxidation by the developing heart. J. Cell Biol., 44:354-360. Wildenthal, K. 1973 Studies of fetal mouse hearts in organ cultures: Metabolic requirements for prolonged function in vitro and the influence of cardiac maturation on substrate utilization. J. Molec. Cellular Cardiology, 5.

97-99.

METABOLISM OF CULTURED HEART CELLS Williamson, J. R., E. T. Browning and M. S. Olson 1968 Interrelations between fatty acid oxidation and the controt of gluconeogenesis in perfused rat liver. Adv. Enz. Reg., 6: 67-100. Wittels, B., and R. Bressler 1963 Lipid metabolism in the newborn heart. J. Clin. Invest., 44: 1639-1646.

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Fatty acid and glucose oxidation by cultured rat heart cells.

Fatty Acid and Glucose Oxidation by Cultured Rat Heart Cells MIRIAM D. ROSENTHAL ' AND JOSEPH B. WARSHAW Division of Perinatal Medicine, Departments o...
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