3ouwwl

of Molecular

and Cellular

Cardiology

(1979)

11, 893-915

Inhibition of Fatty Acid Oxidation in Normal and Hypoxic Perfused Rat Hearts by 2-Tetradecylglycidic Acid* F. J. PEARCE,

J. FORSTER, G. DELEEUW, AND G. F. TUTWILERf

Department of Biochemistry and Biophysics, and SDepartment of Biochemistry, McNeil (Received

6 December

J. R. WILLIAMSONt

University of Pennsylvania, Philadelphia, Pennsylvania, Laboratories, Ft. Washington, Pennsylvania, U.S.A. 1978,

accepted

15 January

1979)

F. J. PEARCE, J. FORSTER, J. R. WILLIAMSON AND G. F. TUT~ILER. Inhibition of Fatty Acid Oxidation in Normal and Hypoxic Perfused Rat Hearts by P-Tetradecylglycidic Acid. 3ournal of Molecular andCellular Cardiology (1979) 11,893915. Studies with a new fatty acid oxidation inhibitor, P-tetradecylglycidic acid, showed that half-maximal inhibition of oleate oxidation occurred at concentrations of 3 x 10-s M. P-Tetradecylglycidic acid also inhibited endogenous fatty acid oxidation, but had little effect on octanoate oxidation. The inhibition of long chain fatty acid oxidation produced by 2-tetradecylglycidic acid depended on its time exposure to the hearts. At low work loads it had no effect on left ventricular pressure development or aortic output, but was inhibitory at high work loads when the capacity for glucose oxidation was exceeded. The relationship of oleate concentration (with 2% albumin present) to pyridine nucleotide fluorescence and to the stimulation of oxygen consumption was examined using hearts perfused with glucose. Pyridine nucleotide fluorescence and oxygen consumption were both increased, with half-maximal effects occurring at 0.08 mM and 0.2 rnM oleate, respectively. Oleate addition, both in the absence and presence of insulin, increased oxygen consumption to a greater degree than expected from the theoretical decrease of the P/O ratio associated with fatty acid oxidation. At fatty acid/albumin molar ratios of 2.5 or greater, contractility was impaired and oxygen consumption increased even further. Pyridine nucleotidcs remained reduced indicating the absence of an uncoupling effect on oxidative phosphorylation. The oxygen wasting effect of fatty acids is interpreted as caused by long chain fatty acyl-CoA formation and hydrolysis. In working heart preparations made hypoxic by lowering the per&sate oxygen content to about 35%, addition of 1 nm oleate to the per&ate resulted in a reversible decrease in left ventricular pressure development and aortic output. Perfusion with 5 x 10-s M L-tetradecylglycidic acid for 40 min before oleate addition prevented this deleterious effect of oleate during hypoxia. Photographs of the surface pyridine nucleotide fluorescence showed that addition of oleate to the hypoxic heart resulted in an increase in the size and number of anoxic areas. Subsequent addition of 10-s M 2-tetradecylglycidic acid caused the anoxic areas to disappear. These results indicate that the deleterious effects of fatty acids on the hypoxic myocardium are due to an increased oxygen demand resulting from accelerated fatty acid oxidation. WORDS: Fatty acid oxidation; rat heart; Tetradecylglycidic acid.

KJZY

Pyridine

nucleotide

fluorescence;

Hypoxia;

Perfused

*This work was supported by NIH grant HL 14461, HL 18708 and GM 00092. tRequest.s for reprints should be sent to: Dr John R. Williamson, University of Pennsylvania, Department of Biochemistry and Biophysics, School of Medicine G3, Philadelphia, Pennsylvania 19104. 0022-2828/79/090893+24

$02.00/O

0 1979 Academic

Press Inc.

(London)

Limited

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F. J. PEARCE

ET-AL.

1. Introduction It is well established that free fatty acids are the most important respiratory substrate for the fully aerobic mammalian heart. However, there have also been numerous studies which indicate that free fatty acids may be detrimental to the function of the comprised heart. Under ischemic conditions, free fatty acids have been reported to induce arrythmias [S, 17, 261, to exacerbate enzyme release [7] and to depress myocardial contractility [IO, 11, 38, 491. Physiological levels of fatty acids do not have these effects in the normal aerobic heart. The mechanism(s) responsible for the detrimental effects of free fatty acids in hypoxia is still poorly understood. Proposed mechanisms include the possibilities, (a) that high intracellular levels of free fatty acids may bind intracellular calcium and limit the amount of calcium available to the myofibrils [IO, II], (b) that increased fatty acyl-CoA levels may inhibit the adenine nucleotide translocator [39] or, (c) that free fatty acids may uncouple oxidative phosphorylation [Z, 311. Either of the latter two mechanisms would lower the availability of ATP to the myofibrils. An additional possibility is that fatty acids may increase the mass of anoxic tissue under ischemic or hypoxic conditions by increasing the demand for oxygen. In order to produce the same amount ofATP, oxygen consumption would be expected to increase by 13% if the heart switched from oxidizing only glucose to exclusive oxidation of fatty acids. In fact, free fatty acids have been reported to stimulate the oxygen consumption of the aerobic dog [19, 201 and rat [3] heart by much more than 15%, indicating that other mechanisms such as uncoupling of oxidative phosphorylation or increased substrate recycling ATPase activity may be involved. The present study examines the relationship of free fatty acid concentration to the stimulation of oxygen consumption and NADf oxidation-reduction state in the aerobic heart. The contribution of fatty acid oxidation to the free fatty acid induced depression of myocardial function during hypoxia is assessed using a newly reported inhibitor of fatty acid oxidation, 2-tetradecylglycidic acid. P-Tetradecylglycidic acid (TDGA)* is an oral hypoglycemic agent [45] which has been reported to inhibit oxidation of long chain but not short chain fatty acids in isolated rat hepatocytes [43] and rat hemidiaphragm muscle [46]. As a result of this inhibition, gluconeogenesis and ketogenesis are inhibited in rat hepatocytes [43] and glucose oxidation by hemidiaphragms is accelerated [44, 461. These characteristics make it a useful tool for the study of the relationships between carbohydrate and fatty acid oxidation as well as the mechanisms responsible for the deleterious effects of elevated fatty acid concentrations in hearts under hypoxic or ischemic conditions. In this paper we describe the effects of tetradecylglycidic acid on fatty acid oxidation and contractility in perfused rat hearts performing different amounts of work. Is is also shown that addition of fatty acids to hearts perfused with restricted *Chemical

Abstracts

Name:

2-Tetradecyloxirane

carboxylic

acid.

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oxygen delivery causes an increase in the amount of anoxic tissue as a result of the fatty acid induced increase of respiration. These changes were reversed by addition of tetradecylglycidic acid.

2. Materials

and Methods

Working and Langendorff heart preparations

Hearts from 300 to 350 g male Sprague-Dawley rats were rapidly excised following decapitation and the aorta was quickly cannulated [41]. Retrograde perfusion was started from a reservoir placed 80 cm above the heart while the left atrium and pulmonary artery were cannulated. Both the inferior and the superior vena cavae were tied off at the points where they entered the right atrium in order to minimize fluid leakage. Left atria1 filling was provided from a reservoir placed 12 cm above the left atrium and the left ventricle ejected fluid against an afterload of 80 cm of water. A 3 ml air space located immediately above the aortic cannula provided elasticity in the system. Unless otherwise stated, hearts were paced at a frequency of 300 beatslmin. Aortic pressurewas measured with a Statham P23 Db pressuretransducer connected to the aortic outflow tract by means of a T-joint. Left ventricular pressure measurementswere made with a Statham P23 Db pressuretransducer which was connected to a 23 guage needle inserted through the left ventricular wall. Effluent fluid from the pulmonary artery was pumped at a constant rate through a chamber fitted with a Yellow Springs oxygen electrode. Extracorporeal electromagnetic flow meters were placed in the pulmonary artery outflow tract to monitor coronary flow, and in the aortic outflow tract to measure left ventricular function. Oxygen consumption was calculated from the solubility of oxygen in Krebs bicarbonate medium at 37°C (0.936 mM), the coronary flow rate, and the difference between the influent (x0.95) and effluent oxygen concentrations. For the oleate titration experiments, in which changes in oxygen consumption were very small, hearts were perfused through the aorta by buffer pumped at a constant rate of 13 ml/min (Langendorff preparation). A bubble trap located just before the aorta served to dampen the flow oscillations due to the peristaltic pump. A stirred, air-tight Plexiglass chamber (volume = 1.5 ml) placed between the pulmonary artery and the oxygen electrode served to dampen fluctuations in the effluent Peg. Hearts were placed at a rate slightly greater than their natural beating rate.

Perfusion medium

The basic perfusion fluid was Krebs bicarbonate medium containing 2.5 lll~ Gas+, 5 mM glucose and 2% (w/v) fatty acid free albumin (Sigma Chemical Company)

896

P. J. PEARCE

ET AL.

to which all additions were made. Insulin was added to this medium at a concentration of 10 mu/ml where indicated. The buffer was filtered through 0.8 pm Millipore filters before use. Equilibration of the perfusion medium with 95% oxygen and 5% carbon dioxide was achieved using a Silastic tubing oxygenator which maintained the fluid at 37°C. Millipore filters (5 pm) were incorporated into the perfusion system as an additional precaution against particulate contamination.

Measurement

of jyridine

nucleotideJuorescence

Surface pyridine nucleotide fluorescence was measured with a 3-way light guide which allowed simultaneous excitation of pyridine nucleotides at 366 nm, and measurementsof fluorescence (470 nm) and reflected excitation light as described by Chance et al. [4]. The light guide was placed lightly against the surface of the heart and was shielded from extraneous light by a light-tight enclosure. The apex of the heart rested lightly on a mesh of Silastic tubing near the bottom of the enclosure. This arrangement minimized movement artifacts in the fluorescence records due to the beating of the heart against the light guide. The intensity of the excitation light generated by the mercury arc lamp was adjusted so that continuous exposure of the heart for 2 h caused no significant decreasein the signal produced by a 3 min period of anoxia (95% Nz, 5% COZ). The relative fluorescenceintensity is expressedin one of two ways: (a) as a percentage of the initial fluorescent signal which is arbitrarily assigneda value of 0.5 V or (b) as a percentage of the difference between the initial fluorescencesignal and the maximal fluorescencesignal obtained after a 3 min anoxic period. Measurements between hearts were standardized by using the initial fluorescencesignal obtained after a 15 min stabilization period with 5 mM glucose and 2% (w/v) albumin in the perfusion medium. Since additions of oleate causedno change in the amount of reflected excitation light, no correction of the fluorescencesignal was necessary.However, reflectance wasroutinely monitored and was useful in detecting any aberrations in the fluorescence traces due to movement artifacts. Initial experiments using the working heart preparation showed that additions of oleate resulted in biphasic changesin the pyridine nucleotide fluorescence. This was avoided in the subsequent experiments using the constant flow perfusion technique by increasing the volume of the recirculating reservoir from 120 to 400 ml and by constantly stirring this reservoir to avoid mixing artifacts which otherwise occur upon addition of the oleate stock solution. Fluorescence photography, as initially described by Barlow and Chance [I] and subsequently modified by Steenbergen et al. [4r], was employed to visualize pyridine nucleotide fluorescence from the entire ventricular surface. A xenon flash provided excitation light, with wavelength greater than 380 nm removed by a Corning CS7-60 glassfilter. A wratten 2A filter (410 to 3000 nm) placed over the

FATTY

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camera lens removed reflected excitation light and allowed the fluorescence to be recorded photographically. The xenon flash was phased to occur during diastole.

Measurement

offatty acid oxidation

Tracer amounts of [9, lo-sH]palmitic acid (New England Nuclear Corporation) were added to the stock solution of oleate to yield a specific activity of approximately 0.3 @/~moI. The final perfusate oleate concentration was 1 mM. Hearts were perfused for 10 min with 150 ml of recirculated Krebs bicarbonate medium containing 5 mM glucose and 2% (w/v) fatty acid free albumin to allow the hearts to stabilize. Tetradecylglycidic acid additions were made to the reservoir at various times before the addition of the stock oleate solution containing [9, lo-sH]palmitate. Aliquots were taken from the reservoir at the times specified for measurements of 3H counts in the water. Each aliquot was frozen in the top of a Thunberg tube with a saturated dry ice-acetone mixture and the tubes evacuated. The Thunberg tubes were kept in the dry ice-acetone mixture for 2 h and a sample of the distillate taken for scintillation counting in Handifluor (Mallinckrodt Inc.). Fatty acid oxidation for each time point was calculated from measurements of the tritiated water release and the specific activity of the perfusate fatty acids.

Reagents Stock solutions of oleate complexed to albumin were prepared in the following manner. A 30% (w/v) solution of essentially fatty acid free bovine serum albumin (Sigma Chemical Company) in Ca s+-free Krebs bicarbonate medium was first filtered through a 0.8 pm Millipore filter to remove the residual charcoal particles left from the defatting procedure. The stock solution was dialyzed 3 times against Krebs bicarbonate medium without glucose or calcium for a total of 24 h. Oleic acid (Chromatopure, Applied Science Laboratories) was converted to its sodium salt and dissolved in Gas+-free Krebs bicarbonate medium containing 20% (w/v) fatty acid free bovine serum albumin warmed to 37°C. The stock solution prepared in this way was filtered before use through a 0.65 pm Millipore filter to remove microcrystals of sodium oleate not complexed to the albumin. Stock solutions were assayed by the method of Laurel1 and Tibbling [Ia] and had a fatty acid concentration of approximately 40 mM. This gave an oleate/albumin ratio of 13.8 which is nearly identical to the maximum fatty acid/albumin ratio of 13.5 found by Spector et al. [40]. Octanoic acid (Sigma Chemical Company) was also converted to its sodium salt and dissolved in the same manner as oleate. 2-Tetradecylglycidic acid, TDGA, (McNeil Laboratories) was prepared in a solution of 1.5 mg/ml in medium identical in composition to that used to dissolve oleate. Additions of fatty

898

ET AL.

F. J. PEARCE

acids and TDGA were made over a period of 1 to 2 min to a stirred reservoir of recirculated medium. Crystalline bovine insulin free of preservatives and glucagon was provided by Dr Walter E. Shaw, Eli Lilly Corporation and was stored in a stock solution of 100 U/ml in 0.6% acetic acid. 3. Results Effects of Z-tetradecylglycidic acid (TDGA) acid oxidation and contractility

on fatp

The rate of fatty acid oxidation by the working perfused rat heart was assessed by measuring the rate of sH release from [9, lo-sH]palmitate into water with 5 mM glucose, 1 mM oleate and 2% (w /v ) alb umin present in the recirculating perfusion medium. The data shown by the closed circles in Figure 1 illustrate that under control conditions, fatty acid oxidation was linear at a rate of 98 f 8 pmol/g dry wt/h. The oxygen consumption ofthese hearts was4670 f 342 patom/g dry wt/h, which corresponds to a relatively low work situation [IS, 531 but higher than that of Langendorff preparations. Since 1 pmol of oleate requires 51 patoms of oxygen for complete oxidation, all of the oxygen consumption could be accounted for by the oxidation of exogenous fatty acids. Preliminary studies suggested that the

60

0

5

20

35 Time

FIGURE 1. Inhibition of fatty acid oxidation by perfused as working heart preparations with 5 rn~ 10 min (O), 18 min (x), and 34 min (A) before palmitate. The control rate of fatty acid oxidation circles (a), where each point represents the mean

50

65

(min)

tetradecylglycidic acid (TDGA). Hearts were glucose, 2% albumin and 10-s M TDGA for addition of 1 mu oleate containing [9, lo-sH] with no TDGA present is shown as the solid f S.E.M. of four hearts.

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effectiveness of TDGA as an inhibitor of exogenous fatty acid oxidation depended both on its concentration and on the length of time the hearts were exposed to the inhibitor before the fatty acids were added. The effect of the pre-exposure time to 10-S M TDGA is illustrated in Figure 1. Under the conditions of this experiment, 10 min of perfusion with TDGA before addition of the fatty acids resulted in an 807; inhibition of fatty acid oxidation, while complete inhibition was produced when TDGA was added 30 min prior to oleate and [sH]palmitate. Figure 2 shows the effect of TDGA concentration on the percentage inhibition of fatty acid oxidation. The inhibitor was added to the perfusion medium 10 min before the addition of 1 mM oleate. Under these conditions, half-maximal inhibition

0.5

1.0

2

5 [TDGA]

IO

20

x IO-~

M

50

100

FIGURE 2. Effect of tetradecylglycidic acid (TDGA) concentration on fatty acid oxidation. Hearts were perfused as working heart preparations with 5 nm glucose, 2% albumin, and various concentrations of TDGA for 10 min before addition of 1 mM oleate containing [9, IO-WJpalmitate. Rates of fatty acid oxidation are expressed as a percentage of the control rate, which was 98 pmol/g dry wt/h.

of fatty acid oxidation was obtained at 3 to 4 x 10-s M TDGA. Despite complete inhibition of exogenous fatty acid oxidation at the highest concentration of drug used, there was no deleterious effect on the mechanical function of the heart, indicating that TDGA had no direct toxic effect under these conditions (but see below). In the above experiments glucose was present as substrate in addition to oleate, hence as oxidation of fatty acids became inhibited, oxidation of glucose increased. Enhanced rates of fatty acid oxidation are characterized by an increased steadystate level of reduction of the mitochondrial pyridine nucleotides. Previous work has established that the oxidation-reduction state of the pyridine nucleotides in intact organs can be assessed by surface fluorometry [5, 9, 35, 371. Application of this technique to a study of the inhibitory effects of TDGA on fatty acid oxidation in hearts perfused with 5 mu glucose and 2% albumin is illustrated in Figure 3.

900

F. J. PEARCE

(a)

ET AL.

Control

0. I rnhf

octonoote

I b 1 [TDGA]

FIGURE 3. Effects of oleate and in the absence (a) and presence (b) as working heart preparations with The results are expressed as percent the tissue with 5 mr.4 glucose as the

octanoate on pyridine nucleotide fluorescence of hearts perfused of 10-s M tetradecylglycidic acid (TDGA). Hearts were perfused 5 nm glucose and 2% albumin before the addition of fatty acids. of the full scale reading obtained from the initial fluorescence of only substrate.

In Figure 3(a), two successive additions of 0.1 mM oleate produced increments of increased pyridine nucleotide fluorescence, showing that the effects of fatty acid on the NADf oxidation-reduction state was concentration dependent. Subsequent addition of 0.1 mM octanoate caused an even larger increase of fluorescence, indicating that the capacity for P-oxidation was not saturated. In Figure 3(b), the heart was perfused with 10-s M TDGA for 10 min prior to the first addition of fatty acid. Under these conditions, the fluorescence response to 0.1 mM oleate was almost completely abolished, while the response to 0.1 mM octanoate was not affected. The percentage inhibition of the oleate- and octanoate-induced increase of pyridine nucleotide fluorescence as a function of TDGA concentration (with a 10 min period of pre-exposure), is shown in Figure 4. The effect of oleate (0.1 mM) was completely inhibited at 10-s M TDGA, with half-maximal inhibition occurring at 3 x 10-s M (cf. Figure 2). Very little inhibition of the octanoate response was observed at all concentrations of TDGA. These findings are in accordance with the drug’s postulated site of action at the mitochondrial external palmitylcarnitine transferase [43, 44, 461. Thus, since octanoate forms octanoyl-CoA directly in the mitochondria and does not require carnitine or palmitylcarnitine transferase for its oxidation, TDGA has no effect on its oxidation. Figure 5 (a) illustrates that 0.2 mM oleate followed by 1O-s M TDGA had no effect on the left ventricular pressure development of hearts perfused under the same conditions as those above. In Figure 5(b) it is shown that TDGA reversed the

FATTY

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OXIDATION

2

4 [TDGA]

901

IN RAT HEART

6

8

IO

x IO-~ M

FIGURE 4. Effect of tetradecylglycidic acid (TDGA) concentration on the fatty acid induced reduction of pyridine nucleotides. Hearts were perfused as working heart preparations with various concentrations of TDGA added 10 min before addition of 0.1 nm oleate or 0.1 mM octanoate. The (a) induced pyridine results are expressed as the percent inhibition of the oleate- (0) or octanoatenucleotide reduction caused by the presence of 10-s M TDGA.

(0)

0.2 rnM

cleate

FIGURE 5. Effect of tetradecylglycidic acid (TDGA) on left ventricular pressure (a) and pyridine nucleotide fluorescence (b) in the presence of oleate. This heart was perfused as a standard working heart preparation with 5 mu glucose and 2% albumin and allowed to stabilize for 15 min before addition of 0.2 mu oleate. After about 2 min, IO-5 M TDGA was also added. Pyridine nucleotide fluorescence changes are expressed as percent of the full scale reading obtained from the initial fluorescence of the tissue surface with 5 mu glucose as the only substrate.

902

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El

AL.

reduction of pyridine nucleotides induced by oleate, and gradually decreased the pyridine nucleotide fluorescence level to below the initial control value. This relative oxidation of the pyridine nucleotides suggests that both exogenous and endogenous fatty acid oxidation are inhibited by TDGA. Since cardiac performance was not affected by the inhibition of fatty acid oxidation, it is apparent that at this work level, oxidation of alternative substrates (i.e. glucose and glycogen) was sufficient to meet the energy demand. Furthermore, it is evident that the pyridine nucleotide fluorescence level responds to substrate concentration effects at the dehydrogenases and not to altered flux through the respiratory chain. At higher work levels, however, TDGA (10-s M) addition resulted in a deterioration of cardiac function as shown by the decreased systolic aortic pressure [Figure 6(a)]. There was also a relative oxidation of the pyridine nucleotides [Figure 6(b)]. This latter change may be taken to reflect an inhibition of fatty acid oxidation. The data depicted in Figure 6 was obtained with a heart perfused with (a)

IO+W TDGA

--j

(b)

1

5

min+

T

Reduction

FIGURE 6. Effect of 10-s M tetradecylglycidic acid (TDGA) on aortic pressure (a) pyridine nuclcotide fluorescence (b) in a high working heart. This heart was perfused glucose at a left atrial filling pressure of 10 cm of HsO. The work level was adjusted restricting the aortic cannula and pacing at a rate of 400 beats/mm. Pyridine nucleotide changes are expressed as the percent of the full scale reading obtained from the initial of the tissue surface with 5 nns glucose as the only substrate.

and surface with 5 msr by partially fluorescence fluorescence

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5 mM glucose as the only exogenous substrates, and with a higher work load obtained by partially constricting the aortic outflow. This resulted in an oxygen consumption which was about 60% greater than that of the heart shown in Figure 5 (i.e. 6600 vs 4200 patom/g dry wt/h). It is apparent that under these conditions of increased energy demand, fatty acid oxidation was indispensible for the maintenance of ventricular function and that oxidation of glucose could not increase sufficiently to meet the increased energy requirements, as illustrated also by the work of Kobayashi and Neely [ 161. Relationship between oleate-induced increases of oxygen consumption and pyridine nucleotidejuorescence with constant flow perfusion

The results of a typical experiment in which increasing concentrations of oleate were added to a heart perfused with a medium containing 5 mM glucose and 2% albumin are shown in Figure 7. The heart was perfused by retrograde flow through the aorta at a constant flow rate of 13 ml/min, asdetermined by a peristaltic pump. The volume of recirculating perfusion fluid was large (400 ml) in order to minimize changesof oleate concentration at the lower values causedby its oxidation. Oxygen uptake was measured simultaneously with the pyridine nucleotide fluorescence from the surface of the left ventricle. No external work was performed by the heart, and the control rate of oxygen consumption was about 2800 patom/g dry wt/h. A 30 min period of perfusion with 5 mM glucose prior to oleate addition allowed the

’ reduction

0.02

0.04

0.08

0.12

0.24

0.36

0.460.60

mM oleate FIGURE 7. Effect of oleate concentration on the surface pyridine nucleotide fluorescence (a) and oxygen consumption (b) in a “nonworking” heart. The coronary circulation was perfused via the aorta at a constant flow rate of 13 ml/min with medium containing 5 mu glucose and 2% albumin. The perfusion pressure at this flow rate was 80 cm water. After a 20 min stabilization period, the oleate concentration was increased in a stepwise manner by addition of 40 mu stock oleate complexed with albumin to a 400 ml recirculated reservoir. The pyridine nucleotidc fluorescence in (a) is expressed as percent of the full scale reading obtained from the initial fluorescence of tissue surface. The control oxygen consumption in (b) is indicated by the dotted line in the bottom trace.

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heart to achieve a stable metabolic state. As shown in Figure 7, a significant increase of pyridine nucleotide fluorescence occurred with 0.04 mM oleate, but no increase of oxygen consumption could be detected at oleate concentrations below 0.12 mM. At this oleate concentration the fluorescence change was almost maximal. A small decrease of oxygen consumption occurred with oleate concentrations below 0.1 mM. This may be the consequence of a direct effect of fatty acids on the vascular tone. Thus, in some hearts the aortic pressure decreased slightly, which resulted in a small decrease of the work load. These effects, however, were very small and it is clear that the NADf oxidation-reduction state was more sensitively affected than the oxygen consumption by exogenous fatty acids. The results of a series of hearts perfused under similar conditions to those of Figure 7 are shown in Figure 8. In order to normalize the pyridine nucleotide fluorescence data between different hearts, the changes induced by oleate addition are presented as a percentage of the maximal fluorescence change observed during anoxia [Figure 8(a)]. Figure 8(b) shows the absolute change of oxygen consumption after oleate addition. The closed circles correspond to hearts perfused with 5 mM glucose prior to oleate addition while data represented by the open circles were obtained with hearts perfused with 5 mM glucose and 10-s units/ml of insulin. The dashed line in Figure 8(c) shows perfusion pressure changes, while the solid line shows changes of left ventricular pressure. Since the presence of insulin had no effect on pressure changes induced by oleate, the data obtained with and without insulin were combined in Figure 8(c). Oleate addition in the absence of insulin caused an increase of pyridine nucleotide fluorescence at the lowest fatty acid concentration used (cf. Figure 7). Addition of 1 mM octanoate at levels of oleate which saturated the pyridine nucleotide fluorescence led to no further reduction of the pyridine nucleotides, indicating that flux through the P-oxidation pathway was already saturated (data not shown). In the presence of insulin, the pyridine nucleotides were more reduced under control conditions, and more than 0.3 mM oleate was required to cause a further reduction of NADf. The increments of oxygen consumption after oleate addition were greater in the presence than in the absence of insulin. However, the control rate of oxygen consumption in hearts perfused with glucose and insulin was an average of 177 j, 40 patom/g dry wt/h lower than that observed in hearts perfused with glucose alone (2440 & 200 patom/dry wt/h). Therefore at oleate concentrations up to 0.8 mM, the oxygen consumption was similar in the absence and presence of insulin. These relative changes of oxygen consumption relate to the effect of insulin in promoting glucose oxidation at the expense of endogenous or exogenous fatty acid oxidation (see discussion). Of particular interest in the data shown in Figure 8 is the finding that at oleate concentrations above 0.8 mM (i.e. at fatty acid/albumin molar ratios greater than 2.5), there was a disproportionate increase of oxygen consumption, which coincided with a decrease of left ventricular pressure development. Under these conditions, therefore, exogenous fatty acids were deleterious to myocardial performance.

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(al

P

A, /

&

0.2

0.4

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0.6

0.8

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mlY o1oote

FIGURE 8. The effect of oleate concentration on surface pyridine nucleotide fluorescence (a), oxygen consumption (b), and in (c) perfusion pressure (dotted line) and left ventricular pressure (solid line). Hearts were perfused by constant flow perfusion and the same experimental protocol was followed as described in Figure 7. In (a) and (b) data given by the closed circles were obtained in the absence of insulin, and those given by the open circles in the presence of insulin. Insulin was added after the 20 min stabilization period at a concentration of 10 mu/ml. The pyridine nucleotide fluorescence changes are expressed as the percent of the differences in signal obtained from a 3 min anoxic period at the end of the experiment and the initial signal obtained after the 20 min stabilization period. Five hearts were perfused without insulin and three with insulin. Each point represents the mean value of at least three measurements.

Negative inotropic effect of oleate in hypoxic hearts and its prevention by 2-tetrade&glycidic acid These studies were designed to test the hypothesis that increased fatty acid oxidation may depress myocardial function during hypoxia by increasing the oxygen demand. Experiments were performed using working heart preparations perfused

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with 5 mM glucose and 2% albumin. Hearts were made hypoxic by decreasing the oxygen content of the gas mixture equilibrating the perfusion fluid from 95% to 35% saturation. With this protocol, coronary flow rates increased from about 15 to 20 ml/min. The effects of oleate addition during hypoxia on the functional parameters of left ventricular pressure and aortic output were measured, as illustrated in Figure 9(a). In the hypoxic heart the available oxygen was all consumed and the oxygen uptake decreased from 4230 to 3200 patom/g dry wt/h. Left ventricular pressure fell by about lOoh, while the aortic output almost ceased. Under these conditions, the rate of oxidative energy metabolism was clearly insufficient to maintain cardiac function at its normal aerobic level. Subsequent addition of oleate decreased left ventricular pressure only slightly, but cardiac function was impaired even further, as shown by the reversal of the aortic flow to retrograde perfusion. Within the time framework of the experiment shown in Figure 9, the negative inotropic effect of oleate was reversible. Figure 9 (b) shows the results of a similar experiment but with a heart perfused with 5 mM glucose and 5 x 10-s M TDGA for 40 min prior to the onset of hypoxia. The arterial oxygen tension was lowered more gradually in this experiment, and

FIGURE 9. Effect of oleate on myocardial function during hypoxia. In (a) the heart was perfused as a working heart preparation with 5 nxs glucose and 2% albumin. Hypoxia was induced by lowering the influent Pas to 250 rnmHg. After two sequential additions of 0.5 mre oleate, washout of the fatty acid was accomplished with medium containing 5 rn~ glucose and 2% albumin. In (b) the per&ion conditions were the same as in (a) with the exception that 5 x 10-s M tetradecylglycidic acid (TDGA) was present in the per&ate for 44) min before the onset of hypoxia.

on the surface pyridine nucleotide PLATE 1 (0uerZeaf). Effect of tetradecylglycidic acid (TDGA) fluorescence during hypoxia. Hearts were perfused as working heart preparations with 5 rn~ glucose and 2% albumin before inducing hypoxia by decreasing the Pas. The pyridine nucleotide fluorescence photographs are sequentially arranged and show: (a) the control, normoxic condition; (b) after 10 min of hypoxia, Pas = 420 mmHg; 5 min after addition of 0.5 rnM oleate. Photographs (d), (e), and (f) were taken 17, 33 and 48 min after addition of 10-s M TDGA, respectively.

[facing

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the left ventricular pressure and aortic output correspondingly fell more slowly. Subsequent addition of oleate had no effect on the left ventricular pressure or aortic output, indicating that the negative inotropic effect of oleate observed in Figure 9(a) was not caused simply by the presence of oieate but required its oxidation. It is evident from the work of Steenbergen et al. [41, 421 that hypoxia in the perfused rat heart is associatedwith heterogeneousoxygenation, such that islands or zonesof anaerobic tissuedevelop which are surrounded throughout the thickness of the ventricle wall by well-oxygenated tissue.This phenomenon can be visualized by taking photographs of the heart using an intense homogeneous light flash at wavelengths that will excite fluorescence emission from NADH in the epicardial cell layers of the tissue. The number and size of anoxic areas visualized by this technique provides a sensitive index of tissueoxygenation since they are determined by the balance between oxygen supply and demand. The results of application of this technique to the study of the effects of oleate and TDGA on the oxygenation state of the hypoxic heart is shown in Plate 1. Plate 1(a) is a picture of the control well-oxygenated heart which shows that the surface of the left ventricle appears uniform, except for major vesselswhich appear dark and the atrium which is light. Photograph 1(b) was taken 10 min after development of hypoxia. The degree of hypoxia was slightly lesssevere than that illustrated in Figure 9(a). White areasare seen on the ventricle, which are caused by high NADH fluorescence, and correspond to anoxic areas of tissue. Photograph 1(c) was taken 5 min after the addition of 0.5 mM oleate to the same heart. Two effects are observed: first the grey background tone of the ventricle is lighter, indicating a greater degree of NAD+ reduction in the aerobic tissue; second there is an increase in the number and size of the anoxic zones. TDGA (10-s M) was subsequently added to the perfusate and photographs l(d), l(e) and l(f) were taken after 17, 33 and 48 min, respectively. These photographs show that there was a gradual darkening of the background tone indicating a lower level of NADH in the aerobic tissue and a disappearance of the white anoxic areas. Comparison of 1(f) and 1(b) showsthat after 48 min of perfusion with TDGA, the heart had fewer anoxic areas than were present before oleate was added. Inhibition of endogenous fatty acid oxidation may explain this improvement. During the time TDGA was present in the perfusate medium there was a slight improvement of left ventricular pressuredevelopment and aortic output. These results indicate that inhibition of fatty acid oxidation during hypoxia decreased the massof anoxic tissue present.

4. Discussion Eficts

of Ptetradecylglycidic

acid (TDGA)

on fat@ acid oxidation

The methyl ester of 2-tetradecylglycidic acid has been found to be an orally effective antiketogenic and hypoglycemic agent in rats, dogs and mice [4.5]. Results

908

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ET AL.

of studies using isolated rat hepatocytes [43] and rat hemidiaphragms [46] have indicated that the blood glucose lowering effect of TDGA is the result of increased peripheral glucose utilization and decreased hepatic gluconeogenesis. These changes are thought to be secondary to inhibition of long chain fatty acid oxidation. A study of the effects of this compound on the myocardium is of interest both for the evaluation of its potential in the treatment of diabetes and as a tool in the exploration of mechanisms relating to the metabolic and functional consequences of fatty acid metabolism. The advantages of TDGA over previously used inhibitors of fatty acid oxidation are: (1) that it is very specific in its action, inhibiting only long chain fatty acid oxidation and (2) it is effective at concentrations much lower than those required for other fatty acid oxidation inhibitors such as pent-4-enoic acid or cr-bromopalmitate [46]. This latter characteristic minimizes the potential for interference due to metabolism of the inhibitor itself which has recently been observed with pent-4-enoic acid [I,?, 131. To date, information concerning the mechanism by which TDGA inhibits long chain fatty acid oxidation has been derived from isolated rat hepatocytes and mitochondria, and rat diaphragm, where the site of inhibition has been reported to occur at the external long chain acyl-carnitine transferase. This conclusion was based in part on the finding that TDGA inhibited the oxidation of long chain fatty acids and ketogenesis, while the oxidation of palmitylcarnitine, short chain fatty acids, and tricarboxylic acid cycle intermediates were unaffected [43, 441. The data presented here concerning the effects of TDGA on the perfused rat heart were found to be consistent with the results previously obtained using other tissues [43-461. The working perfused rat heart exhibited a concentration-dependent inhibition of isotopically labeled long chain fatty acid oxidation by TDGA with a Kt of about 3 x 10-s M, which was very similar to that observed in rat hepatocytes and rat diaphragm [43, 461. In addition, the degree of inhibition at a particular TDGA concentration depended on the length of time the heart was exposed to the compound before exogenous fatty acids were added. Although conclusive evidence is not yet available, preliminary findings suggest that tetradecylglycidyl-CoA is the actual inhibitor of the long chain acylcarnitine transferase [G. Tutwiler, unpublished observations]. The pre-exposure time probably represents the time required for successful competition of TDGA with the naturally occurring fatty acids before maximal inhibiting concentrations of tetradecylglycidyl-CoA are formed. The effect of TDGA on fatty acid oxidation was also investigated from measurements of the pyridine nucleotide fluorescence from the surface of the heart. The dose-dependent relationship of TDGA concentration to the inhibition of the reduction caused by oleate was nearly identical to that obtained with the direct isotopic method for measuring fatty acid oxidation rate. Essentially no effects of TDGA were observed on the octanoate-induced increase of pyridine nucleotide fluorescence. Taken together, these results provide strong evidence for the inhibi-

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OXIDATION

IN RAT

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909

tory effect of TDGA on carnitine-dependent long chain fatty acid oxidation in the heart. Net oxidation of the pyridine nucleotides after addition of TDGA suggested that endogenous as well as exogenous fatty acid oxidation was inhibited by the drug.

RelationshiP between fat0 acid induced increases in oxygen consum@tion and pyridine nucleotidefluorescence Oram et al. [29] using [U-W]palmitate complexed with 3% albumin showed that palmitate uptake and oxidation increased with cardiac work and that a higher palmitate concentration was required to saturate fatty acid oxidation at the higher work loads. This fact explains the lower sensitivity of the pyridine nucleotide fluorescence to oleate between the data in Figure 3 with a moderately working heart (oxygen uptake of 4600 patom/ dry wt/h) and the data in Figures 7 and 8 with hearts performing no external work (oxygen uptake of 2400 yatom/g dry wt/h) . The different sensitivities of respiration and pyridine nucleotide fluorescence to increasing oleate concentrations and the effects of insulin shown in Figure 8 can best be explained on the basis of the known interactions between glucose and fatty acid oxidation [24,25,33,34]. Previous studies have shown that in the Langendorff rat heart preparation perfused with glucose alone, glucose oxidation accounts for only about 25% of the oxygen uptake, the remainder being derived from endogenous triglyceride and fatty acids [52]. Under these conditions the pyridine nucleotides are relatively highly oxidized. Addition of exogenous fatty acids even at concentrations below 0.1 mM increases substrate availability to the mitochondrial dehydrogenases with the result that the NADH/NAD+ ratio increases. The lack of a significant increase of respiration at fatty acid concentrations below 0.1 mM suggest that exogenous fatty acids are replacing endogenous fatty acids as respiratory fuel. At higher fatty acid concentrations, glycolysis and glucose oxidation become inhibited [23, 24, 28, 331. Inhibition of glycolysis is attributed primarily to an inhibition of phosphofructokinase by citrate [24, 331. Citrate accumulates in hearts in response to an elevation of acetyl-CoA levels [32, 511. Pyruvate dehydrogenase activity is also decreased by fatty acids, presumably as a result of elevated NADH and acetyl-CoA levels [15, 481. An increase of acetyl-CoA [25, 291 and citrate [25] only occurs with fatty acid concentration above 0.2 mM, thereby supporting the conclusion that inhibition of glycolysis, at least with relatively low working hearts, occurs after the fatty acid induced increase of NADH has reached a maximal level. In the presence of insulin, glucose oxidation accounts for between 60 and 90% of the oxygen consumption, so that oxidation of endogenous fatty acids is essentially suppressed [16, 36, 521. The increased substrate availability relative to the insulin-free controls increases the steady state level of NAD+ reduction. After addition of fatty acids, further reduction of NAD+ together with substantial increases of respiration only occur with fatty acid concentrations greater

910

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AL.

than 0.2 mM, which are associated with increased rates of fatty acid oxidation at the expense of glucose oxidation [23, 28, 29, 331. Although the increase of pyridine nucleotide fluorescence after fatty acid addition originates primarily from reduction of mitochondrial dehydrogenases, it is of interest that the lactate/pyruvate ratio in the perfusion fluid of hearts perfused with glucose under moderate work conditions increased from 4 to 14 after addition of fatty acids. This increase was accounted for by an increased production of lactate, with pyruvate remaining approximately constant, and probably reflects inhibition of pyruvate dehydrogenase. It may be noted that an increase of the mitochondrial NADH/NAD+ ratio should correspond to an increase of the cytosolic phosphorylation potential [ATP/ADP . Pi] [S, 471. Th is in turn will tend to cause a decrease, rather than an increase, of the cytosolic NADH/NAD+ ratio, if near equilibrium of glyceraldehyde-3-P dehydrogenase and P-glycerate kinase is maintained [X7]. Possibly reducing equivalents are transported out of the mitochondria in the form of malate [I41 combined with an entry of oxalacetate [30], as a consequence of alterations of the anion concentration gradients across the mitochondrial membrane favorable to these processes.

SigniJiance

of fat9

acid induced stimulation

of respiration

A small stimulation of oxygen consumption may be expected if the heart switches from carbohydrate to fatty acid oxidation since the ratio of ATP produced to oxygen consumed is about 13% less for fatty acid than for carbohydrate oxidation. Thus, for complete oxidation of oleate, the overall ATP/O ratio is 2.82 compared to 3.17 for glucose oxidation. Taking as the starting point the control situation of “non-working” hearts perfused with 5 mM glucose, the mean rate of respiration was 2440 patom/g dry wt/h, which corresponds to a rate of ATP generation of 8000 pmol/g dry wt/h. If the rate ofATP generation is assumed to remain constant, the transition from 25% to SO-90% carbohydrate utilization induced by insulin should cause a 5 to 8% decrease of oxygen uptake. This compares favorably with the observed decrease of 7%. Addition of fatty acids in the absence of insulin can cause a maximum increase of fatty acids already account fatty acid oxidation of only 25% (b ecause endogenous for 75%), which corresponds to a 3% increase of respiration. Likewise, addition of fatty acids in the presence of insulin can be assumed to cause conversion from 60 to 90% carbohydrate to 100% fatty acid oxidation, and respiration should increase by 7 to 11%. After addition of 0.6 mM oleate and 2% albumin (free fatty acid/ albumin molar ratio of 1.8), the oxygen uptake was increased by 11 o/0 in the absence of insulin and by 24% in its presence. In both cases this is more than expected solely from the decrease of the ATP/O ratio. On the assumption that the mitochondria remain fully coupled, the theoretical extra ATP production corres-

FATTY

ACID

OXIDATION

IN RAT

HEART

9iI

ponds to 530 and 740 pmol/g dry wt/h, respectively, in the absence and presence of insulin. Since there is no evidence that energy expenditure for contraction changes, the extra ATP production must be associated with a biosynthetic process or an ATP-consuming “futile” cycle. Triglyceride synthesis in heart is stimulated by the addition of exogenous fatty acids [28], and insulin [33]. The extra energy requirement (1 mole of triglyceride requires 7 moles of ATP) may thus be associated with increased fatty acyl-CoA formation, combined with energy wastage resulting from hydrolysis of the fatty acyl-CoA by the long chain fatty acyl-CoA hydrolases since triglyceride formation alone cannot account for the extra ATP usage. In contrast to the above results, which are in basic agreement with those of Oram et al. [29], at fatty acid/albumin molar ratios above 2.5 the percentage increase of oxygen consumption was much higher (up to 50%) and both the perfusion pressure and left ventricular pressure declined. The magnitude of this stimulation of respiration is in agreement with that found by Challoner and Steinberg [3] in non-working perfused rat hearts with fatty acid/albumin molar ratios of 4. Fatty acids are known uncoupling agents of mitochondrial oxidative phosphorylation [2, 311, and the large stimulation of respiration by fatty acids in the perfused heart has been interpreted as being caused by an uncoupling action [3, 211. If increased proton permeability of the mitochondrial membrane is the mechanism for the increasedrespiration, an oxidation of the mitochondrial pyridine nucleotides is expected as the classicalresponseto uncoupling agents. As seenfrom Figure 8, this does not occur. Alternatively, it is more likely that the theoretical ATPjO ratio is maintained, but that further activation of an ATP-consuming futile cycle such as fatty acyl-CoA formation and hydrolysis takes place. Unfortunately, information on the regulation of fatty acyl-CoA hydrolases is currently lacking. In any event, high fatty acid concentrations at a fatty acid/albumin molar ratio in the range of 2.5 to 4 (depending on the cardiac work) appear to be toxic to the heart. Whether this is caused by inadequate energy availability to support optimal contractility, to inhibitory effects of long chain fatty acyl-CoA, or to impairment of intracellular Cazf interaction with the myofibrils requires further investigation.

Fatty acid effects on myocardial contractility

in the hypoxic state

From the studies of Opie [27J, and others [IO, II, 17, 22, 271, it is clear that high fatty acid concentrations can have a deleterious effect on hearts already compromised by oxygen insufficiency. Henderson et al. [IO, II] attempted to evaluate the mechanism of the negative inotropic effect of fatty acids by useof the fatty acid oxidation inhibitor pent-4-enoic acid in rat papillary muscle [II] and intact perfused rat heart [IO]. These authors found that pent-4-enoic acid caused a depression of contractility similar in degree to that caused by linoleic acid. This finding led them to conclude that the depressionmust have been mediated directly

912

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ETAL.

by elevated intracellular concentrations of free fatty acids or their acyl-CoA derivatives. It was suggested that increased binding of intracellular calcium and thereby a limited calcium availability to the myofibrils would result. However, recent studies of pent-4-enoic acid metabolism in the heart have indicated that pent-4-enoic acid can be oxidized, and that it stimulates the oxygen consumption by about 15% [12, 131. In view of these more recent findings the interpretation of the conclusions of Henderson et al. [IO, II] must be re-evaluated. The use in the present study of a new fatty acid oxidation inhibitor, 2-tetradecylglycidic acid (TDGA), offers a unique opportunity to re-examine the effects of fatty acids on contractility in the hypoxic heart. Unlike pent-4-enoic acid, TDGA is not an oxidizable substrate and does not stimulate oxygen consumption. Furthermore, except with high working hearts, TDGA has no effect on contractility, unlike pent-4-enoic acid. The ability of TDGA to prevent the oleate-induced depression of left ventricular pressure development and aortic output in hypoxic hearts together with its demonstrated ability to effectively inhibit fatty acid oxidation, indicates that the deleterious effect of oleate specifically depends on its oxidation and is not simply due to its presence. An alternative explanation for the hypodynamic effect of fatty acids is that they cause the hypoxic heart to become further compromised as a result of a stimulation of oxygen consumption in localized areas of tissue at the expense of oxygen delivery to other areas. The technique of pyridine nucleotide surface fluorescence photography has previously shown that hypoxic condition is characterized by a heterogeneous pattern of anoxic and aerobic tissue, and that the size and number of the anoxic zones correlate with the excess of oxygen demand over oxygen delivery [41, 421. The photographs in Plate 1 show that the size and number of the anoxic areas in the hypoxic heart are increased by oleate addition, reflecting the increase in oxygen demand. This observation indicates that the oleate-induced depression of contractility results from the increase in the mass of non-contracting anoxic tissue which arises from the increased oxygen demand. Subsequent inhibition of long chain fatty acid oxidation by TDGA caused the anoxic areas to disappear. This finding supports the proposal that oxidation of fatty acids is the basis for the deleterious effects of fatty acids in hypoxic myocardium.

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Inhibition of fatty acid oxidation and in normal and hypoxic perfused rat hearts by 2-tetradecylglycidic acid.

3ouwwl of Molecular and Cellular Cardiology (1979) 11, 893-915 Inhibition of Fatty Acid Oxidation in Normal and Hypoxic Perfused Rat Hearts by 2...
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