Regulation of Fatty Acid Utilization in Isolated Perfused Rat Hearts

Abstract Regulation of fatty acid utilization was studied in the isolated, perfused rat heart. The effects of increasing the concentration of perfusate fatty acid and the level of ventricular pressure development on the rates of fatty acid uptake and oxidation and on the tissue levels of CoA and carnitine derivatives were determined. In hearts that were developing low levels of ventricular pressure, the rate of palmitate uptake was proportional to its concentration in the perfusate as the concentration was raised from 0 to 0.6 mm bound to 3% albumin. The faster rate of uptake was associated with only a slight increase in the tissue content of long chain acyl-CoA, acylcarnitine, acetyl-CoA, and acetylcarnitine. Palmitate utilization appeared to be limited by the rate of fatty acid uptake or activation. As the concentration of palmitate was increased from 0.6 to 1.2 mm, the rate of uptake did not increase further; oxygen consumption and 14CO2 production from [U-14C]palmitate increased only slightly, and large amounts of acyl-CoA and acylcarnitine derivatives accumulated in the tissue. These observations indicated that the rate of fatty acid uptake at high levels of exogenous palmitate was limited by the rate of acetyl-CoA oxidation through the citric acid cycle. Fatty acid activation and transfer of the acyl unit from acyl-CoA to acylcarnitine may have been limited by high acetyl-CoA to CoA and acetylcarnitine to carnitine ratios. It was estimated that the concentration of CoA and carnitine decreased to below the level needed for optimal rates of fatty acid activation and oxidation. The mass-action ratio for the carnitine palmityltransferase was constant and independent of the exogenous fatty acid concentration. The mass-action ratio for the carnitine acetyltransferase was shifted toward acetyl-CoA formation as the level of palmitate was raised. A possible role of the carnitine acetyltransferase system in coupling the rate of flux through the citric acid cycle with fatty acid activation and acyl transfer is discussed. Increased ventricular pressure development resulted in (a) a faster rate of oxidative phosphorylation as indicated by increased oxygen consumption (b) an acceleration of the citric acid cycle as indicated by a large increase in CO2 production, and (c) an increase in uptake and β oxidation of palmitate. Acceleration of the citric acid cycle was associated with a decrease in the tissue content of fatty acids, acyl-CoA, acetyl-CoA, and acetylcarnitine and an increase in the levels of acylcarnitine, free CoA, and free carnitine. When high levels of exogenous palmitate were present, the increase in CoA and carnitine could have accelerated the rates of fatty acid activation and acyl transfer from acyl-CoA to acylcarnitine and thus increased the rate of palmitate uptake. With concentrations of palmitate below 0.6 mm, the changes in CoA and carnitine were probably too small to account for the increased fatty acid uptake. The tissue content of long chain acylcarnitine increased with acceleration of oxidative metabolism even though the levels of both acyl-CoA and acetyl-CoA decreased. The mass-action ratio for the carnitine palmityltransferase system shifted toward acylcarnitine formation. In contrast to palmitate, oxidation of octanoate was fast enough to maintain high levels of acetyl-CoA when fatty acid oxidation was accelerated by increased cardiac work. These results suggested that the rate of translocation of acyl units across the inner mitochondrial membrane limited the rate of long chain fatty acylcarnitine oxidation at high levels of ventricular pressure development.


Regulation
of fatty acid utilization was studied in the isolated, perfused rat heart.
The effects of increasing the concentration of perfusate fatty acid and the level of ventricular pressure development on the rates of fatty acid uptake and oxidation and on the tissue levels of CoA and carnitine derivatives were determined.
In hearts that were developing low levels of ventricular pressure, the rate of palmitate uptake was proportional to its concentration in the perfusate as the concentration was raised from 0 to 0.6 mM bound to 3% albumin.
The faster rate of uptake was associated with only a slight increase in the tissue content of long chain acyl-CoA, acylcarnitine, acetyl-CoA, and acetylcarnitine. Pahnitate utilization appeared to be limited by the rate of fatty acid uptake or activation.
As the concentration of palmitate was increased from 0.6 to 1.2 mM, the rate of uptake did not increase further; oxygen consumption and 14C02 production from [U-14C]palmitate increased only slightly, and large amounts of acyl-CoA and acylcarnitine derivatives accumulated in the tissue.
These observations indicated that the rate of fatty acid uptake at high levels of exogenous palmitate was limited by the rate of acetyl-CoA oxidation through the citric acid cycle. Fatty acid activation and transfer of the acyl unit from acyl-CoA to acylcarnitine may have been limited by high acetyl-CoA to CoA and acetylcarnitine to carnitine ratios.
It was estimated that the concentration of CoA and carnitine decreased to below the level needed for optimal rates of fatty acid activation and oxidation. The mass-action ratio for the carnitine palmityltransferase was constant and independent of the exogenous fatty acid concentration.
The mass-action ratio for the carnitine acetyltransferase was shifted toward acetyl-CoA formation as the level of palmitate was raised. A possible role of the carnitine acetyltransferase system in coupling the rate of flux through the citric acid cycle with fatty acid activation and acyl transfer is discussed.
Increased ventricular pressure development resulted in (a) a faster rate of oxidative phosphorylation as indicated by increased oxygen consumption (b) an acceleration of the citric acid cycle as indicated by a large increase in COz production, and (c) an increase in uptake and p oxidation of palmitate. Acceleration of the citric acid cycle was associated with a decrease in the tissue content of fatty acids, acyl-CoA, acetyl-CoA, and acetylcarnitine and an increase in the levels of acylcarnitine, free CoA, and free carnitine. When high levels of exogenous palmitate were present, the increase in CoA and carnitine could have accelerated the rates of fatty acid activation and acyl transfer from acyl-CoA to acylcarnitine and thus increased the rate of palmitate uptake.
With concentrations of palmitate below 0.6 m&I, the changes in CoA and carnitine were probably too small to account for the increased fatty acid uptake.
The tissue content of long chain acylcarnitine increased with acceleration of oxidative metabolism even though the levels of both acyl-CoA and acetyl-CoA decreased.
The mass-action ratio for the carnitine palmityltransferase system shifted toward acylcarnitine formation. In contrast to palmitate, oxidation of octanoate was fast enough to maintain high levels of acetyl-CoA when fatty acid oxidation was accelerated by increased cardiac work.
These results suggested that the rate of translocation of acyl units across the inner mitochondrial membrane limited the rate of long chain fatty acylcarnitine oxidation at high levels of ventricular pressure development.
The importance of fatty acids as substrates for energy mctabo lism in heart muscle is well established.
From 60 to 90% 01" the total oxidative metabolism was accounted for by oxidation of fatty acids under a variety of conditions (l-3).
Fatty acids were oxidized in preference to carbohydrate (4-7). Oxidation of fatty acids or ketone bodies inhibited the utilization of both extracellular glucose and tissue glycogen (S-11).
This inhibition developed at the levels of glucose transport (6, lo), phosphofructokinase (12)(13)(14), glycogcn phosphorylase (II), and pyruvate dehydrogenase (15). Although fatty acids represent an important and preferred substrate for energy metabolism in heart muscle, the mechanisms that regulate the utilization of this substrate are poorly understood.
The rate of fatty acid uptake was shown to depend on the fatty acid to albumin molar ratio in the plasma (16-N).
As this ratio \vas increased, fatty acid uptake and oxidation increased, and a larger fraction of the extracted fatty acid was recovered in tissue lipids (19). At an> fatty acid to albumin ratio, however, the rate of uptake dependcd upon the metabolic state of the tissue. Lptakc was increased by treating the tissue with epinephrine and was decreased by anoxia (2,19,20). The rate of uptake (6) and con-5299 by guest on March 24, 2020 http://www.jbc.org/ Downloaded from 5300 version of [r4C]palmitate to r4C02 (21) was accelerated by increased ventricular pressure development in isolated rat hearts.
The purpose of the present study was to identify the mechanisms that (a) increased fatty acid uptake as the concentration of exogenous palmitate was raised, (b) limited fatty acid oxidation at higher concentrations of exogenous palmitatc, and (c) accelerated fatty acid uptake and oxidation in association with an increase in the rate of osidative phosphorylation secondary to increased ventricular pressure development.
To determine the rate-cont.rolling steps of fatty acid utilization, the tissue levels of acyl-Cod and acylcarnitine derivatives, acetyl-Coh and acetylcarnitinc, and CoA and rarnitine were measured. Transient and steady state changes in the levels of these intermediates and in the rates of palmitate uptake and oxidation were determined after addition of palmitate to the perfusate and after increasing the lcvcl of ventricular pressure development.

METHODS
PerJusion Technique-Hearts from 200-to 250-g male rats were perfused by the Langendorff procedure as described earlier (22). Ventricular pressure development was increased by raising the aortic perfusion pressure.
In this preparation, peak systolic ventricular pressure was from 10 to 40 mm Hg greater than the aortic perfusion pressure over the range of from 50 to 140 mm ITg aortic pressure.
The animals were fasted overnight prior to use. The perfusate was Krebs-Henseleit bicarbonate buffer gassed with 95% 02-5% COZ. The buffer contained glucose (II mM), bovine serum albumin (3%), and the concentrations of fatty acid indicated in the figures. The fatty acid was bound to albumin by solubilizing the free acid as the potassium salt and by injecting the salt mixture into the warm albumin medium.
The albumin-fatty acid complex was dialyzed overnight against a large volume of buffer and filtered through a Riillipore filter (8 cc) before use.
Estimation oj Rates oj O2 Consumption, CO2 Production, and Uptake and p Oxidation of Pa&tale-Osygen consumption was estimated by measuring the difference in arterial and venous Paz and the rate of coronary flow. Coronary effluent was collected without exposure to air by cannulating the pulmonary artery and collecting the effluent under heptane. In the Langendorff preparation, the only fluid returned to the right atrium and ventricle was coronary effluent. Paz was measured with a Clark electrode (Radiometer model PI-I&I 27). 14C02 production from [ U-i4C]palmitate was estimated in coronary effluent that was collected in the same way as for Pop measurements. -1 sample of the perfusate was acidified, the J4C02 released was collected in Hyamine, and the radioactivity was determined in toluene scintillator. For the measurement of i4C02 production and O2 uptake, the perfusate was collected after a single pass through the heart.
Steady state rates of fatty acid uptake were estimated by measuring the disappearance of fatty acids from the perfusate during 15 or 20 min of perfusion.
The perfusate concentration of fatty acids was determined either by the microtitration method described by Noble (23) or by gas chromatography.
For estimation of the transient rate OP palmitate uptake, hearts were perfused with [ U-i%]palmitate, and uptake was estimated from the decrease in radioactivity that occurred during the perfusions. The average rate of uptake during 20 min of perfusion was the same when uptake was determined as disappearance of titratable fatty acid or decrease in perfusatc [ U-i4C]palmitate, indicating that cschangc of labeled with unlabclcd fatty acids in the tissue did not significantly infiueiicc the rate of uptake.
Measuring the decrease in [ U-r4C]palmitate, however, allowed the transient rates of uptake to be followed over short time intervals.
The rate of @ osidation of exogenous [U-i4C]palmitate was determined by measuring the appearance of label in acid-soluble intermediates (acetyl-CoA, acetglcarnitine, and citric acid cycle intermediates) and in COz. The amount of label in the acidsoluble intermediates was determined on 6% perchloric acid extracts of the tissue. The extract was washed twice with heptane (1 ml/2 ml of extract).
The heptane phase was separated by mild centrifugation and was removed by aspiration. By this method more than 95y0 of key citric acid cycle intermediates that were added to the unwashed extract were recovered in the aqueous phase. The amount of label that appeared in the perfusate in compounds other than CO, or palmitate was less than 5% of the total label in the acid-soluble fraction.
Estimation of Tissue Levels of Mehbolic In.termediates-At the end of perfusion, hearts used for estimates of metabolic intermediates were frozen with a Wollenberger clamp maintained at the temperature of liquid nitrogen, powdered in a percussion mortar, and extracted with cold 6% perchloric acid. The extract was neutralized with KOH and used for the analysis of acetyl-Co.4, acetylcarnitine, free CoA, and free carnitine. The perchloric acid precipitate was washed with 0.6% perchloric acid and used for the analysis of long chain acyl-CoA and acylcarnitine derivatives.
The samples were assayed for acetyl-CoA, acetylcarnitine, and CoA within 1 hour after making the extract.
The assays were performed in a Zeiss fluorometer.
Acetyl-CoA was assayed by the citrate synthase method as described by Herrera and Freinkel (24). CoA was assayed by the oc-ketoglutarate dehydrogenase method of Garland et al. (25), and carnitine was determined by combining the CoA assay with a carnitine acetyltransferase and citrate synthase method of Pearson et al. (26). Long chain acyl-CoA and acglcarnitinc were assayed as free CoA and carnitine after alkaline hydrolysis of the washed perchloric acid precipitate obtained from the tissue homogenates (27). Long chain acyl-CoA was hydrolyzed at pH 11 to 12 for 15 min at 55" in the presence of 10 InM dithiothreitol.
Long chain acylcarnitine was hydrolyzed at pH 12.5 to 13.0 for 2 hours at 70". These methods are specific for CoA and carnitine derivatives with chain lengths greater than IO carbons.
Estimation of Tissue Free Fatty Acids-A sample of frozen tissue powder was homogenized with cold CHCl&H,OH (2:l). After centrifugation at 10,000 X g for 15 min, the supernatant was flash-evaporated and then desiccated to complete dryness. The residue was dissolved in CHCl, and run through a small silicic acid column (0.5 g of silicate per column) to remove phospholipids.
The columns were washed several times with CHC13, and the combined washes for each sample were dried in a water bath at 60" under a stream of XZ. The residue was dissolved in 0.2 ml of CHCl-CH30H (2:1), and the free fatty acids were separated from other lipid components by thin layer chromatography.
The fatty acids were eluted with CHCls and methylated with R-F3 (28). The methyl esters were quantitated with a Hewlett-Packard model 120 gas chromatograph (DEGS 20 column).
C16, Ci8, Ci~:i, and C18:2 comprised approximately 90% of the total tissue fatty acids. The other minor peaks were not used in calculating tissue fatty acid. Pentadecanoate (Cl,) was added to the initial homogenate to act, as a carrier and internal standard.
To determine the amount of tissue fatty acids that were present in the vascular space, IzjI-albumin was added to the buffer and its distribution in the tissue was determined. A small portion of the powdered tissue was weighed and the protein was dissolved in formic acid. A sample of the solution (0.2 ml) was added to 10 ml of dioxanc sciutillator, neutralized with SI-IsOH, and couuted for IpsI in a liquid scintillation counter. The perfusate concentration of 12jI-albumin was determined by precipitating the albumin with 6% perchloric acid, dissolving the protein in formic acid, and counting an aliquot of the solution. The size of the albumin space in milligrams per g of tissue was calculated as: lzZI cpm per g of tissue lejI cpm per ml of perfusate 'l'hc conccutration of perfusate fatty acid n-as determined by the Dole (29) extraction procedure.
The upper heptane phase was removed and dried, and fatty acids were rneasurcd by gas chromatography as described above. The amount of fatty acid present in t.hc vascular space in micromoles per g of dry tissue was calculated as: micromoles of fatty acid per ml of perfusate x milliliters of albumin space per g of tissue. With these measurcments, the levels of tissue fatty acids wcrc corrected for those bound to albumin iu the vascular space.

Ejects of Pal&ate
Concentration and Ventricular Pressure Development on Rate of Fatty Acid Uptake, 02 Conszhmption, and 'TO2 Production from [ U-14C]Palmitate-'rlIc rate of palmitate uptake increased as its concentration in the perfusate was raised at both low aud high levels of ventricular pressure development (Fig. 1). The iucrease in uptake with concentration was not linear, however, and the rate leveled off when the concentration of palmitate was about 0.6 and 0.9 mM in hearts developing 50 and 100 mm Hg ventricular pressure, respectively. Raising the pressure significantly increased the rate of uptake at all palmitate concentrations studied. These results indicated that the rate of fatty acid utilization was a function of its concentration only when the exogenous concentration n-as low. At high concentrations, the rate of uptake was limited by intracellular processes. An increase in ventricular pressure accelerated the rate of uptake and caused the process to become saturated at a higher concentration of exogenous palmitatc. 1. Effects of perfusate palmitate and ventricular pressure development on the rate of palmitate uptake. The rate of palmitate uptake by hearts perfused at 50 (---) and 100 (---) mm Hg ventricular pressure development was estimated by measuring the disappearance of pzllniitate from the perfusate during 20 min of perfusion.
Thirty milliliters of buffer containing glucose (11 mM), albumin (3?;), and the concentration of palmitate shown in the figure were recirculated through the heart for the 20.min perfusion period.
Each value represents the mean f S.E.M. for six The cffcct of iucrcased presaurc on pahnitatc uptake ITas associated with a faster rate of oxidativc 1~1iosplior~latioii as indicated by a large increase in osygcn c~orlsunll)tioil. Fig. 2 shows the transient and steady state changes in osygcn consumption that resulted from increasing I-rntricular 1)rcssurc from 60 to 120 n-m Hg in Hearts perfused with buffer rolltaining glucose (11 ml{) aud either 0, 0.4, or 1.2 n1~ palmitate.
A rapid inc&rcasc in oxygen consumption occurred as \-elltriclular pressure tlcvclopmcnt was raised. '1'1~ rate of osygcw consumption was somewhat higher at both 1~~1s of pressure tloT-elopmerit when palmitatc was included in the pcrfusatc.
Oxidation of fatty acids, as determined by 14C02 production from [ U-W]palmitate, n-as somcn-hat faster at 1.2 than at 0.4 11131 palmitate when the level of ventricular pressure was maintained at 60 mm IIg (Fig. 3). The rate of '?CO, production increased rapidly during the first few minutes after addition of llalmitate to tlic pcrfusatc.
The rate of production lcvelcd off in hearts perfused with 0.4 mar palmitatc but continued to increase slon-ly with 1.2 m&I. liaising the level of pressure tierelopment at either zero time or after 6 min of perfusion with palmitate resulted in a large increase in COs production from the fatty acid. At this higher level of ventricular pressure, 14COa production was the same with both concentrations of palmitatc, indicating that the rates of oxidation of acetyl units produced from esogcnous palmitate were the same at both 0.4 and 1.2 maI palmitate. These data indicated that the rate of flux of labeled acetyl units through the citric acid cycle was increased only slightly by raising the csogenous concentration of palmitatc when the level of ventricular pressure was maintained at 60 mm Hg. An increase in pressure-accelerated oxidation of fatty acids and, corresponding to the large increase in oxygen consumption, flus through the citric acid cycle \vas increased 3-to 4-fold. i<Jects

of Increased Ventricular Pressure and Exogenous
Palwi- 2. Effects of fatty acid concentration and ventricular pressure development on the rate of oxygen consumption.
Hearts were perfused for 10 min with buffer containing glucose (11 mM) as the only exogenous substrate prior to switching t,o a perfusate that contained either glucose alone (O ), glucose plus 0.4 m&f palmitate (O), or glucose plus 1.2 m&c palmitate (A). Perfusion with these subst,rates was continued for 16 min at 60 mm Hg peak ventricular pressure development (-).
after 6 min at 60 mm Hg, ventricular pressure development was increased to 120 mm Hg, and the rate of oxygen consumption was follolved for an additional 10 min (---  ---) or 120 (---) mm Hg, and '4C0, production xas measured at the times indicated. Each value represents t)he mean of 6 to 12 determinations.
tate on Tissue Content of CoA, Carnitine, and Their Acyl Derivatioes after G Olin OJ" I'erjusion--The major product of fatty acid oxidation iu heart muscle is acetyl-CoA.
Since the rates of ketogenesis and lipogenesis arc very low, the predominant fate of xcet'yl-Coh is oxidation through the citric acid cycle or transfer of the acetyl unit to carnitine. Therefore, the tissue content of CoX, carnitine, and their acyl derivatives was determined in order to localize the steps in fatty acid oxidation that limited palmit'ate uptake at high concentrations and that accelerated uptake with illcreased ventricular pressure development. The lcrels of these intermediates were measured after 6 min of perfusion with pahnitate.
Other studies demonstrated that maximum increases in the tissue content of acetyl-CoA and citric acid cycle intermediates occurred within 6 min after introducing palmitate in the perfusate (15).
At 60 mm Hg vent&Jar pressure, both acetyl-CoA and acetylcarnit'ine, accumulated as the concentration of palmitate was raised (Fig. 4). Over the range of 0 to 0.5 rnivI palmitatc, only a small accumulation of acetyl-CoA occurred, whereas the increase in acetylrarnitine was more pronounced. The ratio of acctylcaruitinc to acetyl-CoA was about 15:l. As the concentration of palmitate was raisrd above 0.5 mM at the low level of pressure development, reIatively Iargc amounts of acetyl-CoA accumulated in the tissue. In this case, the acetylcarnitine to acetyl-Co-1 ratio decreased to about 10 : 1. The tissue content of CoA and rarnitine decreased as their acetyl derivatives accumulated.
The increase in the level of acetyl derivatives and the decrease in Cal and carnitine were associated with a leveling off in the rate of palmitate uptake, suggesting that the rate of acetyl-Co-4 oxidation through the citric acid cycle limited the rate of fatty arid oxidation.
vented a large accumulation of acetyl-CoA (Fig. 4). The levels of acetylcarnitine were also significantly lower at the higher pressure.
This decrease in acctyl units associated with an increase in 14C02 production indicated that the rate of flux through the citric acid cycle was accelerated by increased pressure and that the rate of oxidation of palmitate was not sufficient to maintain high levels of acetyl-CoA.
The faster rate of acetyl-Cob oxidation resulted in higher levels of CoA a-hen the concentration of palmitate was above 0.6 ITIM; but, at lower palmitate concentrations, the level of CoA was significantly reduced.
The levels of carnitinc mere higher with increased pressure at palmitate concentrations greater than 0.3 mM. In hearts that were developing 60 mm Hg ventricular pressure, the levels of both long chain acyl-CoA and acylcarnitine derivatives increased as the concentration of palmitate was raised from 0 to 1.2 m&f (Fig. 4). The levels of these intermediates did not increase further as palmitate was raised above 1.2 InM (data not shown).
Acyl-CoA increased by only SO%, while the level of acylcarnitine increased by about 3009,.
Raising the concentration of palmitatc from 0 to 0.3 mM increased the levels of these intermediates only slightly but caused the largest increase in palmitate upt,akc. The largest rise in acylcarnitine occurred at the palmitate concentrations where acetyl-CoA also accumulated.
Increasing the level of cardiac work reduced acyl-CoA slightly, but produced a 30% increase in acylcarnitine. This increase in acglcarnitine associated Tyith a faster rate of fatty acid oxidation and decreases in both long and short chain CoA limited -11' ductil,n of acetyl-Co;1 n-hen osidative phosphorylation was accelerated.
To tlctcrmille if carnitine-dependent processes limited the rate of I)roductioll of acetyl-Coh from palmitate, the effects of increased ventricular pressure on the rntc of fatty acid uytakc and the tissue content of acetyl-Cob were determined with octanoate as substrate.
Oxidation of long chain fatty acids by isolated heart mitocshondria has been shoTT-n to be completely dependent on the presence of carnitine, whereas medium chain length fatty acids can be oxidized in the absence of carnitine (30, 31). Table  I shows cornparativc rates of fatty acid uptake and tissue levels of acctyl-CoA in hearts perfused with high concentrations of either I'almitate or octanoate.
Increasing the concentration of octanoatc from 1.5 to 5.0 mM did not increase the rate of its uptake at either 60 or 120 mm Hg. The rate of octanoate uptake was approsimatcly twice that for palmitate at similar perfusate concentrations.
Increasing the pressure from 60 to 120 mm Hg produced a large increase in the rate of uptake of both octanoate and palmitatc.
The level of acetyl-CoA decreased with palmitate as substrate but not with octanoatc, indicating that productioll of acetyl-CoA from octanoate, but not from palmitate, was fast enough to keep pace with the increased rate of flus through the citric acid cycle. These results suggested that carnitinedependent processes limited acetyl-Cob production from palmitatc when the rate of fatty acid oxidation was accelerated.
Transient JSJ'ects of Increased Ventricular Pressure Development and Concentration of Palmitate on Tissue Levels 0~" Acyl-CoA and Acylcarnitine Derivatives-Transient changes in the tissue content of CoA and carnitinr and their acyl derivatives after addit'ion of either 0.4 or 1.2 mM pslmit'atc t'o the perfusate and after increasing ventricular pressure development are shown in Fig. 5. The le\-els of long chain acyl derivatives increased rapidly and rcachcd a maximum within about 2 min after introducing palmitate into the perfusate.
The increase in acylcarnitine was larger thau the increase in acyl-CoX at all time periods studied.
Acylcarnitine decreased between 4 and 10 min of perfusion and appcarcd to reach a steady state after 10 min. The level of acyl-Co;\ decreased rapidly between 4 and 6 min in hearts that were I)c,rfuscd with 1.2 m&x palmitate and then continued to IIearts were perfused for 10 min with Krebs-Henseleit bicarbonate buffer containing glucose (11 mu) before switching to perfusion with brlf'l'er containing glucose and the concentration of fatty acid shown in the table. Perfusion with the fatty acids was continued for an additional 15 min. The rates of fatty acid disappearance from the perfusate were measllred over the 15.min period and the tissue levels of acetyl-CoA were measured in hearts that w-cre qlliclc frozen at the end of 15 min. decrease at a slower rate. Kith 0.4 mM palmitate, the levels of both intermediates decrcascd after 2 min of perfusion. The tissue content of acetyl-CoA and acetylcarnitinc increased rapidly after addition of 1.2 m&I palmitate, reached a maximum within 4 to 6 min, and then slowly declined.
The levels of both CoA and carnitinc decreased to a minimum level within 2 min. With 0.4 mM palmitate, the acet,yl derivatives increased to a maximum within 2 min and then declined.
At this low concentration of fatty acid, ace@-CoA increased by about 80% in comparison to a 5-fold increase with 1.2 mM palmitate.
The level of acetylcarnitine, on the other hand, increased to the same extent within 2 rnin with either 0.4 or 1.2 m&t palmitatc, but the level dcclincd more rapidly at the lower concentration of fatty acid. With 0.4 m&x palmitate, the early rise in acetylcarnitine was much larger in magnitude than was the rise in acetyl-CoA. The levels of both Cob and carnitine reached a minimum within 2 min after adding 0.4 mM palmitate and then slowly increased as the level of their acetyl and acyl derivatives declined.
Wth low levels of palmitatc, excess acctyl units were prcfcrcntially stored as the carnitine derivative in both the 6-min (Fig. 4)   in the transient studies (Fig. 5). At the high level of palmitate, proportionally more of t,hc acetyl units accumulated as the Cob dcri\-ativc.
Inc~reasing the level of ventricular pressure development yroduced a rapid decrease in the tissue content of the acetyl derivatives and of the long chain CoX derivatives, whereas the level of long chain acylcarnitine increased (Fig. 5). Kith 1.2 mM palmitatc, raising the level of I-entricular pressure resulted in higher levels of both CoA and carnitine.
Kith the lower concentration of palmitate, there was only a transient increase in carnitine and the levels of Cod decreased. The tissue content of CoX was therefore dependent on the levels of acetyl-(~0-1, long chain acyl-Co& and some other acid-soluble Coil derivative.
The amount of total Coh present as acetyl-CoA, acyl-CoA, and Coh is shown in Fig. 6. The sum of these three metabolites increased as the concentration of palmitate was raised and decreased as the ventricular pressure was raised from 60 to 120 mm Hg. These results indicated that the level of some other acid-soluble CoA derivative increased rapidly as the pressure was raised at both 0.4 and 1.2 mM palmitate.
As reported elsewhere (32), changes in the level of succinyl-Cob under the conditions used in the present experiments could account for the observed changes in this arid-soluble CoA. This increase in the acid-soluble CoA derivative with increased pressure resulted in lower levels of CoA when exogenous pahnitate was less than 0.6 rnM (Figs. 4 and 5). TTTith higher concentrations of palmitate, the decrease in acetyl-CoX more than compensated Ventricular Pressure Development on Tissue Content oj" Free Fatty Acids-111 order to further characterize the cffccts of increased pressure on palmitate uptake, the tissue level of free fatty acids was estimated.
At 60 mm Hg, the tissue content of fatty acids increased 2-to 3-fold after addition of 0.4 mM palmitatc to the perfusate (upper panel, Fig. 7). Raising the level of cardiac work greatly reduced the tissue content of fatty acids withill 2 min. This effect n-as e\-en more pronounced after 10 min.
The data presented in the upper T panel of Fig. 6 represent the sum of all C16, Cj8, CjgI1, and C1s,? fatty acids and do not distinguish between intracellular fatty acids and those bound to proteins in the interstitial space. Since palmitate was the only exogenous fatt'y acid provided, changes in the tissue content of fatty acids other than palmitate may be a better indicator of the changes that occurred in the intracellular pool. At 60 mm Hg, the tissue content of non-palmitate fatty acids was only slightly higher T&en 0.4 mM palmitate was added ISearts were perfused as described in Fig. 5  At 60 mm Hg, the rate of uptake was maximum within 2 min after addition of palmitate, and uptake exceeded the rate of /3 oxidation (Fig. 8).
As a result, acyl-CoA and acylcarnitinc accumulated in the tissue during the early perfusion t'imes. The rate of /3 oxidation was rapid during the first 2 min, decreased betlveen 2 and 6 min as acetyl-CoA accumulated, and then slowly increased over the next 10 min of perfusion as the level of acetyl-Co9 decreased (Fig. 5). The minimum rate of /I oxidation at 6 rnin corresponded to the maximum acetyl-CoA to CoA ratio in the tissue (Fig. 5). To determine rates of /J oxidation (Panel B), hearts were perfused as described in Fig. 1 9. Effects of palmitate concentration and ventricular pressure development on the mass-action ratio of carnitine palmityltransferases and carnitine acetyltransferases.
The ratios of substrates to products for the respective transferases ?vere plotted according to the equation: The slope (m) in this equation is related to the mass-action ratio as defined by: Mass-action ratio = [acyl-CoA] [carnitine] [acylcarnitine] [CoA] The acyl-CoA to CoA and acylcarnitine to carnitine ratios xere calculated from the curves in Fig. 4 at intervals of 0.1 rnM palmitate. The plots represent the effects of increasing the concentration of palmitate from 0 to 1.2 rnM in hearts that were developing 60 (--) or 120 (---) mm Hg ventricular pressure.
For the carnitine palmityltransferase system, the slopes were 9.7 and 6.2 at 60 and 120 mm Hg, respectively.
For the carnitine acetyltransferase system, the slope at 60 mm Hg increased from 0.7 to 1.4 as the concentration of palmitate was raised. the carnitine acetyltransferase system was shifted toward formation of acetyl-CoA. Raising the level of pressure development had little, if any, effect on the mass-action ratio for this enzyme system. At the higher concentrations of palmitate, the ratio may have been decreased slightly.
Khen these ratios were calculatcd from the transient data, the slol~es of the curves Tvere linear with perfusion time but again shifted toward acetyl-CoA formation at the higher level of palmitate (Fig. 10). to product ratios for these enzymes were plotted as described in Fig. 9. The expanded scale &set in Pa/Lel B was added because the values for hearts perfused with low concentrations of palmitate and high levels of cardiac work were too small to be included with the others.
The slope of the line in the inset is the same as the slope of the line representing hearts perfused with 0.4 rnM palmitate but developing 60 mm Hg ventricular pressure. For the carnitine palmityltransferase, the slopes were 9.4 and 5.7 at 60 and 120 mm Hg, respectively. For t,he carnitine acetyltransferase system, the slopes at 60 mm Hg were 0.45 and 1.1 at 0.4 and 1.2 rnM palmitate, respectively.

DISCUSSIOS
The rate of fatty acid utilization by most tissues is largel) dependent on its concentration in the plasma (16)(17)(18)35). The correlation between concentration and rate of uptake is especially evident in adipose tissue and liver where a major fate of fatty acids is storage as neutral lipids.
The rate of uptake by heart muscle was also concentration-dependent (3, I@, but osidation, rather than storage as complex lipids, was the more prominent fate of fatty acids (11,15,18). Increasing the fatty acid to albumin ratio resulted in higher tissue levels of fatty acids, acy-CoA, and acylcarnitine derivatives and lower levels of fire Co4 and carnitine (15).
The over-all rate of fatty acid utilization by heart muscle should be determined primarily by the supply of exogenous fatty acid and by the energy demands of the tissue. At a constant rate of energy utilization, increased supply of fatty acids would be expected to have a limited ability to accelerat,e fatty acid uptake. The upper limit would be reached when the supply of fatty acids exceeds the capacity of the cells to bind the fatty acids and to convert acyl units to COZ, to complex lipids, or to metabolic intermediates.
Binding of fatty acids and conversion of acyl units to metabolic intermediates could have only a small, transient effect, with the major determinant of uptake being oxidation to COZ.
In the present study, the rate of fatty acid ut.ilization was limited by either the rate of uptake or activation when the esogenous concentration of palmitate was low. As the concentration was raised from 0 to about 0.4 mnI, fatty acid uptake increased proportionately, but t,he tissue content of acylcarnitine, acyl-CoX, and acetyl-CoA remained relatively unchanged, and 5307 the levels of fret CoA and carnitine remained high.
In hearts developing low levels of ventricular pressure, the capacity of the cells to oxidize fatty acids and to convert acyl units to complex lipids was saturated at concentrations greater than about 0.6 InM. As the level of exogenous palmitate was raised from 0.6 to 1.2 mM, large amounts of acyl-CoA and acylcarnitine derivatives accumulated in the tissue, but no further increase in uptake w-as observed.
The rate of palmitate oxidaton at high concentrations was limited by the rate of acetyl-CoA oxidation through the citric acid cycle. This conclusion was based on the observations that w-hen perfusate palmitate was raised from 0.4 to 1.2 mM the rates of oxygen consumption and W02 production from [U-14C]palmitate were increased only slightly, while the level of acetyl-Co24 increased 5-fold.
Flux through the citric acid cycle has been shown to be geared to the rate of oxidative phosphorylation (36). This coupling is thought to occur through feedback control of the cycle by changes in the levels of high energy phosphates and NADH.
Since oxygen consumption was maintained at a constant rate in the present study by controlling the level of ventricular pressure development, the rate of flux through the citric acid cycle would be expected to remain fairly constant as the concentration of palmitate was raised. Sfter addition of 1.2 mM palmitate to the perfustate, the rates of uptake and p oxidation were fastest during the first 2 min, and they exceeded the rate of W04 production from palmitate.
As a result, acetyl-CoA and acetylcarnitine accumulated in the tissue. Fatty acid uptake was about twice as fast as /3 oxidation, and both long chain acyl-CoA and acylcarnitine accumulated. Associated with the high levels of tissue acetyl-CoA and acetylcarnitine and low levels of CoA and carnitine, the rates of both fl oxidation and palmitate uptake decreased.
The rate of uptake may have been limited by a decreased supply of CoA and carnitine for fatty acid activation and acyl transfer.
Interpretation of the data for whole tissue contents of CoA derivatives is complicated by the fact that these derivatives exist in at least two pools, and that the carnitine palmityl-and acetyltransferase systems consist of at least two enzymes arranged in series (see model in Fig. 11). The changes that were observed in the whole tissue content of CoA derivatives may have occurred primarily in one compartment and could, therefore, greatly influence interpretation of the results. In heart muscle, fatty acids are activated on the sarcoplasmic reticulum and the outer mitochondrial membrane (41). The inner mitochondrial membrane appears to be impermeable to carnitine and CoA derivatives as well as to free carnitine and Cob (42, 43). Carnitine and its deriatives are thought to be located exclusively outside the inner membrane.
Coh and its acyl derivatives, however, are compartmentalized in both the cytosol and mitochondrial matrix.
Oxidation of long chain fatty acids by heart mitochondria is completely carnitine-dependent (30,31), and translocation of the fatty acyl unit across the inner mitochondrial membrane requires prior transfer from acyl-CoA to acylcarnitine.
This transfer is catalyzed by a carnitine palmityltransferase (Enxyme I, Fig. 11) which appears to be located on the outer aspect of the inner mitochondrial membrane (44). Acyl translocation across the inner membrane and transfer from extramitochondrial acylcarnitine to matrix CoA is catalyzed by a second carnitine palmityltransferase (Enzyme II, Fig. 11) which is more tightly associated with the inner membrane.
Intramitochondrial acetyl-CoA produced by fi oxidation is then either oxidized through the citric acid cycle or the acetyl unit is transferred to acetylcarnitine in the extramitochondrial spaces by a carnitine acetyltransferase (Enzyme B, Fig. 11). In the cytosol, acetyl units may be trans- The model was first nronosed bv Fritz and Yue (37) and later modified by Yates and 'Capland (38). It includes 'the observation that carnitine (Cn) and its long chain (FACn) and acetyl (A&n) derivatives are located exclusively in the extramitochondrial space, whereas CoA and its long chain (FACoA) and acetyl (AcCoA) derivatives exist in at least two pools (cytosolic and mitochondrial matrix  Fig. 11). In the present study, the increase in both acetylcarnitine and acetyl-CoA as the concentration of palmitate was raised to above 0.6 InM indicated that the level of acetyl-CoA increased in both the mitochondrial matrix and cytosol. Therefore, the level of CoA probably decreased in both spaces. The intracellular volume of heart muscle is approximately 2.5 ml per g of dry tissue (6, 11). Assuming equal distribution of CoA between cytosolic and matrix spaces, the concentration of this metabolite decreased from 0.11 to 0.03 mM as the level of palmitate was raised from 0 to 1.2 mM in hearts developing 60 mm Hg ventricular pressure. This decrease in CoA could have limited the rate of fatty acid activation which would therefore account for the leveling off in the rate of palmitate uptake at concentrations above 0.6 rnbr. Fritz et al. (30) demonstrated that oxidation of palmitate by whole heart homogenates was stimulated a-fold by an increase in CoA from 0.01 to 0.1 mM at a constant level of carnitinc.
The levels of CoA estimated in the present study fall within this concentration range. The K, for CoA of the microsomal fatty acid activating enzymes in liver was 0.03 to 0.05 mM (45,46). Preliminary data from this laboratory indicated that the apparent K, of the heart enzymes was between 0.03 and 0.04 mM. Therefore, the level of cytosolic CoL4 in the intact heart may have decreased to a value near its K, for fatty acid activation as the concentration of palmitate was raised. The mitochondrial matrix space can be estimated to be about 0.4 ml per g dry weight (47). When this space was subtracted from the intracellular volume, the concentration of carnitine, which is exclusively cytosolic, was calculated to have decreased from 1.8 to 0.6 mM as the concentration of palmitate m-as raised from 0 to 1.2 IIIM. Pande (48) demonstrated that 1.5 mM carnitine produced a maximum rate of acyl-CoA oxidation by isolated rat heart mitochondria.
In the present study, the concentration 530s of carilitine was below this level at fatt,y acid concentrations greater than 0.4 mM, Therefore, accumulation of acetyl-CoA and acetylcarnitine and the reduced levels of CoA and carnitine secondary to saturation of the citric acid cycle were probably responsible for limiting the rate of fatty acid utilization at high levels of palmitate.
Any change in the rates of oxidative phosphorylation and flux through the citric acid cycle that would increase the tissue content of CoA and carnitine should have an effect on the rate of fatty acid uptake.
In the present study, the rate of oxidative phosphorylation was increased by raising the level of mechanical work that was performed by the heart.
Since the ti ssue levels of ATP and creatinine phosphate have been shown not to change under the conditions imposed in this study (II), osygen consumption was measured as an index of the rate of oxidative phosphorylation. The rate of oxygen consumption was increased more than 2-fold by raising the level of ventricular pressure from 60 to 120 mm Hg. The small increase in oxygen consumption when palmitate was present probably reflected the lower P:O ratio that results from osidation of fatty acid as compared to oxidation of glucose. The theoretical ATP yield per oxygen consumed is 3.2 for complete oxidation of glucose compared to 2.8 for palmitate.
Oxidation of long chain fatty acids has been shown to inhibit glucose utilization in heart muscle, with oxidation of fatty acids accounting for as much as 90y0 of the oxygen consumed (11). Therefore, under identical rates of ATP production, oxygen consumption would be expected to increase by about 13% with a shift from glucose to palmitate oxidation.
Associated with the increase in oxidative phosphorylation, flux through the citric acid cycle was accelerated as demonstrated by a 3-to 4-fold increase in the rate of 14COZ production from [U-Wlpalmitate.
When palmitate was present at concentrations greater than 0.6 mrvr, the tissue levels of both acetyl-CoA and acetylcarnitine were greatly reduced, and the levels of CoA and carnitine were increased.
In hearts that were perfused with 1.2 m&z palmitate, the rate of p oxidation was rapidly stimulated, and t,he tissue levels of long chain acyl-CoA decreased to a minimum within 1 min following the increase in oxygen consumption.
Uptake of palmitate was accelerated, but this change was not evident until about 4 min. This slower response of uptake as compared to fl oxidation corresponded to a slow rise in tissue CoA and carnitine.
The increase in palmitate uptake could have resulted from higher levels of CoA and carnitine and, consequently, faster rates of fatty acid act.ivation and acyl transfer to carnitine. The tissue levels of long chain acyl carnitine increased in association with the rise in free carnitine.
Therefore, coupling of fatty acid uptake to oxidative phosphorylation may occur through changes in the levels of CoA and carnitine secondary to control of t)he citric acid cycle and oxidation of acetyl-CoA and acetylcarnitine.
These results suggest that the carnitine acetyltransferase system plays an important role in coupling the rate of fatty acid uptake to the activity of the citric acid cycle. This may explain why the activity of this enzyme system is higher in tissues that are predominantly oxidative (49). By transferring acetyl units that are formed in the mitochondrial matrix to cytosolic CoA and carnitine, the carnitine acetyltransferases may integrate the rate of oxidation of acetyl units through the citric acid cycle and the rate of activation of fatty acids in the cytosol.
With low levels of exogenous pahnitate, acetyl units accumulated as acetylcarnitine while acetyl-CoA levels remained very low. However, the mass-action ratio for the transferase enzymes shifted toward acetyl-CoA formation as the level of exogenous palmitate was increased.
These results suggest that some intracellular effector that was dependent on the exogenous fatty acid concentration shifted the mass-action ratio for the carnitine acetyltransferases toward acetyl-CoA formation.
Higher tissue levels of long chain acyl-CoA, a competitive inhibitor of these enzymes with respect to acetylcarnitine and carnitine (50), may have accounted for this effect. The result of this shift would be to limit the rate of fatty acid activation and to aid in the inhibition of /JI oxidation at high levels of palmitate by increasing the acetyl-CoA to CoA ratio.
In the presence of low levels of palmitate, changes in the tissue content of CoA and carnitine did not appear to be of sufficient magnitude to account for the increase in fatty acid uptake. With 0.4 mM palmitate, fatty acid activation and/or acyl transfer from acyl-CoA to carnitine may have been stimulated by other mechanisms.
This was suggested by the large reduction in tissue fatty acids and the rise in acylcarnitine that were associated with a faster rate of palmitate uptake and oxidation.
Under this condition, the tissue content of CoA decreased and there was only a small, transient increase in carnitine.
There is evidence that fatty acid activating enzymes in the liver may be controlled by factors other than availability of substrates (51). The decrease that was observed in whole tissue CoA, however, may not reflect the direction of changes in the cytosolic content of this metabolite. Most of the decrease may have occurred within the mitochondrial matrix since a parallel increase in succinyl CoA has been reported to occur under similar conditions (32). Therefore, it is possible that the level of CoA in the cytosolic compartment did increase enough with increased ventricular pressure to account for the stimulation of fatty acid activation.
At low rates of oxygen consumption, fatty acid utilization appeared to be limited by the rate of uptake or activation at low exogenous concentrations and by flux through the citric acid cycle when high levels of palmitate were present. When oxygen consumption was increased by raising ventricular pressure, the rates of the citric acid cycle and fatty acid uptake were accelerated and the limiting step for fatty acid utilization was shifted to oxidation of acylcarnitine.
The tissue content of acylcarnitine increased even though the rate of /I oxidation was accelerated and the levels of acyl-CoA and acetyl-CoA both decreased.
These results indicated that either translocation of acyl units across the inner mitochondrial membrane or p oxidation had a limited capacity to produce acety-CoA.
When flux through the citric acid cycle was accelerated, the capacity to produce acetyl-CoA was not great enough to maintain high tissue levels of this intermediate. Since oxidation of octanoate, which may bypass the carnitinedependent translocation step (31, 31), maintained high levels of acetyl-CoA as the rate of fatty acid utilization was more than doubled, the capacity of p oxidation did not appear to be exceeded by raising the level of cardiac work.
If the capacity of acyl translocation (Enzyme II in Fig. 11) became limiting for acetyl-CoA production at the high level of pressure, a decrease in the mass-action ratio for the carnitine palmityltransferase system would be expected.
From the plot of the ratios of acyl-CoA to CoA versus acylcarnitine to carnitine, the mass-action ratio for this enzyme system did appear to decrease with acceleration of palmitate oxidation.
Since this plot defines the mass-action ratio in the direction of acyl-CoA production, this apparent decrease in ratio could have resulted from (a) a stimulation of the outer transferase by factors other than availability of substrates which would have increased acylcarnitine production, or (b) a decrease in the mass-action ratio for the imler transferase (Fig.  11). A stimulation of the outer transferase seems unlikely since