Competition Between Lactate and Fatty Acids as Sources of ATP in the Isolated Working Rat Heart

J Mol Cell Cardiol 29, 2725–2733 (1997)

Competition Between Lactate and Fatty Acids as Sources of ATP in the Isolated Working Rat Heart Brett O. Scho¨nekess Department of Pharmacology, The University of Alberta, Edmonton, Canada (Received 1 April 1997, accepted in revised form 19 June 1997) B. O. S¨ . Competition Between Lactate and Fatty Acids as Sources of ATP in the Isolated Working Rat Heart. Journal of Molecular and Cellular Cardiology (1997) 29, 2725–2733. Fatty acid oxidation is generally considered the major source of energy in the heart, although lactate oxidation can be a major contributor to ATP production, depending on the concentration and availability of other competing substrates. In this study, isolated working rat hearts were used to directly determine the relationship between lactate and fatty acid oxidation to overall ATP production from exogenous sources. A range of lactate from 0.5 to 8.0 m lactate was added to hearts perfused with buffer containing 5.5 m glucose, and either 0.4 or 1.2 m palmitate over a 100 min period. Rates of glycolysis, glucose oxidation, lactate oxidation, and palmitate oxidation were determined. In the presence of 0.5 m lactate and 0.4 m palmitate, lactate oxidation provided 17% of the ATP production and palmitate oxidation provided 68%, with the remainder coming from glucose oxidation and glycolysis. In the presence of 0.4 m palmitate, an increase in lactate from 0.5 to 8.0 m increased the steady state rates of lactate oxidation from 1239±236 to 5247±940 nmol/min/g dry weight, respectively. The contribution of lactate oxidation to total ATP production increased to 37%, with palmitate oxidation now contributing only 52% of the total ATP produced. At 8.0 m lactate and 1.2 m palmitate, lactate oxidation contributed 13% of the total ATP production, while palmitate oxidation contributed 81%. This data demonstrates that under near physiological conditions of lactate (0.5 m) and fatty acids (0.4 m), the preferred energy substrate of the heart remains to be fatty acids, and that only at high levels of lactate, such as can be observed during exercise or severe stress, does lactate oxidation become a significant source of ATP production.  1997 Academic Press Limited K W: Lactate oxidation; Fatty acids; ATP; Glucose; Energy metabolism; Isolated working rat heart.

Introduction The mammalian heart can use a variety of substrates for the production of ATP. Fatty acids, lactate, glucose, and to a lesser extent ketone bodies and amino acids, can all be metabolized to produce ATP (Saddik and Lopaschuk, 1991; van der Vusse and de Groot, 1992; Allard et al., 1994). The extent to which these exogenous sources contribute to ATP production varies, and depends on the availability and presence of competing substrates. Fatty acids are known to be the primary source of ATP in the heart (Neely and Morgan, 1974; Saddik and Lopaschuk, 1991). However, there exists evidence

that suggests lactate may in fact play a substantial role in the production of ATP. Indeed, studies have suggested that lactate is the primary source for ATP production in the heart (Drake et al., 1980; van der Vusse and de Groot, 1992), or can contribute substantially to ATP production under extreme circumstances (Spitzer and Spitzer, 1972; Scott et al., 1972; Spitzer, 1974) and during exercise (Gertz et al., 1988; Stanley, 1991). It has also been suggested that an inverse relationship exists between lactate and fatty acid use (Drake et al., 1980). This is supported by the findings of Bielefeld et al. (1985) which shows that 5 m lactate could inhibit 1 m palmitate oxidation by 40%. However, Liu and

Please address all correspondence to: Dr Brett O. Scho¨nekess, Dept of Medical Biochemistry, 3330 Hospital Dr. NW, Calgary, Alberta, Canada, T2N 4N1.

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Spitzer (1978) found that lactate oxidation can be stimulated by fatty acids in isolated myocytes. It has also been shown that after a period of ischemia, the dog heart shifts to a greater oxidation of exogenous free fatty acids, although the exogenous lactate concentration increases (Mickle et al., 1986). Many in vivo tracer studies, in humans and dogs, have shown a gambit of fates for lactate. Some suggest lactate release occurs only during stress, such as ischemia or hypoxia (Mazer et al., 1990; Guth et al., 1990; Kaijser et al., 1993) or haemorrhagic shock (Spitzer and Spitzer, 1972). Other studies suggest that the healthy human heart is a lactate consumer (Opie, 1968), with lactate release occurring in the presence of concurrent lactate uptake (Gertz et al., 1981; Massie et al., 1994). It is clear from the vast literature on lactate and fatty acids that no definitive description of the contribution of lactate and fatty acids to ATP production in the aerobic isolated working heart has been made. The present study was designed to clearly demonstrate the contribution of lactate and fatty acids to ATP production in the isolated working rat heart. A protocol in which a stepwise increase in lactate concentration was used, and the concentration of free fatty acids was set at either 0.4 or 1.2 m palmitate. This allowed for the investigation of a range of conditions which included a near physiological state such as 0.5 m lactate and 0.4 m palmitate, as well as states representing exercise and severe stress (8.0 m lactate and 1.2 m palmitate). The effects of lactate and fatty acids on both glycolysis and glucose oxidation was also measured. It was found that lactate will contribute significantly towards ATP production when the exogenous fatty acid concentration is near physiological. However, when the exogenous supply of fatty acids is high such as during extreme stress, lactate oxidation does not contribute as significantly to total ATP production, and never becomes the primary source of ATP.

Materials and Methods Materials D-[5-3H]glucose, -[U-14C]glucose, -[U-14C]lactate, and [9,10-3H]palmitate were purchased from Du Pont-New England Nuclear. Bovine serum albumin (fraction V) was obtained from Boehringer Mannheim, Germany. Hyamine Hydroxide (methyl-

benzethonium; 1 M in methanol solution) was obtained from Sigma Chemical Company. Dowex 1X4 anion exchange resin (200–400 mesh chloride form) was obtained from Bio-Rad. ACS counting scintillant was purchased from Amersham Canada Ltd. Ecolite counting scintillant was obtained from ICN Biomedicals Canada Ltd. All other chemicals were reagent grade.

Perfusion protocols Male Sprague–Dawley rats (250–300 g) were anaesthetized with a 60 mg/kg i.p. injection of pentobarbitol sodium. Hearts were excised and immediately cannulated in an aortic retrograde perfusion via the Langendorff method. The left atrium was subsequently cannulated to allow for the working heart mode. The perfusion pressures were set at an 11.5 mmHg left atrial preload and 80 mmHg aortic afterload. The perfusion buffer used was a modified Krebs–Henseleit buffer (pH 7.4) gassed with a 95% O2–5% CO2 air mixture, with a free Ca2+ concentration of 1.25 m, and 5.5 m glucose. Alterations in fatty acid content of the perfusion buffer depended on the perfusion series in which either 0.4 or 1.2 m palmitate was prebound to 3% (w/v) bovine serum albumin, which was present in all perfusion buffers. Perfusate lactate concentration was increased every 20 min over a 100 min period, such that the concentration of lactate in the perfusion buffer was increased stepwise from 0.0 to 0.5, 2.0, 5.0 and finally 8.0 m. One series of perfusions contained either 3H/14Cglucose to measure glycolysis and glucose oxidation respectively, whereas a parallel series had 14C-lactate and 3H-palmitate in the perfusion buffer to measure lactate and palmitate oxidation respectively. Peak systolic pressure (PSP) and heart rate (HR) was recorded on a Grass 79-D physiograph with a Spectramed p 23XL pressure transducer. Oxygen consumption was measured in a separate series of hearts, in which the pulmonary artery was cannulated and the coronary flow directed through a YSI oxygen probe. Total cardiac output was measured by a Tansonics in-line flow probe being placed in the preload line, which feeds into the left atrium. Another Transonics in-line flow probe was placed in the afterload line and measured the aortic output past the coronary arteries. Therefore, the difference in preload and afterload flows was equal to the coronary flow, and this value was used for the calculation of oxygen consumption. These hearts

ATP Production From Lactate and Fatty Acids

were also perfused for 100 min with 5.5 m glucose, 1.25 m Ca2+, in a modified Krebs–Henseleit buffer that contained either 0.4 or 1.2 m palmitate, and 0.5 or 8.0 m lactate. Therefore four separate substrate combinations were used for oxygen consumption measurements.

Measurement of glycolysis and glucose oxidation Glycolysis was measured by determining the amount of 3H2O produced (which is liberated from [5-3H]glucose at the enolase step of glycolysis). Separation of 3H2O from [3H/14C]glucose was achieved as described earlier (Saddik and Lopaschuk, 1991; Allard et al., 1994). Measurement of glucose oxidation was achieved by quantitatively collecting 14CO2 produced (which is liberated from [U-14C]glucose at the level of the pyruvate dehydrogenase complex and in the TCA cycle) as described previously (Saddik and Lopaschuk, 1991; Allard et al., 1994).

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method of calculating ATP production assumes that 100% of the NADH and FADH2 produced, is used for the production of the proton gradient across the inner mitochondrial membrane, and that there is no loss of this gradient prior to ATP synthesis by ATP synthase. A recent publication by Brand et al. (1994) suggests that there may be a considerable loss of the proton motive force across the inner mitochondrial membrane in isolated hepatocytes and skeletal muscle. The substantial rate of proton leak can therefore affect the efficiency of oxidative phosphorylation and the production of ATP. Brand et al. (1994) further suggest that proton leak and the resulting respiration driven proton pumping may be the single most important consumer of free energy in animals. Although there is no evidence as to the effects of proton leak in myocardial mitochondria, the substantial ATP demand of the working heart may actually decrease the importance of this phenomenon.

Statistical analysis Measurement of lactate and palmitate oxidation Lactate oxidation was measured by a similar method as for the determination of glucose oxidation. Namely 14CO2 was quantitatively measured by collecting and measuring the amount of 14CO2 gas released and trapped by the hyamine hydroxide trap, and by measuring the amount 14CO2 present in the perfusion buffer in the form of H2CO3 (aq) bicarbonate (Allard et al., 1994). Palmitate oxidation was measured by separating the [3H]-palmitate from 3H2O present in the perfusion buffer samples as described previously (Saddik and Lopaschuk, 1991; Allard et al., 1994). The spill-over of [14C]lactate into the aqueous phase of the extract was taken into account by subtracting the amount of spill-over counts from the 14C counting window of the scintillation counter into the 3H counting window (Allard et al., 1994).

Calculation of ATP production Steady state rates of ATP production from substrate metabolism were calculated as follows: 1 mol of glucose passing through glycolysis forms 2 mol of ATP; 1 mol of glucose passing through glucose oxidation forms 36 mol of ATP; 1 mol of lactate passing through lactate oxidation forms 18 mol of ATP; and 1 mol of palmitate passing through palmitate oxidation forms 129 mol ATP. This

The data are represented as the mean±.. When comparing two group means, Student’s unpaired two-tailed t-test was used. Multiple comparisons and comparisons within groups was made by ordinary ANOVA, using a Student–Newman–Keuls post test. A value of PΖ0.05 was regarded as significant.

Results Myocardial function of hearts perfused with 0.4 or 1.2 mM palmitate and increasing concentrations of lactate Mechanical function expressed as heart rate (HR)×peak systolic pressure (PSP) is shown in Figure 1. As the lactate concentration was increased in the perfusion buffer, an apparent dose dependant depression in mechanical function at 5 m lactate occurred. After 80 min of perfusion, the mechanical function declined until it reached statistical significance in both the 0.4 and 1.2 m palmitate groups at 100 min (8.0 m lactate). The decline in mechanical function in the 0.4 m palmitate group was primarily due to a decrease in PSP, whereas both PSP and HR declined in the 1.2 m palmitate group. Whether or not the high lactate concentration was responsible for the depression in

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Figure 1 Mechanical function of rat hearts perfused with increasing concentrations of lactate and 0.4 (Β) or 1.2 m (Χ) palmitate. Values are the mean±.., n= 15 (0.4 m palmitate group), n=19 (1.2 m palmitate group). ∗ significantly different from 0.0 m lactate (PΖ0.05); † significantly different from 0.5 m lactate (PΖ0.05); ‡ significantly different from 2.0 m lactate (PΖ0.05); § significantly different from 5.0 m lactate (PΖ0.05).

mechanical function seen at 100 min (8.0 m lactate) compared to 80 min (5.0 m lactate) could not be delineated from this series of hearts. Therefore, perfusions were carried out in which hearts were exposed to either 0.5 or 8.0 m lactate, and 0.4 or 1.2 m palmitate for the entire 100 min period perfusion. These results, displayed in Figure 2, show a similar depression in function over the 100 min period, but now it appears to be a time-dependent phenomenon. All groups, except the 8.0 m lactate and 1.2 m palmitate group, actually showed significant depression in mechanical function at 100 min compared to 40 min of perfusion. Hearts supplied with 0.4 m palmitate (and either 0.5 or 8.0 m lactate) had significantly depressed mechanical function at 100 min, compared to hearts supplied with 1.2 m palmitate (and either 0.5 or 8.0 m lactate).

Rates of glycolysis and oxidative metabolism Glycolytic rates at 0.5 or 8.0 m lactate, and 0.4 or 1.2 m palmitate are shown in Figure 3(a). As the lactate concentration increased to 2.0, 5.0 and finally 8.0 m, rates of glycolysis increased stepwise until they were significantly accelerated in the 8.0 m lactate and 1.2 m palmitate group (1565±232 at 0.0 m, 2105±386 at 0.5 m,

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Figure 2 Mechanical function of rat hearts perfused over 100 min with 0.5 (open symbols) or 8.0 m (closed symbols) lactate, and 0.4 (circles) or 1.2 m (triangles) palmitate. Values represent mean±.., n=7 (0.4 m palmitate and 0.5 m lactate), n=6 (1.2 m palmitate and 0.5 m lactate), n=6 (0.4 m palmitate and 8.0 m lactate), and n=5 (1.2 m palmitate and 8.0 m lactate). ∗ significantly different from 10 min time point (PΖ0.05); † significantly different from 20 min time point (PΖ0.05); ‡ significantly different from 40 min time point (PΖ0.05); § significantly different from 60 min time point (PΖ0.05); ¶ significantly different from 1.2 m palmitate group (PΖ0.05).

2534±672 at 2.0 m, 3249±469 at 5.0 m, and 4031±678 nmol/min/g dry weight at 8.0 m lactate). This is different from what was seen at 0.4 m palmitate, in which glycolysis was near “maximal” (Scho¨nekess et al., 1995), no matter what the lactate concentration was (3275±461 at 0.0 m, 3165±460 at 0.5 m, 3408±641 at 2.0 m, 4154±982 at 5.0 m, and 3652±807 nmol/min/g dry weight at 8.0 m lactate). Glucose oxidation was only mildly influenced by both increases in lactate and palmitate in these perfusions [Fig. 3(b)]. At 0.4 m palmitate, glucose oxidation did not change significantly as the concentration of lactate increased (387±59 at 0.0 m, 375±96 at 0.5 m, 321±26 at 2.0 m, 550±140 at 5.0 m, and 517±39 nmol/min/g dry weight at 8.0 m lactate). At 1.2 m palmitate, rates of glucose oxidation did not change (364±21 at 0.0 m, 312±80 at 0.5 m, 352±72 at 2.0 m, 345±115 at 5.0 m, and 307±66 nmol/ min/g dry weight at 8.0 m lactate). Only at 8.0 m lactate was a significant depression seen in the rates of glucose oxidation in hearts perfused with 1.2 m palmitate compared to those perfused with 0.4 m palmitate. Rates of lactate oxidation showed dramatic

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Figure 3 Steady state rates of (a) glycolysis, (b) glucose oxidation, (c) lactate oxidation, and (d) palmitate oxidation in rat hearts perfused with 0.5 or 8.0 m lactate and 0.4 (Φ) or 1.2 m (Ε) palmitate. Values are the mean±.., n=7 (0.5 and 8.0 m lactate and 0.4 m palmitate) and n=5–6 (0.5 and 8.0 m lactate and 1.2 m palmitate) for glycolysis; n=6 for all groups measuring glucose oxidation; n=7–8 (0.5 and 8.0 m lactate and 0.4 m palmitate) and n=11–12 (0.5 and 8.0 m lactate and 1.2 m palmitate) for lactate oxidation; n=6–7 (0.5 and 8.0 m lactate and 0.4 m palmitate) and n=12 (0.5 and 8.0 m lactate and 1.2 m palmitate) for palmitate oxidation. ∗ significantly different from 0.4 m palmitate group (PΖ0.05). † significantly different from 0.5 m lactate group (PΖ0.05).

changes when the exogenous lactate supply was modulated, and a considerable effect was also seen by altering fatty acid supply [Fig. 3(c)]. At 0.4 m palmitate, the oxidation of lactate increased considerably as the concentration of lactate increased (1239±236 at 0.5 m, 3376±606 at 2.0 m, 4694±803 at 5.0 m, and 5247±940 nmol/min/ g dry weight at 8.0 m lactate). An increase in the rates of lactate oxidation also occurred at 1.2 m palmitate as the lactate concentration increased (341±49 at 0.5 m, 1027±124 at 2.0 m, 2103±410 at 5.0 m, and 2535±527 nmol/min/ g dry weight at 8.0 m lactate). It can be seen that increasing the supply of lactate to 8.0 m, resulted in a greater than four-fold increase in rates of lactate

oxidation in the presence of 0.4 m palmitate. At 1.2 m palmitate, increasing exogenous lactate also resulted in a dramatic increase in rates of lactate oxidation. However, the presence of 1.2 m palmitate significantly suppressed lactate oxidation at both 0.5 and 8.0 m lactate compared to hearts perfused with 0.4 m palmitate [Fig. 3(c)]. At 0.4 m palmitate, rates of palmitate oxidation actually increased as the lactate concentration increased (687±50 at 0.0 m, 703±79 at 0.5 m, 952±148 at 2.0 m, 1007±144 at 5.0 m, and 1025±267 nmol/min/g dry weight at 8.0 m lactate). Rates of palmitate oxidation also increased at 1.2 m palmitate as the concentration of lactate increased (1128±108 at 0.0 m, 1462±121 at

0.5 m, 1573±128 at 2.0 m, 2177±115 at 5.0 m, and 2194±196 nmol/min/g dry weight at 8.0 m lactate). Figure 3(d) shows the rates of palmitate oxidation for the perfusions at 0.5 or 8.0 m lactate and 0.4 and 1.2 m palmitate. At a concentration of 0.5 m lactate, rates of palmitate oxidation doubled when exogenous palmitate increased from 0.4 to 1.2 m. A similar phenomenon was seen at 8.0 m lactate; namely, palmitate oxidation increased almost two-fold from the 0.4 m palmitate group to the 1.2 m palmitate group. Only in the 1.2 m palmitate group was an apparent lactate induced increase in fatty acid oxidation statistically significant, (from 1462±121 to 2193±196 nmol/min/g dry weight).

Myocardial rates of ATP production in hearts exposed to increasing concentrations of lactate and 0.4 or 1.2 mM palmitate Total rates of ATP production in hearts perfused with 0.5 or 8.0 m lactate, and 0.4 or 1.2 m palmitate is shown in Figure 4(a) and percentage of total ATP production is shown in Figure 4(b). When hearts were perfused with only 0.4 m palmitate and 5.5 m glucose, the majority of ATP was derived from exogenous fatty acids (81%). When lactate was added to the perfusion system, it supplied about 17% of the ATP produced from exogenous sources. Furthermore, an increase from 0.5 to 8.0 m lactate, resulted in a dramatic increase in the contribution of lactate to total rates of ATP synthesis (from 17–37%). The contribution of lactate to ATP production, occurred in a stepwise manner as the lactate concentration increased during the perfusion (17% at 0.5 m, 30% at 2.0 m, 35% at 5.0 m, and 37% at 8.0 m lactate). Although lactate did significantly contribute to overall rates of ATP production, it never became the primary energy source. There was also an increase in the contribution of lactate oxidation to ATP production in the 1.2 m palmitate group [Figs 4(a) and (b)], as the lactate concentration went from 0.5–8.0 m. A similar stepwise increase in lactate oxidation and its contribution to ATP production occurred at the intermediate concentrations of lactate (3% at 0.5 m, 8% at 2.0 m, 11% at 5.0 m, 13% at 8.0 m lactate). The end result was similar to what was seen at 0.4 m palmitate, in that the majority of ATP in the 1.2 m palmitate group was derived from fatty acids, even at 8.0 m lactate.

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Figure 4 ATP production expressed as (a) steady state rates and (b) as a percentage of total ATP produced in rat hearts perfused with 0.5 or 8.0 m lactate, and 0.4 or 1.2 m palmitate. ATP production rates were calculated from the data shown in Figure 3 (calculations described in Materials and Methods). (Φ) glycolysis; (∆) glucose oxidation; (Γ) lactate acidation; (Ε) palmitate oxidation.

Myocardial rates of oxygen consumption in hearts exposed to increasing concentrations of lactate and 0.4 or 1.2 mM palmitate From Figure 4(a) it can be seen that total rates of ATP production increased as the exogenous supply of lactate and palmitate increased. Looking at the 0.5 m lactate group, there was a 58% increase in total rates of ATP production as exogenous fatty acid availability increased. To see if this also resulted in an increase in the rates of O2 consumption, O2 consumption was measured during 100 min perfusions with 0.5 or 8.0 m lactate and 0.4 or 1.2 m palmitate. O2 consumption remained steady over 100 min of perfusion in hearts exposed to 0.5 or 8.0 m lactate and 1.2 m palmitate (Fig. 5). A steady decline in O2 consumption was seen in the 8.0 m lactate and 0.4 m palmitate group, which was statistically significant between 10 and

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Discussion

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Figure 5 Oxygen consumption of rat hearts perfused over 100 min with 0.5 (open symbols) or 8.0 m (closed symbols) lactate, and 0.4 (circles) or 1.2 m (triangles) palmitate. Values represent mean±.., n=7 (0.4 m palmitate and 0.5 m lactate), n=6 (1.2 m palmitate and 0.5 m lactate), n=6 (0.4 m palmitate and 8.0 m lactate), and n=5 (1.2 m palmitate and 8.0 m lactate). ∗ significantly different from 10 min time point (PΖ0.05). † significantly different from 20 min time point (PΖ0.05).

20 min, compared to between 80 and 100 min of perfusion. This decline in O2 consumption in the 0.4 m palmitate group may be due to the decrease in function seen over the 100 min perfusion. However, O2 consumption normalized for mechanical function showed no decrease or difference among groups over the 100 min period (data not shown). O2 consumption in the 0.4 m palmitate group in the presence of 0.5 and 8.0 m lactate was 39.1±4.9 v 44.6±4.0 lmol/min/g dry weight, respectively (40 min time point). Calculating rates of O2 consumption from the steady state rates of glucose, lactate and palmitate oxidation at 0.4 m palmitate, O2 consumption should be 22.14±2.03 and 42.39±6.77 lmol/min/g dry weight at 0.5 and 8.0 m lactate, respectively. In the 1.2 m palmitate group O2 consumption in the presence of 0.5 and 8.0 m lactate was 43.2±5.7 v 50.4±13.5 lmol/min/g dry weight, respectively (40 min time point). Calculating the rates of O2 consumption here, suggests that rates should be 36.5±2.83 and 59.91±4.8 lmol/min/g dry weight at 0.5 and 8.0 m lactate, respectively. Differences in these calculated rates of O2 consumption and the actual measured values may come from the contribution of endogenous carbon sources (glycogen and triacylglycerol) to overall O2 consumption (Saddik and Lopaschuk, 1991; Goodwin and Taegtmeyer 1995; Henning et al., 1996).

Extracellular lactate can be taken up and oxidized by the isolated working rat heart (Allard et al., 1994). There have been a number of studies that have shown that there exists a net chemical extraction of lactate in human hearts (Gertz et al., 1981, 1988), and lactate release during ischemia (Guth et al., 1990), increased work states (Massie et al., 1994), and hypoxia (Mazer et al., 1990). In fact, the presence of lactate and fatty acids does have an effect on the rate of utilization of both lactate and fatty acids (Liu and Spitzer, 1978), as well as the utilization of glucose (Saddik and Lopaschuk, 1991). Drake et al. (1980) suggested that when the normal dog heart was exposed to levels of lactate above 4.5 m, lactate was the preferred exogenous energy source for the heart, even when levels of fatty acids or glucose are elevated. What the present study has demonstrated is that lactate oxidation was effectively inhibited by increased amounts of exogenous fatty acids. Lactate oxidation also dramatically increased as the availability increased, almost four- to five-fold in the 0.4 m palmitate group, and seven- to eight-fold in the 1.2 m palmitate group. In absolute terms, rates of lactate oxidation were considerably depressed in the 1.2 m palmitate group compared to the 0.4 m palmitate group. When rates of ATP production were calculated from steady state rates of substrate oxidation and glycolysis, lactate was found to contribute significantly at low fat (0.4 m palmitate). The contribution of lactate to total ATP production increased from 17% at 0.5 m lactate to 37% at 8.0 m, with the majority of ATP production from exogenous sources, coming from fatty acid oxidation (81% at 0.5 m and 52% at 8.0 m lactate). At high fat (1.2 m palmitate), the contribution of lactate to total ATP production was dramatically reduced to 3% at 0.5 m and 13% at 8.0 m lactate. Therefore, it is clear that only at low levels of fatty acids (i.e. 0.4 m palmitate) does lactate contribute a relatively large proportion of the isolated working rat hearts ATP supply. It is difficult to reconcile why increased total rates of ATP production were seen. When O2 consumption was measured, the actual rates of O2 consumed were higher than the rates calculated from the steady state rates of exogenous substrate oxidized in the 0.5 m lactate and 0.4 m palmitate group, but agreed reasonably well in the other groups. The fact that endogenous sources of energy for ATP production are contributing to overall O2 consumption, may be a possible explanation for

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what occurs in the 0.5 m lactate and 0.4 m palmitate group. It has previously been shown that endogenous triacylglycerols can contribute significantly to the production ATP, especially when exogenous levels of fatty acids are low (Saddik and Lopaschuk, 1991; Lopaschuk and Saddik, 1992). Myocardial glycogen is also a source of glucose that can contribute to aerobic ATP production (Goodwin and Teagtmeyer, 1995; Henning et al., 1996). However, whether or not an increase in the availability of exogenous energy sources caused a shift in the use of endogenous sources could not clearly be delineated by this study. A question of myocardial efficiency in these hearts comes to mind when the amount of ATP produced per unit mechanical function performed is compared. The appearance of hearts perfused with 0.4 m palmitate being more efficient than hearts perfused with 1.2 m palmitate, may be due to the contribution of endogenous triacylglycerol and glycogen to ATP production. A substrate-induced acceleration of ATP production may also be an explanation for this increase in ATP production when there was an increased availability of exogenous substrates (Katz et al., 1989). There was no change in the amount of oxygen consumed when normalized per unit mechanical function for any of the groups used for this study. However, there was an increase in the calculated amount of ATP produced, suggesting there may be an increased efficiency between substrate oxidation and ADP phosphorylation. This is unlikely, since the data suggests that in this preparation, the increased fatty acid and lactate availability actually caused an acceleration in ATP production similar to that seen by Katz et al. (1989). Alternatively, there may be an uncoupling of mitochondrial respiration from the oxidative metabolism of carbon substrates, which would support the proton leak theory proposed by Brand et al. (1994). However, there is not enough evidence from the current study to conclude that the calculations for ATP production are invalid. The depression in function that occurred may be due to the presence of lactate interfering with intracellular pH regulation, and a competition of H+ with myofibril binding of Ca2+. It has been shown that a lactate/H+ co-transport system exists in the heart (Mann et al., 1985; Trosper and Philipson, 1987, 1989; Poole et al., 1990; Wang et al., 1993). If a high extracellular lactate concentration was present, it may inhibit lactate/H+ transport and, thus, cause an increased acidification of the myocytes that effectively competes with intracellular Ca2+ at the level of myofibril contraction, and decreases force production (Thompson et al.,

1990; Liu et al., 1993). Myocardial efficiency may also be altered in these hearts, as an increased acidification may lead to increased Na+/H+ and Na+/Ca2+ exchange (Tani and Neely, 1989) with an increased use of ATP for the maintenance of ion gradients. Since rates of substrate oxidation were high under all perfusion conditions, an altered mechanical efficiency or altered efficiency in the use of ATP produced by the hearts in this isolated working heart preparation, may be the cause of the depression in mechanical function which was seen at the end of the perfusion protocol. Cardiac output decreased over the 100-min period of perfusion in the oxygen consumption component of this study. For example, in the 8.0 m lactate and 1.2 m palmitate group, a decrease in cardiac output occurred from 40±4 at 10 min to 14±7 ml/min at 100 min with no change in coronary flow (14±5 at 10 min to 15±6 ml/min at 100 min). This suggests that the decrease in the mechanical function was not due to an altered coronary flow or supply of energy substrate and oxygen, but may have been due to other properties of the preparation. It can be concluded from this study that ATP production in the isolated working rat heart supplied with lactate and fatty acids results primarily from the oxidation of exogenous fatty acids. Lactate will contribute significantly to ATP production, only when the exogenous concentration of lactate is high, and the exogenous concentration of fatty acids are low. Minimal effect of varying concentrations of lactate exists on rates of glycolysis and glucose oxidation, and there seems to be a greater effect by the presence of fatty acids on glucose metabolism than by lactate.

Acknowledgements The author would like to thank Heather Fraser, Drs N. Davies, W. Cole, R. Loutzenhiser, and M. Walsh for their encouragement and support. BOS was a graduate student trainee of the Alberta Heritage Foundation for Medical Research and the Heart and Stroke Foundation of Canada at the time of this study, and currently holds a Post-doctoral Fellowship from the Alberta Heritage Foundation for Medical Research.

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