Effect of Endotoxin and Platelet-activating Factor on Lipid Oxidation in the Rat Heart

J Mol Cell Cardiol 29, 1915–1926 (1997)

Effect of Endotoxin and Plateletactivating Factor on Lipid Oxidation in the Rat Heart Xin Wang and Rhys D. Evans Nuffield Department of Anaesthetics, University of Oxford, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK (Received 17 December 1996, accepted in revised form 17 March 1997) X. W  R. D. E. Effect of Endotoxin and Platelet-activating Factor on Lipid Oxidation in the Rat Heart. Journal of Molecular and Cellular Cardiology (1997) 29, 1915–1926. The effects of endotoxin and platelet-activating factor (PAF) administered in vivo and in vitro on lipid oxidation by isolated perfused working rat heart were investigated and compared. Endotoxin administered in vivo decreased oleate oxidation in perfused hearts and caused increased accumulation of lipid in myocardial tissue; this was accompanied by a dose-dependent inhibition of cardiac mechanical function. There was no effect on triolein (chylomicron) oxidation. By contrast, PAF administered in vivo caused a small (non-significant) increase in the oxidation rate of oleate and triolein, and also increased myocardial lipoprotein lipase activity. Cardiac mechanical function was not inhibited by PAF—indeed pretreatment of animals by PAF administered in vivo protected the heart from the decline in function associated with subsequent fatty acid perfusion. Administration of endotoxin during perfusion in vitro did not affect oleate or triolein oxidation and cardiac mechanical function was unchanged; PAF administered in vitro caused an early increase in oleate oxidation and a later increase in triolein oxidation. Administration of both endotoxin and PAF in vitro increased myocardial lipoprotein lipase activity. PAF is unlikely to be responsible for all of the effects of endotoxin on cardiac lipid metabolism.  1997 Academic Press Limited K W: Platelet-activating factor, PAF; Lipid metabolism; Myocardium; Lipoprotein lipase; Endotoxin.

Introduction The myocardium has a high workload with a large energetic demand; it can utilize a wide variety of substrates, including carbohydrates and lipids [triacylglycerols (TAG), non-esterified fatty acids (NEFA) and ketone bodies]. The regulation of substrate utilization by the heart is not fully understood, although it is likely to involve humoral factors, substrate signals and substrate supply. The use of an in vitro model of the perfused heart has allowed these factors to be independently investigated, and development of the “working heart” (Neely and Rovetto, 1975; Taegtmeyer et al., 1980), in which the left atrium is separately cannulated, permitting anterograde filling of the

left ventricle with aortic ejection, and hence development of wall tension and hydraulic work, closely resembles the in vivo state. Using this model, lipid utilization by the heart has been investigated under varying conditions of workload and hormonal milieu (Taegtmeyer et al., 1980). Whilst NEFA may be a preferred substrate for isolated working rat heart (Neely and Morgan, 1974), fatty acids are histotoxic at high concentrations and TAG utilization is dependent on the activity of the regulatory enzyme lipoprotein lipase (LPL; EC 3.1.1.34), present on the luminal endothelial surface of the tissue. Subsequent fatty acid oxidation is dependent on both the capacity and activation state of the b-oxidation system of the heart, the latter regulated by ma-

Please address all correspondence to: R. D. Evans, Nuffield Department of Anaesthetics, University of Oxford, Radcliffe Infirmary, Woodstock Road, Oxford OX2 6HE, UK.

0022–2828/97/071915+12 $25.00/0

mc970430

 1997 Academic Press Limited

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X. Wang and R. D. Evans

lonyl-CoA content and hence activity of carnitine palmitoyl transferase I (CPT I) (Saddik et al., 1993). Substrate supply and utilization, notably lipid metabolism, is known to be affected during pathological systemic inflammatory states including sepsis syndrome (Evans and Williamson, 1991). Lipopolysaccharides from the cell walls of Gramnegative bacteria have direct and widespread bioactive properties and act as the proximal stimulus for the release of more distal pathological mediators of inflammation, including the peptide cytokines [tumor necrosis factor a (TNFa), interleukin-1 (IL1) and interleukin-6 (IL-6) (Evans et al., 1989)] and lipid mediators such as platelet-activating factor (PAF) (Evans et al., 1991a). These substances are released by host inflammatory cells and/or endothelium, and mediate many of the changes associated with the disease state. PAF causes hypertriglyceridemia and hyperglycaemia; it increases hepatic lipogenesis and glycogenolysis (Evans et al., 1990, 1991b) and causes hypoketonemia in starved rats in vivo (Hiraide et al., 1992). Furthermore, these substances are known to have complex and redundant interrelationships and many exert profound effects on cardiovascular physiology (Holland et al., 1991; Schulz et al., 1995)—PAF causes coronary vasoconstriction in dog heart (Kenzora et al., 1984) and is negatively inotropic in guinea pig hearts (Robertson et al., 1988). Myocardial mechanical function is impaired in sepsis and PAF may be involved in this effect. Endotoxin depresses cardiac mechanical performance (Decking et al., 1995; Tao and McKenna, 1994), although an early enhancement of myocardial contractility has been reported in endotoxic dogs (Kober et al., 1985) and ex vivo rat hearts (Rumsey et al., 1988). The effect of endotoxin on myocardial lipid metabolism is poorly documented—this study aims to examine this and relate effects to concomitant cardiac mechanical performance. Since many effects of endotoxin are indirect, via endogenous inflammatory mediators, including PAF (Qi and Jones, 1990; Salari et al., 1990; Dobrowsky et al., 1991; Mulder et al., 1993; Kawamura et al., 1994), it was decided to also examine the potential role of PAF in any such changes in myocardial lipid metabolism. Since changes in vivo may be secondary to release of yet more distal mediators, an in vitro heart model was chosen, with a low dose of PAF to minimize changes in myocardial work and coronary flow.

Materials and Methods The investigation was performed in accordance with the Home Office Guidance On The Operation Of The Animals (Scientific Procedures) Act 1986 published by HMSO, London, UK.

Animals Male Wistar rats (350–450 g) were fed ad libitum on a chow diet comprising (by weight) approximately 52% carbohydrate, 21% protein and 4% fat; the residue was non-digestible material (Special Diet Services, Witham, Essex, UK). Animals had free access to drinking water and were maintained at an ambient temperature of 20±2°C with a 12 hlight/12 h-dark cycle (light from 07.30 h). Groups of animals were injected subcutaneously with lysoPAF (“lyso-PAF in vivo”; 25 lg/kg body weight in 0.25% fatty acid-free bovine serum albumin; 0.2 ml), PAF (“PAF in vivo”; 25 lg/kg body weight, same vehicle and volume) 60 min before killing, or endotoxin (“endotoxin in vivo”; 0.1 lg/kg or 1 mg/ kg body weight, same vehicle and volume) 3 h before killing. Other groups were not pretreated (in vitro administrations).

Chemicals [9,10-3H]oleic acid and glycerol tri[9,103 H]oleate were obtained from Amersham International, Amersham, Bucks, UK; PAF (L-aphosphatidylcholine, b-acetyl-c-0-alkyl), lyso-PAF (L-a-lysophosphatidylcholine,c-0-alkyl), endotoxin (lipopolysaccharide isolated from Escherichia coli serotype 055:B5) and other biochemicals were obtained from Sigma Chemical Co., Poole, Dorset, UK.

Isolated perfused working heart preparation All experiments were commenced between 10.00 and 11.00 h. The heart was perfused through the left atrium (anterograde) in “working” mode by the Taegtmeyer et al. (1980) modification of the method of Neely and Morgan (1975): animals were anaesthetized with sodium pentobarbitone (60 mg/kg body weight) i.p.; a thoracotomy was performed, the heart was rapidly excised and briefly placed in ice-cold Krebs–Henseleit bicarbonate saline. The heart was then cannulated via the aorta (<2 min

Myocardial Lipid Oxidation

from excision) and perfused retrogradely through the coronary arteries in “Langendorff” mode, whilst lung, mediastinal, and pericardiac brown adipose tissue were excised, right pulmonary arteriotomy performed, and the left atrium separately cannulated. A recirculating Krebs–Henseleit bicarbonate buffer solution containing CaCl2 1.3 m, glucose 10 m and fatty-acid free bovine serum albumin 2.0% (w/v) was used in all groups. The perfusate was filtered through a 5 lm cellulose nitrate filter (Millipore, Bedford, MA, USA), gassed with 02:CO2 (95:5) and maintained at 37°C; the prime volume was 250 ml, and the first 50 ml of perfusate were discarded to free the circuit of blood cells, after which the apparatus was switched to the “working” mode and cardiac perfusion maintained through the left atrium. Afterload was maintained at 100 cm H2O and preload (atrial filling pressure) at 15 cm H2O. Peak systolic pressure (PSP) and heart rate (HR) were measured by a calibrated pressure transducer (Druck Ltd, Groby, Leics., UK) connected to a side arm of the aortic cannula, and recorded on a two channel chart recorder (Lectromed, Jersey, CI, UK). Aortic flow rate (AFR) was measured by a timed collection of perfusate ejected through the aortic line, and coronary flow rate (CFR) was measured by a timed collection of perfusate effluent dripping from the heart. Cardiac output (CO) was calculated as (CFR+AFR). Hydraulic work (HW) was calculated as (CO×mean aortic pressure/heart weight). After an initial 15 min stabilization period, lipid substrate (see below) was added slowly (2 min) to the reservoir (Time 0). CFR, AFR, PSP and HR were measured at time 0 and at 10 min intervals for 80 min. Effector (lyso-PAF, PAF, endotoxin; see below) was added slowly to the reservoir immediately after the t=40 min measurements (in vitro addition groups). After the final (t=80 min) measurements, the heart was rapidly excised, freeze-clamped in light alloy tongs cooled in liquid nitrogen, and weighed.

Preparation of lipid substrates 3

H-labelled sodium oleate (specific activity 485 mCi/ mmol) was prebound to 10% (w/v) fatty acid-free bovine serum albumin and added to the perfusate, to give a final NEFA concentration of 1.1 m (oleate groups; final albumin concentration of 2.75% (w/v) in perfusate). 3 H-labelled triolein in the form of chylomicrons

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was prepared from rats using a thoracic duct cannulation technique essentially as described by Bezman-Tarcher et al. (1965). Briefly, anaesthetized rats had a polyethylene catheter inserted into the lower thoracic duct via an extra-peritoneal loin incision and externalized to continuously collect chyle; a percutaneous gastrostomy was also performed. The animals were maintained in a restraining cage for 12 h with free access to food and water, but were given additional i.v. fluid replacement (tail vein). After this initial recovery period, 3H-labelled triolein (1.0 g; 22 mCi) was administered into the stomach, and chyle was collected for the subsequent 12 h. 3H-Chylomicrons were isolated by washing with bovine serum albumin solution and centrifugation. Thin-layer chromatography of the 3H-chylomicrons showed that over 95% of the label was in the triacylglycerol band. Chylomicrons were suspended in 4% (w/v) fatty-acid free bovine serum albumin and triacylglycerol content was assayed with an enzymatic colorimetric test kit (Boehringer Mannheim, GmbH, Lewes, Sussex, UK); 3H-chylomicrons were added to the perfusate reservoir to give a final concentration of 0.4 m TAG (chylomicron groups).

Measurement of lipid oxidation rate Lipid oxidation rate was estimated by measuring 3 H2O appearance in the perfusate (Evans and Wang, 1997). Briefly, aliquots of perfusate were removed at 10 min intervals (except during 40–50 min of perfusion when samples were taken at 2 min intervals), and subjected to Folch lipid extraction with chloroform:methanol and water. An aliquot of the water phase was counted for radioactivity. This method has been compared with an alternative method of estimating lipid oxidation rate by collecting 14CO2 evolved from [14C]-lipid substrate ([14C]NEFA). Good agreement was found between the two methods, and in each case 3H2O production was found to be linear over the 80 min experimental period (r>0.92 for each experiment; data not shown). In some experiments, “ghost” perfusions were performed in which the apparatus was set up as described above, but no heart was attached: following administration of either 3H-oleate (n=3) or 3H-chylomicrons (n=3) to the recirculating perfusate prime, serial sampling of perfusate aliquot and Folch extraction demonstrated no appearance of 3H2O (r>0.94; data not shown). In further experiments, lipid oxidation rate was varied either by addition of dichloroacetate (1 m), an inhibitor of pyruvate

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dehydrogenase kinase, or by increasing the myocardial workload (preload adjusted to 20 cm H2O and afterload adjusted to 140 cm H2O), both interventions performed at 40 min perfusion. Dichloroacetate decreased lipid oxidation, whilst increased cardiac workload increased lipid oxidation rate as measured by this method (data not shown).

Incorporation of exogenous lipid into myocardial lipid Frozen myocardium was ground to powder and 3Hmyocardial lipids were extracted from an aliquot by extraction with chloroform:methanol (Folch). After repeated washing, the lipids were re-solubilized in chloroform and separated by silica gel G thin-layer chromatography, using a hexane-diethylether-acetic acid system (Goldfarb and Pitot, 1971). Lipid classes were counted for 3H-radioactivity.

Lipoprotein lipase activity Myocardial total tissue LPL activity was estimated in duplicate in acetone/ether-dried tissue powders by using a 3H-labelled triolein substrate emulsion containing starved rat serum as a source of activating apoprotein CII to maximize LPL detection (Nilsson-Ehle and Schotz, 1976); the serum was pretreated by heating to 65°C to inactivate nonspecific plasma lipases. Radioactivity in evolved fatty acids was counted following extraction in methanol/chloroform/heptane (Nilsson-Ehle and Ekman, 1977). Results are expressed as nmol of fatty acid released/min/mg of acetone dried powder.

Statistics Results are expressed as mean values±... with numbers of experiments in parentheses. Statistical analysis was performed by one-way analysis of variance (ANOVA) for repeated measurements, or by Student’s t-test with Bonferroni correction for multiple comparisons, where appropriate. Statistical significance was set at P<0.05.

Results Treatment of rats with endotoxin or PAF caused visible signs of malaise with hypokinesis, piloerection and hyperventilation; however, no an-

imals died during this period. Administration of high dose endotoxin 3 h prior to cardiac excision caused a 30% decrease in fatty acid (oleate) oxidation rate when the heart was subsequently perfused ex vivo, compared to lyso-PAF injected controls; this was not seen at lower endotoxin dosage and oxidation of triacylglycerol (triolein) in the form of chylomicrons was not affected (Table 1). Conversely, PAF injection into rats before heart isolation and perfusion, at a dose sufficient to cause hypertriglyceridemia (Table 1; Evans et al., 1990, 1991b), did not alter oleate oxidation, and a tendency to increased triolein oxidation rate compared to hearts from lyso-PAF treated animals was not significant (Table 1). Administration of endotoxin directly in vitro to perfused hearts isolated from untreated rats did not alter oleate or triolein (chylomicron) oxidation rate after 40 min (Table 2). PAF added at low dosage (2 n) to the perfusate of non-pretreated hearts also had no effect on oleate oxidation but higher dosage PAF (200 n) in vitro caused a transient (10 min) doubling of oleate oxidation with a later (40 min) stimulation of triolein oxidation rate (Table 2). Endotoxin administration in vivo did not affect cardiac LPL activity measured in total tissue extract after the perfusion period; however, PAF treatment in vivo caused a significant increase in cardiac LPL activity following perfusion compared to hearts from lyso-PAF-treated controls (Table 3). Addition of endotoxin in vitro to the perfusate did cause a doubling of the heart LPL activity compared to hearts perfused with lyso-PAF (Table 3). PAF added in high concentration to the perfusate caused a brief increase in heart LPL activity— in separate experiments hearts perfused with oleate had LPL activity of 2.31±0.19 nmol fatty acid released/ min/mg acetone-dried tissue (n=5) 10 min after addition of PAF 200 n to the perfusate (P<0.01 compared to 1.46±0.15 nmol fatty acid released/ min/mg acetone-dried tissue (n=5) 10 min after lyso-PAF addition). Labelled lipid content of myocardial tissue was analysed by lipid class (Tables 4 and 5); endotoxin pretreatment in vivo of animals caused a dosedependent increase in labelled tissue lipid in all lipid classes examined, irrespective of perfusate lipid supplement (oleate or triolein). PAF injected into rats in vivo had no significant effect on tissue [3H] lipid accumulation following perfusion with either fatty acid or triacylglycerol (Table 4). Endotoxin administration to the heart perfusate in vitro caused a small but significant increase in [3H]cholesterol, NEFA and TAG accumulation with both lipid substrates, but PAF added in vitro to the heart perfusate

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Myocardial Lipid Oxidation Table 1 Effect of endotoxin and PAF administered in vivo on lipid oxidation rate in isolated perfused rat heart Substrate

Treatment

n

Plasma triacylglycerol (mg/100 ml)

Oxidation rate (nmol fatty acid/min/g wet weight)

Oleate Oleate Oleate Oleate

lyso-PAF 25 lg/kg Endotoxin 0.1 lg/kg Endotoxin 1 mg/kg PAF 25 lg/kg

7 3 4 7

49.0±4.0 45.8±9.4 84.2±11.0‡ 100.3±10.5†

151.7±7.0 149.5±11.4 105.8±24.0∗ 173.2±12.8

Triolein-chylomicrons Triolein-chylomicrons Triolein-chylomicrons Triolein-chylomicrons

lyso-PAF 25 lg/kg Endotoxin 0.1 lg/kg Endotoxin 1 mg/kg PAF 25 lg/kg

6 5 3 5

43.4±7.0 55.8±5.6 78.4±10.1 99.5±15.6†

15.5±1.8 16.2±1.9 16.7±2.1 30.5±12.8

Fed animals were treated (i.p. injection) 3 h before heart excision and perfusion. All groups had glucose 10 m as co-substrate. For further details see text. Values are mean±... Significant differences by ANOVA between endotoxin- and PAF-treated animals, and lyso-PAF controls are indicated: ∗ P<0.05; † P<0.01; ‡ P=0.07.

Table 2 Effect of endotoxin and PAF administered in vitro on lipid oxidation rate in isolated perfused rat heart Oxidation rate (nmol/fatty acid/min/g wet weight) Substrate

Treatment

n Pre-treatment (0–40 min)

Early posttreatment (40–50 min)

Oleate

lyso-PAF 200 n Endotoxin 1 lg/ml PAF 2 n PAF 200 n

6 146.5±22.1

112.1±47.3

5 147.5±16.3

126.9±36.3

5 150.3±18.7 6 163.6±10.6

171.9±51.4 294.1±37.3‡

Oleate Oleate Oleate Trioleinchylomicrons Trioleinchylomicrons Trioleinchylomicrons

lyso-PAF 200 n Endotoxin 1 lg/ml PAF 200 n

Change (%)

Late posttreatment (50–80 min)

Change (%)

−10.3±27

124.4±16.0

−1.4±21.5

−16.6±20

123.3±30.8

−14.6±29.8

140.3±26.3 143.1±17.7

−4.5±19.2 −9.7±13.6

20.9±5.9 98.8±39.3†

7

17.0±3.5

16.1±2.7

−9.2±11

20.2±3.1

21±7.9

5

16.3±3.4

20.9±4.5

33.1±2.5

22.5±1.8

50.6±17.7

8

17.0±4.1

17.5±3.1

10.1±3.5

30.6±9.0

63.6±12.9∗

Hearts from fed animals were excised and perfused with glucose 10 m and lipid substrate as indicated; lipid oxidation rate was measured before and after treatment addition to the perfusate at 40 min. For further details see text. Values are mean±... Significant difference by ANOVA between endotoxin- and PAF-treated animals, and lyso-PAF controls are indicated: ∗ P<0.05; † P<0.01; significant differences before and after treatment is indicated: ‡ P<0.05.

again had no effect on tissue lipid accumulation (Table 5). Mechanical function of the heart was measured throughout the perfusion period. Control (lyso-PAFtreated) hearts perfused with oleate showed a gradual decline in mechanical performance throughout the perfusion period, with a halving of aortic flow and decreased cardiac output and hydraulic work (Figs 1 and 3). However, addition of TAG (trioleinchylomicrons) to the perfusate instead of oleate, maintained aortic flow, and cardiac output and hydraulic work, throughout the perfusion period at baseline values (Figs 2 and 4). Administration of endotoxin to rats in vivo caused

a dose-dependent (inhibition) of cardiac performance, severe at high endotoxin dosage: despite the higher dosage (1 mg/kg body weight) of endotoxin causing no deaths after 3 h, 50% of hearts excised from these pretreated animals could not be made to “work” (i.e. could not maintain an aortic flow against an afterload of 100 cm H2O) and were excluded. The remainder were included for study, but even these hearts generated extremely low aortic flow rates. Thus, aortic flow ex vivo was significantly decreased by endotoxin treatment and declined further during perfusion, regardless of lipid substrate present in the perfusate (Figs 1 and 2). Hydraulic work following endotoxin treatment in

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Table 3 Effect of endotoxin and PAF on lipoprotein lipase activity in isolated perfused rat heart Substrate

Treatment

n

Heart LPL activity (nmol fatty acid released/min/mg acetone-dried tissue)

Oleate Oleate Oleate Oleate

lyso-PAF 25 lg/kg in vivo Endotoxin 0.1 lg/kg in vivo Endotoxin 1 mg/kg in vivo PAF 25 lg/kg in vivo

7 4 3 6

1.42±0.21 1.41±0.12 1.90±0.60 3.35±0.58†

Oleate Oleate Oleate Oleate Oleate Oleate

lyso-PAF 200 n in vitro lyso-PAF 200 n in vitro short exposure Endotoxin 1 lg/ml in vitro PAF 2 n in vitro PAF 200 n in vitro PAF 200 n in vitro short exposure

6 5 5 5 6 5

1.51±0.04 1.46±0.15 2.82±0.53∗ 2.21±0.13 1.23±0.10 2.31±0.19†

Triolein-chylomicrons Triolein-chylomicrons Triolein-chylomicrons Triolein-chylomicrons

lyso-PAF 25 lg/kg in vivo Endotoxin 0.1 lg/kg in vivo Endotoxin 1 mg/kg in vivo PAF 25 lg/kg in vivo

6 5 4 5

1.38±0.21 1.75±0.09 2.57±0.63§ 2.60±0.21∗

Triolein-chylomicrons Triolein-chylomicrons Triolein-chylomicrons

lyso-PAF 200 n in vitro Endotoxin 1 lg/ml in vitro PAF 200 n in vitro

7 5 8

1.53±0.13 3.29±0.43‡ 1.96±0.14∗

For details see legends to Tables 1 and 2 and text. Values are mean±... Significant differences by ANOVA between endotoxinand PAF-treated, and lyso-PAF controls are indicated: ∗ P<0.05; † P<0.01; ‡ P<0.001; § P=0.08.

vivo also declined during perfusion with oleate (Fig. 1), but not with triolein (Fig. 2). Interestingly, PAF administration at a pathological but sub-lethal dose in vivo prevented the decrease in aortic flow and mechanical function observed when hearts were perfused with oleate (Fig. 1); PAF pretreatment had no effect on hearts perfused with trioleinchylomicrons (Fig. 2). Neither PAF nor endotoxin added to the perfusate affected the decline in aortic flow and mechanical function associated with oleate perfusion (Fig. 3). Similarly, the preserved cardiac performance observed during triolein perfusion was not affected by in vitro additions of either PAF or endotoxin (Fig. 4).

Discussion Despite intensive investigation, the physiological regulation of lipid substrate utilization by the heart remains uncertain. Fatty acids are considered to be the favoured energetic fuel for myocardial contraction, suppressing (though incompletely) cardiac glucose utilization (Neely and Morgan, 1974); circulating NEFA concentrations are limited but substantial quantities of fatty acids can be assimilated by the heart for oxidation through hydrolysis of circulating TAG, in the form of liverderived very-low-density lipoprotein (VLDL) and

gut-derived chylomicrons. This hydrolysis is catalysed by LPL; although LPL is considered to be regulatory and rate-limiting for tissue TAG uptake and subsequent fate (Olivecrona and BengtssonOlivecrona, 1993), the control mechanisms in heart are unclear, though distinct from the reciprocal humoral control observed in adipose tissue and lactating mammary gland (Olivecrona and Bengtsson-Olivecrona, 1993). In the present experiments, TAG oxidation was low compared to oleate oxidation, since the chylomicrons were apoCII-poor; an approximate doubling of chylomicrontriolein oxidation rate can be obtained by addition of “starved” serum (unpublished work), but this compromises the defined nature of the perfusate. Endotoxin has widespread metabolic activities and may affect cardiac lipid metabolism by a variety of mechanisms—it may act directly on the myocardium, or indirectly in vivo via the release of intermediary mediators (endocrine, paracrine), or by alteration of prevailing substrate milieu or cardiac mechanical function. There is evidence for all of these factors. In the present work, endotoxin was demonstrated to decrease oleate oxidation when given in vivo but not in vitro, suggesting an indirect mechanism. This may be due to decreased fatty acid transport or to impaired intracellular NEFA oxidation—the latter is suggested by the concomitant increase in lipid accumulation within cardiac tissue; similar results have been reported in

Myocardial Lipid Oxidation

Aortic flow (ml/min/g wet weight)

40 ‡

35 30 25 20

† †

15 10 5 0

Cardiac output (ml/min/g wet weight)

70 ‡

60 50 *

40

*

30 20 10

(mmHg

Hydraulic work ml/min/g wet weight)

0 ‡

2500 2000

*

1500 1000 500 * 0 –20

0

20

40 60 Time (min)

80

100

Figure 1 Effect of endotoxin and PAF in vivo on mechanical function in isolated oleate-perfused rat hearts. For details see text. LPS, endotoxin. Significant differences between baseline (0 min) and subsequent measurements within groups are indicated: ∗ P<0.05; † P<0.01. Significant difference between lyso-PAF and PAF groups at each time point is indicated: ‡ P<0.05. (Φ) lyso-PAF; (∆) LPS 0.1 lg/kg; (Γ) LPS 1.0 mg/kg; (Ε) PAF 25 lg/ kg.

dog cardiomyocytes which showed increased incorporation of [14C]palmitate into phospholipids, tri- and di-acylglycerols and NEFAs after 2 h (Liu, 1982), and decreased palmitate oxidation (Liu and Spitzer, 1977) despite unaltered hexadecanol transport (Liu et al., 1981). Further support for a defect in intracellular fatty acid oxidation is provided by the observation that endotoxin decreases glycogen and 3-hydroxybutyrate content (Kuttner et al., 1981) and high energy phosphates and, hence, energy charge in rat heart (Raymond and Gordey, 1989), although this is not invariably found (Rumsey et al., 1988), and myocardial utilization

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of glucose, lactate and NEFA substrates has been shown not to be altered in a Langendorff preparation from septic rats (McDonough et al., 1986); methodological differences may be important. Decreased oleate oxidation in hearts from animals given endotoxin in vivo may simply be secondary to their severely obtunded mechanical status, since oxidation rates correlate with cardiac work (see Lopaschuk et al., 1994). However, it has proved difficult to relate endotoxin-induced effects on cardiac mechanical function directly to cardiac oxidative metabolism: sublethal endotoxemia caused an increased metabolic rate (O2 consumption) in isolated rat hearts perfused with glucose/pyruvate (Rumsey et al., 1988), but unchanged cardiac oxidative metabolism in dogs (D’Orio et al., 1986) and decreased myocardial O2 extraction capacity in pigs (Herbertson et al., 1995) have been reported. The lack of effect of endotoxin in vitro on oleate oxidation in the present studies may be a reflection of the relatively brief exposure of the isolated rat heart since lipid was starting to accumulate (Table 5). Differences in timing and dosage may also explain the finding of increased LPL activity in hearts treated with endotoxin in vitro (Table 3). This is surprising in terms of previous observations reporting modestly decreased heart LPL activity in vivo following very high dose [1–5 mg/kg (Bagby et al., 1987); [30 mg/kg body weight (Gouni et al., 1993)] endotoxin treatment for prolonged periods [18 h (Bagby et al., 1987); 7–24 h (Gouni et al., 1993)] with no change in heart LPL mRNA (Gouni et al., 1993). However, polymicrobial sepsis (Scholl et al., 1984) and burn injury (Bagby et al., 1981) both increase cardiac LPL activity, and may represent a mechanism to attempt to assimilate circulating substrate, in view of the increased cardiac workload in early sepsis (not seen in this study) and also the concomitant increase in intracellular lipid derived from exogenous triolein seen to accumulate in these hearts (Table 5). Measurement of LPL activity in acetone/ether-dried tissue powders may be criticized for assaying total tissue enzyme, not the physiologically relevant (heparin-releasable) portion on the endothelial luminal surface. However, LPL on the myocyte plasma membrane is active (Rodrigues et al., 1992), and the heart contains small but important endogenous TAG stores; intramyocyte LPL may play a role in intracellular TAG hydrolysis. These observations also support other recent evidence of direct in vitro effects of endotoxin on isolated myocardium and myocytes, including expression of Ca2+-independent nitric oxide synthase with release of nitric oxide (Schulz et al., 1995), and TNFa release (Kapadia et al., 1995).

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Table 4 Effect of endotoxin and PAF administered in vivo on incorporation of [3H]labelled lipid substrates into myocardial tissue lipids 3

H incorporation into extractable tissue lipids (lmol/g wet weight)

Substrate

Treatment

n

Oleate

lyso-PAF 25 lg/kg Endotoxin 0.1 lg/kg Endotoxin 1.0 mg/kg PAF 25 lg/kg

4 0.027±0.006

Oleate Oleate Oleate Trioleinchylomicrons Trioleinchylomicrons Trioleinchylomicrons Trioleinchylomicrons

lyso-PAF 25 lg/kg Endotoxin 0.1 lg/kg Endotoxin 1.0 mg/kg PAF 25 lg/kg

Cholesterol

Cholesterol ester

Non-esterified fatty acid

Triacylglycerols

Total

0.029±0.009

0.225±0.072

1.90±0.451

2.18±0.531

4

0.34±0.02∗

0.11±0.03

1.62±0.2

7.43±1.46∗

9.50±1.33∗

4

0.97±0.16‡

0.31±0.13‡

2.42±0.64∗

32.2±2.3‡

39.3±3.65‡

6 0.020±0.004

0.020±0.005

1.08±0.19

1.34±0.2

0.220±0.04

4 0.014±0.0019 0.011±0.0024 0.033±0.0084 0.375±0.109

0.433±0.118

5 0.006±0.002

0.008±0.0005

0.10±0.02

0.29±0.05

0.40±0.08

3 0.060±0.02∗

0.007±0.001

0.83±0.38∗

3.33±1.2†

4.08±1.69†

5 0.034±0.02

0.014±0.007

0.06±0.03

0.46±0.11

0.57±0.13

For further details see legends to Tables 1 and 2 and text. Results are mean±... Differences between endotoxin/PAF-treated animals and lyso-PAF controls are indicated: ∗ P<0.05; † P<0.01; ‡ P<0.001.

Table 5 Effect of endotoxin and PAF administered in vitro on incorporation of [3H]labelled lipid substrates into myocardial tissue lipids 3

H incorporation into extractable tissue lipids (lmol/g wet weight)

Substrate

Treatment

n

Oleate

lyso-PAF 200 n Endotoxin 1.0 lg/ml PAF 2 n PAF 200 n

Oleate Oleate Oleate Trioleinchylomicrons Trioleinchylomicrons Trioleinchylomicrons

lyso-PAF 200 n Endotoxin 1.0 lg/ml PAF 200 n

Cholesterol

Cholesterol ester

Non-esterified fatty acid

Triacylglycerols

Total

6 0.032±0.006

0.027±0.006

0.189±0.01

2.00±0.31

2.25±0.30

5 0.101±0.01∗

0.040±0.01

1.05±0.3∗

5.58±1.1∗

6.62±2.3∗

5 0.038±0.027 6 0.019±0.005

0.014±0.009 0.020±0.006

0.683±0.16 0.201±0.023

3.17±0.7 1.60±0.24

3.88±0.71 1.84±0.25

7 0.016±0.006

0.011±0.002

0.030±0.007

0.459±0.09

0.515±0.11

6 0.053±0.01∗

0.014±0.004

0.220±0.05‡

0.900±0.2∗

8 0.013±0.002

0.007±0.001

0.016±0.003

0.357±0.05

1.18±0.23† 0.389±0.06

For further details see legends to Tables 1 and 2 and text. Results are mean±... Differences between endotoxin/PAF-administered hearts and lyso-PAF controls are indicated: ∗ P<0.05; † P<0.01; ‡ P<0.001.

The inhibitory effect of endotoxin on cardiac contractile function in vivo is well documented (Qi and Jones, 1990; Dobrowsky et al., 1991; Mulder et al., 1993; Kawamura et al., 1994) and was confirmed in the present study: high dose (1 mg/ kg) endotoxin significantly decreased cardiac mechanical function (Fig. 1), which is possibly a reflection of the inhibition of fatty acid oxidation at this dosage (Table 1). Recent work has demonstrated the ability of endotoxin to directly inhibit contractile

function of cardiomyocytes in vitro (Starr et al., 1995; Tao and McKenna, 1994) after 6 h incubation (Starr et al., 1995); the lack of significant effect of endotoxin administration in vitro on cardiac mechanical function (Figs 3 and 4) may again be due to the brief exposure, and agrees with previous studies under comparable conditions (Salari et al., 1990); the preserved fatty acid oxidation (Table 2) under these conditions may be responsible. Endotoxin stimulates secretion of a cascade of

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Myocardial Lipid Oxidation 35 Aortic flow (ml/min/g wet weight)

40 30 *

20 10

30 25 20

10

0 60 Cardiac output (ml/min/g wet weight)

50 40 30 20 10

Hydraulic work ml/min/g wet weight)

0 3000 2500 2000 1500 1000 500 0 –20

0

20

40 60 Time (min)

80

100

*

* *

5

70 60

*

15

0

(mmHg

(mmHg

Hydraulic work ml/min/g wet weight)

Cardiac output (ml/min/g wet weight)

Aortic flow (ml/min/g wet weight)

50

50 *

40

*

30 20 10 0 2500 2000 *

1500

*

1000 500 0 –20

0

20

40 60 Time (min)

80

100

Figure 2 Effect of endotoxin and PAF in vivo on mechanical function in isolated triolein-perfused rat hearts. For details see text. LPS, endotoxin. Significant differences between baseline (0 min) and subsequent measurements within groups are indicated: ∗ P<0.05. (Φ) lyso-PAF; (∆) LPS 0.1 lg/kg; (Γ) LPS 1.0 mg/kg; (Ε) PAF 25 lg/ kg.

Figure 3 Effect of endotoxin and PAF in vitro on mechanical function in isolated oleate-perfused rat hearts. For details see text. LPS, endotoxin. Significant differences between baseline (0 min) and subsequent measurements within groups are indicated: ∗ P<0.05. (Φ) lyso-PAF; (∆) LPS 1 lg/ml; (Γ) PAF 2 n; (Ε) PAF 200 n.

inflammatory mediators, several of which are known to affect lipid metabolism in a variety of tissues (Evans and Williamson, 1991); of these, certain cytokines, and the structurally-unrelated PAF, are also known to alter cardiac mechanical function and are candidate mediators of the indirect effects of endotoxin in vivo. TNFa [known to be released by endotoxin within myocardium (Kapadia et al., 1995)] has no effect on heart LPL (Hulsmann et al., 1988), whilst IL-1 decreases myocardial LPL activity in vitro (Friedman et al., 1991). Pretreatment of animals with a PAF-receptor antagonist prevents endotoxin-induced cardiac mechanical dysfunction (Baum et al., 1990; Dobrowsky et al., 1991; Kawamura et al., 1994)

and PAF injection causes endotoxin-like effects on cardiac mechanical function (Salari et al., 1990; Mulder et al., 1993), but the effects of PAF on cardiac lipid metabolism are unknown. We thus investigated PAF, which, at a dose we have previously shown in the Langendorff model, does not affect coronary flow rate or diastolic function, but modestly decreases contractility (+dP/dtmax; to about 90% of controls) with glucose as sole substrate (Munoz et al., 1995). In triolein/glucoseperfused working hearts, PAF had no effect on cardiac mechanical function; however, hearts perfused with oleate/glucose (at a relatively high fatty acid concentration—1.1 m—but with apparently adequate NEFA pre-binding to albumin) showed a

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X. Wang and R. D. Evans

Aortic flow (ml/min/g wet weight)

50 40 30 20 10

(mmHg

Hydraulic work ml/min/g wet weight)

Cardiac output (ml/min/g wet weight)

0 70 60 50 40 30 20 10 0 3000 2500 2000 1500 1000 500 0 –20

0

20

40 60 Time (min)

80

100

Figure 4 Effect of endotoxin and PAF in vitro on mechanical function in isolated triolein-perfused rat hearts. For details see text. LPS, endotoxin. (Φ) lyso-PAF; (∆) LPS 1 lg/ml; (Ε) PAF 200 n.

gradual decline in aortic flow and, hence, cardiac output, which may be related to detergent-like effects of the fatty acid itself or viscosity/Ca2+binding effects of the albumin. This decline was not altered by addition of PAF in vitro, but was prevented by PAF administered in vivo beforehand (Figs 1 and 2). This is intriguing, as the dosage of PAF given to the animal (25 lg/kg body weight) is sufficient to produce obvious signs of malaise (piloerection, thirst, diarrhoea, hyperventilation, asthenia), consistent with its putative role as a pathological mediator, and also to induce hypertriglyceridemia [Table 1; (Evans et al., 1990)] and hyperglycaemia, stimulate hepatic lipogenesis, and decrease plasma insulin concentration, yet insufficient to cause hepatic vasoconstriction (as evidenced by unaltered

hepatic glycogen content) (Evans et al., 1990, 1991a). The mechanism whereby this apparently deleterious pretreatment in vivo “protects” the heart during subsequent high-dose fatty acid perfusion is unclear, but may involve a “preconditioning” action affecting membrane or ion-channel status; however, the same PAF dosage (25 lg/kg) did not significantly affect heart total LPL activity nor accumulation by this tissue of [14C]lipid from an orally administered [14C]triolein load, despite widespread effects on lipid metabolism in other tissues (Evans et al., 1991b), including increased lipolysis with increased plasma NEFA (Holland et al., 1991). Since flows and pressures were unchanged compared to controls in perfused hearts following PAF treatment, any observed changes in lipid metabolism following additions of PAF are not secondary to changes in cardiac work. Thus, PAF significantly increased the oxidation rate of triolein compared to lyso-PAF treated controls, but did not alter oleate oxidation in a system capable of increasing and decreasing lipid oxidation rates (Fig. 3). The likely explanation for this difference between triolein and oleate oxidation rates is an effect of PAF on cardiac LPL activity, and PAF significantly increased LPL in chylomicron/glucose perfused hearts (Table 1). Surprisingly, PAF did not increase LPL in oleate/ glucose perfused hearts, indeed a small but significant decrease was observed (Table 1). This observation raises the issue of the putative role of the substrate itself in regulation or responsiveness of tissue LPL activity [administration of PAF in vivo caused hypertriglyceridemia in the present experiments (Table 1)]. Some insight into this is provided by the observations of the inverse relationship between plasma TAG as presented in vivo to the enzyme located on the luminal surface of the tissue endothelium, and tissue LPL activity (Friedman et al., 1979; Stam and Hulsmann, 1984; Rodrigues et al., 1992) and the presence in the lipoprotein substrate of the enzyme activator apoprotein CII— possibly the chylomicron particle influences the responsiveness of the tissue LPL activity to exogenous effectors. [3H]-Labelled myocardial lipids were measured to assess the tissue fate of the exogenous lipid substrate (Tables 4 and 5). More lipid of all classes examined was present in oleate-perfused hearts than chylomicron-perfused hearts, as expected, since the former does not involve access through LPL. The unchanged lipid profile seen in PAF-exposed hearts with both NEFA and TAG as substrate argues against a myocardial fatty acid assimilation or oxidation defect (PAF increased oxidation of both) and further suggests preserved integrity of the intra-

Myocardial Lipid Oxidation

cardiomyocyte lipolysis-re-esterification pathways. However, markedly increased accumulation of labelled lipid in hearts treated with endotoxin is in good agreement with previous findings (Liu and Spitzer, 1977) and suggests an intracellular accumulation of total unlabelled lipid; such an effect would alter the pool size of lipid and could explain observed changes in lipid oxidation rate (Table 1). Although intracellular lipid mass was not measured in the present study, previous work by the same group found no significant change in dog myocardial NEFA, TAG or phospholipid content after 4 h endotoxin exposure in vivo (Liu and Spitzer, 1977) despite 100–200% increased accumulation of labelled exogenous [14C]palmitate by myocytes at 2 h of endotoxin exposure (Liu, 1982). This suggests that tissue lipid pool size was constant in the present experiments and changes in lipid oxidation were not secondary to this. PAF has therefore been shown to have significant effects on cardiac lipid metabolism at doses too low to alter mechanical performance. These effects do not mimic those of endotoxin under the present experimental conditions, suggesting that PAF is not involved in the changes in myocardial lipid metabolism observed during sepsis/endotoxemia.

Acknowledgements The authors are grateful to Drs D. H. Williamson and G. F. Gibbons, and Mr R. Hems, Metabolic Research Laboratory, University of Oxford, for helpful discussions and technical assistance, and to the Wellcome Trust and the British Journal of Anaesthesia for financial support.

References B GJ, M HI, S JA, S JJ, 1981. Increased tissue lipoprotein lipase activity in guinea pigs following burn trauma. Circ Shock 8: 131–136. B GJ, C CB, M RR, 1987. Triacylglycerol kinetics in endotoxic rats with suppressed lipoprotein lipase activity. Am J Physiol 253: E59–E64. B TD, H SO, F HS, L CA, F MP, 1990. Endotoxin-induced myocardial depression in rats: effect of ibuprofen and SDZ 64-688, a platelet activating factor receptor antagonist. J Surg Res 48: 629–634. B-T A, O S, R DS, 1965. The removal of triglyceride fatty acids from the incubation of the supradiaphragmatic portion of the rat. Proc Roy Soc B 162: 411–426. D’O V, -A D, V S, F A, J J, M R, 1986. Lack of defective cardiac oxidative metabolism in intact dogs subjected to a prolonged

1925

low-dose infusion of Escherichia coli endotoxin. Circ Shock 18: 43–52. D UK, F CW, G A, S J, 1995. Endotoxin-induced contractile dysfunction in guinea pig hearts is not mediated by nitric oxide. Am J Physiol 268: H2460–H2465. D RT, V RD, O NC, 1991. Effect of SRI 63-675 on hemodynamics and blood PAF levels during porcine endotoxemia. Am J Physiol 260: H1455–H1465. E RD, W X, 1997. Measurement of fatty acid oxidation in isolated perfused rat heart. Biotech Tech 11: 127–132. E RD, W DH, 1991. Signals, mechanisms, and function of the acute lipid response to sepsis. Biochem Cell Biol 69: 320–321. E RD, A JM, W DH, 1989. Metabolic effects of tumour necrosis factor-a (cachectin) and interleukin-1. Clin Sci 77: 357–364. E RD, I V, W DH, 1990. Metabolic effects of platelet-activating factor in rats in vivo. Stimulation of hepatic glycogenolysis and lipogenesis. Biochem J 269: 269–272. E RD, L P, W DH, 1991a. Plateletactivating factor and its metabolic effects. Prostagl Leukotr Essentl Fatty Acids 44: 1–10. E RD, I V, W DH, 1991b. Effect of platelet-activating factor on lipid metabolism in rats in vivo. Origin of the hypertriglyceridaemia. Biochem J 280: 541–543. F G, S O, S Y, 1979. Stimulation of rat heart lipoprotein lipase activity by 4-aminopyrazolo[3, 4-d]pyrimidine-induced reduction of plasma triacylglycerol. FEBS Lett 100: 371–373. F G, B V, C-S T, 1991. Recombinant human interleukin-1 suppresses lipoprotein lipase activity, but not expression of lipoprotein lipase mRNA in mesenchymal rat heart cell cultures. Biochim Biophys Acta 1089: 83–87. G S, P HC, 1971. Improved assay of 3hydroxy-3-methyl glutaryl coenzyme A reductase. J Lipid Res 12: 512–515. G I, E J, C L, 1993. Endotoxin-induced hypertriglyceridemia is mediated by suppression of lipoprotein lipase at a post-transcriptional level. J Lipid Res 34: 139–146. H MJ, W HA, R JA, I K, W KR, 1995. Myocardial oxygen extraction ratio is decreased during endotoxemia in pigs. J Appl Physiol 79: 479–486. H A, E RD, W DH, 1992. Plateletactivating factor causes hypoketonaemia in starved rats. Clin Sci 83: 495–501. H MR, M TM, Z GA, P SM, 1991. Cardiovascular effects of platelet-activating factor. Trends Cardiovasc Med 1: 117–121. H WC, D M-L, DW LEA, P NLM, 1988. Cardiac lipoprotein lipase: effects of lipopolysaccharide and tumor necrosis factor. Mol Cell Biochem 79: 137–145. K S, L J, T-A G, B HH, M TS, M DL, 1995. Tumor necrosis factor-a gene and protein expression in adult feline myocardium after endotoxin administration. J Clin Invest 96: 1042–1052. K M, K T, T Z, F S, T M, N K, 1994. Effects of TCV-309,

1926

X. Wang and R. D. Evans

a novel PAF antagonist, on circulatory shock and hematological abnormality induced by endotoxin in dogs. J Lipid Mediat Cell Signal 9: 255–265. K JL, P JE, B SR, L LG, 1984. Effects of acetyl glyceryl ether of phosphorylcholine (platelet activating factor) on ventricular preload, afterload, and contractility in dogs. J Clin Invest 74: 1193– 1203. K PM, T JX, R RM, 1985. Increased myocardial contractility during endotoxin shock in dogs. Am J Physiol 249: H715–H722. K RE, A FO, S W, 1981. Glucocorticoid effect on glycolytic intermediates in septic rat heart. Adv Shock Res 5: 103–111. L MS, 1982. Increased incorporation of 14C-palmitate into tissue lipids by isolated heart myocytes in endotoxic shock. Adv Shock Res 7: 43–49. L MS, S JJ, 1977. In vitro effects of E. coli endotoxin on fatty acid and lactate oxidation in canine myocardium. Circ Shock 4: 181–190. L MS, L WM, S JJ, 1981. Influence of Escherichia coli endotoxin on palmitate, glucose, and lactate utilization by isolated dog heart myocytes. Prog Clin Biol Res 62: 115–121. L GD, B DD, G J, I T, S BO, 1994. Regulation of fatty acid oxidation in the mammalian heart in health and disease. Biochim Biophys Acta 1213: 263–276. MD KH, H JJ, L CH, S JJ, 1986. Substrate utilization and high energy phosphate levels of hearts from hyperdynamic septic rats. Circ Shock 18: 161–170. M MF,  L AA,  K AA, S PG, B AA,   B GC, T LG, 1993. Systemic and regional hemodynamic changes during endotoxin or platelet-activating factor (PAF)induced shock in rats. Circ Shock 41: 221–229. M HR, E RD, M SCU, F¨  P, 1995. Platelet-activating factor does not affect diastolic function in isolated rat hearts. Cardiovasc Res 30: 1028– 1032. N JR, M HE, 1974. Relationship between carbohydrate and lipid metabolism and the energy balance of heart muscle. Ann Rev Physiol 36: 413–459. N JR, R MJ, 1975. Techniques for perfusing isolated rat hearts. Methods Enzymol 39: 43–60. N-E P, E R, 1977. Rapid, simple and specific assays for lipoprotein lipase and hepatic lipase. Artery 3: 197–209. N-E P, S MC, 1976. A stable radioactive substrate emulsion for assay of lipoprotein lipase. J Lipid Res 17: 536–541.

O T, B-O G, 1993. Lipoprotein lipase and hepatic lipase. Curr Opin Lipidol 4: 187–196. Q M, J SB, 1990. Contribution of platelet activating factor to hemodynamic and sympathetic responses to bacterial endotoxin in conscious rats. Circ Shock 32: 153–163. R RM, G J, 1989. The effect of hypodynamic shock on myocardial high energy phosphates in the rat. Cardiovasc Res 23: 200–204. R DA, W DY, L CO, L R, 1988. Negative inotropic effect of platelet-activating factor: association with a decrease in intracellular sodium activity. J Pharmacol Exp Ther 245: 124–128. R B, S MR, S DL, 1992. Long term incubation of cardiac myocytes with oleic acid and very-low-density lipoprotein reduces heparin-releasable lipoprotein lipase activity. Mol Cell Biochem 116: 33–37. R WL, K L, W DF, E M, 1988. Myocardial metabolism and coronary flow: effects of endotoxemia. Am J Physiol 255: H1295– H1304. S M, G J, W LA, L GD, 1993. Acetyl-CoA carboxylase regulation of fatty acid oxidation in the heart. J Biol Chem 34: 25836–25845. S H, D M, W A, 1990. Comparative hemodynamics and cardiovascular effects of endotoxin and platelet-activating factor in rat. Circ Shock 32: 189– 207. S RA, L CH, B GJ, 1984. Hypertriglyceridemia and its relation to tissue lipoprotein lipase activity in endotoxemic, Escherichia coli bacteremic, and polymicrobial septic rats. J Surg Res 37: 394–401. S R, P DL, C R, M S, O PM, L GD, 1995. The role of nitric oxide in cardiac depression induced by interleukin-1 beta and tumour necrosis factor-alpha. Br J Pharmacol 114: 27–34. S H, H WC, 1984. Effects of hormones, amino acids and specific inhibitors on rat heart heparin-releasable lipoprotein lipase activities during long term perfusion. Biochim Biophys Acta 794: 72–82. S RG, L AS, P GC, S CE, A FL, 1995. Direct action of endotoxin on cardiac muscle. Shock 3: 380–384. T H, H R, K HA, 1980. Utilization of energy-providing substrates in the isolated working rat heart. Biochem J 186: 701–711. T S, MK TM, 1994. In vitro endotoxin exposure induces contractile dysfunction in adult rat cardiac myocytes. Am J Physiol 267: H1745–H1752.