Materials And Methods

2.1. Preparation of Lipid Substrates

(i) 3H-Labelled sodium oleate was prebound to fatty acid-free bovine serum albumin and added to the heart perfusate to give a final concentration of 1.1 mM (NEFA group), (ii) 3H-Labelled triolein in the form of rat VLDL was prepared by rat liver perfusion. Fasted rats were pre-treated 15 h prior to experiment with endotoxin (from E. coli serotype 055 :B5) 100|ig/kg body wt. or saline (control) intraperitoneally; they were anaesthetised, the portal vein and right atrium were cannulated without heparin and the inferior vena cava was ligated. The liver was perfused in situ with a recirculating solution comprising Waymouth's medium supplemented with glucose, amino acids, washed red cells and [3H]oleate prebound to fatty acid-free albumin, gassed with02:C02 (95:5) at 37 °C. The perfusate was ultracentrifuged at 144,500 g to separate d < 1.006 layer. Thin-layer chromatography (TLC) of the 3H-VLDL showed that > 95% of the label was in the triacylglycerol band. VLDL were suspended in fatty-acid free bovine serum albumin, TAG content was assayed (Boehringer test kit) and added to the heart perfusate to give a final concentration of 0.4 mM TAG.

2.2. Isolated Perfused Working Heart Preparation

Hearts from fed rats were perfused in "working" mode by the Taegtmeyer et al.1 modification of the method of Neely and Morgan. The heart was excised, the aorta cannulated (<2min from excision) and perfused retrogradely through the coronary arteries in "Langendorff" mode whilst lung, mediastinal, and peri-cardiac brown adipose tissue were excised, pulmonary arteriotomy performed, and the left atrium cannulated. The apparatus was switched to "working" mode and cardiac perfusion maintained through the left atrium (anterograde). A recirculating Krebs-Henseleit bicarbonate buffer solution containing CaCl2 1.3mM, glucose lOmM and fatty-acid free bovine serum albumin 2% (w/v) was filtered and gassed with 02: C02(95:5) at 37°C. Afterload was 100cmH20 and preload (atrial filling pressure) 15cmH20. After 15 min stabilisation lipid substrate (v.s.) was added (2min) (Time "0"). Peak systolic pressure (PSP) and heart rate (HR) were measured by pressure transducer. Aortic flow rate (AFR) and coronary flow rate (CFR) were measured by timed collections of perfusate. Measurements were made at time 0 and at 10 min intervals for 60 min. Cardiac output (CO) was calculated as (CFR + AFR). Hydraulic work (HW) was calculated as (CO x mean aortic pressure heart wt.). Rate-pressure product = PSP x HR. After t = 60min heparin (5U/ml) was added and at t = 62 min a perfusate sample was taken for LPL assay, the heart was rapidly excised, freeze-clamped in light alloy tongs cooled in liquid nitrogen, and weighed.

2.3. Measurement of Lipid Oxidation Rate

Timed perfusate samples were extracted (Folch) with chloroform/methanol/water; the aqueous supernatant phase was counted for 3H20 radioactivity as described2 (TAG oxidation rate), the organic infranatant phase was dried, resolubilised in ethanol and assayed for TAG (TAG uptake rate).

2.4. Incorporation of Exogenous Lipid into Myocardial Lipid

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