Comp. Biochem. Physiol. Vol. 83B, No. I, pp. 191-196, 1986 0305-0491/86 $3.00 + 0.00 Printed in Great Britain © 1986 Pergamon Press Ltd PARTIAL PROTECTION AGAINST ERUCOYL-CARNITINE INHIBITION IN HAMSTER BROWN-ADIPOSE-TISSUE MITOCHONDRIA IS DUE ,TO HIGH CoA LEVELS: A COMPARISON WITH RAT BROWN-ADIPOSE-TISSUE MITOCHONDRIA STEFAN ALEXSON, JAN NEDERGAARD and BARBARA CANNON The Wenner-Gren Institute, University of Stockholm, Biologihus F3, S-10691 Stockholm, Sweden (Tel: 08-16-4125) (Received 11 April 1985) A~tract--l. Brown-adipose-tissue mitochondria isolated from golden hamsters were found to contain more CoA per mg protein than rat brown-fat mitochondria, and after incubation with erucoyl-carnitine, a higher free CoA level remained, than in rat mitochondria. 2. In accordance with the suggestion (Alexson et al. (1985) Biochim. biophys. Acta 834, 149-158) that the inhibitory effect of erucoyl-carnitine on brown-fat mitochondrial respiration is entirely due to CoA sequestration, hamster mitochondria (with more CoA) were less sensitive to erucoyl inhibition than were rat mitochondria. 3. Thus, increased mitochondrial CoA levels may augment the ability of animals to withstand the detrimental effects of a high erucoyl ester content of the diet. INTRODUCTION The ingestion of certain fatty acids, notably erucic acid, may lead to detrimental effects in certain tissues [for reviews see Beare-Rogers (1977) and Bremer and Norum (1982)]. Especially, mitochondrial oxidation may be inhibited. Brown adipose tissue [see Nedergaard and Lind- berg (1982) and Cannon and Nedergaard (1985) for general reviews on the tissue] accumulates, from the circulation a very large fraction of all different types of lipids ingested [see, for example, Sj6rgren et al. (1977), Appelgren and R6ssner (1980), Ahrn6 et al. (1982), Becket et al. (1983, 1985) and Becker (1984)]. This is also true for erucic acid (Martinelli et al., 1976), and brown adipose tissue may therefore be especially prone to erucic acid inhibition. We have therefore recently investigated the effects of erucoyl-carnitine on brown-adipose-tissue mito- chondria of the rat (Alexson et aL, 1985). We con- cluded that the ability of erucoyl esters to inhibit the oxidation of different CoA-linked substrates in mito- chondria could probably be fully explained as the result of sequestration of free CoA in the form of poorly metabolizable erucoyl-CoA esters (Alexson et aL, 1985). In order to examine the general validity of this mechanism we have here investigated the correlation between CoA levels and erucoyl-carnitine inhibition in two species: hamster and rat. We have especially investigated whether a high matrix CoA level could have a protective effective against the inhibition by erucoyl esters. MATERIALS AND METHODS Preparation of brown-adipose-tissue mitochondria For this study, brown-adipose-tissue mitochondria were mainly prepared from adult golden hamsters (Mesocricetus auratus) of both sexes. The hamsters had been individually housed at 5 + I°C, 8 hr light/16 hr dark, for at least 3 weeks, and had had free access to water and food (a sunflower-seed based mixture). For some experiments, mitochondria were prepared from the brown fat of adult cold-acclimated female Sprague-Dawley rats. The mitochondria were prepared as described by Cannon and Lindberg (1979) and Alexson et al. (1985). The pellet was finally suspended in lfl0mM KCI and 20mM Tes [K-(N- tris(hydroxymethyl)-9-methyl- 2-aminoethane- sulphonate] (pH7.2) (final protein concentration about 45 mg/ml). Mitochondrial respiration Mitochondrial respiration was measured polar- ographically as earlier described (Alexson and Cannon, 1984) with a Clark type electrode in a medium consisting of I00 mM KCI, 20 mM Tes (pH 7.2), 1 mM EDTA (ethylene diaminetetraacetic acid), 2raM MgC12, 4mM potassium phosphate, 3 mM malate and 0.7 mg of mitochondrial pro- tein in a total volume of 1 ml. The waterbath temperature was 25°C. Measurement of mitoehondrial CoA, acetyl-CoA and long- chain acyl-CoA contents These measurements were performed as earlier described (Alexson et al., 1985). Briefly, the mitochondria were incu- bated in the same medium as used in the respiratory studies (+ 3 mM dithiothreitol), with further additions as indicated. The acyl-CoA esters of the mitochondrial incubations were then separated into acid-soluble and -insoluble esters (Tubbs and Garland, 1969), and (after hydrolysis) the CoA content of both fractions was determined by an enzymatic cycling procedure according to Veloso and Veech (1975) and Allred and Guy (1969). In certain experiments, acetyl-CoA was measured accord- ing to Allred and Guy (1969) after preincubation of 50/~1 samples for I0 rain at room temperature with 2 #mol N- ethylmaleimide (which eliminates all free CoA), followed by reacting the excess N-ethylmaleimide with addition of 4 #mol dithioerythritol. CoA and a mixture of acetyl-CoA plus CoA were used as standards. 191