14C]palmitate uptake in isolated rat liver mitochondria: effects of fasting, diabetes mellitus, and inhibitors of carnitine acyltransferase.

J M Amatruda,D H Lockwood,S Margolis,L A Kiesow

doi:10.1016/s0022-2275(20)41268-4

Abstract

The rapid association of Na-[16-(14)C]palmitate with isolated rat liver mitochondria was measured by an oil separation method. This association was time and temperature-dependent and was absolutely dependent on the presence of exogenous ATP and CoASH and partially dependent on exogenous carnitine. Carnitine dependence was enhanced at lower concentrations of [(14)C]palmitate. At 6.5 micro M [(14)C]palmitate (molar ratio of palmitate to albumin equal to 0.54), the rate of association was linear for 20 sec and was increased more than 100% in the presence of carnitine. Carnitine-dependent association was inhibited by 2-bromopalmitate, an inhibitor of carnitine acyltransferase I, but not by (+)-octanoylcarnitine, a presumed inhibitor of carnitine acyltransferase II. The association of [(14)C]palmitate with mitochondria was enhanced from 190 to 330% in mitochondria isolated from fasted animals and from 160 to 230% in mitochondria isolated from diabetic, ketotic animals as compared to control animals. The enhanced association with mitochondria from fasted animals was inhibited by 2-bromopalmitate. These studies demonstrate a method of evaluating fatty acid association with mitochondria which, because of its dependence on carnitine and carnitine acyltransferase I activity, most likely represents true uptake into mitochondria. Furthermore, these studies indicate that the carnitine-dependent uptake of fatty acids into mitochondria is enhanced in the two ketotic states evaluated and that the carnitine acyltransferase system may be a regulatory site in ketone body production.

Highlights

The rapid association of Na-[16-14C]palmitate with isolated rat liver mitochondria was measured by an oil separation method
Much interest has been directed toward the carnitine acyltransferase system and consequent free fatty acids (FFA) uptake into mitochondria as a possible regulatory site of ketone body production
We describe a method for the direct measurement of carnitine-responsive FFA uptake in isolated rat liver mitochondria and present the first direct evidence that fatty acid uptake is enhanced in mitochondria isolated from ketotic animals

Summary

MATERIALS AND METHODS

Male Sprague-Dawley rats, fed ad libitum and weighing approximately 200 g, were used for all experiments. Uptake as reported represents the total [14C]palmitateassociated with the mitochondria in the presence of ATP, CoASH, and carnitine minus the [14C]palmitateassociated with mitochondria in the absence of ATP, CoASH, and carnitine This procedure automatically corrects for the amount of [14C]palmitatetrapped outside the mitochondria in the mitochondrial pellet. The quantity of mitochondrial protein sedimented by our method was the same in the presence or absence of the oil mixture for centrifugations of 45 sec and 4 min. The microfuge tubes were placed in rubber adaptors which were filled to the top of the microfuge tube with water to prevent deformation of the tube Under these conditions, the mitochondrial protein sedimented is the same as that sedimented following a 14,000 g centrifugation for 45 sec in the Beckman microfuge.

RESULTS

Findings

DISCUSSION