Measurement of carnitine palmitoyltransferase I in hepatocyte monolayers.

Long-chain fatty acyl-groups are translocated across the mitochondrial inner membrane as acyl-carnitine esters. Camitine palmitoyltransferase I (CPT I) on the outer mitochondrial membrane, catalyzes the formation of palmitoylcarnitine from palmitoyl-CoA and camitine. In rat liver mitochondria CPT I is inhibited by malonyl-CoA concentrations in the physiological range. Inhibition is reversible and can be overcome by increasing [palmitoyl-CoA]. Alterations in nutritionalhormonal status of the animal changes hepatic [malonyl CoA], as well as the sensitivity of CPT I to malonyl-CoA inhibition; both mechanisms may contribute to the elevated CPT I activity observed in mitochondria from starved rats [l]. Due to the sensitivity of the enzyme to malonyl-CoA inhibition, CPT I is now thought to play a crucial role in the control of 0-oxidation [21. CPT I activity is determined i n isolated rat liver mitochondria by measuring the formation of palmitoyl[3H]camitine from [3H]carnitine and palmitoyl-CoA. Recently CPT I activity has been assayed radiochemically in freshly isolated hepatocytes, permeabilized with either filipin [3] or digitonin [4,5], and the short-term effect of hormones on enzyme activity investigated. Guzman and Geelen reported that glucagon increased CPT I activity [4] and Stephens and Harris [3] found that CAMP also increased CPT I activity but had no effect on its sensitivity to inhibition. Boon and Zammit [5] found that CPT I activity, nor its sensitivity to inhibition by malonyl-CoA were altered by pretreatment with glucagon or insulin. Cultured hepatocytes provide a system in which both the long-term and short-term effect of hormones and inhibitors on CPT I activity can be measured. In this study we describe a radiochemical assay that measures CPT I activity in situ, in permeablized rat hepatocyte monolayers. Etomoxir (2[6(4chloro-phenoxy)hexyl]oxirane-2-carboxylate), the CoA-ester of which strongly inhibits CPT I [6], and malonyl-CoA were used to characterize enzyme activity. Hepatocytes were isolated from male Albino Wistar rats by collagenase perfusion of livers and were cultured as in [7]. After overnight culture i n serum-free medium containing 10 nM dexamethasone hepatocytes were incubated with varying concentrations of etomoxir, as required. Inhibitor was then removed from the cells, hepatocytes were washed with 150 mM NaCI, then incubated with medium A containing 50 mM imidazole, 70 mM KC1, 80 mM sucrose, 1 mM EGTA, 2 mM MgCl,, 1mM DTT, 1 mM KCN, 1 mM ATP, 0.1% BSA, pH 7.1, supplemented with 0.05 mdml digitonin at 37.C. After 5 minutes the permeabilization medium was aspirated from the cells and was replaced with medium A with malonyl-CoA as needed. The assay was started by the addition of 50 pM palmitoyl-CoA plus 0.5 mM carnitine containing [3H]carnitine (2.5 pCipmo1). After 3 minutes incubation with substrate at 37C the reaction was terminated by the addition of HCl (final concentration 1 M). Cells were then disrupted by sonication and the palmitoyl-[3H]carnitine formed was extracted with n-butanol and counted. Permeabilization of hepatocyte monolayers by hgitonin was assessed by measuring the release of the cytoplasmic enzyme lactate dehydrogenase. Optimal conditions for permeabilization of cells were incubation of cells for 5 minutes with 0.05 mdml digitonin at 370C, this caused approximately 45% lactate dehydrogenase activity to be released, with minimal release of glutamate dehydrogenase (a mitochondria1 marker enzyme) and CPT activity from the hepatocytes. CPT I activity measured in hepatocytes permeabilized in this manner was linear for at least 3 minutes at 37C. Increasing digitonin concentrations above 0.05 mdml increased both lactate dehydrogenase and glutamate dehydrogenase release from the hepatocytes and increased the malonyl-CoA insensitive CPT acitivty that is assumed to be CPTII. To test the validity of the assay we used malonyl-CoA and etomoxir to inhibit CPT I activity. Camitine palmitoylmsferase (CPT) activity in the absence of inhibitor was 0.95 0.19 nmoVmin per mg protein (mean 5 S.E.M., n=7) and in the presence of 100 p M malonyl-CoA activity was decreased to 0.20 + 0.03 nmoVmin per mg protein. This degree of inhibition of CPT activity (78%) by malonyl-CoA suggests that most of the measured activity was due to CPT I. This was confirmed by using etomoxir to inhibit CPT I, CPT activity was progressively decreased by increasing concentrations of etomoxir (Figure 1).