American Journal of Clinical Nutrition, Vol 26, 814-822, Copyright © 1973 by The American Society for Clinical Nutrition, Inc.

Subcellular localization of acetoacetyl-CoA deacylase and its role in acetoacetate synthesis

¹ Associate Professor of Medicine, Creighton University
² Professor of Medicine, Columbia University College of Physicians and Surgeons, New York, N. Y.

We have shown that acetoacetyl-CoA deacylase activity resides in both the mitochondria and supernatant of rat liver cells. Mitochondrial damage was not responsible for supernatant deacylase activity. This was shown in two ways. First, failure to demonstrate appreciable activity of the mitochondrial marker, glutamic dehydrogenase, in supernatant fractions, and second, lack of change in the specific activity of supernatant deacylase when varying concentrations of sucrose were used to prepare homogenates. The validity of the assay used in these studies was verified by experiments showing that preincubation with 10^–3 m iodoacetamide inhibits the enzymes involved in acetoacetate production via the HMG-CoA pathway. Lesser concentrations of iodoacetamide or lack of preincubation with the inhibitor do not completely inhibit HMG-CoA pathway enzymes while concentrations of iodoacetamide in excess of 10^–3 m inhibit acetoacetyl-CoA deacylase. Failure to enhance the disappearance of acetoacetyl-CoA by addition of various substrates excluded other potential pathways for acetoacetate production in the assay used in these studies. Finally, fasting is associated with increased specific activity as well as increased total activity of rat liver mitochondrial acetoacetyl-CoA deacylase. Neither diabetes nor diabetes plus fasting was associated with increased mitochondrial deacylase activity, and insulin failed to restore the stimulatory effect of fasting on mitochondria from rats. Fasting or diabetes had no effect on supernatant deacylase activity.