Dietary fat types differently modulate the activity and expression of mitochondrial carnitine/acylcarnitine translocase in rat liver.

The carnitine/acylcarnitine translocase (CACT), an integral protein of the mitochondrial inner membrane, belongs to the carnitine-dependent system of fatty acid transport into mitochondria, where beta-oxidation occurs. CACT exchanges cytosolic acylcarnitine or free carnitine for carnitine in the mitochondrial matrix. The object of this study was to investigate in rat liver the effect, if any, of diets enriched with saturated fatty acids (beef tallow, BT, the control), n-3 polyunsaturated fatty acids (PUFA) (fish oil, FO), n-6 PUFA (safflower oil, SO), and mono-unsaturated fatty acids (MUFA) (olive oil, OO) on the activity and expression of CACT. Translocase exchange rates increased, in parallel with CACT mRNA abundance, upon FO-feeding, whereas OO-dietary treatment induced a decrease in both CACT activity and expression. No changes were observed upon SO-feeding. Nuclear run-on assay revealed that FO-treatment increased the transcriptional rate of CACT mRNA. On the other hand, only in the nuclei of hepatocytes from OO-fed rats splicing of the last intron of CACT pre-mRNA and the rate of formation of the 3'-end were affected. Overall, these findings suggest that compared to the BT-enriched diet, the SO-enriched diet did not influence CACT activity and expression, whereas FO- and OO-feeding alters CACT activity in an opposite fashion, i.e. modulating its expression at transcriptional and post-transcriptional levels, respectively.

Experimental lipid storage myopathy. A quantitative ultrastructural and biochemical study.

A lipid storage myopathy was induced in rats treated with daily doses of 2.5 g/kg brominated vegetable oil. As in human lipid storage myopathies, type I fibers were selectively severely affected, the lipid deposits were surrounded by mitochondria and the mitochondrial fraction of the affected fibers was increased. The oxidation of [U-14C]palmitate, [1(-14)C]octanoate and beta-[3(-14)C]hydroxybutyrate was significantly depressed but [1(-14)C]palmitate, as well as labeled pyruvate and succinate were oxidized at normal rates. The activities of long-chain, medium-chain and short-chain carnitine acyltransferases and the muscle carnitine levels were normal. The lipid storage is attributed to impaired beta oxidation of medium-chain and short-chain fatty acyl residues. An approach to the investigation of those human lipid storage myopathies not due to defects in the carnitine system is suggested.

Quantitation of water-soluble acylcarnitines and carnitine acyltransferases in rat tissues.

The water-soluble acylcarnitines isolated from rat heart, skeletal muscle, liver, and testis have been characterized. The following acyl residues derived from the acylcarnitine fraction were found: acetyl, propionyl, isobutyryl, butyryl, alpha-methylbutyryl, isovaleryl, tiglyl, caproyl, beta-methylcrotonyl and methacrylyl. The amounts of these acylcarnitines in heart, liver, testis and skeletal muscle from fed rats were determined. Acetylcarnitine was the most abundant acylcarnitine; however, appreciable quantities of propionyl-, isobutyryl-, isovaleryl-, and tiglyl-carnitine were found. The levels of carnitine octanyltransferse, carnitine acetyltransferase and carnitine palmityltransferase activities were determined in several tissues. In addition, carnitine isovaleryltransferase and isobutyryltransferase activities were measured in heart, skeletal muscle, liver, testis and kidney. In all instances the specific activity of isobutyryltransferase was similar to the specific activity of acetyltransferase. The results are consistent with the proposal that carnitine is involved in the catabolism of branched-chain amino acids.

The effect of starvation on obese mice.

Plasma level of total and acylcarnitine and the activities of carnitine acetyltransferase (CAT) and carnitine palmitoyltransferase (PCT) in liver and CAT in brown fat were determined in young obese (ob/ob) mice and their littermates during starvation. Plasma levels of acylcarnitine and beta-hydroxybutyrate rose equally in both groups. Total carnitine levels, however, decreased in lean and rose in obese animals. Hepatic PCT and phosphoenolpyruvate carboxykinase activities rose more in lean than obese mice and brown fat CAT activity decreased in the obese group. Fatty acid synthetase activity decreased equally in the liver in obese mice and their lean littermates.

Neonatal hyperammonemia caused by a defect of carnitine-acylcarnitine translocase.

Carnitine-acylcarnitine translocase deficiency is a newly recognized inborn error of metabolism that involves transport of long-chain fatty acids into mitochondria, which in turn impairs mitochondrial beta-oxidation, and ketogenesis. We report a new familial example; the affected twins had neonatal distress, hyperammonemia, and transient intracardiac conduction defects. Clinical and biochemical analysis of both our patients and the two previously reported patients revealed that this inherited defect could be manifested during the neonatal period without any of the signs classically associated with fatty oxidation defects. In contrast, all four patients had sustained and "isolated" hyperammonemia, which could be misinterpreted as being caused by urea cycle defects. We conclude that carnitine-acylcarnitine translocase deficiency is a potential differential diagnosis in neonates with unexplained neonatal hyperammonemia. Cardiac and muscle involvement may represent further early pivotal symptoms.

Kinetic properties of carnitine palmitoyltransferase I in cultured neonatal rat cardiac myocytes.

An understanding of the mechanism of malonyl-CoA interaction with carnitine palmitoyltransferase (CPT-I) in isolated mitochondria is complicated by membrane fragmentation and CPT-II exposure. Using cultured neonatal rat cardiac myocytes, as in situ model was developed to measure CPT-I. In the cardiac cells treated with 5 microM digitonin, CPT-II contamination of CPT activity is 0.62% as quantitated by citrate synthase activity present in damaged myocytes under assay conditions. Moreover, the sensitivity of myocyte CPT-I to malonyl-CoA, its substrate preference for decanoyl-CoA and the affinity of CPT-I for l-carnitine (0.19 mM) are comparable with similar measurements published for isolated cardiac mitochondrial membranes. There is no evidence in the cells for contamination of CPT-I activities by extramitochondrial sources, in particular, the sarcoplasmic reticulum (SR). The presence of carnitine octanoyltransferase (COT) is not detected either in the cells or in preparations of adult SR from which COT is subsequently isolated. With these control measurements, the inhibition kinetics of CPT-I in the cardiac cells in situ maintains a partial competitive pattern which is more pronounced with decanoyl-CoA than with palmitoyl-CoA as substrate. The presence of a malonyl-CoA/long chain acyl-CoA binding site on CPT-I, distinct from the inhibitory site, has previously been proposed. Existence of this binding region is consistent with partial inhibition kinetics so that malonyl-CoA at this site could modify the CPT-high-affinity malonyl-CoA inhibitory interaction, producing acylcarnitine even at high malonyl-CoA concentrations in the cell. These findings may help to explain, in part, the inability to suppress completely beta-oxidation in the heart where malonyl-CoA may be 50 to 100 times the estimated values of its Ki.

Sudden neonatal death in carnitine transporter deficiency.

A newborn infant died suddenly and unexpectedly on day 5 of life. Postmortem investigations led to a suspicion of carnitine transporter deficiency, a diagnosis supported by the finding that both parents are heterozygotes for this disorder. The fasting stress caused by poor breast-feeding with no formula supplements and, possibly, the vegetarian diet of the mother were likely the critical factors leading to neonatal death, an outcome previously not described in this disorder.