Carnitine Acetyltransferase Activity Research Articles

Stimulation of carnitine acetyltransferase in PC12 cells by nerve growth factor: Relationship to choline acetyltransferase stimulation

The activity of carnitine acetyltransferase (acetyl-CoA:L-carnitine O-acetyltransferase) was found to be at least 50-fold higher than that of choline acetyltransferase in PC12 cells. Nerve growth factor stimulated both enzymes in a parallel manner with respect to concentration of NGF and culture time. The stimulation of both enzymes was completely inhibited by 10 microM 6-thioguanine, an inhibitor of protein kinase N. Results are discussed with reference to the hypothesis that the two enzymes may be functionally related in neuronal cells.

Effects of Prolonged Treatment with Phthalate Ester on Rat Liver

Rats were fed a diet containing 2%, 0.2% or 0.02% di(2-ethylhexyl)phthalate (DEHP) for a period of 102 weeks. Only the 2% diet caused a substantial decrease in body weight. Both peroxisomal palmitoyl-CoA dehydrogenase and mitochondrial carnitine acetyltransferase activities were greatly induced by exposure to the highest dose of phthalate ester, reaching maximal plateau values after about 20 weeks of treatment. The diet containing 0.2% DEHP increased both activities slowly, but continuously, and at the end of the two-year period these increases were almost comparable to those obtained with the highest dose. Even the lowest dose gave a slowly increase in these activities. Both microsomal NADPH-cytochrome c reductase activity and the level of cytochrome P-450 were increased initially by exposure to 2% DEHP, but returned almost or completely to the control level after about 30 weeks of exposure. Depending on the dose of DEHP in the diet, peroxisomal catalase activity was elevated above the control level during the first year of treatment but was about the same as in the control animals during the second year. A substantial decrease in peroxisomal urate oxidase activity was observed throughout the entire experimental period. When treatment was ceased after one year, all activities returned to the control values within 2-3 weeks. These results demonstrate the complex nature of the effects caused by prolonged treatment with DEHP with cumulative increases at low doses.

Characterization of the hepatic responses to the short-term administration of ciprofibrate in several rat strains: Co-induction of microsomal cytochrome P-450 IVA1 and peroxisome proliferation

The influence of ciprofibrate, a potent oxyisobutyrate derivative, on several hepatic enzyme parameters was studied in five rat strains following a 14-day treatment period. Ciprofibrate-dependent hepatomegaly was observed at two dose levels (2 and 20 mg/kg) in all rat strains examined. A 10- to 15-fold induction in the 12-hydroxylation of lauric acid with a less marked 1.5- to 5-fold induction of 11-hydroxylation was observed in treated animals. This dose-dependent increase in fatty acid hydroxylase activity was associated with a maximal 10-fold increase in the specific content of cytochrome P-450 IVA1 isoenzyme apoprotein, as assessed immunochemically using an ELISA technique. The activities of the cytochrome P-450 I (IA1 and IA2) and II (IIB1 and IIB2) families, as measured by ethoxyresorufin- O-deethylase and benzphetamine- N-demethylase activities respectively, were decreased on treatment. In the mitochondria, monoamine oxidase activity was significantly decreased at the higher dose level whereas α-glycerophosphate dehydrogenase activity was elevated. Total carnitine acetyltransferase activity (mitochondrial and peroxisomal) and peroxisomal β-oxidation were markedly increased at both dose levels in all strains examined. Cytosolic glutatnione peroxidase activity, measured using both t-butylhydroperoxide and hydrogen peroxide as substrates, was decreased on treatment to approximately 50% of the control value. In treated animals, a marked increase in mRNA levels coding for cytochrome P-450 IVA1 and the peroxisomal bifunctional protein of the fatty acid β-oxidation spiral was observed. However, mRNA levels coding for glutatnione peroxidase appeared unchanged following ciprofibrate administration, in contrast to the above-noted decrease of glutathione peroxidase enzyme activity. Taken collectively, our results have further substantiated a close association between the induction of microsomal cytochrome P-450 IVA1, peroxisomal β-oxidation and total carnitine acetyltransferase activity in rat liver, and have formed a conceptual basis for the rationalization of the chronic toxicity of peroxisome proliferators in this species.

Differential action of thyroid hormones and chemically related compounds on the activity of UDP-glucuronosyltransferases and cytochrome P-450 isozymes in rat liver

The effect of thyroid hormones and chemically related compounds, on the activity of UDP-glucuronosyltransferases (EC 2.4.1.17) and cytochrome P-450-dependent monooxygenases in rat liver microsomes was investigated. The animals were thyroidectomized and treated with different doses of the drugs for 3 weeks. Opposite effects were observed depending on the isoenzyme of UDP-glucuronosyltransferase considered. While 3,3′,5-triiodo- l-thyronine, 3,3′,5-triiodothyroacetic acid, 3,3′,5-triiodothyropropionic acid, isopropyldiiodothyronine and l- and d-thryoxine strongly increased 4-nitrophenol glucuronidation in a dose-dependent fashion, they decreased markedly bilirubin glucuronidation. However, the activity toward nopol, a monoterpenoid alcohol, was not significantly changed regardless of which compound or dose was used. Variation of UDP-glucuronosyltransferase observed with 4-nitrophenol and bilirubin was related to the thyromimetic effect of the drugs estimated from the increase in α-glycerophosphate dehydrogenase. Thyronine and 3,5-diiodo- l-tyrosine, which did not enhance this activity, also failed to affect glucuronidation. Variations in UDP-glucuronosyltransferase activity were more likely due to changes in protein expression rather than changes in enzyme latency, since lipid organization of the microsomal membrane, as estimated from the mean anisotropy of 1,6-diphenyl-1,3,5-hexatriene by fluorescence polarization was not significantly modified by the drug administration. Although some of the drugs could significantly decrease the triacylglycerol and cholesterol contents in plasma, all failed to affect lauric acid hydroxylation. The activities of catalase, palmitoyl-CoA dehydrogenase (CN − insensitive) and carnitine acetyltransferase in the fraction enriched in peroxisomes were also not significantly affected by treatment with the thyroid hormone LT 3. In contrast, the activity of 7-ethoxycoumarine O-deethylase was increased by large doses of thyronine and by 3,3′,5′-triiodothyropropionic acid. The concentration of total cytochrome P-450 was decreased in a dose-dependent fashion by all the compounds used, except thyronine. Finally, significant correlations were observed between glucuronidation of bilirubin and 4-nitrophenol and the content in cytochrome P-450. This suggests a possible coordinate regulation of the two processes, which depends on the physicochemical characteristics of the thyroid hormones and related compounds.

Pathways to acetyl-CoA formation inCandida albicans

The supply of acetyl units from the mitochondrion to the cytosol of Candida albicans appears to be dependent only upon the activity of carnitine acetyltransferase (CAT). The enzyme ATP: citrate lyase (ACL), the major source of acetyl units in oleaginous yeasts, is absent from C. albicans in both the mycelial and yeast forms. There appears to be no other active translocation of acetate or acetyl groups except via the action of carnitine acetyltransferase.

Pathways to acetyl-CoA formation in Candida albicans

The supply of acetyl units from the mitochondrion to the cytosol of Candida albicans appears to be dependent only upon the activity of carnitine acetyltransferase (CAT). The enzyme ATP: citrate lyase (ACL), the major source of acetyl units in oleaginous yeasts, is absent from C. albicans in both the mycelial and yeast forms. There appears to be no other active translocation of acetate or acetyl groups except via the action of carnitine acetyltransferase.

Carnitine acetyltransferase activity is not changed with age in rat brain and human platelets

Carnitine acetyltransferase activity was studied in different brain regions of the rat and in human platelets. The values of K m for carnitine and for acetyl-CoenzymeA (acetyl-CoA) were similar in cortex, hippocampus and striatum of the rat and in human platelets, suggesting that carnitine acetyltransferase might be a useful peripheral marker of its central activity. The activity of this enzyme was similar in the cortex and hippocampus of young and old rats. Furthermore, the determination of carnitine acetyltransferase activity in platelets from healthy volunteers showed no significant difference with age.

Species and strain sensitivity to the induction of peroxisome proliferation by chloroacetic acids

B6C3F 1 mice and Sprague-Dawley rats were provided drinking water containing 6–31 m m (1–5 g/liter) trichloroacetic acid (TCA), 8–39 m m (1–5 g/liter) dichloroacetic acid (DCA), or 11–32 m m (1–3 g/liter) monochloroacetic acid (MCA) for 14 days. TCA and DCA, but not MCA, increased the mouse relative liver weight in a dose-dependent manner. Rat liver weights were not altered by TCA or DCA treatment, but were depressed by MCA. Hepatic peroxisome proliferation was demonstrated by (1) increased palmitoyl-CoA oxidase and carnitine acetyl transferase activities, (2) appearance of a peroxisome proliferation-associated protein, and (3) morphometric analysis of electron micrographs. Mouse peroxisome proliferation was enhanced in a dose-dependent manner by both TCA and DCA, but only the high DCA concentration (39 m m) increased rat liver peroxisome proliferation. MCA was ineffective in both species. Three other mouse strains (Swiss-Webster, C3H, and C57BL 6 ) and two strains of rat (F344 and Osborne-Mendel) were examined for sensitivity to TCA. TCA (12 and 31 m m) effectively enhanced peroxisome proliferation in all mouse strains, especially the C57BL 6 . A more modest enhancement in the Osborne-Mendel (288%) and F344 rat (167%) was seen. Dosing F344 rats with 200 mg/kg TCA in water or corn oil for 10 days increased peroxisome proliferation 179 and 278%, respectively, above the vehicle controls. These studies demonstrate that the mouse is more sensitive than the rat with respect to the enhancement of liver peroxisome proliferation by TCA and DCA and suggest that if peroxisome proliferation is critical for the induction of hepatic cancer by TCA and DCA, then the rat should be less sensitive or refractory to tumor induction.

Clofibrate induces carnitine acyltransferases in periportal and perivenous zones of rat liver and does not disturb the acinar zonation of gluconeogenesis

Clofibrate induces hypertrophy and hyperplasia and marked changes in the activities of various enzymes in rat liver. We examined the effects of treatment of rats with clofibrate on enzyme induction and on rates of metabolic flux in hepatocytes isolated from the periportal and perivenous zones of the liver. Clofibrate induced the activities of carnitine acetyltransferase (90-fold), carnitine palmitoyltransferase (3-fold) and NADP-linked malic enzyme (3-fold) to the same level in periportal as in perivenous hepatocytes, suggesting that these enzymes were induced uniformly throughout the liver acinus. Increased rates of palmitate metabolism and ketogenesis after clofibrate treatment were associated with: a more oxidised mitochondrial redox state; diminished responsiveness to glucagon and loss of periportal/perivenous zonation. Despite the marked liver enlargement and hyperplasia caused by clofibrate, the normal periportal/perivenous zonation of alanine aminotransferase and gluconeogenesis was preserved in livers of clofibrate-treated rats, indicating that clofibrate-induced hyperplasia does not disrupt the normal acinar zonation of these metabolic functions.

2-Ethylhexanoic acid inhibits urea synthesis and stimulates carnitine acetyltransferase activity in rat liver mitochondria.

Adult male 3-month-old Wistar rats were given 0, 100 mg/l, 1, 5 or 10 g/l 2-ethylhexanoic acid in their drinking water for 20 days. Their daily consumption of contaminated water was measured and compared with the free acid found in their 24-h urine samples. The excretion was dose and time dependent. At the termination of the experiment, liver mitochondrial carnitine acetyltransferase activity was induced dose dependently and the citrulline synthesis in the urea cycle inhibited. Our results compare very well with the toxicity of a structural congener of the 2-ethylhexanoic acid, i.e. valproate, an antiepileptic drug.

Altered hepatic fatty acid metabolism in endotoxicosis: effect of L-carnitine on survival

The activities of palmitoyl-coenzyme A (CoA) synthetase, carnitine acetyltransferase (CAT), and carnitine palmitoyltransferase (CPT) and the levels of ketone bodies, reduced coenzyme A (CoASH), carnitine, and their esters, which are involved in fatty acid metabolism, in rat liver and plasma were measured after the administration of Escherichia coli lipopolysaccharide (LPS). We also studied the effect of L-carnitine treatment before LPS administration on survival and on hepatic fatty acid metabolism. The activities of CAT and CPT and the concentrations of ketone bodies, CoA, and carnitine derivatives (except for malonyl-CoA) declined in the liver after LPS administration. The activity of palmitoyl-CoA synthetase was changed little after LPS administration, and the level of hepatic malonyl-CoA increased significantly, suggesting that LPS causes activated fatty acids to undergo esterification and lipogenesis rather than oxidation. Treatment of rats with L-carnitine before LPS greatly increased the survival rate, but did not affect enzymes that metabolize fatty acids, CoA, or carnitine derivatives in the liver. Further studies are necessary to elucidate the mechanism of the effect of carnitine on post-LPS survival.

Effects of scopolamine treatment and lesion of the nucleus basalis upon various cholinergic markers in the rat brain cortex

The effect of chronic scopolamine on muscarinic receptors in rat frontoparietal cortex was investigated by measuring the specific binding of [(3)H](?)quinuclidinyl benzilate and [ (3)H ](+)-cis- methyldioxolane . Administration of the muscarinic antagonist, scopolamine (10 mg/kg/day i.p.) for 21 days, produced a significant increase of the density of muscarinic receptors by 20% when using [(3)H](?)quinuclidinyl benzilate, without modifying the affinity for this ligand. However, this chronic treatment left the binding of [ (3)H ](+)-cis- methyldioxolane unchanged. After bilateral lesion with ibotenic acid at the level of nucleus basalis of Meynert, there was a significant decrease of about 50% in the high affinity uptake of choline and choline acetyltransferase activity in the frontoparietal cortex. Carnitine acetyltransferase activity in this region was significantly decrease by 15% in lesioned rats compared to sham control rats. Under these conditions, the specific binding of [ (3)H ](+)-cis- methyldioxolane was significantly decreased while the binding of [(3)H](?)quinuclidinyl benzilate was not modified. From these results, it would seem, on the one hand, that the muscarinic receptors labelled by [ (3)H ](+)-cis- methyldioxolane are located at least partially presynaptically and are regulated differently from those labelled by [(3)H](?)quinuclidinyl benzilate since they are insensitive to a chronic treatment with scopolamine. On the other hand, the results of cortical carnitine acetyltransferase obtained here suggest that this enzyme is partially localized to neurons arising from the region of the nucleus basalis.

Ultrastructural localisation of Carnitine acetyltransferase activity in mitochondria of rat myocardium

Ultrastructural localisation of Carnitine acetyltransferase activity in mitochondria of rat myocardium

Gas chromatographic determination of carnitine in tissues

Carnitine [4-(N-trimethylamino)-3-hydroxybutyrate, (I)] is determined inhigh concentrations in skeletal musculature, heart, and epididymis, and in lower concentrations in the liver, brain kidneys, and blood [4, 7]. At the present time, it is known that (I) is an active carrier in the transport of fatty acids across the internal mitochondrial membranes to the sites of 8-oxidation [7]; (I) is also a most important factor in the conversion of acetyl CoA in the cells of the sex glands [5], and is a regulator of the catabolism of branched aminoacids in the skeletal musculature [3, i0]. Methods for the quantitative determination of (I) in the free and esterified form have been described [6], whereby the most sensitive and specific is the determination of the enzyme activity of carnitine acetyl transferase [8, 12] which requires work with the radioactive :~C-acetyl CoA. The gas chromatographic analysis of (I) is applied significantly less often [ii].

Biosynthesis of soluble carnitine acetyltransferases from the yeast Candida tropicalis

Soluble carnitine acetyltransferase from Candida tropicalis is synthesized as a 76 kDa precursor, which is monomeric and possesses no or very little carnitine acetyltransferase activity. Maturation of the enzyme begins with proteolytic processing of the 76 kDa precursor to 64 and 57 kDa subunits. The processed subunits subsequently associate into two kinds of active oligomers; the 57 kDa subunits are assembled into a tetramer and the 64 kDa subunits into an octamer. Formation of these oligomers depends apparently on growth conditions, since both oligomers were present in cells grown in continuous culture, but cells grown batchwise contained only the tetrameric form of carnitine acetyltransferase.

Effects of ciprofibrate and 2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate (POCA) on the distribution of carnitine and CoA and their acyl-esters and on enzyme activities in rats. Relation between hepatic carnitine concentration and carnitine acetyltransferase activity.

The effects of feeding the peroxisome proliferators ciprofibrate (a hypolipidaemic analogue of clofibrate) or POCA (2-[5-(4-chlorophenyl)pentyl]oxirane-2-carboxylate) (an inhibitor of CPT I) to rats for 5 days on the distribution of carnitine and acylcarnitine esters between liver, plasma and muscle and on hepatic CoA concentrations (free and acylated) and activities of carnitine acetyltransferase and acyl-CoA hydrolases were determined. Ciprofibrate and POCA increased hepatic [total CoA] by 2 and 2.5 times respectively, and [total carnitine] by 4.4 and 1.9 times respectively, but decreased plasma [carnitine] by 36-46%. POCA had no effect on either urinary excretion of acylcarnitine esters or [acylcarnitine] in skeletal muscle. By contrast, ciprofibrate decreased [acylcarnitine] and [total carnitine] in muscle. In liver, ciprofibrate increased the [carnitine]/[CoA] ratio and caused a larger increase in [acylcarnitine] (7-fold) than in [carnitine] (4-fold), thereby increasing the [short-chain acylcarnitine]/[carnitine] ratio. POCA did not affect the [carnitine]/[CoA] and the [short-chain acylcarnitine]/[carnitine] ratios, but it decreased the [long-chain acylcarnitine]/[carnitine] ratio. Ciprofibrate and POCA increased the activities of acyl-CoA hydrolases, and carnitine acetyltransferase activity was increased 28-fold and 6-fold by ciprofibrate and POCA respectively. In cultures of hepatocytes, ciprofibrate caused similar changes in enzyme activity to those observed in vivo, although [carnitine] decreased with time. The results suggest that: (1) the reactions catalysed by the short-chain carnitine acyltransferases, but not by the carnitine palmitoyltransferases, are near equilibrium in liver both before and after modification of metabolism by administration of ciprofibrate or POCA; (2) the increase in hepatic [carnitine] after ciprofibrate or POCA feeding can be explained by redistribution of carnitine between tissues; (3) the activity of carnitine acetyltransferase and [total carnitine] in liver are closely related.

Interactions of inhibitors of carnitine palmitoyltransferase I and fibrates in cultured hepatocytes.

Culture of rat hepatocytes with etomoxir, an inhibitor of carnitine palmitoyltransferase I (CPT I), for 48 h, resulted in increased carnitine acetyltransferase (CAT) activity (74%), a marked decrease in CPT activity (82%) measured in detergent extracts, and increased activities of glucose-6-phosphate dehydrogenase (227%) and fructose-1,6-bisphosphatase (65%). Changes in CAT and CPT activities were not observed after 4 h culture with etomoxir. When hepatocytes were cultured with etomoxir and benzafibrate (a hypolipidaemic analogue of clofibrate) for 48 h, etomoxir prevented the 5-fold increase in CAT activity caused by bezafibrate, whereas bezafibrate suppressed the increase in glucose-6-phosphate dehydrogenase and fructose-bisphosphatase caused by etomoxir. However, bezafibrate did not prevent the suppression of CPT activity by etomoxir. Etomoxir inhibited palmitate beta-oxidation and ketogenesis after short-term (0-4 h) and long-term (48 h) exposure, but it caused accumulation of triacylglycerol in hepatocytes only after short-term exposure (0-4 h). These effects of etomoxir on fatty acid metabolism and suppression of CPT (after 48 h) were similar in periportal and perivenous hepatocytes, but the increases in CAT and glucose-6-phosphate dehydrogenase activities were higher in periportal than in perivenous cells. The effects of CPT I inhibitors on CAT activity and long-term suppression of CPT activity are probably mediated by independent mechanisms.

Effects of cyclophosphamide on selected cytosolic and mitochondrial enzymes in the epididymis of the rat.

The anticancer and immunosuppressive drug cyclophosphamide is extensively used in clinical practice and is known to alter fertility in man. We showed previously that treatment of male rats with low daily doses of cyclophosphamide over a 9-week period caused fetal malformations, a high rate of postimplantation loss and affected epididymal and sperm histology. In the present study, five biochemical measures of epididymal function were used to characterize further the effects of cyclophosphamide on the epididymis. For 1, 3, 6, or 9 weeks, adult Sprague-Dawley rats were gavage-fed daily with saline (control), 5.1 (low dose), or 6.8 (high dose) mg/kg of cyclophosphamide. The specific activities of the two glycolytic enzymes aldolase and lactate dehydrogenase (LDH), the mitochondrial enzyme succinate dehydrogenase, the cytosolic enzyme carnitine acetyltransferase and the lysosomal enzyme acid phosphatase were determined in cytosolic and mitochondrial subcellular fractions from four segments of the epididymis. Cyclophosphamide caused decreases in protein concentrations in all segments of the epididymis only after 6 weeks of treatment with the high dose. The specific activities of aldolase, LDH and succinate dehydrogenase did not differ from control with respect to dose or duration of treatment. In contrast, there were significant effects of cyclophosphamide on carnitine acetyltransferase and acid phosphatase specific activity. After 1 week of treatment, there was a transient dose-related decrease in the specific activity of carnitine acetyltransferase, which was most striking for the corpus epididymidis (76% of control), but which did not differ from control after 3, 6, and 9 weeks. After 6 weeks of treatment with the high dose of cyclophosphamide, carnitine acetyltransferase specific activity in the initial segment and the corpus epididymidis was elevated to 165 and 140%, respectively, as compared with the 1-week high dose values. The specific activity of acid phosphatase did not differ from control after 1 and 9 weeks of treatment. At 3 and 6 weeks, however, there was a dose-related increase in acid phosphatase specific activity for all regions of the epididymis that was most marked in the cauda after the 6-week treatment (140% of control). Therefore, low dose, daily treatment of male rats with cyclophosphamide not only alters specific enzymes in specific segments of the epididymis, but acts in a dose- and time-dependent manner. It is possible that these changes could be mediated by direct, toxic effects of the drug on the epithelium or be secondary to alterations in the spermatozoa as a result of the treatment.

Failure of the peroxisome proliferator WY-14,643 to induce unscheduled DNA synthesis in rat hepatocytes following in vivo treatment.

Peroxisome proliferator hepatocarcinogens lack genotoxic activity in numerous in vitro assays using non-target cells which do not respond with peroxisome proliferation. Therefore, the effect of in vivo treatment with WY-14,643 on DNA repair was quantitated in rat hepatocytes, the target cell for carcinogenesis. Palmitoyl CoA oxidase and carnitine acetyltransferase activities in isolated hepatocytes were elevated by WY-14,643 (50 mg/kg/day by gavage for up to 5 consecutive days) and by WY-14,643 (0.1%) or di(2-ethyl-hexyl)phthalate (DEHP) (1.2%) feeding (for up to 28 days), indicating peroxisome proliferation had occurred. DNA repair as unscheduled DNA synthesis (UDS) was measured autoradiographically as net nuclear grains following thymidine incorporation in primary hepatocyte cultures. Treatment of rats with WY-14,643 (gavage or feeding) or DEHP (feeding) did not induce UDS. Addition of 2-acetylaminofluorene to replicate cultures demonstrated that WY-14,643 or DEHP treatment did not prevent repair response. Additional cultures were treated with H2O2 (0.8 mM H2O2 3x at 1-h intervals) to evaluate the ability of UDS to detect any repair which may be induced by peroxisomal metabolism. H2O2 did not induce UDS at this concentration, nor did it prevent 2-acetylaminofluorene-induced repair. UDS was, however, observed in a separate experiment using a higher concentration of H2O2. In summary, a highly carcinogenic peroxisome proliferator did not induce UDS in the target cell for carcinogenesis in spite of peroxisome proliferation following in vivo treatment.

Valproic acid-induced increase in carnitine acetyltransferase in rat hepatocytes is not due to an induction of peroxisomes.

Valproic acid induced a dose-dependent increase in carnitine acetyltransferase (CAT) activity in rat hepatic mitochondrial fractions isolated by differential centrifugation. An increase in CAT and carnitine palmitoyltransferase (CPT) also occurred in cultured rat hepatocytes in a concentration-and time-dependent fashion. A maximal increase of 8-fold in the activity of CAT and 2-fold in the activity of CPT was induced by 3 mM valproic acid in 72 h. Valproic acid had no effect on cytochrome P-450 levels in cultured rat hepatocytes. Electron-microscopic examination of rat hepatocytes showed that there was no increase in the number of peroxisomes but there was a marked proliferation of mitochondria in parallel with an increase in glutathione level and succinic dehydrogenase in the liver cells after incubation with valproic acid in vitro.