Restoration of the properties of carnitine palmitoyltransferase I in liver mitochondria during re-feeding of starved rats.
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ABSTRACT:
SUBMITTER: Grantham BD
PROVIDER: S-EPMC1147307 | biostudies-other | 1986 Oct
REPOSITORIES: biostudies-other
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Project description:A new carnitine palmitoyltransferase (CPT) was purified to homogeneity from bovine liver mitochondria which were 96% free of peroxisomal contamination, as judged by catalase and glutamate dehydrogenase activities. The enzyme is easily removed from mitochondria, without the use of detergent. It is monomeric (Mr 63,500), unlike other preparations of CPT from mitochondria, and is most active with myristoyl-CoA and palmitoyl-CoA. The Km values are between 0.8 and 4 microM for a range of substrates from hexanoyl-CoA to stearoyl-CoA; these are much lower than values reported for other purified CPT preparations. The Km for L-carnitine is 185 microM measured with palmitoyl-CoA, and does not vary greatly with the chain length. This is also lower than the values reported for other CPT preparations, but higher than those cited for the medium-chain transferases. Kinetic and inhibitor studies were consistent with a rapid-equilibrium random-order mechanism. 2-Bromopalmitoyl-CoA, which is an inhibitor of the outer CPT, inhibited the enzyme competitively with palmitoyl-CoA as the variable substrate, when added without preincubation. If the enzyme was preincubated with 2-bromopalmitoyl-CoA and carnitine, the activity did not reappear after gel filtration of the protein. The inhibitor was bound in a 1:1 stoichiometry per subunit of enzyme.
| S-EPMC1147987 | biostudies-other
Project description:1. A continuously recording and sensitive fluorimetric assay is described for carnitine palmitoyltransferase. This assay has been applied to whole or disintegrated mitochondria and to soluble protein fractions. 2. When rat liver mitochondria had been disintegrated by ultrasound, the specific activity of carnitine palmitoyltransferase was 15-20m-units/mg of protein. Only one-fifth of this activity was assayable (with added substrates) before mitochondrial disintegration. 3. It is concluded that there are two carnitine palmitoyltransferase activities in rat liver mitochondria, of which one (type I) is relatively superficial in location and catalyses an acyl-group transfer between added CoA and carnitine, whereas the other (type II) is less superficial and catalyses an acyl-group transfer in unbroken mitochondria between added carnitine and intramitochondrial CoA. The existence of two distinct carnitine palmitoyltransferases was predicted by Fritz & Yue (1963). 4. In unbroken mitochondria, type I transferase is accessible to the inhibitor 2-bromostearoyl-CoA whereas the type II transferase is inaccessible. 5. A major part of the total carnitine palmitoyltransferase activity of rat liver mitochondria is membrane-bound and of type II. 6. These observations, when considered in conjunction with the penetration of mitochondria by CoASH or carnitine, indicate that the type II transferase is attached to the inner mitochondrial membrane.
| S-EPMC1179386 | biostudies-other
Project description:Cellular senescence is a fundamental biological process that has profound implications in cancer development and therapeutics, but the underlying mechanisms remain elusive. Here we show that carnitine palmitoyltransferase 1C (CPT1C), an enzyme that catalyzes carnitinylation of fatty acids for transport into mitochondria for ?-oxidation, plays a major role in the regulation of cancer cell senescence through mitochondria-associated metabolic reprograming. Metabolomics analysis suggested alterations in mitochondria activity, as revealed by the marked decrease in acylcarnitines in senescent human pancreatic carcinoma PANC-1 cells, indicating low CPT1C activity. Direct analyses of mRNA and protein show that CPT1C is significantly reduced in senescent cells. Furthermore, abnormal mitochondrial function was observed in senescent PANC-1 cells, leading to lower cell survival under metabolic stress and suppressed tumorigenesis in a mouse xenograft model. Knock-down of CPT1C in PANC-1 cells induced mitochondrial dysfunction, caused senescence-like growth suppression and cellular senescence, suppressed cell survival under metabolic stress, and inhibited tumorigenesis in vivo. Further, CPT1C knock-down suppressed xenograft tumor growth in situ. Silencing of CPT1C in five other tumor cell lines also caused cellular senescence. On the contrary, gain-of-function of CPT1C reversed PANC-1 cell senescence and enhanced mitochondrial function. This study identifies CPT1C as a novel biomarker and key regulator of cancer cell senescence through mitochondria-associated metabolic reprograming, and suggests that inhibition of CPT1C may represent a new therapeutic strategy for cancer treatment through induction of tumor senescence.
| S-EPMC5864250 | biostudies-literature
Project description:The release of carnitine palmitoyltransferase (CPT) activity from rat liver mitochondria by increasing concentrations of digitonin was studied for mitochondrial preparations from fed, 48 h-starved and diabetic animals. A bimodal release of activity was observed only for mitochondria isolated from starved and, to a lesser degree, from diabetic rats, and it appeared to result primarily from the enhanced release of approx. 40% and 60%, respectively, of the total CPT activity. This change in the pattern of release was specific to CPT among the marker enzymes studied. For all three types of mitochondria there was no substantial release of CPT concurrently with that of the marker enzyme for the soluble intermembrane space, adenylate kinase. These results illustrate that the bimodal pattern of release of CPT reported previously for mitochondria from starved rats [Bergstrom & Reitz (1980) Arch. Biochem. Biophys. 204, 71-79] is not an immutable consequence of the localization of CPT activity on either side of the mitochondrial inner membrane. Sequential loss of CPT I (i.e. the overt form) from the mitochondrial inner membrane did not affect the concentration of malonyl-CoA required to effect fractional inhibition of the CPT I that remained associated with the mitochondria. The results are discussed in relation to the possibility that altered enzyme-membrane interactions may account for some of the altered regulatory properties of CPT I in liver mitochondria of animals in different physiological states.
| S-EPMC1152629 | biostudies-other
Project description:Hepatic carnitine palmitoyltransferase (CPT) turnover was studied in control and in non-ketotic hyperglycaemic streptozotocin-diabetic rats. The degradation constant (kd) and half-life (t1/2) did not appear to be altered by mild diabetes. The hepatic CPT (micrograms/g of liver) was not increased by the mild, non-ketotic, diabetes. However, the total hepatic CPT (micrograms/liver) was 37% greater in the diabetic animals, owing to the increased liver weight. This resulted from a 40% increase in the synthesis constant (ks). Hepatic CPT activity (total detergent-solubilized) and translation rates were measured in fed, starved (48 h), non-ketotic diabetic, ketotic diabetic and diethylhexyl phthalate (DEHP)-treated rats. CPT activity (m units/mg of mitochondrial protein) was not significantly increased with non-ketotic diabetes (44% increase, but non-significant), but was increased approx. 2-fold with starvation and ketotic diabetes, and 3.5-fold with DEHP treatment. CPT expressed as units/liver was increased non-significantly (23%) in non-ketotic and starved rats, similar to the turnover study, but was significantly increased with ketotic diabetes and with DEHP treatment. mRNA-translation activity for CPT was elevated in all states to a somewhat greater extent than was activity. It was concluded that protein synthesis as a product of increased CPT-mRNA translation activity is a major means of long-term regulation.
| S-EPMC1148328 | biostudies-other
Project description:Muscle carnitine palmitoyltransferase I is predominant in the heart, but the liver isoform (liver carnitine palmitoyltransferase I [L-CPT1]) is elevated in hearts with low long chain fatty acid oxidation, such as fetal and hypertrophied hearts.This work examined the effect of acute L-CPT1 expression on the regulation of palmitate oxidation and energy metabolism in intact functioning rat hearts for comparison with findings in hypertrophied hearts.L-CPT1 was expressed in vivo in rat hearts by coronary perfusion of Adv.cmv.L-CPT1 (L-CPT1, n=15) vs. phosphate-buffered saline (PBS) infusion (PBS, n=7) or empty virus (empty, n=5). L-CPT1 was elevated 5-fold at 72 hours after Adv.cmv.L-CPT1 infusion (P<0.05), but muscle carnitine palmitoyltransferase I was unaffected. Despite similar tricarboxylic acid cycle rates, palmitate oxidation rates were reduced with L-CPT1 (1.12 ± 0.29 ?mol/min per gram of dry weight, mean±SE) vs. PBS (1.6 ± 0.34). Acetyl CoA production from palmitate was reduced with L-CPT1 (69 ± 0.02%; P<0.05; PBS=79 ± 0.01%; empty=81 ± 0.02%), similar to what occurs in hypertrophied hearts, and with no difference in malonyl CoA content. Glucose oxidation was elevated with L-CPT1 (by 60%). Surprisingly, L-CPT1 hearts contained elevated atrial natriuretic peptide, indicating induction of hypertrophic signaling.The results link L-CPT1 expression to reduced palmitate oxidation in a nondiseased adult heart, recapitulating the phenotype of reduced long chain fatty acid oxidation in cardiac hypertrophy. The implications are that L-CPT1 expression induces metabolic remodeling hypertrophic signaling and that regulatory factors beyond malonyl CoA in the heart regulate long chain fatty acid oxidation via L-CPT1.
| S-EPMC3537878 | biostudies-literature
Project description:The activation of overt carnitine palmitoyltransferase activity that occurs when rat liver mitochondria are incubated at near-physiological temperatures and ionic strengths was studied for mitochondria obtained from animals in different physiological states. In all instances, it was found to be due exclusively to an increase in the catalytic capacity of the enzyme and not to an increase in affinity of the enzyme for palmitoyl-CoA. The enzyme in mitochondria from fed animals always showed a larger degree of activation than that in mitochondria from starved animals. This was the case even for mitochondria (e.g. from fed diabetic animals) in which the kinetic characteristics of carnitine palmitoyltransferase were more similar to those for the enzyme in mitochondria from starved rats. Glucagon treatment of rats before isolation of the mitochondria did not affect the characteristics either of the kinetic parameters of overt carnitine palmitoyltransferase or of its activation in vitro.
| S-EPMC1147841 | biostudies-other
Project description:Brain-specific carnitine palmitoyltransferase-1 (CPT-1c) is implicated in CNS control of food intake. In this article, we explore the role of hypothalamic CPT-1c in leptin's anorexigenic actions. We first show that adenoviral overexpression of CPT-1c in hypothalamic arcuate nucleus of rats increases food intake and concomitantly up-regulates orexigenic neuropeptide Y (NPY) and Bsx (a transcription factor of NPY). Then, we demonstrate that this overexpression antagonizes the anorectic actions induced by central leptin or compound cerulenin (an inhibitor of fatty acid synthase). The overexpression of CPT-1c also blocks leptin-induced down-regulations of NPY and Bsx. Furthermore, the anorectic actions of central leptin or cerulenin are impaired in mice with brain CPT-1c deleted. Both anorectic effects require elevated levels of hypothalamic arcuate nucleus (Arc) malonyl-CoA, a fatty acid-metabolism intermediate that has emerged as a mediator in hypothalamic control of food intake. Thus, these data suggest that CPT-1c is implicated in malonyl-CoA action in leptin's hypothalamic anorectic signaling pathways. Moreover, ceramide metabolism appears to play a role in leptin's central control of feeding. Leptin treatment decreases Arc ceramide levels, with the decrease being important in leptin-induced anorectic actions and down-regulations of NPY and Bsx. Of interest, our data indicate that leptin impacts ceramide metabolism through malonyl-CoA and CPT-1c, and ceramide de novo biosynthesis acts downstream of both malonyl-CoA and CPT-1c in mediating their effects on feeding and expressions of NPY and Bsx. In summary, we provide insights into the important roles of malonyl-CoA, CPT-1c, and ceramide metabolism in leptin's hypothalamic signaling pathways.
| S-EPMC3111271 | biostudies-literature
Project description:Treatment of female rats with ethynyloestradiol decreased both the activity of carnitine palmitoyltransferase and its Ki value for malonyl-CoA compared with pair-fed control rats. In starved rats, carnitine palmitoyltransferase activity and its Ki value for malonyl-CoA were both elevated as expected. Oestrogen treatment had no effect on carnitine palmitoyltransferase activity in the starved state, but the Ki value for malonyl-CoA was substantially decreased by oestrogen. Treatment with a more physiological concentration of ethynyloestradiol (15 micrograms/kg body wt. per day) also produced a decrease in the Ki value for malonyl-CoA. These data suggest that the oestrogen may produce its anti-ketogenic effect by increasing the affinity of carnitine palmitoyltransferase for its physiological inhibitor, malonyl-CoA.
| S-EPMC1147025 | biostudies-other
Project description:Liver mitochondria prepared by differential centrifugation are contaminated by significant quantities of peroxisomes and microsomal fractions. 'Easily solubilized carnitine palmitoyltransferase' prepared from liver mitochondria is thought to originate from the outer surface of the mitochondrial inner membrane. We have characterized the carnitine palmitoyltransferase activities of freeze-thaw extracts of rat liver mitochondrial preparations. Chromatography on Sephadex G-100 yields two broad peaks of carnitine decanoyltransferase activity: one eluted at the end of the void volume, which can be removed (precipitated) by ultracentrifugation; the second peak represents the soluble activity and is eluted at an Mr near 70,000. The activity in the soluble peak is precipitated by an antibody raised against carnitine octanoyltransferase purified from mouse liver peroxisomes. In contrast, antibody raised against carnitine palmitoyltransferase purified from liver mitochondrial membranes had no effect (P. Brady & L. Brady, personal communication). The carnitine acyltransferase activities of the Mr-70,000 peak in the presence or absence of Tween 20 showed maximum activity with decanoyl-CoA and about one-third of this activity with palmitoyl-CoA, similar to peroxisomal carnitine octanoyltransferase. These data show that 7500 g preparations of liver mitochondria isolated by differential centrifugation are enriched by peroxisomal carnitine octanoyltransferase (approx. 20% of the protein of the fraction is peroxisomal) and indicate that this enzyme may be the one reported as 'overt' or 'easily solubilized' mitochondrial carnitine palmitoyltransferase.
| S-EPMC1148689 | biostudies-other