Carnitine palmitoyltransferase in liver and five extrahepatic tissues in the rat. Inhibition by DL-2-bromopalmitoyl-CoA and effect of hypothyroidism.

Mitochondria were isolated from rat adult liver, foetal liver, kidney cortex, heart, skeletal muscle and interscapular brown adipose tissue. DL-2-Bromopalmitoyl-CoA inhibited the overt form of carnitine palmitoyltransferase (CPT1) in heart, skeletal muscle and brown adipose tissue, with an IC50 value (concentration giving 50% inhibition) of 1.3-1.6 microM. By contrast, the IC50 value for inhibition of the kidney or adult liver enzyme was 0.08-0.1 microM. CPT1 in near-term foetal liver differed from that in adult liver in that the IC50 for inhibition by 2-bromopalmitoyl-CoA was 0.57 microM. It is suggested that there may be tissue-specific forms of the catalytic entity of CPT1 and that foetal liver may contain a mixture of adult liver- and muscle-type enzymes. In rats made hypothyroid by administration of propylthiouracil and an iodine-deficient diet, hepatic CPT1 activity was decreased by 83%. However, CPT1 activity in extrahepatic tissues showed no adaptive decrease in hypothyroidism.     

The localization of palmitoyl-CoA: Carnitine palmitoyltransferase in rat liver

1. The distribution of palmitoyl-CoA:carnitine palmitoyltransferase has been studied in subcellular fractions of rat liver. By using two different estimations for the enzyme activity and by differential centrifugation and linear sucrose density gradient centrifugation, the enzyme is shown to be localized both in mitochondria and microsomes.

2. The mitochondrial palmitoyl-CoA: carnitine palmitoyltransferase is localized in the inner membrane plus matrix fraction.

3. During palmitate oxidation by isolated mitochondria, in the presence of a physiological concentration of carnitine, palmitoylcarnitine accumulates. From this and experiments with sonicated mitochondria, it is concluded that the capacities of long-chain fatty acid activation and of palmitoyl-CoA:carnitine palmitoyltransferase in vitro by far exceed the capacity of fatty acid oxidation.     

Carnitine palmitoyltransferase 1C: From cognition to cancer

Carnitine palmitoyltransferase 1 (CPT1) C was the last member of the CPT1 family of genes to be discovered. CPT1A and CPT1B were identified as the gate-keeper enzymes for the entry of long-chain fatty acids (as carnitine esters) into mitochondria and their further oxidation, and they show differences in their kinetics and tissue expression. Although CPT1C exhibits high sequence similarity to CPT1A and CPT1B, it is specifically expressed in neurons (a cell-type that does not use fatty acids as fuel to any major extent), it is localized in the endoplasmic reticulum of cells, and it has minimal CPT1 catalytic activity with l-carnitine and acyl-CoA esters. The lack of an easily measurable biological activity has hampered attempts to elucidate the cellular and physiological role of CPT1C but has not diminished the interest of the biomedical research community in this CPT1 isoform. The observations that CPT1C binds malonyl-CoA and long-chain acyl-CoA suggest that it is a sensor of lipid metabolism in neurons, where it appears to impact ceramide and triacylglycerol (TAG) metabolism. CPT1C global knock-out mice show a wide range of brain disorders, including impaired cognition and spatial learning, motor deficits, and a deregulation in food intake and energy homeostasis. The first disease-causing CPT1C mutation was recently described in humans, with Cpt1c being identified as the gene causing hereditary spastic paraplegia. The putative role of CPT1C in the regulation of complex-lipid metabolism is supported by the observation that it is highly expressed in certain virulent tumor cells, conferring them resistance to glucose- and oxygen-deprivation. Therefore, CPT1C may be a promising target in the treatment of cancer. Here we review the molecular, biochemical, and structural properties of CPT1C and discuss its potential roles in brain function, and cancer.     

Carnitine palmitoyltransferase. Activation by palmitoyl-CoA and inactivation by malonyl-CoA

Extraction of rat liver mitochondria twice with 0.5% Triton X-100 in a salt-free medium leaves less than 10% of the carnitine palmitoyltransferase membrane bound. The remaining membrane-bound enzyme is inhibited virtually completely by 10 microM malonyl-CoA. Preincubation of the extracted membranes with palmitoyl-CoA and salts (KCI) for several minutes activates the enzyme and makes it increasingly insensitive to malonyl-CoA. Addition of malonyl-CoA to the preincubation reverses this desensitization. In albumin-containing media salts also decrease the binding of palmitoyl-CoA to albumin and stimulate carnitine palmitoyltransferase by increasing substrate availability in free solution. The reverse reaction shows accelerated desensitization by palmitoylcarnitine and resensitization by malonyl-CoA.     

Carnitine and derivatives in rat tissues

1. Free carnitine, acetylcarnitine, short-chain acylcarnitine and acid-insoluble carnitine (probably long-chain acylcarnitine) have been measured in rat tissues. 2. Starvation caused an increase in the proportion of carnitine that was acetylated in liver and kidney; at least in liver fat-feeding had the same effect, whereas a carbohydrate diet caused a very low acetylcarnitine content. 3. In heart, on the other hand, starvation did not cause an increase in the acetylcarnitine/carnitine ratio, whereas fat-feeding caused a decrease. The acetylcarnitine content of heart was diminished by alloxan-diabetes or a fatty diet, but not by re-feeding with carbohydrate. 4. Under conditions of increased fatty acid supply the acid-insoluble carnitine content was increased in heart, liver and kidney. 5. The acylation state of carnitine was capable of very rapid change. Concentrations of carnitine derivatives varied with different methods of obtaining tissue samples, and very little acid-insoluble carnitine was found in tissues of rats anaesthetized with Nembutal. In liver the acetylcarnitine (and acetyl-CoA) content decreased if freezing of tissue samples was delayed; in heart this caused an increase in acetylcarnitine. 6. Incubation of diaphragms with acetate or dl-β-hydroxybutyrate caused the acetylcarnitine content to become elevated. 7. Perfusion of hearts with fatty acids containing an even number of carbon atoms, dl-β-hydroxybutyrate or pyruvate resulted in increased contents of acetylcarnitine and acetyl-CoA. Accumulation of these acetyl compounds was prevented by the additional presence of propionate or pentanoate in the perfusion medium; this prevention was not due to extensive propionylation of CoA or carnitine. 8. Perfusion of hearts with palmitate caused a severalfold increase in the content of acid-insoluble carnitine; this increase did not occur when propionate was also present. 9. Comparison of the acetylation states of carnitine and CoA in perfused hearts suggests that the carnitine acetyltransferase reactants may remain near equilibrium despite wide variations in their steady-state concentrations. This is not the case with the citrate synthase reaction. It is suggested that the carnitine acetyltransferase system buffers the tissue content of acetyl-CoA against rapid changes.     

Carnitine palmitoyltransferase II deficiency: Diagnosis by molecular analysis of blood

Four missense mutations have been reported to be associated with the typical, adult form of carnitine palmitoyltransferase II (CPT II) deficiency: Three amino acid substitutions (R631C, P50H and D553N) appear to be rare, while the S113L mutation was found to be common in a group of European patients with CPT II deficiency.We analyzed genomic DNA from 20 American patients with recurrent episodes of myoglobinuria as well as DNA from 10 normal controls in order to determine the frequency of the reported missense mutations in our patient population.The three previously described rare mutations were not found in our group of patients. The S113L mutation was found in 19 of our patients: 5 patients were homozygous, 14 patients were heterozygous.Given the high frequency of this mutation in our series of patients we concluded that the clinical diagnosis of CPT II deficiency can be confirmed by a 'blood test' without resorting to a muscle biopsy.     

Carnitine palmitoyltransferase: separation of enzyme activity and malonyl-CoA binding in rat liver mitochondria

Carnitine palmitoyltransferase activity and malonyl-CoA binding capacity have been studied in Triton X-100 extracts and membrane residues of rat liver mitochondria. Rat liver mitochondria extracted twice with 0.5% Triton X-100 in a salt-free medium showed increased specific binding of [2-14C]malonyl-CoA when compared with intact mitochondria. High malonyl-CoA binding required the presence of salts and was inhibited by albumin. Further solubilization of the membrane residues in the Triton/KCl medium and subsequent hydroxylapatite chromatography gave a complete separation of carnitine palmitoyltransferase and malonyl-CoA binding. The results show that malonyl-CoA binds to mitochondrial component(s) which is different from and more difficult to extract from the mitochondrial membrane than most of the carnitine palmitoyltransferase.     

Characterization of serine palmitoyltransferase in normal human tissues.

Sphingolipids serve as structural elements of cells and as lipid second messengers. They regulate cellular homeostasis, mitogenesis, and apoptosis. Sphingolipid signaling may also be important in various pathophysiologies such as vascular injury, inflammation, and cancer. Serine palmitoyltransferase (SPT) catalyzes the condensation of serine with palmitoyl-CoA, the first, rate-limiting step in de novo sphingolipid biosynthesis. This integral microsomal membrane protein consists of at least two subunits, SPT1 and SPT2. In this study we analyzed the expression of SPT1 and SPT2 in normal human tissues. Strong SPT1 and SPT2 expression was observed in pyramidal neurons in the brain, in colon epithelium, and in mucosal macrophages. However, SPT2 expression was more prominent than SPT1 in the colon mucosal macrophages, the adrenomedullary chromaffin cells and endothelium, and in the uterine endothelium. SPT2 was localized in both nuclei and cytoplasm of the adrenomedullary chromaffin cells, whereas SPT1 was primarily cytoplasmic. These observations link enhanced SPT expression to proliferating cells, such as the lung, stomach, intestinal epithelium, and renal proximal tubular epithelium, and to potentially activated cells such as neurons, chromaffin cells, and mucosal macrophages. A baseline expression of SPT, established by this study, may serve as a measure for aberrant expression in various disease states.     

Carnitine palmitoyltransferase in the heart is controlled by a different mechanism than the hepatic enzyme

Diminished sensitivity of hepatic carnitine palmitoyltransferase to inhibition by malonyl-CoA in the fasting and diabetic states is a well-recognized aspect of the regulatory mechanism forhepatic fatty acid oxidation. Inhibition of myocardial carnitine palmitoyltransferase by malonyl-CoA may play an important role in regulation of fatty acid oxidation in the heart, but there has been a discrepancy in data relating to changes in malonyl-CoA sensitivity of the myocardial carnitine palmitoyltransferase during fasting. Analysis of malonyl-CoA inhibition of myocardial carnitine palmitoyltransferase in fasting and fed states under a variety of conditions has indicated that under no condition could any difference be found in malonyl-CoA sensitivity that was attributable to fasting. Proteolysis of the outer carnitine palmitoyltransferase led to artifactual changes in sensitivity due to the appearance of partial inhibition. We have concluded that the sensitivity of myocardial carnitine palmitoyltransferase to malonyl-CoA does not change during fasting. Changes in fatty acid oxidation in the heart are probably due to changes in malonyl-CoA concentrations or to other inhibitors. (Mol Cell Biochem 116: 39–45, 1992)     

Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 form a complex in the inner mitochondrial membrane

Carnitine/acylcarnitine translocase and carnitine palmitoyltransferase 2 are members of the carnitine system, which are responsible of the regulation of the mitochondrial CoA/acyl-CoA ratio and of supplying substrates for the ß-oxidation to mitochondria. This study, using cross-Linking reagent, Blue native electrophoresis and immunoprecipitation followed by detection with immunoblotting, shows conclusive evidence about the interaction between carnitine palmitoyltransferase 2 and carnitine/acylcarnitine translocase supporting the channeling of acylcarnitines and carnitine at level of the inner mitochondrial membrane.     

Carnitine palmitoyltransferase 2: New insights on the substrate specificity and implications for acylcarnitine profiling

Over the last years acylcarnitines have emerged as important biomarkers for the diagnosis of mitochondrial fatty acid β-oxidation (mFAO) and branched-chain amino acid oxidation disorders assuming they reflect the potentially toxic acyl-CoA species, accumulating intramitochondrially upstream of the enzyme block. However, the origin of these intermediates still remains poorly understood. A possibility exists that carnitine palmitoyltransferase 2 (CPT2), member of the carnitine shuttle, is involved in the intramitochondrial synthesis of acylcarnitines from accumulated acyl-CoA metabolites. To address this issue, the substrate specificity profile of CPT2 was herein investigated. Saccharomyces cerevisiae homogenates expressing human CPT2 were incubated with saturated and unsaturated C2–C26 acyl-CoAs and branched-chain amino acid oxidation intermediates. The produced acylcarnitines were quantified by ESI-MS/MS. We show that CPT2 is active with medium (C8–C12) and long-chain (C14–C18) acyl-CoA esters, whereas virtually no activity was found with short- and very long-chain acyl-CoAs or with branched-chain amino acid oxidation intermediates. Trans-2-enoyl-CoA intermediates were also found to be poor substrates for CPT2. Inhibition studies performed revealed that trans-2-C16:1-CoA may act as a competitive inhibitor of CPT2 (K_i of 18.8 μM). The results obtained clearly demonstrate that CPT2 is able to reverse its physiological mechanism for medium and long-chain acyl-CoAs contributing to the abnormal acylcarnitines profiles characteristic of most mFAO disorders. The finding that trans-2-enoyl-CoAs are poorly handled by CPT2 may explain the absence of trans-2-enoyl-carnitines in the profiles of mitochondrial trifunctional protein deficient patients, the only defect where they accumulate, and the discrepancy between the clinical features of this and other long-chain mFAO disorders such as very long-chain acyl-CoA dehydrogenase deficiency.     

Carnitine palmitoyltransferase 1A prevents fatty acid-induced adipocyte dysfunction through suppression of c-Jun N-terminal kinase

The adipocyte is the principal cell type for fat storage. CPT1 (carnitine palmitoyltransferase-1) is the rate-limiting enzyme for fatty acid β-oxidation, but the physiological role of CPT1 in adipocytes remains unclear. In the present study, we focused on the specific role of CPT1A in the normal functioning of adipocytes. Three 3T3-L1 adipocyte cell lines stably expressing hCPT1A (human CPT1A) cDNA, mouse CPT1A shRNA (short-hairpin RNA) or GFP (green fluorescent protein) were generated and the biological functions of these cell lines were characterized. Alteration in CPT1 activity, either by ectopic overexpression or pharmacological inhibition using etomoxir, did not affect adipocyte differentiation. However, overexpression of hCPT1A significantly reduced the content of intracellular NEFAs (non-esterified fatty acids) compared with the control cells when adipocytes were challenged with fatty acids. The changes were accompanied by an increase in fatty acid uptake and a decrease in fatty acid release. Interestingly, CPT1A protected against fatty acid-induced insulin resistance and expression of pro-inflammatory adipokines such as TNF-α (tumour necrosis factor-α) and IL-6 (interleukin-6) in adipocytes. Further studies demonstrated that JNK (c-Jun N terminal kinase) activity was substantially suppressed upon CPT1A overexpression, whereas knockdown or pharmacological inhibition of CPT1 caused a significant enhancement of JNK activity. The specific inhibitor of JNK SP600125 largely abolished the changes caused by the shRNA- and etomoxir-mediated decrease in CPT1 activity. Moreover, C2C12 myocytes co-cultured with adipocytes pre-treated with fatty acids displayed altered insulin sensitivity. Taken together, our findings have identified a favourable role for CPT1A in adipocytes to attenuate fatty acid-evoked insulin resistance and inflammation via suppression of JNK.     

Carnitine palmitoyltransferase 2: analysis of membrane association and complex structure with a substrate analog

The mitochondrial membrane-associated carnitine palmitoyltransferase system is a validated target for the treatment of type 2 diabetes mellitus. To further facilitate structure-based drug discovery, we determined the crystal structure of rat CPT-2 (rCPT-2) in complex with the substrate analogue palmitoyl-aminocarnitine at 1.8A resolution. Biochemical analyses revealed a strong effect of this compound on rCPT-2 activity and stability. Using a computational approach we examined the membrane association of rCPT-2. The protein interacts with the membrane as a functional monomer and the calculations confirm the presence of a membrane association domain that consists of layers of hydrophobic and positively charged residues.     

Activation and detoxification of bromobenzene in extrahepatic tissues

Bromobenzene causes hepatic and extrahepatic toxicity in rats. Toxicity is related to the presence of covalently bound material in these tissues. A major bromobenzene metabolite, p-bromophenol, has been shown to give rise to covalently bound material in liver, lung and kidney $\text{in vivo}$, but is not toxic. p-Bromophenol is formed from bromobenzene in liver, lung and kidney microsomes and is subsequently metabolized to 4-bromocatechol and covalently bound material. Bromobenzene-3, 4-oxide generated $\text{in situ}$ by liver microsomes, is detoxified by kidney, liver and lung cytosol. The results suggest that the kidney toxicity caused by bromobenzene is probably not mediated by either bromobenzene-3, 4-oxide or the reactive metabolites of p-bromophenol. In contrast, bromobenzene-3, 4-oxide may play a role in the lung toxicity observed after bromobenzene administration. However, the covalently bound material found in extrahepatic tissues may be derived from both bromobenzene-3, 4-oxide or the reactive metabolites of p-bromophenol, which may be formed directly by these tissues or transported there from the liver.