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Marilyn D Resh - One of the best experts on this subject based on the ideXlab platform.
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hedgehog acyltransferase promotes uptake of palmitoyl coa across the endoplasmic reticulum membrane
2019Co-Authors: James J Asciolla, Marilyn D ReshAbstract:Summary Attachment of palmitate to the N terminus of Sonic hedgehog (Shh) is essential for Shh signaling. Shh palmitoylation is catalyzed on the luminal side of the endoplasmic reticulum (ER) by Hedgehog acyltransferase (Hhat), an ER-resident enzyme. Palmitoyl-coenzyme A (CoA), the palmitate donor, is produced in the cytosol and is not permeable across membrane bilayers. It is not known how Palmitoyl-CoA crosses the ER membrane to access the active site of Hhat. Here, we use fluorescent and radiolabeled Palmitoyl-CoA probes to demonstrate that Hhat promotes the uptake of Palmitoyl-CoA across the ER membrane in microsomes and semi-intact cells. Reconstitution of purified Hhat into liposomes provided further evidence that Palmitoyl-CoA uptake activity is an intrinsic property of Hhat. Palmitoyl-CoA uptake was regulated by and could be uncoupled from Hhat enzymatic activity, implying that Hhat serves a dual function as a palmitoyl acyltransferase and a conduit to supply Palmitoyl-CoA to the luminal side of the ER.
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biochemical characterization of a palmitoyl acyltransferase activity that palmitoylates myristoylated proteins
1995Co-Authors: Luc Berthiaume, Marilyn D ReshAbstract:Abstract Dynamic regulation of signal transduction by reversible palmitoylation-depalmitoylation cycles has been recently described. However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thioesterases involved. Here, we describe an assay for the palmitoyl acyltransferase (PAT) that palmitoylates “myrGlyCys” containing members of the Src family of protein tyrosine kinases (PTKs). Since N-myristoylation of Fyn PTK, a member of the Src family, has been shown to be a prerequisite for palmitoylation, a new single plasmid vector that allows overexpression of myristoylated Fyn substrate in Escherichia coli was developed. Purified myristoylated protein substrates were incubated with iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of bovine brain lysates. Transfer of radiolabel to the Fyn substrate was detected by SDS-polyacrylamide gel electrophoresis and autoradiography. This assay was used to partially purify and characterize PAT activity from bovine brain. Here, we demonstrate that PAT is a membrane-bound enzyme, which palmitoylates myristoylated Fyn substrates containing a cysteine residue in position three. The PAT activity attached palmitate to Fyn proteins via a thio-ester linkage and exhibited a fatty acyl CoA preference for long chain fatty acids. It is likely that palmitoylation of Fyn and other Src family members by PAT regulates PTK localization and signaling functions.
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biochemical characterization of a palmitoyl acyltransferase activity that palmitoylates myristoylated proteins
1995Co-Authors: Luc Berthiaume, Marilyn D ReshAbstract:Abstract Dynamic regulation of signal transduction by reversible palmitoylation-depalmitoylation cycles has been recently described. However, further understanding of fatty acylation reactions has been hampered by our lack of knowledge about the specific transferases and thioesterases involved. Here, we describe an assay for the palmitoyl acyltransferase (PAT) that palmitoylates “myrGlyCys” containing members of the Src family of protein tyrosine kinases (PTKs). Since N-myristoylation of Fyn PTK, a member of the Src family, has been shown to be a prerequisite for palmitoylation, a new single plasmid vector that allows overexpression of myristoylated Fyn substrate in Escherichia coli was developed. Purified myristoylated protein substrates were incubated with iodopalmitoyl CoA, a palmitoyl CoA analog, in the presence of bovine brain lysates. Transfer of radiolabel to the Fyn substrate was detected by SDS-polyacrylamide gel electrophoresis and autoradiography. This assay was used to partially purify and characterize PAT activity from bovine brain. Here, we demonstrate that PAT is a membrane-bound enzyme, which palmitoylates myristoylated Fyn substrates containing a cysteine residue in position three. The PAT activity attached palmitate to Fyn proteins via a thio-ester linkage and exhibited a fatty acyl CoA preference for long chain fatty acids. It is likely that palmitoylation of Fyn and other Src family members by PAT regulates PTK localization and signaling functions.
Klaas Krab - One of the best experts on this subject based on the ideXlab platform.
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Modular Kinetic Analysis of the Adenine Nucleotide Translocator–Mediated Effects of Palmitoyl-CoA on the Oxidative Phosphorylation in Isolated Rat Liver
2015Co-Authors: Marijke J. Wagner, Hans V Westerhoff, Klaas KrabAbstract:To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we ap-plied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential () and the fraction of matrix ATP as intermediates. We found that 5 mol/l Palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect ef-fects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate ac-ceptor were in excess, and the mitochondrial “work” flux was allowed to vary, Palmitoyl-CoA decreased phos-phorylation flux by 38 % and the fraction of ATP in the medium by 39%. increased by 15 mV, and the fractio
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metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl coa effects
2006Co-Authors: Hans V Westerhoff, Jolita Ciapaite, Gerco Van Eikenhorst, Stephan J L Bakker, Michaela Diamant, Robert J Heine, Klaas KrabAbstract:Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Delta psi) and cytosolic ATP concentrations. We here show that 5 mu M Palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (QH(2)/Q) by 42 +/- 9%, Delta psi by 13 +/- 1 mV (9%), and the intramitochondrial ATP/ADP ratio by 352 +/- 34%, and decreases the extramitochondrial ATP/ADP ratio by 63 +/- 4% in actively phosphorylating mitochondria. The latter reduction is expected to translate into a 24% higher extramitochondrial AMP concentration. Furthermore, Palmitoyl-CoA induced concentration-dependent H2O2 formation, which can only partly be explained by its effect on Delta psi. Although all measured fluxes and intermediate concentrations were affected by Palmitoyl-CoA, modular kinetic analysis revealed that this resulted mainly from inhibition of the ANT. Through Metabolic Control Analysis, we then determined to what extent the ANT controls the investigated mitochondrial properties. Under steady-state conditions, the ANT moderately controlled oxygen uptake (control coefficient C = 0.13) and phosphorylation (C = 0.14) flux. It controlled intramitochondrial (C = -0.70) and extramitochondrial ATP/ADP ratios (C = 0.23) more strongly, whereas the control exerted over the QH(2)/Q ratio (C = -0.04) and Delta psi (C = -0.01) was small. Quantitative assessment of the effects of Palmitoyl-CoA showed that the mitochondrial properties that were most strongly controlled by the ANT were affected the most. Our observations suggest that long-chain acyl-CoA esters may contribute to cellular dysfunction in obesity and type 2 diabetes through effects on cellular energy metabolism and production of reactive oxygen species.
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modular kinetic analysis of the adenine nucleotide translocator mediated effects of palmitoyl coa on the oxidative phosphorylation in isolated rat liver mitochondria
2005Co-Authors: Jolita Ciapaite, Hans V Westerhoff, Marijke J. Wagner, Gerco Van Eikenhorst, Stephan J L Bakker, Michaela Diamant, Robert J Heine, Klaas KrabAbstract:To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Δψ) and the fraction of matrix ATP as intermediates. We found that 5 μmol/l Palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial “work” flux was allowed to vary, Palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Δψ increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Δψ increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Δψ and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of Palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator.
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application of modular control analysis to inhibition of the adenine nucleotide translocator by palmitoyl coa
2002Co-Authors: Jolita Ciapaite, Gerco Van Eikenhorst, Klaas KrabAbstract:Modular kinetic analysis was used to characterize inhibition of adenine nucleotide translocation by Palmitoyl-CoA in isolated rat-liver mitochondria. To this purpose, oxidative phosphorylation has been divided into two modules with the fraction of matrix ATP as linking intermediate. The adenine nucleotide translocator is the matrix ATP-consuming module and the remainder of oxidative phosphorylation (ATP synthesis, respiratory chain and transport of phosphates and respiratory substrate) is the matrix ATP-producing module. We found that Palmitoyl-CoA inhibits ATP-consuming module (ANT) and has no effect on ATP-producing module. There were no significant differences between kinetic curves obtained with oligomycin and myxothiazol, inhibitors that have opposite effect on membrane potential, suggesting that the use of the fraction of matrix ATP as the only intermediate is a good approximation. A new method has been used to determine the fraction of ATP in the mitochondrial matrix.
Gregory J Cooney - One of the best experts on this subject based on the ideXlab platform.
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excess lipid availability increases mitochondrial fatty acid oxidative capacity in muscle evidence against a role for reduced fatty acid oxidation in lipid induced insulin resistance in rodents
2007Co-Authors: Nigel Turner, Clinton R Bruce, Susan M Beale, Kyle L Hoehn, Trina So, Michael S Rolph, Gregory J CooneyAbstract:A reduced capacity for mitochondrial fatty acid oxidation in skeletal muscle has been proposed as a major factor leading to the accumulation of intramuscular lipids and their subsequent deleterious effects on insulin action. Here, we examine markers of mitochondrial fatty acid oxidative capacity in rodent models of insulin resistance associated with an oversupply of lipids. C57BL/6J mice were fed a high-fat diet for either 5 or 20 weeks. Several markers of muscle mitochondrial fatty acid oxidative capacity were measured, including 14C-palmitate oxidation, Palmitoyl-CoA oxidation in isolated mitochondria, oxidative enzyme activity (citrate synthase, β-hydroxyacyl CoA dehydrogenase, medium-chain acyl-CoA dehydrogenase, and carnitine palmitoyl-transferase 1), and expression of proteins involved in mitochondrial metabolism. Enzyme activity and mitochondrial protein expression were also examined in muscle from other rodent models of insulin resistance. Compared with standard diet–fed controls, muscle from fat-fed mice displayed elevated palmitate oxidation rate (5 weeks +23%, P < 0.05, and 20 weeks +29%, P < 0.05) and increased Palmitoyl-CoA oxidation in isolated mitochondria (20 weeks +49%, P < 0.01). Furthermore, oxidative enzyme activity and protein expression of peroxisome proliferator–activated receptor γ coactivator (PGC)-1α, uncoupling protein (UCP) 3, and mitochondrial respiratory chain subunits were significantly elevated in fat-fed animals. A similar pattern was present in muscle of fat-fed rats, obese Zucker rats, and db/db mice, with increases observed for oxidative enzyme activity and expression of PGC-1α, UCP3, and subunits of the mitochondrial respiratory chain. These findings suggest that high lipid availability does not lead to intramuscular lipid accumulation and insulin resistance in rodents by decreasing muscle mitochondrial fatty acid oxidative capacity.
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acyl coa inhibition of hexokinase in rat and human skeletal muscle is a potential mechanism of lipid induced insulin resistance
2000Co-Authors: A L Thompson, Gregory J CooneyAbstract:There are strong correlations between impaired insulin-stimulated glucose metabolism and increased intramuscular lipid pools; however, the mechanism by which lipids interact with glucose metabolism is not completely understood. Long-chain acyl CoAs have been reported to allosterically inhibit liver glucokinase (hexokinase IV). The aim of the present study was to determine whether long-chain acyl CoAs inhibit hexokinase in rat and human skeletal muscle. At subsaturating glucose concentrations, 10 micromol/l of the three major long-chain acyl-CoA species in skeletal muscle, palmitoyl CoA (16:0), oleoyl CoA (18:1, n = 9), and linoleoyl CoA (18:2, n = 6), reduced hexokinase activity of rat skeletal muscle to 61 +/- 3, 66 +/- 7, and 57 +/- 5% of control activity (P
Jolita Ciapaite - One of the best experts on this subject based on the ideXlab platform.
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metabolic control of mitochondrial properties by adenine nucleotide translocator determines palmitoyl coa effects
2006Co-Authors: Hans V Westerhoff, Jolita Ciapaite, Gerco Van Eikenhorst, Stephan J L Bakker, Michaela Diamant, Robert J Heine, Klaas KrabAbstract:Inhibition of the mitochondrial adenine nucleotide translocator (ANT) by long-chain acyl-CoA esters has been proposed to contribute to cellular dysfunction in obesity and type 2 diabetes by increasing formation of reactive oxygen species and adenosine via effects on the coenzyme Q redox state, mitochondrial membrane potential (Delta psi) and cytosolic ATP concentrations. We here show that 5 mu M Palmitoyl-CoA increases the ratio of reduced to oxidized coenzyme Q (QH(2)/Q) by 42 +/- 9%, Delta psi by 13 +/- 1 mV (9%), and the intramitochondrial ATP/ADP ratio by 352 +/- 34%, and decreases the extramitochondrial ATP/ADP ratio by 63 +/- 4% in actively phosphorylating mitochondria. The latter reduction is expected to translate into a 24% higher extramitochondrial AMP concentration. Furthermore, Palmitoyl-CoA induced concentration-dependent H2O2 formation, which can only partly be explained by its effect on Delta psi. Although all measured fluxes and intermediate concentrations were affected by Palmitoyl-CoA, modular kinetic analysis revealed that this resulted mainly from inhibition of the ANT. Through Metabolic Control Analysis, we then determined to what extent the ANT controls the investigated mitochondrial properties. Under steady-state conditions, the ANT moderately controlled oxygen uptake (control coefficient C = 0.13) and phosphorylation (C = 0.14) flux. It controlled intramitochondrial (C = -0.70) and extramitochondrial ATP/ADP ratios (C = 0.23) more strongly, whereas the control exerted over the QH(2)/Q ratio (C = -0.04) and Delta psi (C = -0.01) was small. Quantitative assessment of the effects of Palmitoyl-CoA showed that the mitochondrial properties that were most strongly controlled by the ANT were affected the most. Our observations suggest that long-chain acyl-CoA esters may contribute to cellular dysfunction in obesity and type 2 diabetes through effects on cellular energy metabolism and production of reactive oxygen species.
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modular kinetic analysis of the adenine nucleotide translocator mediated effects of palmitoyl coa on the oxidative phosphorylation in isolated rat liver mitochondria
2005Co-Authors: Jolita Ciapaite, Hans V Westerhoff, Marijke J. Wagner, Gerco Van Eikenhorst, Stephan J L Bakker, Michaela Diamant, Robert J Heine, Klaas KrabAbstract:To test whether long-chain fatty acyl-CoA esters link obesity with type 2 diabetes through inhibition of the mitochondrial adenine nucleotide translocator, we applied a system-biology approach, dual modular kinetic analysis, with mitochondrial membrane potential (Δψ) and the fraction of matrix ATP as intermediates. We found that 5 μmol/l Palmitoyl-CoA inhibited adenine nucleotide translocator, without direct effect on other components of oxidative phosphorylation. Indirect effects depended on how oxidative phosphorylation was regulated. When the electron donor and phosphate acceptor were in excess, and the mitochondrial “work” flux was allowed to vary, Palmitoyl-CoA decreased phosphorylation flux by 38% and the fraction of ATP in the medium by 39%. Δψ increased by 15 mV, and the fraction of matrix ATP increased by 46%. Palmitoyl-CoA had a stronger effect when the flux through the mitochondrial electron transfer chain was maintained constant: Δψ increased by 27 mV, and the fraction of matrix ATP increased 2.6 times. When oxidative phosphorylation flux was kept constant by adjusting the rate using hexokinase, Δψ and the fraction of ATP were not affected. Palmitoyl-CoA increased the extramitochondrial AMP concentration significantly. The effects of Palmitoyl-CoA in our model system support the proposed mechanism linking obesity and type 2 diabetes through an effect on adenine nucleotide translocator.
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application of modular control analysis to inhibition of the adenine nucleotide translocator by palmitoyl coa
2002Co-Authors: Jolita Ciapaite, Gerco Van Eikenhorst, Klaas KrabAbstract:Modular kinetic analysis was used to characterize inhibition of adenine nucleotide translocation by Palmitoyl-CoA in isolated rat-liver mitochondria. To this purpose, oxidative phosphorylation has been divided into two modules with the fraction of matrix ATP as linking intermediate. The adenine nucleotide translocator is the matrix ATP-consuming module and the remainder of oxidative phosphorylation (ATP synthesis, respiratory chain and transport of phosphates and respiratory substrate) is the matrix ATP-producing module. We found that Palmitoyl-CoA inhibits ATP-consuming module (ANT) and has no effect on ATP-producing module. There were no significant differences between kinetic curves obtained with oligomycin and myxothiazol, inhibitors that have opposite effect on membrane potential, suggesting that the use of the fraction of matrix ATP as the only intermediate is a good approximation. A new method has been used to determine the fraction of ATP in the mitochondrial matrix.
Rolf K Berge - One of the best experts on this subject based on the ideXlab platform.
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acyl coa esters antagonize the effects of ligands on peroxisome proliferator activated receptor alpha conformation dna binding and interaction with co factors
2001Co-Authors: Morten Elholm, Rolf K Berge, Claus Jorgensen, Annem Krogsdam, Dorte Holst, Irina Kratchmarova, Martin Gottlicher, Janake Gustafsson, Torgeir Flatmark, Jens KnudsenAbstract:Abstract The peroxisome proliferator-activated receptor α (PPARα) is a ligand-activated transcription factor and a key regulator of lipid homeostasis. Numerous fatty acids and eicosanoids serve as ligands and activators for PPARα. Here we demonstrate thatS-hexadecyl-CoA, a nonhydrolyzable Palmitoyl-CoA analog, antagonizes the effects of agonists on PPARα conformation and function in vitro. In electrophoretic mobility shift assays, S-hexadecyl-CoA prevented agonist-induced binding of the PPARα-retinoid X receptor α heterodimer to the acyl-CoA oxidase peroxisome proliferator response element. PPARα bound specifically to immobilized Palmitoyl-CoA and Wy14643, but not BRL49653, abolished binding. S-Hexadecyl-CoA increased in a dose-dependent and reversible manner the sensitivity of PPARα to chymotrypsin digestion, and theS-hexadecyl-CoA-induced sensitivity required a functional PPARα ligand-binding pocket. S-Hexadecyl-CoA prevented ligand-induced interaction between the co-activator SRC-1 and PPARα but increased recruitment of the nuclear receptor co-repressor NCoR. In cells, the concentration of free acyl-CoA esters is kept in the low nanomolar range due to the buffering effect of high affinity acyl-CoA-binding proteins, especially the acyl-CoA-binding protein. By using PPARα expressed in Sf21 cells for electrophoretic mobility shift assays, we demonstrate that S-hexadecyl-CoA was able to increase the mobility of the PPARα-containing heterodimer even in the presence of a molar excess of acyl-CoA-binding protein, mimicking the conditions found in vivo.
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3-thia fatty acid treatment, in contrast to eicosapentaenoic acid and starvation, induces gene expression of carnitine palmitoyltransferase-II in rat liver
1999Co-Authors: Lise Madsen, Rolf K BergeAbstract:The aim of the present study was to investigate the hepatic regulation and β-oxidation of long-chain fatty acids in peroxisomes and mitochondria, after 3-thia- tetradecylthioacetic acid (C_14-S-acetic acid) treatment. When Palmitoyl-CoA and palmitoyl- l -carnitine were used as substrates, hepatic formation of acid-soluble products was significantly increased in C_14-S-acetic acid treated rats. Administration of C_14-S-acetic acid resulted in increased enzyme activity and mRNA levels of hepatic mitochondrial carnitine palmitoyltransferase (CPT)-II. CPT-II activity correlated with both Palmitoyl-CoA and palmitoyl- l -carnitine oxidation in rats treated with different chain-length 3-thia fatty acids. CPT-I activity and mRNA levels were, however, marginally affected. The hepatic CPT-II activity was mainly localized in the mitochondrial fraction, whereas the CPT-I activity was enriched in the mitochondrial, peroxisomal, and microsomal fractions. In C_14-S-acetic acid-treated rats, the specific activity of peroxisomal and microsomal CPT-I increased, whereas the mitochondrial activity tended to decrease. C_14-S-Acetyl-CoA inhibited CPT-I activity in vitro . The sensitivity of CPT-I to malonyl-CoA was unchanged, and the hepatic malonyl-CoA concentration increased after C_14-S-acetic acid treatment. The mRNA levels of acetyl-CoA carboxylase increased. In hepatocytes cultured from palmitic acid- and C_14-S-acetic acid-treated rats, the CPT-I inhibitor etomoxir inhibited the formation of acid-soluble products 91 and 21%, respectively. In contrast to 3-thia fatty acid treatment, eicosapentaenoic acid treatment and starvation increased the mitochondrial CPT-I activity and reduced its malonyl-CoA sensitivity. Palmitoyl- l -carnitine oxidation and CPT-II activity were, however, unchanged after either EPA treatment or starvation. The results from this study open the possibility that the rate control of mitochondrial β-oxidation under mitochondrion and peroxisome proliferation is distributed between an enzyme or enzymes of the pathway beyond the CPT-I site after 3-thia fatty acid treatment. It is suggested that fatty acids are partly oxidized in the peroxisomes before entering the mitochondria as acylcarnitines for further oxidation.
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mitochondrial 3 hydroxy 3 methylglutaryl coenzyme a synthase and carnitine palmitoyltransferase ii as potential control sites for ketogenesis during mitochondrion and peroxisome proliferation
1999Co-Authors: Lise Madsen, Fausto G Hegardt, Dolors Serra, Guillermina Asins, Alexis Garras, Rolf K BergeAbstract:Thia fatty acids are potent hypolipidemic fatty acid derivatives and mitochondrion and peroxisome proliferators. Administration of 3-thia fatty acids to rats was followed by significantly increased levels of plasma ketone bodies, whereas the levels of plasma non-esterified fatty acids decreased. The hepatic mRNA levels of fatty acid binding protein and formation of acid-soluble products, using both Palmitoyl-CoA and palmitoyl-L-carnitine as substrates, were increased. Hepatic mitochondrial carnitine palmitoyltransferase (CPT) -II and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase activities, immunodetectable proteins, and mRNA levels increased in parallel. In contrast, the mitochondrial CPT-I mRNA levels were unchanged and CPT-I enzyme activity was slightly reduced in the liver. The CoA ester of the monocarboxylic 3-thia fatty acid, tetradecylthioacetic acid, which accumulates in the liver after administration, inhibited the CPT-I activity in vitro, but not that of CPT-II. Acetoacetyl-CoA thiolase and HMG-CoA lyase activities involved in ketogenesis were increased, whereas the citrate synthase activity was decreased. The present data suggest that 3-thia fatty acids increase both the transport of fatty acids into the mitochondria and the capacity of the b-oxidation process. Under these conditions, the regulation of ketogenesis may be shifted to step(s) beyond CPT-I. This opens the possibility that mitochondrial HMG-CoA synthase and CPT-II retain some control of ketone body formation. BIOCHEM PHARMACOL 57;9:1011-1019, 1999. © 1999 Elsevier Science Inc.
