Showing papers on "Acyl-CoA published in 2002"

Critical role for cataplerosis via citrate in glucose-regulated insulin release

Abstract: The molecular mechanisms mediating acute regulation of insulin release by glucose are partially known. The process involves at least two pathways that can be discriminated on basis of their (in)dependence of closure of ATP-sensitive potassium (K+ATP) channels. The mechanism of the K+ATP channel–independent pathway was proposed to involve cataplerosis, the export of mitochondrial intermediates into the cytosol and in the induction of fatty acid–derived signaling molecules. In the present article, we have explored in fluorescence-activated cell sorter (FACS)-purified rat β-cells the molecular steps involved in chronic glucose regulation of the insulin secretory response. When compared with culture in 10 mmol/l glucose, 24 h culture in 3 mmol/l glucose shifts the phenotype of the cells into a state with low further secretory responsiveness to glucose, lower rates of glucose oxidation, and lower rates of cataplerosis. Microarray mRNA analysis indicates that this shift can be attributed to differences in expression of genes involved in the K+ATP channel–dependent pathway, in cataplerosis and in fatty acid/cholesterol biosynthesis. This response was paralleled by glucose upregulation of the transcription factor sterol regulatory element binding protein 1c (SREBP1c) (ADD1) and downregulation of peroxisome proliferator—activated receptor (PPAR)-α and PPAR-β (PPARδ). The functional importance of cataplerosis via citrate for glucose-induced insulin release was further supported by the observation that two ATP-citrate lyase inhibitors, radicicol and (−)-hydroxycitrate, block part of glucose-stimulated release in β-cells. In conclusion, chronic glucose regulation of the glucose-responsive secretory phenotype is associated with coordinated changes in gene expression involved in the K+ATP channel–dependent pathway, in cataplerosis via citrate and in acyl CoA/cholesterol biosynthesis.

Acyl CoA profiles of transgenic plants that accumulate medium‐chain fatty acids indicate inefficient storage lipid synthesis in developing oilseeds

Abstract: Several Brassica napus lines transformed with genes responsible for the synthesis of medium- or long-chain fatty acids were examined to determine limiting factor(s) for the subsequent accumulation of these fatty acids in seed lipids. Examination of a decanoic acid (10:0) accumulating line revealed a disproportionately high concentration of 10:0 CoA during seed development compared to long-chain acyl CoAs isolated from the same tissues, suggesting that poor incorporation of 10:0 CoA into seed lipids limits 10:0 fatty acid accumulation. This relationship was also seen for dodecanoyl (12:0) CoA and fatty acid in a high 12:0 line, but not for octadecanoic (18:0) CoA and fatty acid in a high 18:0 line. Comparison of 10:0 CoA and fatty acid proportions from seeds at different developmental stages for transgenic B. napus and Cuphea hookeriana, the source plant for the medium-chain thioesterase and 3-ketoacyl-ACP synthase transgenes, revealed that C. hookeriana incorporates 10:0 CoA into seed lipids more efficiently than transgenic B. napus. Furthermore, β-oxidation and glyoxylate cycle activities were not increased above wild type levels during seed development in the 8:0/10:0 line, suggesting that lipid catabolism was not being induced in response to the elevated 10:0 CoA concentrations. Taken together, these data suggest that transgenic plants that are engineered to synthesize medium-chain fatty acids may lack the necessary mechanisms, such as specific acyltransferases, to incorporate these fatty acids efficiently into seed lipids.

Isolation and characterization of two fatty acid binding proteins from mouse brain

Abstract: Two fatty acid binding proteins (FABPs) were isolated from Swiss Webster mouse brains. Neither protein cross-reacted with antisera to recombinant liver L-FABP. One protein, designated brain H-FABP, migrated on tricine sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as a single band at 14.5 kDa with pl 4.9. Brain H-FABP bound NBD-stearic acid and cis-parinaric acid with K D values near 0.02 and 0.5 microM, respectively. Brain H-FABP cross-reacted with affinity-purified antisera to recombinant heart H-FABP. The second protein, mouse brain B-FABP, migrated on tricine SDS-PAGE gels as a doublet at 16.0 and 15.5 kDa with pl values of 4.5 and 4.7, respectively. Brain B-FABP bound NBD-stearic and cis-parinaric acid with K D values near 0.01 and 0.7 microM, respectively. The brain B-FABP doublet was immunoreactive with affinity-purified antibodies against recombinant mouse brain B-FABP, but not with affinity-purified antibodies against heart H-FABP. (3H)Oleate competition binding indicated that the two brain FABPs had distinct ligand binding specificities. Both bound fatty acids, fatty acyl CoA, and lysophosphatidic acid. Although both preferentially bound unsaturated fatty acids, twofold differences in specific saturated fatty acid binding were observed. Brain B-FABP and brain H-FABP represented 0.1% and 0.01% of brain total cytosolic protein, respectively. In summary, mouse brain contains two native fatty acid binding proteins, brain H-FABP and brain B-FABP.

Concerted regulation of free arachidonic acid and hormone-induced steroid synthesis by acyl-CoA thioesterases and acyl-CoA synthetases in adrenal cells

Abstract: Although the role of arachidonic acid (AA) in the regulation of steroidogenesis is well documented, the mechanism for AA release is not clear. Therefore, the aim of this study was to characterize the role of an acyl-CoA thioesterase (ARTISt) and an acyl-CoA synthetase as members of an alternative pathway in the regulation of the intracellular levels of AA in steroidogenesis. Purified recombinant ARTISt releases AA from arachidonoyl-CoA (AA-CoA) with a Km of 2 µm. Antibodies raised against recombinant acyl-CoA thioesterase recognize the endogenous protein in both adrenal tissue and Y1 adrenal tumor cells by immunohistochemistry and immunocytochemistry and Western blot. Stimulation of Y1 cells with ACTH significantly stimulated endogenous mitochondrial thioesterases activity (1.8-fold). Nordihydroguaiaretic acid (NDGA), an inhibitor of AA release known to affect steroidogenesis, affects the in vitro activity of recombinant ARTISt and also the endogenous mitochondrial acyl-CoA thioesterases. ACTH-stimulated steroid synthesis in Y1 cells was significantly inhibited by a synergistic effect of NDGA and triacsin C an inhibitor of the AA-CoA synthetase. The apparent IC50 for NDGA was reduced from 50 µm to 25, 7.5 and 4.5 µm in the presence of 0.1, 0.5 and 2 µm triacsin C, respectively. Our results strongly support the existence of a new pathway of AA release that operates in the regulation of steroid synthesis in adrenal cells.

Changes in Cerebral Acyl‐CoA Concentrations Following Ischemia‐Reperfusion in Awake Gerbils

Abstract: Transient global cerebral ischemia affects phospholipid metabolism and features a considerable increase in unesterified fatty acids. Reincorporation of free fatty acids into membrane phospholipids during reperfusion following transient ischemia depends on conversion of fatty acids to acyl-CoAs via acyl-CoA synthetases and incorporation of the acyl group into lysophospholipids. To study the effect of ischemia-reperfusion on brain fatty acid and acyl-CoA pools, the common carotid arteries were tied for 5 min in awake gerbils, after which the ligatures were released for 5 min and the animals were killed by microwave irradiation. Twenty percent of these animals (two of 10) were excluded from the ischemia-reperfusion group when it was demonstrated statistically that brain unesterified arachidonic acid concentration was not elevated beyond the range of the control group. Brain unesterified fatty acid concentration was increased 4.4-fold in the ischemic-reperfused animals, with stearic acid and arachidonic acid increasing the most among the saturated and polyunsaturated fatty acids, respectively. The total acyl-CoA concentration remained unaffected, indicating that reacylation of membrane lysophospholipids is maintained during recovery. However, there was a substantial increase in the stearoyl- and arachidonoyl-CoA and a marked decrease in palmitoyl- and docosahexaenoyl-CoA. These results suggest that unesterified fatty acid reacylation into phospholipids is reprioritized according to the redistribution in concentration of acyl-CoA molecular species, with incorporation of stearic acid and especially arachidonic acid being favored.

Fluorescently labelled bovine acyl-CoA-binding protein acting as an acyl-CoA sensor: interaction with CoA and acyl-CoA esters and its use in measuring free acyl-CoA esters and non-esterified fatty acids.

Abstract: Long-chain acyl-CoA esters are key metabolites in lipid synthesis and beta-oxidation but, at the same time, are important regulators of intermediate metabolism, insulin secretion, vesicular trafficking and gene expression. Key tools in studying the regulatory functions of acyl-CoA esters are reliable methods for the determination of free acyl-CoA concentrations. No such method is presently available. In the present study, we describe the synthesis of two acyl-CoA sensors for measuring free acyl-CoA concentrations using acyl-CoA-binding protein as a scaffold. Met24 and Ala53 of bovine acyl-CoA-binding protein were replaced by cysteine residues, which were covalently modified with 6-bromoacetyl-2-dimethylaminonaphthalene to make the two fluorescent acyl-CoA indicators (FACIs) FACI-24 and FACI-53. FACI-24 and FACI-53 showed fluorescence emission maximum at 510 and 525 nm respectively, in the absence of ligand (excitation 387 nm). Titration of FACI-24 and FACI-53 with hexadecanoyl-CoA and dodecanoyl-CoA increased the fluorescence yield 5.5-and 4.7-fold at 460 and 495 nm respectively. FACI-24 exhibited a high, and similar increase in, fluorescence yield at 460 nm upon binding of C14-C20 saturated and unsaturated acyl-CoA esters. Both indicators bind long-chain (>C14) acyl-CoA esters with high specificity and affinity (K(d)=0.6-1.7 nM). FACI-53 showed a high fluorescence yield for C8-C12 acyl chains. It is shown that FACI-24 acts as a sensitive acyl-CoA sensor for measuring the concentration of free acyl-CoA, acyl-CoA synthetase activity and the concentrations of free fatty acids after conversion of the fatty acid into their respective acyl-CoA esters.

The effect of charge reversal mutations in the α-helical region of liver fatty acid binding protein on the binding of fatty-acyl CoAs, lysophospholipids and bile acids

Abstract: Liver fatty acid binding protein (LFABP) is unique among the various types of FABPs in that it can bind a variety of ligands in addition to fatty acids. LFABP is able to bind long chain fatty acids with a 2:1 stoichiometry and the crystal structure has identified two fatty acid binding sites in the binding cavity. The presumed primary site (site 1 ) involves the fatty acid binding with the carboxylate group buried in the cavity whereas the fatty acid at site 2 has the carboxylate group solvent-exposed within the ligand portal region and in the vicinity of α-helix II. The α-helical region contains three cationic residues, K20, K31, K33 and modelling studies suggest that K31 on α-helix II could make an electrostatic contribution to anionic ligands binding to site 2. The preparation of three charge reversal mutants of LFABP, K20E, K31E and K33E has allowed an investigation of the role of site 2 in ligand binding, particularly those ligands with a bulky anionic head group. The binding of oleoyl CoA, lysophosphatidic acid, lysophosphatidylcholine, lithocholic acid and taurolithocholate 3-sulphate to LFABP has been studied using the α-helical mutants. The results support the concept that such ligands bind at site 2 of LFABP where solvent exposure allows the accommodation of their bulky anionic group. (Mol Cell Biochem 239: 55–60, 2002)

INHIBITION OF CHOLESTEROL ABSORPTION BY SCH 58053 IN THE MOUSE IS NOT MEDIATED VIA CHANGES IN EITHER THE INTESTINAL BILE ACID POOL OR THE EXPRESSION OF mRNA FOR ABCA1, ABCG5 OR ABCG8 IN THE ENTEROCYTE

Abstract: Abbreviations: LDL, low density lipoprotein; ACAT-2, acyl CoA:cholesterol acyltransferase-2; ABCA1, adenosine triphosphate-binding cassette transporter A1; ABCB4, ABC transporter B4 (MDR2/3); ABCG5, ABC transporter G5; ABCG8, ABC transporter G8; ABCB11, ABC transporter B11 (SPGP/BSEP); HMG CoA syn, 3-hydroxy-3-methylglutaryl coenzyme A synthase; HMG CoA red, 3-hydroxy-3-methylglutaryl coenzyme A reductase; DPS, digitoninprecipitable sterols

Tissue Specific Differences in Intramitochondrial Control of β-Oxidation

Abstract: It has become clear that there are important tissue specific differences in the control and regulation of oxidation and that these differences may largely reside with the entry of acyl moieties into the mitochondrion by the carnitine palmitoyltransferase (CPT) /translocase system. CPT I has wide tissue-specific variations in sensitivity to its physiological inhibitor, malonyl-CoA,1 which is probably due to the presence of different isozymes. However, it is possible that there are additional intramitochondrial controls which vary amongst tissues. The individual reactions of the oxidation of long-chain fatty acids within the mitochondrion have been known for many years although their functional and topological relationship is less well known. Recently, it has been found that the long-chain activities of three of the enzymes reside on a single membrane-bound protein (the trifunctional protein,4,5) and, in addition, there exists a fourth acyl-CoA dehydrogenase which is similarly membrane bound. Hence, the possibility of functional organization of oxidation enzyme activities, associated with the inner mitochondrial membrane, must be considered. The organization of oxidation enzymes within the mitochondrion has previously been postulated as a result of the classic studies of Stanley and Tubbs, amongst others, leading to the “leaky hosepipe” model of oxidation. Their work also led to the view that the acyl-CoA dehydrogenases were the “rate-limiting step” for oxidation, as only saturated acyl groups accumulated under well oxygenated conditions. These workers measured acyl groups resulting from hydrolysis of CoA and car-

Techniques for measuring the activity of carboxylic acid:CoA ligase and acyl-CoA:amino acid N-acyltransferase: the amino acid conjugation pathway.

Abstract: A wide variety of xenobiotic carboxylic acids are metabolized to their amino acid conjugates via a pathway that exists primarily in liver and kidney. This conjugation occurs in a two-step pathway catalyzed by two distinct types of enzymes, ligases and transferases. Measurements of acyl-CoA ligase activity include monitoring the rate of appearance of AMP or PPi, or the CoA adduct. N-acyltransferases catalyze formation of an amino acid conjugate from the CoA-activated intermediate, releasing CoA. This reaction is monitored by following the release of free CoA or the disappearance of the acyl-CoA adduct.