α‐Oxidation of 3‐Methyl‐Substituted Fatty Acids in Rat Liver

Abstract

coxidation of 3-methyl-substituted fatty acids in rat liver was studied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of a-oxidation is formic acid, which is then converted to CO,. Rates of a-oxidation identical to those observed in intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fez+, 2-oxoglutarate and ascorbate were added, suggesting that aoxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation by differential and density gradient centrifugation demonstrated that a-oxidation is confined to peroxisomes, which produce formic acid that is converted to CO,, mainly in the cytosol. a-Oxidation in broken cell systems went hand in hand with the formation of a 2-hydroxy-3-methylacyl-CoA ester. Formation of the metabolite was strictly dependent on the presence of the above-mentioned cofactors, was confined to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of a-oxidation in intact cells, indicating that the 2-hydroxyacyl-CoA ester is a bona fide intermediate of a-oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3-methyl fatty acids, 3-methyl fatty acyl-CoAs and their respective 2-hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction.Our experiments demonstrate that a-oxidation is a peroxisomal process that consists of at least three reactions : fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.

IDH1 gene transcription is sterol regulated and activated by SREBP-1a and SREBP-2 in human hepatoma HepG2 cells

“…The subcellular localization of IDH1 in both cytosol and peroxisomes ideally situates it to respond to the lipogenic requirement of the cell. The coproduct of the reaction, ␣-ketoglutarate, which is required as a cosubstrate for the phytanoyl-CoA ␣-hydroxylase reaction (17)(18)(19)(20), is required for the catabolic removal of phytanic acid, a known agonist to PPAR␣ (21,22). PPAR␣ is highly expressed in heart, muscle, kidney, and endothelial cells, but mostly in liver (27,34), where its function is in the catabolism of fatty acids through the regulation of genes encoding mitochondrial and peroxisomal enzymes important for ␤-oxidation (68).…”

Purification, molecular cloning, and expression of 2-hydroxyphytanoyl-CoA lyase, a peroxisomal thiamine pyrophosphate-dependent enzyme that catalyzes the carbon–carbon bond cleavage during α-oxidation of 3-methyl-branched fatty acids

“…Other incubations were terminated by adding 50 l of 1 M K-phosphate, pH 2, and [ 14 C]formate and [1][2][3][4][5][6][7][8][9][10][11][12][13][14] C]formyl-CoA were measured separately by HPLC as described (4). For the analysis of lipid-soluble reaction products, similar incubations with unlabeled 2-hydroxy-3-methylhexadecanoyl-CoA were quenched by adding 0.5 ml of methanol͞glacial acetic acid (98͞2, vol͞vol) and extracted with hexane.…”

“…In the rat, the entire ␣-oxidation pathway appears to be peroxisomal (3,6) (except for the conversion of formate to CO 2 , which occurs mainly in the cytosol). Also in humans, the ␣-oxidation pathway (activation, 2-hydroxylation, cleavage) occurs in peroxisomes (11), but the dehydrogenation of the 2-methyl-branched aldehyde to the corresponding acid may be catalyzed in the endoplasmic reticulum (12).…”

Production of formyl‐CoA during peroxisomal α‐oxidation of 3‐methyl‐branched fatty acids

“…14 C]palmitate) as described before [8], or (for the other substrates with lower specific radioactivity) by collection of the fractions and liquid scintillation counting. Counting efficiency of the online detector was approximately 50%.…”