Phytanic acid α-oxidation in human cultured skin fibroblasts

We studied the oxidation of [1-¹⁴C]phytanic acid, 3-methyl substituted fatty acid, to pristanic acid and ¹⁴CO₂ in human skin fibroblasts. The specific activity for α-oxidation of phytanic acid in peroxisomes was 29- and 124-fold higher than mitochondria and endoplasmic reticulum. This finding demonstrates for the first time the presence of fatty acid α-oxidation enzyme system in peroxisomes.     

Lipid metabolism in peroxisomes in relation to human disease

Peroxisomes were long believed to play only a minor role in cellular metabolism but it is now clear that they catalyze a number of important functions. The importance of peroxisomes in humans is stressed by the existence of a group of genetic diseases in man in which one or more peroxisomal functions are impaired. Most of the functions known to take place in peroxisomes have to do with lipids. Indeed, peroxisomes are capable of 1, fatty acid β-oxidation 2, fatty acid α-oxidation 3, synthesis of cholesterol and other isoprenoids 4, ether-phospholipid synthesis and 5, biosynthesis of polyunsaturated fatty acids. In Chapter 2–6 we will discuss the functional organization and enzymology of these pathways in detail. Furthermore, attentin is paid to the permeability properties of peroxisomes with special emphasis on recent studies which suggest that peroxisomes are closed structures containing specific membrane proteins for trransport of metabolites. Finally, the disorders of peroxisomal lipid metabolism will be discussed.     

Peroxisomes, lipid metabolism, and human disease

In the past few years, much has been learned about the metabolic functions of peroxisomes. These studies have shown that peroxisomes play a major role in lipid metabolism, including fatty acid β-oxidation, etherphospholipid biosynthesis, and phytanic acid α-oxidation. This article describes the current state of knowledge concerning the role of peroxisomes in these processes, especially in relation to various peroxisomal disorders in which there is an impairment in peroxisomal lipid metabolism.     

Peroxisomes contribute to the acylcarnitine production when the carnitine shuttle is deficient

Fatty acid β-oxidation may occur in both mitochondria and peroxisomes. While peroxisomes oxidize specific carboxylic acids such as very long-chain fatty acids, branched-chain fatty acids, bile acids, and fatty dicarboxylic acids, mitochondria oxidize long-, medium-, and short-chain fatty acids. Oxidation of long-chain substrates requires the carnitine shuttle for mitochondrial access but medium-chain fatty acid oxidation is generally considered carnitine-independent. Using control and carnitine palmitoyltransferase 2 (CPT2)- and carnitine/acylcarnitine translocase (CACT)-deficient human fibroblasts, we investigated the oxidation of lauric acid (C12:0). Measurement of the acylcarnitine profile in the extracellular medium revealed significantly elevated levels of extracellular C10- and C12-carnitine in CPT2- and CACT-deficient fibroblasts. The accumulation of C12-carnitine indicates that lauric acid also uses the carnitine shuttle to access mitochondria. Moreover, the accumulation of extracellular C10-carnitine in CPT2- and CACT-deficient cells suggests an extramitochondrial pathway for the oxidation of lauric acid. Indeed, in the absence of peroxisomes C10-carnitine is not produced, proving that this intermediate is a product of peroxisomal β-oxidation. In conclusion, when the carnitine shuttle is impaired lauric acid is partly oxidized in peroxisomes. This peroxisomal oxidation could be a compensatory mechanism to metabolize straight medium- and long-chain fatty acids, especially in cases of mitochondrial fatty acid β-oxidation deficiency or overload.     

Peroxisomal β-oxidation stimulates cholesterol biosynthesis in the liver in diabetic mice

Although diabetes normally causes an elevation of cholesterol biosynthesis and induces hypercholesterolemia in animals and human, the mechanism linking diabetes to the dysregulation of cholesterol biosynthesis in the liver is not fully understood. As liver peroxisomal β-oxidation is induced in the diabetic state and peroxisomal oxidation of fatty acids generates free acetate, we hypothesized that peroxisomal β-oxidation might play a role in liver cholesterol biosynthesis in diabetes. Here, we used erucic acid, a specific substrate for peroxisomal β-oxidation, and 10,12-tricosadiynoic acid, a specific inhibitor for peroxisomal β-oxidation, to specifically induce and suppress peroxisomal β-oxidation. Our results suggested that induction of peroxisomal β-oxidation increased liver cholesterol biosynthesis in streptozotocin-induced diabetic mice. We found that excessive oxidation of fatty acids by peroxisomes generated considerable free acetate in the liver, which was used as a precursor for cholesterol biosynthesis. In addition, we show that specific inhibition of peroxisomal β-oxidation decreased cholesterol biosynthesis by reducing acetate formation in the liver in diabetic mice, demonstrating a crosstalk between peroxisomal β-oxidation and cholesterol biosynthesis. Based on these results, we propose that induction of peroxisomal β-oxidation serves as a mechanism for a fatty acid-induced upregulation in cholesterol biosynthesis and also plays a role in diabetes-induced hypercholesterolemia.     

Mitochondrial β-oxidation of fatty acids in higher plants

The subcellular localization of β -oxidation of fatty acids in plants has been the subject of controversy for many years. Peroxisomes have been accepted as the sole site of fatty acid β -oxidation for the past 15 years, whilst a proposed dual location, in both mitochondria and peroxisomes, has been disputed. Accumulated evidence demonstrating mitochondrial β -oxidation now demands that a dual location for plant β -oxidation must be considered.     

Human metabolism of phytanic acid and pristanic acid

Phytanic acid is a methyl-branched fatty acid present in the human diet. Due to its structure, degradation by β-oxidation is impossible. Instead, phytanic acid is oxidized by -oxidation, yielding pristanic acid. Despite many efforts to elucidate the -oxidation pathway, it remained unknown for more than 30 years. In recent years, the mechanism of -oxidation as well as the enzymes involved in the process have been elucidated. The process was found to involve activation, followed by hydroxylase, lyase and dehydrogenase reactions. Part, if not all of the reactions were found to take place in peroxisomes. The final product of phytanic acid -oxidation is pristanic acid. This fatty acid is degraded by peroxisomal β-oxidation. After 3 steps of β-oxidation in the peroxisome, the product is esterified to carnitine and shuttled to the mitochondrion for further oxidation. Several inborn errors with one or more deficiencies in the phytanic acid and pristanic degradation have been described. The clinical expressions of these disorders are heterogeneus, and vary between severe neonatal and often fatal symptoms and milder syndromes with late onset. Biochemically, these disorders are characterized by accumulation of phytanic and/or pristanic acid in tissues and body fluids. Several of the inborn errors involoving phytanic acid and/or pristanic acid metabolism have been characterized on the molecular level.     

Ricinoleic Acid Catabolism in Peroxisomes

Peroxisomes from castor bean endosperm and mung bean hypocotyl completely degrade ricinoleic acid (12-D-hydroxy-9-cis-octadecenoic acid) to acetyl-CoA. Concomitant NADH formation occurred with a stoichiometry of 9 nmol NADH formed per 1 nmol ricinoleate degraded. At the C₈-intermediate level, where the hydroxy group of ricinoleic acid forms a barrier to β-oxidation, 2-hydroxyoctanoate and 2-oxooctanoate were detected as intermediates. 2-Hydroxyoctanoate was oxidized to 2-oxooctanoate with H₂O₂ producing a reaction exhibiting 1:1 stoichiometry of the products. The peroxisomes appeared to oxidize both isomers of racemic 2-hydroxyoctanoate. 2-Oxooctanoate was metabolized to heptanoyl-CoA (propionyl-CoA and acetyl-CoA) in a NAD-dependent, but ATP-independent, reaction. Heptanoate was not detected as an intermediate. Imidazole, an inhibitor of α-oxidation, did not effect the degradation of ricinoleate or 2-oxooctanoate. Arsenite, an inhibitor of oxidative decarboxylation, inhibited the metabolism of ricinoleate at the C₈-intermediate level, according to the accumulation of 2-oxooctanoate and the stoichiometry of concomitant NADH formation. Arsenite completely inhibited the metabolism of 2-oxooctanoate. It is concluded that the barrier caused by the hydroxy group of ricinoleic acid and prevention of β-oxidation at the C₈-intermediate level, is circumvented by an α-hydroxy acid oxidase reaction followed by an oxidative decarboxylation allowing return to the β-oxidation track.     

Effect of ciprofibrate on the activation and oxidation of very long chain fatty acids

The effect of ciprofibrate, a hypolipidemic drug, was examined in the metabolism of palmitic (C_16:0) and lignoceric (C_24:0) acids in rat liver. Ciprofibrate is a peroxisomal proliferating drug which increases the number of peroxisomes. The palmitoyl-CoA ligase activity in peroxisomes, mitochondria and microsomes from ciprofibrate treated liver was 3.2, 1.9 and 1.5-fold higher respectively and the activity for oxidation of palmitic acid in peroxisomes and mitochondria was 8.5 and 2.3-fold higher respectively. Similarly, ciprofibrate had a higher effect on the metabolism of lignoceric acid. Treatment with ciprofibrate increased lignoceroyl-CoA ligase activity in peroxisomes, mitochondria and microsomes by 5.3, 3.3 and 2.3-fold respectively and that of oxidation of lignoceric acid was increased in peroxisomes and mitochondria by 13.4 and 2.3-fold respectively. The peroxisomal rates of oxidation of palmitic acid (8.5-fold) and lignoceric acid (13.4-fold) were increased to a different degree by ciprofibrate treatment. This differential effect of ciprofibrate suggests that different enzymes may be responsible for the oxidation of fatty acids of different chain length, at least at one or more step(s) of the peroxisomal fatty acid -oxidation pathway.     

A Fox2-Dependent Fatty Acid ?-Oxidation Pathway Coexists Both in Peroxisomes and Mitochondria of the Ascomycete Yeast Candida lusitaniae

It is generally admitted that the ascomycete yeasts of the subphylum Saccharomycotina possess a single fatty acid ß-oxidation pathway located exclusively in peroxisomes, and that they lost mitochondrial ß-oxidation early during evolution. In this work, we showed that mutants of the opportunistic pathogenic yeast Candida lusitaniae which lack the multifunctional enzyme Fox2p, a key enzyme of the ß-oxidation pathway, were still able to grow on fatty acids as the sole carbon source, suggesting that C. lusitaniae harbored an alternative pathway for fatty acid catabolism. By assaying ¹⁴C_α-palmitoyl-CoA consumption, we demonstrated that fatty acid catabolism takes place in both peroxisomal and mitochondrial subcellular fractions. We then observed that a fox2Δ null mutant was unable to catabolize fatty acids in the mitochondrial fraction, thus indicating that the mitochondrial pathway was Fox2p-dependent. This finding was confirmed by the immunodetection of Fox2p in protein extracts obtained from purified peroxisomal and mitochondrial fractions. Finally, immunoelectron microscopy provided evidence that Fox2p was localized in both peroxisomes and mitochondria. This work constitutes the first demonstration of the existence of a Fox2p-dependent mitochondrial β-oxidation pathway in an ascomycetous yeast, C. lusitaniae. It also points to the existence of an alternative fatty acid catabolism pathway, probably located in peroxisomes, and functioning in a Fox2p-independent manner.     

The emerging role of acyl-CoA thioesterases and acyltransferases in regulating peroxisomal lipid metabolism

The importance of peroxisomes in lipid metabolism is now well established and peroxisomes contain approximately 60 enzymes involved in these lipid metabolic pathways. Several acyl-CoA thioesterase enzymes (ACOTs) have been identified in peroxisomes that catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. A number of acyltransferase enzymes, which are structurally and functionally related to ACOTs, have also been identified in peroxisomes, which conjugate (or amidate) bile acid-CoAs and acyl-CoAs to amino acids, resulting in the production of amidated bile acids and fatty acids. The function of ACOTs is to act as auxiliary enzymes in the α- and β-oxidation of various lipids in peroxisomes. Human peroxisomes contain at least two ACOTs (ACOT4 and ACOT8) whereas mouse peroxisomes contain six ACOTs (ACOT3, 4, 5, 6, 8 and 12). Similarly, human peroxisomes contain one bile acid-CoA:amino acid N-acyltransferase (BAAT), whereas mouse peroxisomes contain three acyltransferases (BAAT and acyl-CoA:amino acid N-acyltransferases 1 and 2: ACNAT1 and ACNAT2). This review will focus on the human and mouse peroxisomal ACOT and acyltransferase enzymes identified to date and discuss their cellular localizations, emerging structural information and functions as auxiliary enzymes in peroxisomal metabolic pathways. This article is part of a Special Issue entitled: Metabolic Functions and Biogenesis of Peroxisomes in Health and Disease.     

Phytanic acid alpha-oxidation,new insights into an old problem: a review

Phytanic acid (3,7,10,14-tetramethylhexadecanoic acid) is a branched-chain fatty acid which is known to accumulate in a number of different genetic diseases including Refsum disease. Due to the presence of a methyl-group at the 3-position, phytanic acid and other 3-methyl fatty acids can not undergo beta-oxidation but are first subjected to fatty acid alpha-oxidation in which the terminal carboxyl-group is released as CO(2). The mechanism of alpha-oxidation has long remained obscure but has been resolved in recent years. Furthermore, peroxisomes have been found to play an indispensable role in fatty acid alpha-oxidation, and the complete alpha-oxidation machinery is probably localized in peroxisomes. This Review describes the current state of knowledge about fatty acid alpha-oxidation in mammals with particular emphasis on the mechanism involved and the enzymology of the pathway.     

A role for the human peroxisomal half-transporter ABCD3 in the oxidation of dicarboxylic acids

Peroxisomes play a major role in human cellular lipid metabolism, including fatty acid β-oxidation. Free fatty acids (FFAs) can enter peroxisomes through passive diffusion or by means of ATP binding cassette (ABC) transporters, including HsABCD1 (ALDP, adrenoleukodystrophy protein), HsABCD2 (ALDRP) and HsABCD3 (PMP70). The physiological functions of the different peroxisomal half-ABCD transporters have not been fully determined yet, but there are clear indications that both HsABCD1 and HsABCD2 are required for the breakdown of fatty acids in peroxisomes. Here we report that the phenotype of the pxa1/pxa2Δ yeast mutant, i.e. impaired oxidation of oleic acid, cannot only be partially rescued by HsABCD1, HsABCD2, but also by HsABCD3, which indicates that each peroxisomal half-transporter can function as homodimer. Fatty acid oxidation measurements using various fatty acids revealed that although the substrate specificities of HsABCD1, HsABCD2 and HsABCD3 are overlapping, they have distinctive preferences. Indeed, most hydrophobic C24:0 and C26:0 fatty acids are preferentially transported by HsABCD1, C22:0 and C22:6 by HsABCD2 and most hydrophilic substrates like long-chain unsaturated-, long branched-chain- and long-chain dicarboxylic fatty acids by HsABCD3. All these fatty acids are most likely transported as CoA esters. We postulate a role for human ABCD3 in the oxidation of dicarboxylic acids and a role in buffering fatty acids that are overflowing from the mitochondrial β-oxidation system.     

Indentification of β-oxidation enzymes among peroxisomal polypeptides: Increase in Coomassie blue-stainable protein after clofibrate treatment

Peroxisomes contain enzymes catalyzing the β-oxidation of fatty acids, which have been purified and partially characterized. Hypolipidemic drugs, including clofibrate, cause a marked proliferation of peroxisomes and a striking increase in the activity of their β-oxidation system. We have compared by sodium dodecyl sulfate—polyacrylamide gel electrophoresis the polypeptide patterns of normal and clofibrate-induced peroxisomes and the purified β-oxidation enzymes. The data allow a tentative identification of the β-oxidation enzymes among the peroxisomal polypeptides; these enzymes constitute only a small part of the protein of normal peroxisomes. A subset of peroxisomal polypeptides, including the β-oxidation enzymes, is preferentially increased by clofibrate.     

Bovine seminal ribonuclease: Non-hyperbolic kinetics in the second reaction step

Peroxisomes contain enzymes catalyzing the β-oxidation of fatty acids, which have been purified and partially characterized. Hypolipidemic drugs, including clofibrate, cause a marked proliferation of peroxisomes and a striking increase in the activity of their β-oxidation system. We have compared by sodium dodecyl sulfate—polyacrylamide gel electrophoresis the polypeptide patterns of normal and clofibrate-induced peroxisomes and the purified β-oxidation enzymes. The data allow a tentative identification of the β-oxidation enzymes among the peroxisomal polypeptides; these enzymes constitute only a small part of the protein of normal peroxisomes. A subset of peroxisomal polypeptides, including the β-oxidation enzymes, is preferentially increased by clofibrate.     

Mechanisms for elongation in the biosynthesis of fatty acid components of epi-cuticular waxes

It is known that branched-chain amino acids can serve as precursors to iso- and anteiso-branched components of epi-cuticular waxes. Keto acid deamination products of Val, Leu and Ile are thought to serve as primers which are elongated by fatty acid synthase. However, the origin of elongation carbons has not been studied directly. Nor has the mechanism for formation of odd-carbon-length, straight- or branched-chain, cuticular ester fatty acids or free odd-carbon-length, straight fatty acid components of waxes been characterized. It is not known that α-oxidation of even-length precursors or elongation of odd-length primers is involved in these cases. Here, we present evidence which substantiates the expectation that elongation of branched as well as straight-chain precursors to wax ester acids occurs by fatty acid synthase catalyzed by addition of two carbon units via acetate. Also, we present evidence which indicates that odd-carbon-length acids can result from elongation of odd-carbon-length primers (at least branched), rather than even-length acids shortened by α-oxidation.     

Impairment of 8-iso-PGF2ALPHA isoprostane metabolism by dietary conjugated linoleic acid (CLA)

8-iso-PGF_2α isoprostane (IP) is one of the most-used markers of lipid peroxidation in experimental models and humans. After its formation, it is promptly metabolized to 2,3 dinor (DIN) in peroxisomes.Conjugated linoleic acid (CLA) is preferentially β-oxidized in peroxisomes which may compete with IP, and thereby may affect its metabolism.In order to verify whether CLA is able to influence IP formation and/or metabolism and to explain the mechanism, we challenged rats supplemented with CLA or with triolein (as a control fatty acid), with a single dose of carbon tetrachloride (CCl₄) or of bacterial lipopolysaccharide (LPS). The results showed that IP and its precursor arachidonic acid hydroperoxide, as well as malondialdheyde (MDA), increase significantly in the liver of rats challenged with CCl₄, irrespective of the diet, while in LPS-treated rats only nitrites in liver and isoprostane in plasma increase. On the other hand, the peroxisomal β-oxidation products of IP, the DIN, is significantly lower in the CLA group with respect to control and triolein groups.To further investigate whether this is due to competition between CLA and IP at the cellular level, we incubated human fibroblasts from healthy subjects or patients with adrenoleukodystrophy (ALD), with CLA and/or commercially available IP. The rationale of this approach is based on the deficient peroxisomal β-oxidation of fibroblasts from ALD patients, leading to a reduced formation of DIN. In both normal and ALD cells, the presence of CLA significantly inhibits the formation of DIN from IP.We may conclude that both in vitro and in vivo studies strongly suggest that CLA may impair IP catabolism in peroxisomes. Consequently an increase of IP, as a sole result of CLA intake, cannot be considered as a marker of lipid peroxidation.     

Peroxisome-generated succinate induces lipid accumulation and oxidative stress in the kidneys of diabetic mice

Diabetes normally causes lipid accumulation and oxidative stress in the kidneys, which plays a critical role in the onset of diabetic nephropathy; however, the mechanism by which dysregulated fatty acid metabolism increases lipid and reactive oxygen species (ROS) formation in the diabetic kidney is not clear. As succinate is remarkably increased in the diabetic kidney, and accumulation of succinate suppresses mitochondrial fatty acid oxidation and increases ROS formation, we hypothesized that succinate might play a role in inducing lipid and ROS accumulation in the diabetic kidney. Here we demonstrate a novel mechanism by which diabetes induces lipid and ROS accumulation in the kidney of diabetic animals. We show that enhanced oxidation of dicarboxylic acids by peroxisomes leads to lipid and ROS accumulation in the kidney of diabetic mice via the metabolite succinate. Furthermore, specific suppression of peroxisomal β-oxidation improved diabetes-induced nephropathy by reducing succinate generation and attenuating lipid and ROS accumulation in the kidneys of the diabetic mice. We suggest that peroxisome-generated succinate acts as a pathological molecule inducing lipid and ROS accumulation in kidney, and that specifically targeting peroxisomal β-oxidation might be an effective strategy in treating diabetic nephropathy and related metabolic disorders.     

Construction of a series of intermediates in the β-oxidation pathway from THA to EPA via DHA in free acid form

β-Oxidation of most fatty acids occurs in the mitochondria. However, β-oxidation for ω-3 polyunsaturated fatty acids (PUFAs) is distinct from abundant fatty acids and occurs in the peroxisomes. Since little is known about peroxisomal β-oxidation, here we report the synthesis of proposed intermediates of ω-3 PUFA β-oxidation steps in free fatty acid form having a conjugated double bond, a β-hydroxyl group, a β-olefin and a β-carbonyl group. These fatty acids can serve as authentic samples for biological experiments.     

过氧化物酶体脂肪酸β氧化

除线粒体外,过氧化物酶体也是真核细胞脂肪酸β氧化分解的重要部位．过氧化物酶体β氧化过程包括氧化、加水、脱氢和硫解4步反应,主要参与极长链、支链脂肪酸等的分解．近年关于过氧化物酶体β氧化的研究活跃,在代谢途径及功能等方面有了新的认识,尤其在对相关代谢酶的研究中取得了较大进展．本文就过氧化物酶体β氧化相关进展作一综述．