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.     

Very long chain fatty acid beta-oxidation by subcellular fractions of normal and Zellweger syndrome skin fibroblasts

Very long chain fatty acid (VLCFA) beta-oxidation was compared in homogenates and subcellular fractions of cultured skin fibroblasts from normal individuals and from Zellweger patients who show greatly reduced numbers of peroxisomes in their tissues. beta-Oxidation of lignoceric (C24:0) acid was greatly reduced compared to controls in the homogenates and the subcellular fractions of Zellweger fibroblasts. The specific activity of C24:0 acid beta-oxidation was highest in the crude peroxisomal pellets of control fibroblasts. Fractionation of the crude mitochondrial and the crude peroxisomal pellets on Percoll density gradients revealed that the C24:0 acid oxidation was carried out entirely by peroxisomes, and the peroxisomal beta-oxidation activity was missing in Zellweger fibroblasts. In contrast to the beta-oxidation of C24:0 acid, the beta-oxidation of C24:0 CoA was observed in both mitochondria and peroxisomes. We postulate that a very long chain fatty acyl CoA (VLCFA CoA) synthetase, which is different from long chain fatty acyl CoA synthetase, is required for the effective conversion of C24:0 acid to C24:0 CoA. The VLCFA CoA synthetase appears to be absent from the mitochondrial membrane but present in the peroxisomal membrane.     

Adrenoleukodystrophy: impaired oxidation of fatty acids due to peroxisomal lignoceroyl-CoA ligase deficiency

Very long chain fatty acids (lignoceric acid) are oxidized in peroxisomes and pathognomonic amounts of these fatty acids accumulate in X-adrenoleukodystrophy (X-ALD) due to a defect in their oxidation. However, in cellular homogenates from X-ALD cells, lignoceric acid is oxidized at a rate of 38% of control cells. Therefore, to identify the source of this residual activity we raised antibody to palmitoyl-CoA ligase and examined its effect on the activation and oxidation of palmitic and lignoceric acids in isolated peroxisomes from control and X-ALD fibroblasts. The normalization of peroxisomal lignoceric acid oxidation in the presence of exogenously added acyl-CoA ligases and along with the complete inhibition of activation and oxidation of palmitic and lignoceric acids in peroxisomes from X-ALD by antibody to palmitoyl-CoA ligase provides direct evidence that lignoceroyl-CoA ligase is deficient in X-ALD and demonstrates that the residual activity for the oxidation of lignoceric acid was derived from the activation of lignoceric acid by peroxisomal palmitoyl-CoA ligase. This antibody inhibited the activation and oxidation of palmitic acid but had little effect on these activities for lignoceric acid in peroxisomes from control cells. Furthermore, these data provide evidence that peroxisomal palmitoyl-CoA and lignoceroyl-CoA ligases are two different enzymes.     

Peroxisomes and peroxisomal functions in muscle. Studies with muscle cells from controls and a patient with the cerebro-hepato-renal (Zellweger) syndrome

In the present study we investigated peroxisomal functions in cultured human muscle cells from control subjects and from a patient with the Zellweger syndrome, a genetic disease characterized by the absence of morphologically distinguishable peroxisomes in liver and kidney. In homogenates of cultured muscle cells from control subjects, catalase is contained within subcellular particles, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity is present and palmitoyl-CoA can be oxidized by a peroxisomal beta-oxidative pathway; these findings are indicative of the presence of peroxisomes in the cells. In homogenates of cultured muscle cells from the patient with the Zellweger syndrome, acyl-CoA:dihydroxyacetonephosphate acyltransferase activity was deficient, peroxisomal beta-oxidation of palmitoyl-CoA was impaired and catalase was not particle-bound. These findings indicate that functional peroxisomes are absent in muscle from patients with the Zellweger syndrome. We conclude that cultured human muscle cells can be used as a model system to study peroxisomal functions in muscle and the consequences for this tissue of a generalized dysfunction of peroxisomes.     

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.     

Characterization of the 70-kDa peroxisomal membrane protein, an ATP binding cassette transporter

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major components of rat liver peroxisomal membranes and belongs to a superfamily of proteins known as ATP binding cassette transporters. PMP70 is markedly induced by administration of hypolipidemic agents in parallel with peroxisome proliferation and induction of peroxisomal fatty acid beta-oxidation enzymes. To characterize the role of PMP70 in biogenesis and function of peroxisomes, we transfected the cDNA of rat PMP70 into Chinese hamster ovary cells and established cell lines stably expressing PMP70. The content of PMP70 in the transfectants increased about 5-fold when compared with the control cells. A subcellular fractionation study showed that overexpressed PMP70 was enriched in peroxisomes. This peroxisomal localization was confirmed by immunofluorescence and immunoelectron microscopy. The number of immuno-gold particles corresponding to PMP70 on peroxisomes increased markedly in the transfectants, but the size and the number of peroxisomes were essentially the same in both the transfectants and the control cells. beta-Oxidation of palmitic acid increased about 2-3-fold in the transfectants, whereas the oxidation of lignoceric acid decreased about 30-40%. When intact peroxisomes prepared from both the cell lines were incubated with palmitoyl-CoA, oxidation was stimulated with ATP, but the degree of the stimulation was higher in the transfectants than in the control cells. Furthermore, we established three Chinese hamster ovary cell lines stably expressing mutant PMP70. In these cells, beta-oxidation of palmitic acid decreased markedly. These results suggest that PMP70 is involved in metabolic transport of long chain acyl-CoA across peroxisomal membranes and that increase of PMP70 is not associated with proliferation of peroxisomes.     

The involvement of fatty acid binding protein in peroxisomal fatty acid oxidation

Rat liver fatty acid-binding protein (FABP) can function as a fatty acid donor protein for both peroxisomal and mitochondrial fatty acid oxidation, since 14C-labeled palmitic acid bound to FABP is oxidized by both organelles. FABP is, however, not detected in peroxisomes and mitochondria of rat liver by ELISA. Acyl-CoA oxidase activity of isolated peroxisomes was not changed by addition of FABP or flavaspidic acid, an inhibitor of fatty acid binding to FABP, nor by disruption of the peroxisomal membranes. These data indicate that FABP may transfer fatty acids to peroxisomes, but is not involved in the transport of acyl-CoA through the peroxisomal membrane.     

A comparative study of straight chain and branched chain fatty acid oxidation in skin fibroblasts from patients with peroxisomal disorders.

The beta-oxidation of stearic acid and of alpha- and gamma-methyl isoprenoid-derived fatty acids (pristanic and tetramethylheptadecanoic acids, respectively) was investigated in normal skin fibroblasts and in fibroblasts from patients with inherited defects in peroxisomal biogenesis. Stearic acid beta-oxidation by normal fibroblast homogenates was several-fold greater compared to the oxidation of the two branched chain fatty acids. The effect of phosphatidylcholine, alpha-cyclodextrin, and bovine serum albumin on the three activities suggests that different enzymes are involved in the beta-oxidation of straight chain and branched chain fatty acids. Homogenates of fibroblasts from patients with a deficiency in peroxisomes (Zellweger syndrome and infantile Refsum's disease) showed a normal ability to beta-oxidize stearic acid, but the oxidation of pristanic and tetramethylheptadecanoic acid was decreased. Concomitantly, 14CO2 production from the branched chain fatty acids by Zellweger fibroblasts in culture (but not from stearic acid) was greatly diminished. The Zellweger fibroblasts also showed a marked reduction in the amount of water-soluble metabolites from the radiolabeled branched chain fatty acids that are released into the culture medium. The data presented indicate that the oxidation of alpha- and gamma-methyl isoprenoid-derived fatty acids takes place largely in peroxisomes in human skin fibroblasts.     

Transport of fatty acids into human and rat peroxisomes. Differential transport of palmitic and lignoceric acids and its implication to X-adrenoleukodystrophy.

The different topology of palmitoyl-CoA ligase (on the cytoplasmic surface) and of lignoceroyl-CoA ligase (on the luminal surface) in peroxisomal membranes suggests that these fatty acids may be transported in different form through the peroxisomal membrane (Lazo, O., Contreras, M., and Singh, I. (1990) Biochemistry 29, 3981-3986), and this differential transport may account for deficient oxidation of lignoceric acid in X-adrenoleukodystrophy (X-ALD) (Singh, I., Moser, A. B., Goldfisher, S., and Moser, H. W. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4203-4207). To define the transport mechanism for these fatty acids through the peroxisomal membrane and its possible implication to lignoceric acid metabolism in X-ALD, we examined cofactors and energy requirements for the transport of palmitic and lignoceric acids in isolated peroxisomes from rat liver and peroxisomes isolated from X-ALD and control fibroblasts. The similar rates of transport of palmitoyl-CoA (87.6 +/- 6.3 nmol/h/mg protein) and palmitic acid in the fatty acid activating conditions (83.4 +/- 5.1 nmol/h/mg protein) and lack of transport of palmitic acid (4% of palmitoyl-CoA transport) when ATP and/or CoASH were removed or substituted by alpha,beta-methyleneadenosine-5'-triphosphate (AMPCPOP) and/or desulfoCoA-agarose from assay medium clearly demonstrate that transport of palmitic acid requires prior synthesis of palmitoyl-CoA by palmitoyl-CoA ligase on the cytoplasmic surface of peroxisomes. The 10-fold higher rate of transport of lignoceric acid (5.3 +/- 0.6 nmol/h/mg protein) as compared with lignoceroyl-CoA (0.41 +/- 0.11 nmol/h/mg protein) and lack of inhibition of transport of lignoceric acid when ATP and/or CoASH were removed or substituted with AMPCPOP or desulfoCoA-agarose suggest that lignoceric acid is transported through the peroxisomal membrane as such. Moreover, the lack of effect of removal of ATP or substitution with AMPOPCP (a nonhydrolyzable substrate) demonstrates that the translocation of palmitoyl-CoA and lignoceric acid across peroxisomal membrane does not require energy. The transport, activation, and oxidation of palmitic acid are normal in peroxisomes from X-ALD. The deficient lignoceroyl-CoA ligase (13% of control) and oxidation of lignoceric acid (10% of control) as compared with normal transport of lignoceric acid into peroxisomes from X-ALD clearly demonstrates that pathogenomonic accumulation of very long chain fatty acids (greater than C22) in X-ALD is due to the deficiency of peroxisomal lignoceroyl-CoA ligase activity.     

Chinese hamster ovary cell mutants defective in peroxisome biogenesis. Comparison to Zellweger syndrome

We have previously reported the isolation of Chinese hamster ovary (CHO) cell mutants that are defective in the biosynthesis of plasmalogens, deficient in at least two peroxisomal enzymes (dihydroxyacetonephosphate (DHAP) acyltransferase and alkyl-DHAP synthase), and in which catalase is not found within peroxisomes (Zoeller, R. A., and Raetz, C. R. H. (1986) Proc. Natl. Acad. Sci. U.S.A. 83, 5170). We now provide further evidence that three such strains are more generally defective in peroxisome biogenesis. Electron microscopic cytochemistry revealed that the mutants did not contain recognizable peroxisomes. However, immunofluorescence microscopy using an antibody directed against peroxisomal integral membrane proteins revealed the presence of peroxisomal membrane ghosts resembling those seen in cells of patients suffering from one of the human peroxisomal disorders, Zellweger syndrome. Immunoblot analyses, using antibodies specific for peroxisomal matrix proteins, demonstrated deficiencies of peroxisomal proteins in the mutant CHO cells that were similar to those in Zellweger syndrome. Fusion of a CHO mutant with fibroblasts obtained from Zellweger patients resulted in restoration of peroxisomal dihydroxyacetonephosphate acyltransferase and peroxisomal acyl-coenzyme A oxidation activities. The hybrid cells also regained the ability to synthesize plasmenylethanolamine. Moreover, normal peroxisomes were seen by immunofluorescence in the hybrid cells. These results indicate that the hybrid cells have recovered the ability to assemble peroxisomes and that, although the mutant CHO cells are biochemically and morphologically very similar to cells from patients with Zellweger syndrome, the genetic lesions are distinct. Our somatic cell mutants should be useful in identifying factors and genes involved in peroxisome biogenesis and may aid the genetic categorization of the various peroxisomal disorders.     

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

Summary Genetic heterogeneity in peroxisome-deficient disorders, including Zellweger's cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1^-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.     

Complementation in Zellweger syndrome: biochemical analysis of newly generated peroxisomes.

The Zellweger syndrome is characterized by a defect which results in the abnormal biogenesis of peroxisomes. As a consequence, metabolic activities associated with peroxisomes such as the oxidation of very long chain fatty acids, the synthesis of plasmalogens, and the catabolism of phytanic and pipecolic acids are impaired. Since this disorder is genetically heterogeneous and several complementation groups are known, we were able to study the normalization of peroxisomal activity during the process of complementation. The restoration of catalase and dihydroxyacetone phosphate acyltransferase activities peaked within 3-4 days postfusion while the oxidation of lignoceric acid was much delayed (7-8 days). Electron microscopy indicated that by 6 days following hybridization, peroxisome structure and density in heterokaryons was comparable to normal control cells. The heterogenous biochemical response during peroxisome normalization could be due to several factors including a possible requirement for restoration of peroxisomal structural integrity for maximum activation of certain metabolic pathways.     

Mitochondria are targets for peroxisome-derived oxidative stress in cultured mammalian cells

Many cellular processes are driven by spatially and temporally regulated redox-dependent signaling events. Although mounting evidence indicates that organelles such as the endoplasmic reticulum and mitochondria can function as signaling platforms for oxidative stress-regulated pathways, little is known about the role of peroxisomes in these processes. In this study, we employ targeted variants of the genetically encoded photosensitizer KillerRed to gain a better insight into the interplay between peroxisomes and cellular oxidative stress. We show that the phototoxic effects of peroxisomal KillerRed induce mitochondria-mediated cell death and that this process can be counteracted by targeted overexpression of a select set of antioxidant enzymes, including peroxisomal glutathione S-transferase kappa 1, superoxide dismutase 1, and mitochondrial catalase. We also present evidence that peroxisomal disease cell lines deficient in plasmalogen biosynthesis or peroxisome assembly are more sensitive to KillerRed-induced oxidative stress than control cells. Collectively, these findings confirm and extend previous observations suggesting that disturbances in peroxisomal redox control and metabolism can sensitize cells to oxidative stress. In addition, they lend strong support to the ideas that peroxisomes and mitochondria share a redox-sensitive relationship and that the redox communication between these organelles is not only mediated by diffusion of reactive oxygen species from one compartment to the other. Finally, these findings indicate that mitochondria may act as dynamic receivers, integrators, and transmitters of peroxisome-derived mediators of oxidative stress, and this may have profound implications for our views on cellular aging and age-related diseases.     

缺氧复氧时肥大心肌细胞凋亡及其与能量代谢途径转换的关系

心肌细胞凋亡是心肌肥大向心力衰竭转化的重要机制,因此,抑制肥大心肌细胞凋亡可能是防治心力衰竭的有 效药物靶点之一。本研究以0．1μmol／L血管紧张素Ⅱ和1 μmol／L去甲肾上腺素刺激培养心肌细胞,复制心肌细胞肥大模 型,用三气孵箱培养。缺氧条件是95％N2和5％CO2,控制氧分压低于5 mmHg以下,8 h后常氧培养,液闪计数法测 定丙酮酸脱氢酶(pyruvate dehydrogenase,PDH)和肉碱脂酰转移酶-1(carnitine palmitoyltransferase 1,CPT-1)活性,糖氧化、 糖酵解、脂肪酸氧化率,以及细胞凋亡百分率,分析肥大心肌细胞能量代谢变化与细胞凋亡间的关系。结果如下:(1)与 常氧培养比较,缺氧8 h后,活化型丙酮酸脱氢酶(PDHa)和CPT-1活性均有显著下降,但复氧早期肥大心肌细胞PDHa活 性有轻度进一步降低(P>0．05),而CPT-1活性却较快恢复。(2)缺氧时,正常和肥大心肌细胞葡萄糖氧化代谢率均有降低[分 别下降(16±0．9)％、(48±1．1)％];复氧时,正常心肌细胞糖氧化代谢较快恢复,而肥大心肌细胞在复氧早期,糖氧化 率进一步降低,此后才逐渐恢复。(3)在缺氧时,肥大心肌细胞糖酵解率仅轻度下降,但在复氧后糖酵解率迅速升高,呈 爆发样达峰值后又逐渐恢复到缺氧前水平。(4)肥大心肌细胞在缺氧后脂肪酸代谢明显降低,但复氧后脂肪酸代谢呈爆发式 上升,并大大高于缺血前的代谢水平。(5)缺氧时肥大心肌细胞凋亡率即显著增加,在复氧早期细胞凋亡率继续大幅度上 升,此后逐渐减少。(6)预先用二氯乙酸处理肥大心肌细胞,可显著逆转缺氧复氧导致的细胞糖氧化受抑、糖酵解和脂肪 酸代谢活化,同时,抑制细胞凋亡的发生。上述结果提示,缺氧复氧后的肥大心肌细胞能量代谢途径转换是导致细胞凋 亡的重要原因。     

L-pipecolaturia in Zellweger syndrome

Purified rat peroxisomes have been reported to oxidize D-pipecolic acid and the pipecolaturia of Zellweger syndrome has been attributed to the absence of peroxisomes. The logical consequences would be excesses of D-pipecolic acid in the urine of patients with Zellweger syndrome. The urine of two patients with Zellweger syndrome has been analyzed by complexing the pipecolic acid to copper-aspartame to separate the L- and D-isomers. L-Pipecolic acid constituted 100% and 78% of the total pipecolic acid in the two urines. The possibility of preferential retention of D-pipecolic acid was excluded by measuring renal excretion in two control subjects following administration of each isomer. The clearance of L-pipecolic acid was 1.1 and 0.2 ml/min and of D-pipecolic acid was 36.4 and 43.6 ml/min. These results do not support the contention that the pipecolaturia of Zellweger syndrome is the direct result of peroxisomal deficiencies.     

Abnormality in Translational Regulation of Catalase Expression in Disorders of Peroxisomal Biogenesis

Abstract: Peroxisomal disorders are a newly described group of inherited neurological diseases. In disorders of peroxisomal biogenesis, e.g., Zellweger syndrome, owing to the lack of peroxisomes, catalase, a peroxisomal enzyme, is found to be present in the cytoplasm instead. We observed higher catalase activity (7.59 ± 0.41 mU/mg of protein) in cultured skin fibroblasts from Zellweger patients than in control fibroblasts (4.45 ± 0.29 mU/mg of protein). Moreover, we also found that the majority of the catalase in Zellweger cells was present in the inactive form. The specific activities following reactivation in Zellweger and control cells were 12.1 and 4.9 mU/mg of protein, respectively. To understand the molecular basis of higher levels of catalase in Zellweger than control cells, we examined the rate of synthesis and turnover of catalase and levels of catalase mRNA and protein levels in Zellweger cells as compared with control cells. The initial rates of synthesis of catalase in Zellweger (1.68 ± 0.15 mU/mg of protein) and control (1.51 ± 0.14 mU/mg of protein) cells were similar. The rates of turnover of catalase in Zellweger (t_1/2 = 47 ± 8 h) and control (t_1/2 = 49 ± 7 h) were also similar. Consistent with the enzyme activity, the levels of catalase protein were higher in Zellweger cells as compared with control cells. On the other hand, there was no difference in the level of catalase mRNA between control and Zellweger cells. Although the rate of synthesis in Zellweger and control cells were initially similar, it was down-regulated to a lower level at ～72 h of culture in control fibroblasts as compared with Zellweger cells, which continued to synthesize catalase at the same rate up to 5 days in culture. The presence of similar levels of mRNA in control and Zellweger cells and continued synthesis of catalase in Zellweger cells at a higher level as compared with control cells suggest a loss of regulation at the translational level.     

Cytochrome P-450 2E1 in Rat Liver Peroxisomes: Downregulation by Ischemia/Reperfusion-Induced Oxidative Stress

Cytochrome P-450 containing enzymes, known to be present in the endoplasmic reticulum and mitochondria, catalyze the oxidation of various compounds. In this study we have used highly purified peroxisomes (>95%) to provide evidence by analytical cell fractionation, enzyme activity, Western blot, and immunocytochemical analysis that cytochrome P-450 2E1 (Cyp 2E1) is present in peroxisomes. Similar specific activities of aniline hydroxylase, a Cyp 2E1-dependent enzyme, in purified peroxisomes (0.72 ± 0.03 nmol/min/mg protein) and microsomes (0.58 ± 0.03 nmol/min/mg protein) supports the conclusion that peroxisomes contain significant amount of Cyp 2E1. This peroxisomal Cyp 2E1 was also induced in acetone-treated rat liver. The status of microsomal and peroxisomal Cyp 2E1 was also examined following ischemia/reperfusion-induced oxidative stress. Ischemia alone had no effect; however, reperfusion following ischemia resulted in decrease in Cyp 2E1 both in microsomes and peroxisomes. This demonstration of cytochrome P-450 2E1 in peroxisomes and its downregulation during ischemia/reperfusion describes a new role for this organelle in cytochrome P-450 related cellular metabolism and in oxidative stress induced disease conditions.     

Very long chain fatty acid beta-oxidation by rat liver mitochondria and peroxisomes

Crude mitochondrial fractions were isolated by differential centrifugation of rat liver homogenates. Subfractionation of these fractions on self-generating continuous Percoll gradients resulted in clearcut separation of peroxisomes from mitochondria. Hexacosanoic acid beta-oxidation was present mainly in peroxisomal fractions whereas hexacosanoyl CoA oxidation was present in the mitochondrial as well as in the peroxisomal fractions. The presence of much greater hexacosanoyl CoA synthetase activity in the purified preparations of microsomes and peroxisomes compared to mitochondria, suggests that the synthesis of coenzyme A derivatives of very long chain fatty acids (VLCFA) is limited in mitochondria. We postulate that a specific VLCFA CoA synthetase may be required to effectively convert VLCFA to VLCFA CoA in the cell. This specific synthetase activity is absent from the mitochondrial membrane, but present in the peroxisomal and the microsomal membranes. We postulate that substrate specificity and the subcellular localization of the specific VLCFA CoA synthetase directs and regulates VLCFA oxidation in the cell.     

Cellular oxidation of lignoceric acid is regulated by the subcellular localization of lignoceroyl-CoA ligases

The acyl-CoA ligases convert free fatty acids to acyl-CoA derivatives, and these enzymes have been shown to be present in mitochondria, peroxisomes, and endoplasmic reticulum. Because their activity is obligatory for fatty acid metabolism, it is important to identify their substrate specificities and subcellular distributions to further understand the cellular regulation of these pathways. To define the role of the enzymes and organelles involved in the metabolism of very long chain (VLC) fatty acids, we studied human genetic cell mutants impaired for the metabolism of these molecules. Fibroblast cell lines were derived from patients with X-linked adrenoleukodystrophy (X-ALD) and Zellweger's cerebro-hepato-renal syndrome (CHRS). While peroxisomes are present and morphologically normal in X-ALD, they are either greatly reduced in number or absent in CHRS. Palmitoyl-CoA ligase is known to be present in mitochondria, peroxisomes, and endoplasmic reticulum (microsomes). We found enzyme-dependent formation of lignoceroyl-CoA in these same organelles (specific activities were 0.32 +/- 0.12, 0.86 +/- 0.12, and 0.78 +/- 0.07 nmol/h per mg protein, respectively). However, lignoceroyl-CoA synthesis was inhibited by an antibody to palmitoyl-CoA ligase in isolated mitochondria while it was not inhibited in peroxisomes or endoplasmic reticulum (ER). This suggests that palmitoyl-CoA ligase and lignoceroyl-CoA are different enzymes and that mitochondria lack lignoceroyl-CoA ligase. This conclusion is further supported by data showing that oxidation of lignoceric acid was found almost exclusively in peroxisomes (0.17 nmol/h per mg protein) but was largely absent from mitochondria and the finding that monolayers of CHRS fibroblasts lacking peroxisomes showed a pronounced deficiency in lignoceric acid oxidation in situ (1.8% of control). In spite of the observation that lignoceroyl-CoA ligase activity is present on the cytoplasmic surface of ER, our data indicate that lignoceroyl-CoA synthesized by ER is not available for oxidation in mitochondria. This organelle plays no physiological role in the beta-oxidation of VLC fatty acids. Furthermore, the normal peroxisomal oxidation of lignoceroyl-CoA but deficient oxidation of lignoceric acid in X-ALD cells indicates that cellular VLC fatty acid oxidation is dependent on peroxisomal lignoceroyl-CoA ligase. These studies allow us to propose a model for the subcellular localization of various acyl-CoA ligases and to describe how these enzymes control cellular fatty acid metabolism.     

OCTN3 is a mammalian peroxisomal membrane carnitine transporter

Carnitine is a zwitterion essential for the beta-oxidation of fatty acids. The role of the carnitine system is to maintain homeostasis in the acyl-CoA pools of the cell, keeping the acyl-CoA/CoA pool constant even under conditions of very high acyl-CoA turnover, thereby providing cells with a critical source of free CoA. Carnitine derivatives can be moved across intracellular barriers providing a shuttle mechanism between mitochondria, peroxisomes, and microsomes. We now demonstrate expression and colocalization of mOctn3, the intermediate-affinity carnitine transporter (Km 20 microM), and catalase in murine liver peroxisomes by TEM using immunogold labelled anti-mOctn3 and anti-catalase antibodies. We further demonstrate expression of hOCTN3 in control human cultured skin fibroblasts both by Western blotting and immunostaining analysis using our specific anti-mOctn3 antibody. In contrast with two peroxisomal biogenesis disorders, we show reduced expression of hOCTN3 in human PEX 1 deficient Zellweger fibroblasts in which the uptake of peroxisomal matrix enzymes is impaired but the biosynthesis of peroxisomal membrane proteins is normal, versus a complete absence of hOCTN3 in human PEX 19 deficient Zellweger fibroblasts in which both the uptake of peroxisomal matrix enzymes as well as peroxisomal membranes are deficient. This supports the localization of hOCTN3 to the peroxisomal membrane. Given the impermeability of the peroxisomal membrane and the key role of carnitine in the transport of different chain-shortened products out of peroxisomes, there appears to be a critical need for the intermediate-affinity carnitine/organic cation transporter, OCTN3, on peroxisomal membranes now shown to be expressed in both human and murine peroxisomes. This Octn3 localization is in keeping with the essential role of carnitine in peroxisomal lipid metabolism.