Peroxisomal Fatty Acid Beta-oxidation Research Articles

ACOX1-mediated peroxisomal fatty acid oxidation contributes to metabolic reprogramming and survival in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is still an incurable disease, with many patients developing resistance to conventional and targeted therapies. To better understand the physiology of CLL and facilitate the development of innovative treatment options, we examined specific metabolic features in the tumor CLL B-lymphocytes. We observed metabolic reprogramming, characterized by a high level of mitochondrial oxidative phosphorylation activity, a low glycolytic rate, and the presence of C2- to C6-carnitine end-products revealing an unexpected, essential role for peroxisomal fatty acid beta-oxidation (pFAO). Accordingly, downmodulation of ACOX1 (a rate-limiting pFAO enzyme overexpressed in CLL cells) was enough to shift the CLL cells' metabolism from lipids to a carbon- and amino-acid-based phenotype. Complete blockade of ACOX1 resulted in lipid droplet accumulation and caspase-dependent death in CLL cells, including those from individuals with poor cytogenetic and clinical prognostic factors. In a therapeutic translational approach, ACOX1 inhibition spared non-tumor blood cells from CLL patients but led to the death of circulating, BCR-stimulated CLL B-lymphocytes and CLL B-cells receiving pro-survival stromal signals. Furthermore, a combination of ACOX1 and BTK inhibitors had a synergistic killing effect. Overall, our results highlight a less-studied but essential metabolic pathway in CLL and pave the way towards the development of new, metabolism-based treatment options.

Proteomics reveals defective peroxisomal fatty acid oxidation during the progression of acute kidney injury and repair

Acute kidney injury (AKI) is characterized by a rapid decrease in renal function with high mortality and risk of progression to chronic kidney disease (CKD). Ischemia and reperfusion injury (IRI) is one of the major causes of AKI. However, the cellular and molecular responses of the kidney to IRI are complex and not fully understood. Herein, we conducted unbiased proteomics and bioinformatics analyses in an IRI mouse model on days 3, 7, and 21, and validated the results using IRI, unilateral ureteral obstruction (UUO), and biopsies from patients with AKI or CKD. The results indicated an obvious temporal expression profile of differentially expressed proteins and highlighted impaired lipid metabolism during the progression of AKI to CKD. Acyl-coenzyme A oxidase 1 (Acox1), the first rate-limiting enzyme of peroxisomal fatty acid beta-oxidation, was then selected, and its disturbed expression in the two murine models validated the proteomic findings. Accordingly, Acox1 expression was significantly downregulated in renal biopsies from patients with AKI or CKD, and its expression was negatively correlated with kidney injury score. Furthermore, in contrast to the decreased Acox1 expression, lipid droplet accumulation was remarkably increased in these renal tissues, suggesting dysregulation of fatty acid oxidation. In conclusion, our results suggest that defective peroxisomal fatty acid oxidation might be a common pathological feature in the transition from AKI to CKD, and that Acox1 is a promising intervention target for kidney injury and repair.

The Regulators of Peroxisomal Acyl-Carnitine Shuttle CROT and CRAT Promote Metastasis in Melanoma

Circulating tumor cells (CTCs) are the key link between a primary tumor and distant metastases, but once in the bloodstream loss of adhesion induces cell death. To identify mechanisms relevant for melanoma CTC survival we performed RNAseq and discovered that detached melanoma cells and isolated melanoma CTCs rewire lipid metabolism by up-regulating fatty acid transport and fatty acid beta-oxidation (FAO) related genes. In melanoma patients high expression of fatty acid transporters and FAO enzymes significantly correlates with reduced progression free and overall survival. Amongst the highest expressed regulators in melanoma CTCs were the carnitine-transferases CROT and CRAT, which control the shuttle of peroxisome derived medium-chain fatty acids (MCFAs) towards mitochondria to fuel mitochondrial FAO. Knockdown of CROT or CRAT and short-term treatment with peroxisomal or mitochondrial FAO inhibitors thioridazine or ranolazine suppressed melanoma metastasis in mice. CROT and CRAT depletion could be rescued by MCFA supplementation, indicating that the peroxisomal supply of fatty acids is crucial for the survival of non-adherent melanoma cells. Our study identifies targeting the fatty acid based cross-talk between peroxisomes and mitochondria as a potential therapeutic opportunity to challenge melanoma progression. Moreover, the discovery of the anti-metastatic activity of the FDA-approved drug ranolazine carries translational potential.

Mechanisms Mediating the Regulation of Peroxisomal Fatty Acid Beta-Oxidation by PPARα.

In mammalian cells, two cellular organelles, mitochondria and peroxisomes, share the ability to degrade fatty acid chains. Although each organelle harbors its own fatty acid β-oxidation pathway, a distinct mitochondrial system feeds the oxidative phosphorylation pathway for ATP synthesis. At the same time, the peroxisomal β-oxidation pathway participates in cellular thermogenesis. A scientific milestone in 1965 helped discover the hepatomegaly effect in rat liver by clofibrate, subsequently identified as a peroxisome proliferator in rodents and an activator of the peroxisomal fatty acid β-oxidation pathway. These peroxisome proliferators were later identified as activating ligands of Peroxisome Proliferator-Activated Receptor α (PPARα), cloned in 1990. The ligand-activated heterodimer PPARα/RXRα recognizes a DNA sequence, called PPRE (Peroxisome Proliferator Response Element), corresponding to two half-consensus hexanucleotide motifs, AGGTCA, separated by one nucleotide. Accordingly, the assembled complex containing PPRE/PPARα/RXRα/ligands/Coregulators controls the expression of the genes involved in liver peroxisomal fatty acid β-oxidation. This review mobilizes a considerable number of findings that discuss miscellaneous axes, covering the detailed expression pattern of PPARα in species and tissues, the lessons from several PPARα KO mouse models and the modulation of PPARα function by dietary micronutrients.

Label-Free Proteomics of the Fetal Pancreas Identifies Deficits in the Peroxisome in Rats with Intrauterine Growth Restriction.

Aim The objective of the present study was to identify differentially expressed proteins (DEPs) in the pancreas of a fetus with intrauterine growth restriction (IUGR) and to investigate the molecular mechanisms leading to adulthood diabetes in IUGR. Methods The IUGR rat model was induced by maternal protein malnutrition. The fetal pancreas was collected at embryonic day 20 (E20). Protein was extracted, pooled, and subjected to label-free quantitative proteomic analysis. Bioinformatics analysis (GO and IPA) was performed to define the pathways and networks associated with DEPs. LC-MS results were confirmed by western blotting and/or quantitative PCR (q-PCR). The principal parameters of oxidative stress-superoxide dismutase (Sod) were determined in blood samples of fetal rats. Results A total of 57 DEPs (27 upregulated, 30 downregulated) were identified with a 1.5-fold change threshold and a p value ≤ 0.05 between the IUGR and the control pancreas. Bioinformatics analysis revealed that these proteins play important roles in peroxisome biogenesis and fission, fatty acid beta-oxidation (FAO), mitotic cell cycle, and histone modification. The peroxin Pex14 was downregulated in the IUGR pancreas as confirmed by western blotting and q-PCR. Pmp70, a peroxisomal membrane protein involved in the transport of fatty acids, was upregulated. Hsd17b4 and Acox1/2, which catalyze different steps of peroxisomal FAO, were dysregulated. Sod plasma concentrations in the IUGR fetus were higher than those in the control, suggesting partial compensation for oxidative stress. Multiple DEPs were related to the regulation of the cell cycle, including reduced Cdk1, Mcm2, and Brd4. The histone acetylation regulators Hdac1/2 were downregulated, whereas Sirt1/3 and acetylated H3K56 were increased in the IUGR fetal pancreas. Conclusion The present study identified DEPs in the fetal pancreas of IUGR rats by proteomic analysis. Downregulation of pancreas peroxins and dysregulation of enzymes involved in peroxisomal FAO may impair the biogenesis and function of the peroxisome and may underlie the development of T2 diabetes mellitus in adult IUGR rats. Disorders of cell cycle regulators may induce cell division arrest and lead to smaller islets. The present data provide new insight into the role of the peroxisome in the development of the pancreas and may be valuable in furthering our understanding of the pathogenesis of IUGR-induced diabetes.

RiboTag translatomic profiling of Drosophila oenocytes under aging and induced oxidative stress

BackgroundAging is accompanied with loss of tissue homeostasis and accumulation of cellular damages. As one of the important metabolic centers, liver shows age-related dysregulation of lipid metabolism, impaired detoxification pathway, increased inflammation and oxidative stress response. However, the mechanisms for these age-related changes still remain unclear. In the fruit fly, Drosophila melanogaster, liver-like functions are controlled by two distinct tissues, fat body and oenocytes. Compared to fat body, little is known about how oenocytes age and what are their roles in aging regulation. To characterize age- and stress-regulated gene expression in oenocytes, we performed cell-type-specific ribosome profiling (RiboTag) to examine the impacts of aging and oxidative stress on oenocyte translatome in Drosophila.ResultsWe show that aging and oxidant paraquat significantly increased the levels of reactive oxygen species (ROS) in adult oenocytes of Drosophila, and aged oenocytes exhibited reduced sensitivity to paraquat treatment. Through RiboTag sequencing, we identified 3324 and 949 differentially expressed genes in oenocytes under aging and paraquat treatment, respectively. Aging and paraquat exhibit both shared and distinct regulations on oenocyte translatome. Among all age-regulated genes, oxidative phosphorylation, ribosome, proteasome, fatty acid metabolism, and cytochrome P450 pathways were down-regulated, whereas DNA replication and immune response pathways were up-regulated. In addition, most of the peroxisomal genes were down-regulated in aged oenocytes, including genes involved in peroxisomal biogenesis factors and fatty acid beta-oxidation. Many age-related mRNA translational changes in oenocytes are similar to aged mammalian liver, such as up-regulation of innate immune response and Ras/MAPK signaling pathway and down-regulation of peroxisome and fatty acid metabolism. Furthermore, oenocytes highly expressed genes involving in liver-like processes (e.g., ketogenesis).ConclusionsOur oenocyte-specific translatome analysis identified many genes and pathways that are shared between Drosophila oenocytes and mammalian liver, highlighting the molecular and functional similarities between the two tissues. Many of these genes were altered in both oenocytes and liver during aging. Thus, our translatome analysis provide important genomic resource for future dissection of oenocyte function and its role in lipid metabolism, stress response and aging regulation.

Quantitative Proteomics and Targeted Fatty Acids Analysis Reveal the Damage of Triptolide in Liver and Kidney.

Triptolide (TP), the major active component in Tripterygium wilfordii Hook. f., is widely used for the treatment of rheumatoid arthritis and autoimmune diseases. However, organ toxicity, especially hepatotoxicity and nephrotoxicity, limits its clinical application. To fully understand the mechanism underlying TP toxicity, iTRAQ-based 2D-LC-MS/MS proteomics is used to detect differentially expressed proteins in the livers and kidneys of mice administered the LD50 dose of TP. Functional annotation revealed that multiple pathways are involved in TP toxicity, including acute-phase response signaling, the antigen presentation pathway, FXR/RXR activation, LPS/IL-1-mediated inhibition of RXR function, and EIF2 signaling.Members of the cytochromeP450 protein family that are involved in fatty acid (FA) metabolism, such as CYP2E1, show significant differences in expression among groups. Additionally, the proteomics data suggested that FAs are involved in TP-induced toxicity. FA analysis is conducted using HPLC-MRM to characterize the differences among various groups exposed to TP for different times. It has been found that 20 FAs in the liver show significant differences in abundance among groups, whereas in the kidneys, six FAs show significant differences in abundance. By integrating the proteomic and targeted FA analyses, it has been found that differently expressed proteins and FAs both participate in pathways including cellular lipolytic activity, peroxisomal fatty acid beta-oxidation, and so on. Our data contribute to understanding the mechanisms underlying TP-induced organ toxicity. The results may help to improve the clinical efficacy and safety of TP in the future.

Adrenoleukodystrophy

Introduction: Adrenoleukodystrophy (ALD) is a disease characterized by the accumulation of very long chain fatty acids in tissues throughout the body. The most severely affected tissues are the myelin in the central nervous system, the adrenal cortex and the Leydig cells in the testes. Clinically, ALD is a heterogeneous disorder, presenting with several distinct phenotypes, and no clear pattern of genotype-phenotype correlation. Case report: Patients S.A. 50 years old, in the third year of life was diagnosed with primary adrenal insufficiency. From the age of 45 he feels, headache, dizziness, bends to the right when walking, night incontinence of urine. Talking to himself, the last year goes to the cemetery every day, occasionally crying without control and remembrance. Condition with spinocerebellar ataxia and a left pyramidal defect with incontinence of urine and psychiatric problems required re-examination. In consultation with neurologist at the Department of Neurology, investigations have shown that patient is suffering from X-linked adrenoleukodystrophy with affected central and peripheral nervous system. Adrenoleukodystrophy (ALD) is caused by mutations in ABCD1, a gene located on the X chromosome that codes for ALD, a peroxisomal membrane transporter protein. The exact mechanism of the pathogenesis of the various forms of ALD is not known. it is a disorder of peroxisomal fatty acid beta oxidation which results in the accumulation of very long chain fatty acids in tissues throughout the body. The most severely affected tissues are the myelin in the central nervous system, the adrenal cortex and the Leydig cells in the testes. Clinically, ALD is a heterogeneous disorder, presenting with several distinct phenotypes, and no clear pattern of genotype-phenotype correlation. As an X-linked disorder, ALD presents most commonly in males, however approximately 50% of heterozygote females show some symptoms later in life. Approximately two-thirds of ALD patients will present with the childhood cerebral form of the disease, which is the most severe form. It is characterized by normal development in early childhood, followed by rapid degeneration to a vegetative state. The other forms of ALD vary in terms of onset and clinical severity, ranging from adrenal insufficiency to progressive paraparesis in early adulthood (this form of the disease is typically known as adrenomyeloneuropathy). Conclusion: In our case hypocorticism was the first sign of X-linked adrenoleukodystrophy. In male patients with hypocorticism X-linked adrenoleukodystrophy should always be excluded as one of the possible causes of primary adrenal insufficiency.

Abstract 14653: Prevention of Diet-induced Metabolic Dysregulation, Inflammation and Atherosclerosis in Ldlr-/- Mice by Treatment With the ACL Inhibitor ETC-1002

ETC-1002 (8-hydroxy-2,2,14,14-tetramethylpentadecanedioic acid) is a novel LDL cholesterol-lowering compound. Preclinical studies revealed that ETC-1002 targets the liver where its metabolite, ETC-1002-CoA, inhibits the synthesis of sterols and fatty acids. The primary mechanism of action is through inhibition of hepatic ATP-citrate lyase (ACL). In the current study we tested the ability of ETC-1002 to prevent diet-induced metabolic dysregulation, inflammation and atherosclerosis in LDL receptor deficient (Ldlr-/-) mice. Ldlr-/- mice fed a high-fat high-cholesterol diet (42% kcal fat; 0.2% cholesterol; HFHC) developed hypercholesterolemia, hypertriglyceridemia, hyperglycemia, hyperinsulinemia, fatty liver and obesity over the 12 week study. ETC-1002 supplementation to the HFHC diet at 3, 10 and 30 mg/kg/d significantly attenuated these metabolic abnormalities in a dose-dependent manner. ETC-1002 + HFHC attenuated plasma triglyceride (up to 64%) and plasma cholesterol concentrations (up to 50%), and improved glucose homeostasis compared to HFHC alone as determined by glucose tolerance tests. Adiposity was attenuated up to 48% with treatment. In liver, ETC-1002 dose dependently prevented cholesterol and triglyceride accumulation up to 75% and 90%, respectively. Hepatic gene expression analysis revealed that treatment significantly reduced the expression of the inflammatory genes Tnfa, Il1b, Ccl3 and Nos2 as well as increased the expression of genes involved in mitochondrial (Cpt1a, 1.3-fold) and peroxisomal (Acox1, 4.1-fold) fatty acid beta-oxidation. ETC-1002 reduced aortic cholesterol ester content by up to 63% and robustly attenuated atherosclerotic lesion development in the aortic sinus by 48%. These studies demonstrate that ETC-1002 effectively prevents plasma and tissue lipid elevations and attenuates the onset of inflammation, leading to the prevention of atherosclerotic lesion development in a mouse model of metabolic dysregulation.

Facile Chemiluminescence Assay for Acyl-CoA Oxidase Activity: Fundamentals and Illustrative Examples

Acyl-CoA oxidase (ACO) is the key enzyme that catalyzes the rate-determining step in the peroxisomal beta-oxidation of fatty acids. ACO catalyzes the oxidation of acyl-CoA with molecular oxygen to produce trans-2,3-dehydroacyl-CoA and H2O2. Given the participation of H2O2 in an enhanced chemiluminescent reaction with luminol, we have developed a novel two-step procedure for the quantitation of ACO activity by recording light emission using a chemiluminescence detector. A number of recommendations on standardizing the reaction conditions for the quantitative measurement of ACO-catalyzed reactions are offered. The proposed method is simple and reliable and has been successfully applied in the rat liver assay. The results indicate that ACO activities increased in the liver of type-2 diabetic rats but showed no significant change in the liver of insulin-resistant rat.

Adrenoleukodystrophy and the mitochondrial connection: clues for supplementing Lorenzo’s oil

The 1992 American film ‘Lorenzo’s Oil’ became a euphemism for finding cures for devastating neurological diseases. Lorenzo’s disease was adrenoleukodystrophy, which is caused by defects in peroxisomal fatty acid beta-oxidation. The defect results in the accumulation of very-long chain fatty acids in several organs, particularly the adrenal cortex, testis and the nervous system. Myelinated tissues are most severely affected, underlying the chief clinical symptoms, which can vary from vegetative state in early childhood to paraparesis in adulthood (Kemp et al. , 2012). The genetic cause of adrenoleukodystrophy was identified as recessive mutations in the X-linked ABCD1 gene (Mosser et al. , 1993), which codes for a peroxisomal membrane transporter responsible for shuttling very long chain fatty acids into peroxisomes. Although mitochondria are major sites of lipid catabolism, these very long chain fatty acids (>22 carbons) cannot be metabolized in mitochondria, and need to gain access to peroxisomes to be degraded. Interestingly, not all males with adrenoleukodystrophy develop CNS demyelination and consequent neuroinflammation, suggesting that modifying factors (genetic, epigenetic and environmental) play a role in the CNS symptoms of adrenoleukodystrophy. Although the pathophysiology of adrenoleukodystrophy is not fully understood, individuals with adrenoleukodystrophy show high levels of saturated very long chain fatty acids—mostly cerotic acid (C26:0)—in affected tissues. Free saturated fatty acids are known inducers of apoptosis and this effect increases with chain length (Artwohl et al. , 2009). Accordingly, exposure of oligodendrocytes and astrocytes to C22:0, C24:0 and C26:0 (but not C16:0) fatty acids caused cell …

A mutation in PEX19 causes a severe clinical phenotype in a patient with peroxisomal biogenesis disorder

Peroxisomal biogenesis disorders (PBD) are groups of inherited neurometabolic disorders caused by defects in PEX genes. We report on a female infant, born to a consanguineous parents (first degree cousins), who presented with inactivity, poor sucking, and hypotonia early in the neonatal period. She had subtle dysmorphic features. Liver function tests were impaired with raised liver enzymes, conjugated and unconjugated hyperbilirubinemia. CT of the brain showed diffuse bilateral changes. She developed seizures with an abnormal EEG. Plasma very long chain fatty acid analysis showed high C26:0 levels and increasedC26:0/C22:0 and C24:0/C22:0 ratios, which is consistent with a PBD. Studies in fibroblasts including plasmalogen biosynthesis, peroxisomal fatty acid alfa and beta oxidation confirmed the diagnosis of PBD. Immunofluoresence microscopy revealed the absence of peroxisomes in fibroblasts. The patient was assigned to the PEX19 complementation group. Subsequent mutation analysis of the PEX19 gene revealed homozygosity for a c.320delA frameshift mutation. The patient had a stormy course with multiple admissions to the pediatric intensive care unit with pneumonia, liver impairment, sepsis, and epilepsy. At 1 year of age she developed metabolic acidosis with normal anion gap, proteinuria, aminoaciduria, and glucosuria consistent with a renal tubular defect. Abdominal ultrasound showed multiple gallstones. Other causes of gallstones like haemoglobinopathy were excluded. So far, only two siblings had been reported with mutations in the PEX19 gene. Our patient showed a previously unrecognized association of gallstones and a renal tubular defect with a PBD.

Mutations in the DBP-Deficiency Protein HSD17B4 Cause Ovarian Dysgenesis, Hearing Loss, and Ataxia of Perrault Syndrome

Perrault syndrome is a recessive disorder characterized by ovarian dysgenesis in females, sensorineural deafness in both males and females, and in some patients, neurological manifestations. No genes for Perrault syndrome have heretofore been identified. A small family of mixed European ancestry includes two sisters with well-characterized Perrault syndrome. Whole-exome sequencing of genomic DNA from one of these sisters revealed exactly one gene with two rare functional variants: HSD17B4, which encodes 17beta-hydroxysteroid dehydrogenase type 4 (HSD17B4), also known as D-bifunctional protein (DBP). HSD17B4/DBP is a multifunctional peroxisomal enzyme involved in fatty acid beta-oxidation and steroid metabolism. Both sisters are compound heterozygotes for HSD17B4 c.650A>G (p.Y217C) (maternal allele) and HSB17B4 c.1704T>A (p.Y568X) (paternal allele). The missense mutation is predicted by structural analysis to destabilize the HSD17B4 dehydrogenase domain. The nonsense mutation leads to very low levels of HSD17B4 transcript. Expression of mutant HSD17B4 protein in a compound heterozygote was severely reduced. Mutations in HSD17B4 are known to cause DBP deficiency, an autosomal-recessive disorder of peroxisomal fatty acid beta-oxidation that is generally fatal within the first two years of life. No females with DBP deficiency surviving past puberty have been reported, and ovarian dysgenesis has not previously been associated with this illness. Six other families with Perrault syndrome have wild-type sequences of HSD17B4. These results indicate that Perrault syndrome and DBP deficiency overlap clinically; that Perrault syndrome is genetically heterogeneous; that DBP deficiency may be underdiagnosed; and that whole-exome sequencing can reveal critical genes in small, nonconsanguineous families.

A peroxisomal ABC transporter promotes seed germination by inducing pectin degradation under the control of ABI5

Seed dormancy is essential for most plants to control the timing of germination. In Arabidopsis thaliana, PED3 is a single-copy gene encoding an ATP-binding cassette transporter that is required for peroxisomal fatty acid beta-oxidation. PED3 is involved in the import of several biologically important molecules into the peroxisome, including very-long-chain fatty acids associated with the breakdown of seed-reserve lipids, and precursors of auxin and jasmonic acid. The germination of ped3 mutants is significantly impaired, suggesting that PED3 regulates dormancy and germination. A transcriptome analysis revealed that many genes containing the core motif of the ABA responsive element (ABRE) in their promoter regions, and the ABA insensitive 5 (ABI5) transcription factor that binds to ABRE, are abnormally up-regulated in imbibed ped3 seeds. Expression of polygalacturonase inhibiting proteins (PGIPs) is also up-regulated specifically in ped3 after imbibition. By contrast, the ped3 abi5 double mutant does not show any of these expression patterns. The results indicate that the abi5 mutation normalizes PGIP expression and rescues the impaired germination phenotype of the ped3 mutant. PGIPs are known to act as inhibitors of polygalacturonases that degrade pectin. The amount of PGIP1 transcript regulates the timing of radicle protrusion. The impaired germination of ped3 could also be rescued by removal of pectin from the seed coat using exogenous polygalacturonase or acidic conditions. Overall, our results suggest that PED3, a peroxisomal ABC transporter, promotes seed germination by suppressing PGIPs under the control of ABI5.

Histone acetylation and steroid receptor coactivator expression during clofibrate‐induced rat hepatocarcinogenesis

Peroxisome proliferators (PPs), non-genotoxic rodent carcinogens, cause the induction of the peroxisomal fatty acid beta-oxidation system, including bifunctional enzyme (BE) and 3-ketoacyl-CoA thiolase (TH), in the liver. GST M1 gene is polymorphic in Sprague-Dawley rats, NC- and KS-type. The KS-type rats showed enhanced susceptibility to ethyl-alpha-chlorophenoxyisobutyrate (clofibrate, CF), one of the PPs. The degree of BE induction was higher in the KS-type and preneoplastic foci developed after 6-8 weeks of treatment, whereas no foci developed in the NC-type. In the preset study, factors involved in different BE inducibility were investigated. There were no differences in hepatic peroxisome proliferator-activated receptor (PPAR) alpha levels between them. Among various coactivators for PPARalpha, only steroid receptor coactivator (SRC)-3 level was higher in the KS-type. To investigate the association between PPARalpha and SRC-3 or other proteins, nuclear extracts from CF-treated livers were applied to a PPARalpha column. In the KS-type, 110, 72, and 42 kDa proteins were bound and these were identified as SRC-3, BE, and TH, respectively. EMSA supported the binding of these proteins to PPARalpha associated to the BE enhancer in CF-treated KS-type, but not in the NC-type. Histone H3 acetylation was increased 11-fold in the KS-type by CF treatment but not in the NC-type. As BE and TH are responsible for acetyl-CoA production and SRC-3 possesses a histone acetyltransferase activity, these results suggest that enhanced BE induction in the KS-type livers is due to acetylation-mediated transcriptional activation and epigenetic mechanisms might be involved in CF-induced rat hepatocarcinogenesis.

UP-1.132: The Value and Limits of Alpha-Methyl-Acyl CoA Racemase (AMACR) in the Diagnosis of Prostate Cancer

Alpha-methyl-acyl CoA racemase (AMACR), which has a significant role in the peroxisomal beta-oxidation of branched-chain fatty acids, is a valuable marker in prostate cancer diagnosis, included in the workup algorithm of difficult cases. This study intends to evaluate the value of immunohistochemical determination of AMACR in the diagnosis of prostate cancer.

Peroxisomal and mitochondrial status of two murine oligodendrocytic cell lines (158N, 158JP): potential models for the study of peroxisomal disorders associated with dysmyelination processes

In some neurodegenerative disorders (leukodystrophies) characterized by myelin alterations, the defect of peroxisomal functions on myelin-producing cells (oligodendrocytes) are poorly understood. The development of in vitro models is fundamental to understanding the physiopathogenesis of these diseases. We characterized two immortalized murine oligodendrocyte cell lines: a normal (158N) and a jimpy (158JP) cell line mutated for the proteolipid protein PLP/DM20. Fluorescence microscopy, flow cytometry, and western blotting analysis allow to identify major myelin proteins (PLP colocalizing with mitochondria; myelin basic protein), oligodendrocyte (CNPase and myelin oligodendrocyte glycoprotein), and peroxisomal markers [adrenoleukodystrophy protein, PMP70, acyl-CoA oxidase 1 (ACOX1), l-peroxisomal bifunctional enzyme, and catalase]. Using electron microscopy, peroxisomes were identified in the two cell lines. Gene expression (ATP-binding cassette, Abcd1, Abcd2, Abcd3, and Acox1) involved in peroxisomal transport or beta-oxidation of fatty acids was evaluated using quantitative PCR. 4-phenylbutyrate treatment increases expression of ACOX1, l-peroxisomal bifunctional enzyme, PLP, myelin oligodendrocyte glycoprotein, and CNPase, mainly in 158N cells. In both cell lines, 4-phenylbutyrate-induced ACOX1 and catalase activities while only Abcd2 gene was up-regulated in 158JP. Moreover, the higher mitochondrial activity and content observed in 158JP were associated with higher glutathione content and increased basal production of reactive oxygen species revealing different redox statuses. Altogether, 158N and 158JP cells will permit studying the relationships between peroxisomal defects, mitochondrial activity, and oligodendrocyte functions.

Metabolomics reveals a novel vitamin E metabolite and attenuated vitamin E metabolism upon PXR activation

Pregnane X receptor (PXR) is an important nuclear receptor xenosensor that regulates the expression of metabolic enzymes and transporters involved in the metabolism of xenobiotics and endobiotics. In this study, ultra-performance liquid chromatography (UPLC) coupled with electrospray time-of-flight mass spectrometry (TOFMS), revealed altered urinary metabolomes in both Pxr-null and wild-type mice treated with the mouse PXR activator pregnenolone 16alpha-carbonitrile (PCN). Multivariate data analysis revealed that PCN significantly attenuated the urinary vitamin E metabolite alpha-carboxyethyl hydroxychroman (CEHC) glucuronide together with a novel metabolite in wild-type but not Pxr-null mice. Deconjugation experiments with beta-glucuronidase and beta-glucosidase suggested that the novel urinary metabolite was gamma-CEHC beta-D-glucoside (Glc). The identity of gamma-CEHC Glc was confirmed by chemical synthesis and by comparing tandem mass fragmentation of the urinary metabolite with the authentic standard. The lower urinary CEHC was likely due to PXR-mediated repression of hepatic sterol carrier protein 2 involved in peroxisomal beta-oxidation of branched-chain fatty acids (BCFA). Using a combination of metabolomic analysis and a genetically modified mouse model, this study revealed that activation of PXR results in attenuated levels of the two vitamin E conjugates, and identification of a novel vitamin E metabolite, gamma-CEHC Glc. Activation of PXR results in attenuated levels of the two vitamin E conjugates that may be useful as biomarkers of PXR activation.

Effect of Polyphenol-Rich Extract from Walnut on Diet-Induced Hypertriglyceridemia in Mice via Enhancement of Fatty Acid Oxidation in the Liver

The kernel pellicles of walnut are rich in ellagitannins with antioxidative activity. A polyphenol-rich extract from walnuts (WP, 45% polyphenol) was prepared and evaluated for its hypolipidemic effect in high fat diet fed mice. Oral administration of WP (100 and 200 mg/kg) significantly reduced liver weight and liver and serum triglycerides (TG). Hepatic beta-oxidation in cytosol, including peroxisome, was enhanced by WP (50-200 mg/kg). mRNA expressions of hepatic peroxisome proliferator-activated receptor (PPAR) alpha and acyl coenzyme A oxidase (ACOX) 1 were enhanced by WP (50-200 mg/kg). With respect to the hypotriglyceridemic mechanism of WP, it suppressed neither olive oil induced serum TG elevation in mice nor oleic acid induced TG accumulation in HepG2 cells. On the other hand, mRNA expressions of PPARalpha, ACOX1, and carnitine palmitoyltransferase (CPT) 1A in HepG2 cells were significantly enhanced by addition of WP (100 microg/mL). Moreover, tellimagrandin I, a polyphenolic constituent in WP, enhanced ACOX1 expression at 1-100 microg/mL. In conclusion, WP was found to possess hypotriglyceridemic activity via enhancement of peroxisomal fatty acid beta-oxidation in the liver. These results suggest that tellimagrandin I is involved in the hypotriglyceridemic mechanism of WP.

Molecular Mechanisms of Alcoholic Fatty Liver

Alcoholic fatty liver is a potentially pathologic condition which can progress to steatohepatitis, fibrosis, and cirrhosis if alcohol consumption is continued. Alcohol exposure may induce fatty liver by increasing NADH/NAD(+) ratio, increasing sterol regulatory element-binding protein-1 (SREBP-1) activity, decreasing peroxisome proliferator-activated receptor-alpha (PPAR-alpha) activity, and increasing complement C3 hepatic levels. Alcohol may increase SREBP-1 activity by decreasing the activities of AMP-activated protein kinase and sirtuin-1. Tumor necrosis factor-alpha (TNF-alpha) produced in response to alcohol exposure may cause fatty liver by up-regulating SREBP-1 activity, whereas betaine and pioglitazone may attenuate fatty liver by down-regulating SREBP-1 activity. PPAR-alpha agonists have potentials to attenuate alcoholic fatty liver. Adiponectin and interleukin-6 may attenuate alcoholic fatty liver by up-regulating PPAR-alpha and insulin signaling pathways while down-regulating SREBP-1 activity and suppressing TNF-alpha production. Recent studies show that paracrine activation of hepatic cannabinoid receptor 1 by hepatic stellate cell-derived endocannabinoids also contributes to the development of alcoholic fatty liver. Furthermore, oxidative modifications and inactivation of the enzymes involved in the mitochondrial and/or peroxisomal beta-oxidation of fatty acids could contribute to fat accumulation in the liver.