Peroxisomal Β-oxidation Enzymes Research Articles

Liver ACOX1 regulates levels of circulating lipids that promote metabolic health through adipose remodeling.

The liver gene expression of the peroxisomal β-oxidation enzyme acyl-coenzyme A oxidase 1 (ACOX1), which catabolizes very long chain fatty acids (VLCFA), increases in the context of obesity, but how this pathway impacts systemic energy metabolism remains unknown. Here, we show that hepatic ACOX1-mediated β-oxidation regulates inter-organ communication involved in metabolic homeostasis. Liver-specific knockout of Acox1 (Acox1-LKO) protects mice from diet-induced obesity, adipose tissue inflammation, and systemic insulin resistance. Serum from Acox1-LKO mice promotes browning in cultured white adipocytes. Global serum lipidomics show increased circulating levels of several species of ω-3 VLCFAs (C24-C28) with previously uncharacterized physiological role that promote browning, mitochondrial biogenesis and Glut4 translocation through activation of the lipid sensor GPR120 in adipocytes. This work identifies hepatic peroxisomal β-oxidation as an important regulator of metabolic homeostasis and suggests that manipulation of ACOX1 or its substrates may treat obesity-associated metabolic disorders.

Tamoxifen upregulates the peroxisomal β-oxidation enzyme Enoyl CoA hydratase and 3-hydroxyacyl CoA hydratase ameliorating hepatic lipid accumulation in mice

Tamoxifen is an estrogen receptor modulator that has been reported to alleviate hepatic lipid accumulation in mice, but the mechanism is still unclear. Peroxisome fatty acid β-oxidation is the main metabolic pathway for the overload of long-chain fatty acids. As long-chain fatty acids are a cause of hepatic lipid accumulation, the activation of peroxisome fatty acid β-oxidation might be a novel therapeutic strategy for metabolic associated fatty liver disease. In this study, we investigated the mechanism of tamoxifen against hepatic lipid accumulation based on the activation of peroxisome fatty acid β-oxidation. Tamoxifen reduced liver long-chain fatty acids and relieved hepatic lipid accumulation in high fat diet mice without sex difference. In vitro, tamoxifen protected primary hepatocytes against palmitic acid-induced lipotoxicity. Mechanistically, the RNA-sequence of hepatocytes isolated from the liver revealed that peroxisome fatty acid β-oxidation was activated by tamoxifen. Protein and mRNA expression of enoyl CoA hydratase and 3-hydroxyacyl CoA hydratase were significantly increased in vivo and in vitro. Small interfering RNA enoyl CoA hydratase and 3-hydroxyacyl CoA hydratase in primary hepatocytes abolished the therapeutic effects of tamoxifen in lipid accumulation. In conclusion, our results indicated that tamoxifen could relieve hepatic lipid accumulation in high fat diet mice based on the activation of enoyl CoA hydratase and 3-hydroxyacyl CoA hydratase-mediated peroxisome fatty acids β-oxidation.

Peroxisomal β-oxidation enzyme, DECR2, regulates lipid metabolism and promotes treatment resistance in advanced prostate cancer

BackgroundPeroxisomes are central metabolic organelles that have key roles in fatty acid homoeostasis. As prostate cancer (PCa) is particularly reliant on fatty acid metabolism, we explored the contribution of peroxisomal β-oxidation (perFAO) to PCa viability and therapy response.MethodsBioinformatic analysis was performed on clinical transcriptomic datasets to identify the perFAO enzyme, 2,4-dienoyl CoA reductase 2 (DECR2) as a target gene of interest. Impact of DECR2 and perFAO inhibition via thioridazine was examined in vitro, in vivo, and in clinical prostate tumours cultured ex vivo. Transcriptomic and lipidomic profiling was used to determine the functional consequences of DECR2 inhibition in PCa.ResultsDECR2 is upregulated in clinical PCa, most notably in metastatic castrate-resistant PCa (CRPC). Depletion of DECR2 significantly suppressed proliferation, migration, and 3D growth of a range of CRPC and therapy-resistant PCa cell lines, and inhibited LNCaP tumour growth and proliferation in vivo. DECR2 influences cell cycle progression and lipid metabolism to support tumour cell proliferation. Further, co-targeting of perFAO and standard-of-care androgen receptor inhibition enhanced suppression of PCa cell proliferation.ConclusionOur findings support a focus on perFAO, specifically DECR2, as a promising therapeutic target for CRPC and as a novel strategy to overcome lethal treatment resistance.

Development of a system adapted for the diagnosis and evaluation of peroxisomal disorders by measuring bile acid intermediates

ObjectiveBile acid intermediates, 3α,7α,12α-trihydroxycholestanoic acid (THCA) and 3α,7α-dihydroxycholestanoic acid (DHCA), are metabolized in peroxisomes. Some peroxisomal disorders (PDs), such as Zellweger spectrum disorder (ZSD), show an accumulation of bile acid intermediates. In particular, ABCD3 deficiency and acyl-CoA-oxidase 2 deficiency are characterized by these metabolite abnormalities. In patients with ZSD, levels of bile acid intermediates can be lowered by a primary bile acid supplementation treatment; therefore, measuring their levels could help evaluate treatment effectiveness. Here, we established a method for the quantitative determination of bile acid intermediates (THCA/DHCA) for differentiating PDs and assessing bile acid treatment. MethodsSerum samples, obtained from patients with several forms of ZSD as well as peroxisomal β-oxidation enzyme deficiencies, were deproteinized and analyzed using liquid chromatography-mass spectrometry. ResultsLevels of the bile acid intermediates increased significantly in patients with Zellweger syndrome (ZS) and slightly in patients with neonatal adrenoleukodystrophy and infantile Refsum disease (IRD), reflecting the severity of these diseases. One patient with ZS treated with primary bile acids for 6 months showed slightly decreased serum DHCA levels but significantly increased serum THCA levels. One patient with IRD who underwent living-donor liver transplantation showed a rapid decrease in serum THCA and DHCA levels, which remained undetected for 6 years. In all controls, THCA and DHCA levels were below the detection limit. ConclusionThe analytical method developed in this study is useful for diagnosing various PD and validating bile acid treatment. Additionally, it can help predict the prognosis of patients with PD and support treatment strategies.

Role of acyl-coenzyme A oxidase 1 (ACOX1) on palmitate-induced inflammation and ROS production of macrophages in large yellow croaker (Larimichthys crocea)

Acyl-coenzyme A oxidase 1 (ACOX1) is the rate-limiting enzyme in peroxisomal β-oxidation, and it plays an essential role in mediating the inflammatory response and reactive oxygen species (ROS) metabolism in mammals. However, the role of ACOX1 in fish has not been completely elucidated. Herein, this study was conducted to investigate the role of large yellow croaker (Larimichthys crocea) ACOX1 (Lc-ACOX1) on palmitate (PA)-induced inflammation and ROS production. In this study, Lc-ACOX1 was cloned and characterized. The full-length CDS of Lc-acox1 was 1986 bp, encoding 661 amino acids. Tissue distribution results showed that the gene expression of Lc-acox1 was the highest in the intestine and the lowest in the spleen. Moreover, results showed that the mRNA expression of Lc-acox1 was upregulated by PA, with elevated pro-inflammatory gene expression, including il-1β, il-6, il-8, tnf-α, cox2 and ifn-γ, as well as ROS content in macrophages of large yellow croaker. Furthermore, the role of Lc-ACOX1 in inflammation induced by PA was investigated by using the ACOX1 inhibitor TDYA. Treatment of macrophages with TDYA reduced the mRNA expression of pro-inflammatory genes induced by PA. Moreover, inhibition of ACOX1 reduced the elevated level of ROS caused by PA and increased the mRNA expression of antioxidant genes. In conclusion, this study first identified that fish ACOX1 was involved in the PA-induced inflammatory response and ROS production.

Cell Type-Selective Loss of Peroxisomal β-Oxidation Impairs Bipolar Cell but Not Photoreceptor Survival in the Retina

Retinal degeneration is a common feature in peroxisomal disorders leading to blindness. Peroxisomes are present in the different cell types of the retina; however, their precise contribution to retinal integrity is still unclear. We previously showed that mice lacking the central peroxisomal β-oxidation enzyme, multifunctional protein 2 (MFP2), develop an early onset retinal decay including photoreceptor cell death. To decipher the function of peroxisomal β-oxidation in photoreceptors, we generated cell type selective Mfp2 knockout mice, using the Crx promotor targeting photoreceptors and bipolar cells. Surprisingly, Crx-Mfp2−/− mice maintained photoreceptor length and number until the age of 1 year. A negative electroretinogram was indicative of preserved photoreceptor phototransduction, but impaired downstream bipolar cell signaling from the age of 6 months. The photoreceptor ribbon synapse was affected, containing free-floating ribbons and vesicles with altered size and density. The bipolar cell interneurons sprouted into the ONL and died. Whereas docosahexaenoic acid levels were normal in the neural retina, levels of lipids containing very long chain polyunsaturated fatty acids were highly increased. Crx-Pex5−/− mice, in which all peroxisomal functions are inactivated in photoreceptors and bipolar cells, developed the same phenotype as Crx-Mfp2−/− mice. In conclusion, the early photoreceptor death in global Mfp2−/− mice is not driven cell autonomously. However, peroxisomal β-oxidation is essential for the integrity of photoreceptor ribbon synapses and of bipolar cells.

Endocrine pheromones couple fat rationing to dauer diapause through HNF4α nuclear receptors.

Developmental diapause is a widespread strategy for animals to survive seasonal starvation and environmental harshness. Diapaused animals often ration body fat to generate a basal level of energy for enduring survival. How diapause and fat rationing are coupled, however, is poorly understood. The nematode Caenorhabditis elegans excretes pheromones to the environment to induce a diapause form called dauer larva. Through saturated forward genetic screens and CRISPR knockout, we found that dauer pheromones feed back to repress the transcription of ACOX-3, MAOC-1, DHS-28, DAF-22 (peroxisomal β-oxidation enzymes dually involved in pheromone synthesis and fat burning), ALH-4 (aldehyde dehydrogenase for pheromone synthesis), PRX-10 and PRX-11 (peroxisome assembly and proliferation factors). Dysfunction of these pheromone enzymes and factors relieves the repression. Surprisingly, transcription is repressed not by pheromones excreted but by pheromones endogenous to each animal. The endogenous pheromones regulate the nuclear translocation of HNF4α family nuclear receptor NHR-79 and its co-receptor NHR-49, and, repress transcription through the two receptors. The feedback repression maintains pheromone homeostasis, increases fat storage, decreases fat burning, and prolongs dauer lifespan. Thus, the exocrine dauer pheromones possess an unexpected endocrine function to mediate a peroxisome-nucleus crosstalk, coupling dauer diapause to fat rationing.

Peroxisomes in the mouse parotid glands: An in-depth morphological and molecular analysis

BackgroundThe parotid gland is a major salivary gland that has important roles in the digestive and immune system. Peroxisomes are ubiquitous, single-membrane-bound organelles that are present in all eukaryotic cells. Peroxisomes help mediate lipid and reactive oxygen species metabolism, as well as polyunsaturated fatty acid, cholesterol and plasmalogen synthesis. Much of the knowledge on peroxisomes has derived from metabolic organs, however no detailed knowledge is available on peroxisomes in the parotid glands. We thus aimed to comprehensively delineate the localization and characterization of peroxisomal proteins in the murine parotid gland. MethodsWe characterized peroxisomes in the acinar and striated duct cells of the murine parotid gland by fluorescence and electron microscopy, as well as protein and mRNA expression analyses for important peroxisomal genes and proteins. ResultsWe found that peroxisomes are present in all cell types of the mouse parotid gland, however, exhibit notable cell-specific differences in their abundance and enzyme content. We also observed that mouse parotid glands contain high levels of peroxisomal β-oxidation enzymes (including Acox1, Mfp2 and Acaa1), catalase and other peroxisomal anti-oxidative enzymes. ConclusionsThis data suggests that peroxisomes are highly abundant in the murine parotid gland and might help to protect against oxidative stress. This comprehensive description of peroxisomes in the parotid gland lays the groundwork for further research concerning their role in the pathogenesis of parotid gland diseases and tumors.

ACOX1, regulated by C/EBPα and miR-25-3p, promotes bovine preadipocyte adipogenesis.

Acyl-coenzyme A oxidase 1 (ACOX1) is the first and rate-limiting enzyme in peroxisomal fatty acid β-oxidation of fatty acids. Previous studies have reported that ACOX1 was correlated with the meat quality of livestock, while the role of ACOX1 in intramuscular adipogenesis of beef cattle and its transcriptional and post-transcriptional regulatory mechanisms remain unclear. In the present study, gain-of-function and loss-of-function assays demonstrated that ACOX1 positively regulated the adipogenesis of bovine intramuscular preadipocytes. The C/EBPα-binding sites in the bovine ACOX1 promoter region at −1142 to −1129 bp, −831 to −826 bp, and −303 to −298 bp were identified by promoter deletion analysis and site-directed mutagenesis. Electrophoretic mobility shift assays (EMSA) and chromatin immunoprecipitation (ChIP) further showed that these three regions are C/EBPα-binding sites, both in vitro and in vivo, indicating that C/EBPα directly interacts with the bovine ACOX1 promoter and inhibits its transcription. Furthermore, the results from bioinformatics analysis, dual luciferase assay, site-directed mutagenesis, qRT-PCR, and Western blotting demonstrated that miR-25-3p directly targeted the ACOX1 3’UTR (3’UTR). Taken together, our findings suggest that ACOX1, regulated by transcription factor C/EBPα and miR-25-3p, promotes adipogenesis of bovine intramuscular preadipocytes via regulating peroxisomal fatty acid β-oxidation.

Developmental and Degenerative Cerebellar Pathologies in Peroxisomal β-Oxidation Deficiency.

The integrity of the cerebellum is exquisitely dependent on peroxisomal β-oxidation metabolism. Patients with peroxisomal β-oxidation defects commonly develop malformation, leukodystrophy, and/or atrophy of the cerebellum depending on the gene defect and on the severity of the mutation. By analyzing mouse models lacking the central peroxisomal β-oxidation enzyme, multifunctional protein-2 (MFP2), either globally or in selected cell types, insights into the pathomechanisms could be obtained. All mouse models developed ataxia, but the onset was earlier in global and neural-selective (Nestin) Mfp2-/- knockout mice as compared to Purkinje cell (PC)-selective Mfp2 knockouts.At the histological level, this was associated with developmental anomalies in global and Nestin-Mfp2-/- mice, including aberrant wiring of PCs by parallel and climbing fibers and altered electrical properties of PCs. In all mouse models, dystrophy of PC axons with swellings initiating in the deep cerebellar nuclei and evolving to the proximal axon, preceded death of PCs. These degenerative features are in part mediated by deficient peroxisomal β-oxidation within PCs but are accelerated when MFP2 is also absent from other neural cell types. The metabolic causes of the diverse cerebellar pathologies remain unknown.In conclusion, peroxisomal β-oxidation is required both for the development and for the maintenance of the cerebellum. This is mediated by PC autonomous and nonautonomous mechanisms.

Metabolic Alterations in the Enoyl-CoA Hydratase 2 Mutant Disrupt Peroxisomal Pathways in Seedlings.

Mobilization of seed storage compounds, such as starch and oil, is required to provide energy and metabolic building blocks during seedling development. Over 50% of fatty acids in Arabidopsis (Arabidopsis thaliana) seed oil have a cis-double bond on an even-numbered carbon. Degradation of these substrates requires peroxisomal fatty acid β-oxidation plus additional enzyme activities. Such auxiliary enzymes, including the enoyl-CoA hydratase ECH2, convert (R)-3-hydroxyacyl-CoA intermediates to the core β-oxidation substrate (S)-3-hydroxyacyl-CoA. ECH2 was suggested to function in the peroxisomal conversion of indole-3-butyric acid (IBA) to indole-3-acetic acid, because ech2 seedlings have altered IBA responses. The underlying mechanism connecting ECH2 activity and IBA metabolism is unclear. Here, we show that ech2 seedlings have reduced root length, smaller cotyledons, and arrested pavement cell expansion. At the cellular level, reduced oil body mobilization and enlarged peroxisomes suggest compromised β-oxidation. ech2 seedlings accumulate 3-hydroxyoctenoate (C8:1-OH) and 3-hydroxyoctanoate (C8:0-OH), putative hydrolysis products of catabolic intermediates for α-linolenic acid and linoleic acid, respectively. Wild-type seedlings treated with 3-hydroxyoctanoate have ech2-like growth defects and altered IBA responses. ech2 phenotypes are not rescued by Suc or auxin application. However, ech2 phenotypes are suppressed in combination with the core β-oxidation mutants mfp2 or ped1, and ech2 mfp2 seedlings accumulate less C8:1-OH and C8:0-OH than ech2 seedlings. These results indicate that ech2 phenotypes require efficient core β-oxidation. Our findings suggest that low ECH2 activity causes metabolic alterations through a toxic effect of the accumulating intermediates. These effects manifest in altered lipid metabolism and IBA responses leading to disrupted seedling development.

Peroxisome-derived lipids regulate adipose thermogenesis by mediating cold-induced mitochondrial fission.

Peroxisomes perform essential functions in lipid metabolism, including fatty acid oxidation and plasmalogen synthesis. Here, we describe a role for peroxisomal lipid metabolism in mitochondrial dynamics in brown and beige adipocytes. Adipose tissue peroxisomal biogenesis was induced in response to cold exposure through activation of the thermogenic coregulator PRDM16. Adipose-specific knockout of the peroxisomal biogenesis factor Pex16 (Pex16-AKO) in mice impaired cold tolerance, decreased energy expenditure, and increased diet-induced obesity. Pex16 deficiency blocked cold-induced mitochondrial fission, decreased mitochondrial copy number, and caused mitochondrial dysfunction. Adipose-specific knockout of the peroxisomal β-oxidation enzyme acyl-CoA oxidase 1 (Acox1-AKO) was not sufficient to affect adiposity, thermogenesis, or mitochondrial copy number, but knockdown of the plasmalogen synthetic enzyme glyceronephosphate O-acyltransferase (GNPAT) recapitulated the effects of Pex16 inactivation on mitochondrial morphology and function. Plasmalogens are present in mitochondria and decreased with Pex16 inactivation. Dietary supplementation with plasmalogens increased mitochondrial copy number, improved mitochondrial function, and rescued thermogenesis in Pex16-AKO mice. These findings support a surprising interaction between peroxisomes and mitochondria regulating mitochondrial dynamics and thermogenesis.

Neuronal Dysfunction and Behavioral Abnormalities Are Evoked by Neural Cells and Aggravated by Inflammatory Microglia in Peroxisomal β-Oxidation Deficiency.

It is becoming evident that microglia, the resident immune cells of the central nervous system (CNS), are active contributors in neurological disorders. Nevertheless, the impact of microgliosis on neuropathology, behavior and clinical decline in neuropathological conditions remains elusive. A mouse model lacking multifunctional protein-2 (MFP2), a pivotal enzyme in peroxisomal β-oxidation, develops a fatal disorder characterized by motor problems similar to the milder form of human disease. The molecular mechanisms underlying neurological decline in men and mice remain unknown. The hallmark of disease in the mouse model is chronic proliferation of microglia in the brain without provoking neuronal loss or demyelination. In order to define the contribution of Mfp2−/− neural cells to development of microgliosis and clinical neuropathology, the constitutive Mfp2−/− mouse model was compared to a neural selective Nestin-Mfp2−/− mouse model. We demonstrate in this study that, in contrast to early-onset and severe microgliosis in constitutive Mfp2−/− mice, Mfp2+/+ microglia in Nestin-Mfp2−/− mice only become mildly inflammatory at end stage of disease. Mfp2−/− microglia are primed and acquire a chronic and strong inflammatory state in Mfp2−/− mice whereas Mfp2+/+ microglia in Nestin-Mfp2−/− mice are not primed and adopt a minimal activation state. The inflammatory microglial phenotype in Mfp2−/− mice is correlated with more severe neuronal dysfunction, faster clinical deterioration and reduced life span compared to Nestin-Mfp2−/− mice. Taken together, our study shows that deletion of MFP2 impairs behavior and locomotion. Clinical decline and neural pathology is aggravated by an early-onset and excessive microglial response in Mfp2−/− mice and strongly indicates a cell-autonomous role of MFP2 in microglia.

Peroxisomal β-oxidation regulates whole body metabolism, inflammatory vigor, and pathogenesis of nonalcoholic fatty liver disease.

Nonalcoholic fatty liver disease (NAFLD), a metabolic predisposition for development of hepatocellular carcinoma (HCC), represents a disease spectrum ranging from steatosis to steatohepatitis to cirrhosis. Acox1, a rate-limiting enzyme in peroxisomal fatty acid β-oxidation, regulates metabolism, spontaneous hepatic steatosis, and hepatocellular damage over time. However, it is unknown whether Acox1 modulates inflammation relevant to NAFLD pathogenesis or if Acox1-associated metabolic and inflammatory derangements uncover and accelerate potential for NAFLD progression. Here, we show that mice with a point mutation in Acox1 (Acox1Lampe1) exhibited altered cellular metabolism, modified T cell polarization, and exacerbated immune cell inflammatory potential. Further, in context of a brief obesogenic diet stress, NAFLD progression associated with Acox1 mutation resulted in significantly accelerated and exacerbated hepatocellular damage via induction of profound histological changes in hepatocytes, hepatic inflammation, and robust upregulation of gene expression associated with HCC development. Collectively, these data demonstrate that β-oxidation links metabolism and immune responsiveness and that a better understanding of peroxisomal β-oxidation may allow for discovery of mechanisms central for NAFLD progression.

MiR-31-5p-ACOX1 Axis Enhances Tumorigenic Fitness in Oral Squamous Cell Carcinoma Via the Promigratory Prostaglandin E2.

During neoplastic development, a multitude of changes in genome-encoded information are progressively selected to confer growth and survival advantages to tumor cells. microRNAs-mRNAs regulatory networks, given their role as a critical layer of robust gene expression control, are frequently altered in neoplasm. However, whether and how these gene perturbations impact metabolic homeostasis remains largely unresolved.Methods: Through targeted miRNA expression screening, we uncovered an oral squamous cell carcinoma (OSCC)-associated miRNAome, among which miR-31-5p was identified based on extent of up-regulation, functional impact on OSCC cell migration and invasion, and direct regulation of the rate-limiting enzyme in peroxisomal β-oxidation, ACOX1.Results: We further found that both miR-31-5p and ACOX1 underpin, in an antagonistic manner, the overall cellular lipidome profiles as well as the migratory and invasive abilities of OSCC cells. Interestingly, the extracellular levels of prostaglandin E2 (PGE2), a key substrate of ACOX1, were controlled by the miR-31-5p-ACOX1 axis, and were shown to positively influence the extent of cell motility in correlation with metastatic status. The promigratory effect of this metabolite was mediated by an elevation in EP1-ERK-MMP9 signaling. Of note, functional significance of this regulatory pathway was further corroborated by its clinicopathologically-correlated expression in OSCC patient specimens.Conclusions: Collectively, our findings outlined a model whereby misregulated miR-31-5p-ACOX1 axis in tumor alters lipid metabolomes, consequently eliciting an intracellular signaling change to enhance cell motility. Our clinical analysis also unveiled PGE2 as a viable salivary biomarker for prognosticating oral cancer progression, further underscoring the importance of lipid metabolism in tumorigenesis.

Specific suppression of microgliosis cannot circumvent the severe neuropathology in peroxisomal β-oxidation-deficient mice.

An important hallmark of various neurodegenerative disorders is the proliferation and activation of microglial cells, the resident immune cells of the central nervous system (CNS). Mice that lack multifunctional protein-2 (MFP2), the key enzyme in peroxisomal β-oxidation, develop excessive microgliosis that positively correlates with behavioral deficits whereas no neuronal loss occurs. However, the precise contribution of neuroinflammation to the fatal neuropathology of MFP2 deficiency remains largely unknown. Here, we first attempted to suppress the inflammatory response by administering various anti-inflammatory drugs but they failed to reduce microgliosis. Subsequently, Mfp2-/- mice were treated with the selective colony-stimulating factor 1 receptor (CSF1R) inhibitor PLX5622 as microglial proliferation and survival is dependent on CSF1R signaling. This resulted in the elimination of >95% of microglia from control mice but only 70% of the expanded microglial population from Mfp2-/- mice. Despite microglial diminution in Mfp2-/- brain, inflammatory markers remained unaltered and residual microglia persisted in a reactive state. CSF1R inhibition did not prevent neuronal dysfunction, cognitive decline and clinical deterioration of Mfp2-/- mice. Collectively, the unaltered inflammatory profile despite suppressed microgliosis concurrent with persevering clinical decline strengthens our hypothesis that neuroinflammation importantly contributes to the Mfp2-/- phenotype.

Effect of Lorenzo's Oil on Hepatic Gene Expression and the Serum Fatty Acid Level in abcd1-Deficient Mice.

Lorenzo's oil is known to decrease the saturated very long chain fatty acid (VLCFA) level in the plasma and skin fibroblasts of X-linked adrenoleukodystrophy (ALD) patients. However, the involvement of Lorenzo's oil in in vivo fatty acid metabolism has not been well elucidated. To investigate the effect of Lorenzo's oil on fatty acid metabolism, we analyzed the hepatic gene expression together with the serum fatty acid level in Lorenzo's oil-treated wild-type and abcd1-deficient mice. The change in the serum fatty acid level in Lorenzo's oil-treated abcd1-defcient mice was quite similar to that in the plasma fatty acid level in ALD patients supplemented with Lorenzo's oil. In addition, we found that the hepatic gene expression of two peroxisomal enzymes, Dbp and Scp2, and three microsomal enzymes, Elovl1, 2, and 3, were significantly stimulated by Lorenzo's oil. Our findings indicate that Lorenzo's oil activates hepatic peroxisomal fatty acid β-oxidation at the transcriptional level. In contrast, the transcriptional stimulation of Elovl1, 2, and 3 by Lorenzo's oil does not cause changes in the serum fatty acid level. It seems likely that the inhibition of these elongation activities by Lorenzo's oil results in a decrease in saturated VLCFA. Thus, these results not only contribute to a clarification of the mechanism by which the saturated VLCFA level is reduced in the serum of ALD patients by Lorenzo's oil-treatment, but also suggest the development of a new therapeutic approach to peroxisomal β-oxidation enzyme deficiency, especially mild phenotype of DBP deficiency.

Abstract 1104: MicroRNA miR-31 regulates oral squamous cell carcinoma cell migration by targeting critical enzyme of peroxisomal lipid metabolism

Abstract MicroRNAs (miRNAs) are small endogenous non-coding RNAs that play important roles in a variety of cellular processes, such as growth, differentiation and metabolic homeostasis. Dysregulation of microRNA has been linked to the development of various types of human diseases, including cancer. Our previous study has identified that miR-31 is significantly up-regulated in oral squamous cell carcinoma (OSCC) tissues. Further gain- and loss-of-function analyses revealed that miR-31 promotes OSCC by enhancing the migration and invasiveness of OSCC cells but not cell proliferation. However, the exact role of miR-31 in OSCC neoplastic development, especially metastasis, has not been explored. Computational microRNA target prediction and pathway analysis showed an enrichment of putative targets in lipid metabolism pathway. In this study, we aimed to investigate the role of miR-31 in the regulation of lipid metabolism and to establish the relationship between lipid metabolism and tumor pathogenesis in OSCC cells. First, we demonstrated that ACOX1, the rate-limiting enzyme in peroxisomal β-oxidation, is the direct target of miR-31. We further analyzed publicly available mRNA and miRNA deep sequencing datasets and found that the expression levels of miR-31 inversely correlate with those of ACOX1. Furthermore, we discovered that depletion of miR-31 significantly decreased the intensity of cellular lipid droplets (LDs), and knockdown of ACOX1 conversely showed an increase of LD formation. These data suggested that miR-31 may regulate lipid metabolism by targeting ACOX1. In cultured OSCC cells, the migration capability and invasiveness were significantly increased after knockdown of ACOX1. We further confirmed that ACOX1 depletion enhances ERK1/2 expression and phosphorylation and increases the expression levels of MMP9 in OSCC cells. Collectively, our findings suggest that the elevated expression of miR-31 in OSCC cells directly contributes to the down-regulated expression of ACOX1, subsequently leading to accumulation of lipid metabolites and elevated OSCC migration and invasion through ERK pathway. Citation Format: Yi-Shiuan Lai, Hsuan Liu, Ting-Wen Chen, Shu-Jen Chen, Hua-Chien Chen, Bertrand Chin-Ming Tan. MicroRNA miR-31 regulates oral squamous cell carcinoma cell migration by targeting critical enzyme of peroxisomal lipid metabolism. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 1104.

Identification of a chronic non-neurodegenerative microglia activation state in a mouse model of peroxisomal β-oxidation deficiency.

The functional diversity and molecular adaptations of reactive microglia in the chronically inflamed central nervous system (CNS) are poorly understood. We previously showed that mice lacking multifunctional protein 2 (MFP2), a pivotal enzyme in peroxisomal β-oxidation, persistently accumulate reactive myeloid cells in the gray matter of the CNS. Here, we show that the increased numbers of myeloid cells solely derive from the proliferation of resident microglia and not from infiltrating monocytes. We defined the signature of Mfp2(-/-) microglia by gene expression profiling after acute isolation, which was validated by quantitative polymerase reaction (qPCR), immunohistochemical, and flow cytometric analysis. The features of Mfp2(-/-) microglia were compared with those from SOD1(G93A) mice, an amyotrophic lateral sclerosis model. In contrast to the neurodegenerative milieu of SOD1(G93A) spinal cord, neurons were intact in Mfp2(-/-) brain and Mfp2(-/-) microglia lacked signs of phagocytic and neurotoxic activity. The chronically reactive state of Mfp2(-/-) microglia was accompanied by the downregulation of markers that specify the unique microglial signature in homeostatic conditions. In contrast, mammalian target of rapamycin (mTOR) and downstream glycolytic and protein translation pathways were induced, indicative of metabolic adaptations. Mfp2(-/-) microglia were immunologically activated but not polarized to a pro- or anti-inflammatory phenotype. A peripheral lipopolysaccharide challenge provoked an exaggerated inflammatory response in Mfp2(-/-) brain, consistent with a primed state. Taken together, we demonstrate that chronic activation of resident microglia does not necessarily lead to phagocytosis nor overt neurotoxicity.

Astaxanthin lowers plasma TAG concentrations and increases hepatic antioxidant gene expression in diet-induced obesity mice.

Non-alcoholic fatty liver disease (NAFLD) is significantly associated with hyperlipidaemia and oxidative stress. We have previously reported that astaxanthin (ASTX), a xanthophyll carotenoid, lowers plasma total cholesterol and TAG concentrations in apoE knockout mice. To investigate whether ASTX supplementation can prevent the development of NAFLD in obesity, male C57BL/6J mice (n 8 per group) were fed a high-fat diet (35%, w/w) supplemented with 0, 0.003, 0.01 or 0.03% of ASTX (w/w) for 12 weeks. The 0.03% ASTX-supplemented group, but not the other groups, exhibited a significant decrease in plasma TAG concentrations, suggesting that ASTX at a 0.03% supplementation dosage exerts a hypotriacylglycerolaemic effect. Although there was an increase in the mRNA expression of fatty acid synthase and diglyceride acyltransferase 2, the mRNA levels of acyl-CoA oxidase 1, a critical enzyme in peroxisomal fatty acid β-oxidation, exhibited an increase in the 0.03% ASTX-supplemented group. There was a decrease in plasma alanine transaminase (ALT) and aspartate transaminase (AST) concentrations in the 0.03% ASTX-supplemented group. There was a significant increase in the hepatic mRNA expression of nuclear factor erythroid 2-related factor 2 and its downstream genes, which are critical for endogenous antioxidant mechanism, in the 0.03% ASTX-supplemented group. Furthermore, there was a significant decrease in the mRNA abundance of IL-6 in the primary splenocytes isolated from the 0.03% ASTX-supplemented group upon lipopolysaccharide (LPS) stimulation when compared with that in the splenocytes isolated from the control group. In conclusion, ASTX supplementation lowered the plasma concentrations of TAG, ALT and AST, increased the hepatic expression of endogenous antioxidant genes, and rendered splenocytes less sensitive to LPS stimulation. Therefore, ASTX may prevent obesity-associated metabolic disturbances and inflammation.