Lipid homeostasis and inflammatory activation are disturbed in classically activated macrophages with peroxisomal ß-oxidation deficiency.

Macrophage activation is characterized by pronounced metabolic adaptation. Classically activated macrophages show decreased rates of mitochondrial fatty acid oxidation and oxidative phosphorylation and acquire a glycolytic state together with their pro-inflammatory phenotype. In contrast, alternatively activated macrophages require oxidative phosphorylation and mitochondrial fatty acid oxidation for their anti-inflammatory function. Although it is evident that mitochondrial metabolism is regulated during macrophage polarization and essential for macrophage function, little is known on the regulation and role of peroxisomal ß-oxidation during macrophage activation. In this study, we show that peroxisomal ß-oxidation is strongly decreased in classically activated bone-marrow-derived macrophages (BMDM) and mildly induced in alternatively activated BMDM. To examine the role of peroxisomal ß-oxidation in macrophages, we used Mfp2-/- BMDM lacking the key enzyme of this pathway. Impairment of peroxisomal ß-oxidation in Mfp2-/- BMDM did not cause lipid accumulation but rather an altered distribution of lipid species with very-long-chain fatty acids accumulating in the triglyceride and phospholipid fraction. These lipid alterations in Mfp2-/- macrophages led to decreased inflammatory activation of Mfp2-/- BMDM and peritoneal macrophages evidenced by impaired production of several inflammatory cytokines and chemokines, but did not affect anti-inflammatory polarization. The disturbed inflammatory responses of Mfp2-/- macrophages did not affect immune cell infiltration, as mice with selective elimination of MFP2 from myeloid cells showed normal monocyte and neutrophil influx upon challenge with zymosan. Together, these data demonstrate that peroxisomal ß-oxidation is involved in fine-tuning the phenotype of macrophages, probably by influencing the dynamic lipid profile during macrophage polarization.

Phytol-induced pathology in 2-hydroxyacyl-CoA lyase (HACL1) deficient mice. Evidence for a second non-HACL1-related lyase.

2-Hydroxyacyl-CoA lyase (HACL1) is a key enzyme of the peroxisomal α-oxidation of phytanic acid. To better understand its role in health and disease, a mouse model lacking HACL1 was investigated. Under normal conditions, these mice did not display a particular phenotype. However, upon dietary administration of phytol, phytanic acid accumulated in tissues, mainly in liver and serum of KO mice. As a consequence of phytanic acid (or a metabolite) toxicity, KO mice displayed a significant weight loss, absence of abdominal white adipose tissue, enlarged and mottled liver and reduced hepatic glycogen and triglycerides. In addition, hepatic PPARα was activated. The central nervous system of the phytol-treated mice was apparently not affected. In addition, 2OH-FA did not accumulate in the central nervous system of HACL1 deficient mice, likely due to the presence in the endoplasmic reticulum of an alternate HACL1-unrelated lyase. The latter may serve as a backup system in certain tissues and account for the formation of pristanic acid in the phytol-fed KO mice. As the degradation of pristanic acid is also impaired, both phytanoyl- and pristanoyl-CoA levels are increased in liver, and the ω-oxidized metabolites are excreted in urine. In conclusion, HACL1 deficiency is not associated with a severe phenotype, but in combination with phytanic acid intake, the normal situation in man, it might present with phytanic acid elevation and resemble a Refsum like disorder.

Peroxisomal 2-Hydroxyacyl-CoA Lyase Is Involved in Endogenous Biosynthesis of Heptadecanoic Acid.

Circulating heptadecanoic acid (C17:0) is reported to be a pathology risk/prognosis biomarker and a dietary biomarker. This pathology relationship has been shown to be reliably predictive even when independent of dietary contributions, suggesting that the endogenous biosynthesis of C17:0 is related to the pathological aetiology. Little is known about C17:0 biosynthesis, which tissues contribute to the circulating levels, and how C17:0 is related to pathology. Hacl1+/- mice were mated to obtain Hacl1-/- and Hacl1+/+ control mice. At 14 weeks, they were anesthetized for tissue collection and fatty acid analysis. Compared to Hacl1+/+, C15:0 was not significantly affected in any Hacl1-/- tissues. However, the Hacl1-/- plasma and liver C17:0 levels were significantly lower: ~26% and ~22%, respectively. No significant differences were seen in the different adipose tissues. To conclude, Hacl1 plays a significant role in the liver and plasma levels of C17:0, providing evidence it can be endogenously biosynthesized via alpha-oxidation. The strong inverse association of C17:0 with pathology raises the question whether there is a direct link between α-oxidation and these diseases. Currently, there is no clear evidence, warranting further research into the role of α-oxidation in relation to metabolic diseases.

RP-HPLC-fluorescence analysis of aliphatic aldehydes: application to aldehyde-generating enzymes HACL1 and SGPL1.

Long-chain aldehydes are commonly produced in various processes, such as peroxisomal α-oxidation of long-chain 3-methyl-branched and 2-hydroxy fatty acids and microsomal breakdown of phosphorylated sphingoid bases. The enzymes involved in the aldehyde-generating steps of these processes are 2-hydroxyacyl-CoA lyase (HACL1) and sphingosine-1-phosphate lyase (SGPL1), respectively. In the present work, nonradioactive assays for these enzymes were developed employing the Hantzsch reaction. Tridecanal (C13-al) and heptadecanal (C17-al) were selected as model compounds and cyclohexane-1,3-dione as 1,3-diketone, and the fluorescent derivatives were analyzed by reversed phase (RP)-HPLC. Assay mixture composition, as well as pH and heating, were optimized for C13-al and C17-al. Under optimized conditions, these aldehydes could be quantified in picomolar range and different long-chain aldehyde derivatives were well resolved with a linear gradient elution by RP-HPLC. Aldehydes generated by recombinant enzymes could easily be detected via this method. Moreover, the assay allowed to document activity or deficiency in tissue homogenates and fibroblast lysates without an extraction step. In conclusion, a simple, quick, and cheap assay for the study of HACL1 and SGPL1 activities was developed, without relying on expensive mass spectrometric detectors or radioactive substrates.

Role of thiamine pyrophosphate in oligomerisation, functioning and import of peroxisomal 2-hydroxyacyl-CoA lyase.

During peroxisomal α-oxidation, the CoA-esters of phytanic acid and 2-hydroxylated straight chain fatty acids are cleaved into a (n-1) fatty aldehyde and formyl-CoA by 2-hydroxyacyl-CoA lyase (HACL1). HACL1 is imported into peroxisomes via the PEX5/PTS1 pathway, and so far, it is the only known peroxisomal TPP-dependent enzyme in mammals. In this study, the effect of mutations in the TPP-binding domain of HACL1 on enzyme activity, subcellular localisation and oligomerisation was investigated. Mutations of the aspartate 455 and serine 456 residues within the TPP binding domain of the human HACL1 did not affect the targeting upon expression in transfected CHO cells, although enzyme activity was abolished. Gel filtration of native and mutated N-His(6)-fusions, expressed in yeast, revealed that the mutations did not influence the oligomerisation of the (apo)enzyme. Subcellular fractionation of yeast cells expressing HACL1 showed that the lyase activity sedimented at high density in a Nycodenz gradient. In these fractions TPP could be measured, but not when mutated HACL1 was expressed, although the recombinant enzyme was still targeted to peroxisomes. These findings indicate that the binding of TPP is not required for peroxisomal targeting and correct folding of HACL1, in contrast to other TPP-dependent enzymes, and suggest that transport of TPP into peroxisomes is dependent on HACL1 import, without requirement of a specific solute transporter.

Slc25a17 Gene Trapped Mice: PMP34 Plays a Role in the Peroxisomal Degradation of Phytanic and Pristanic Acid.

Mice lacking PMP34, a peroxisomal membrane transporter encoded by Slc25a17, did not manifest any obvious phenotype on a Swiss Webster genetic background, even with various treatments designed to unmask impaired peroxisomal functioning. Peroxisomal α- and ß-oxidation rates in PMP34 deficient fibroblasts or liver slices were not or only modestly affected and in bile, no abnormal bile acid intermediates were detected. Peroxisomal content of cofactors like CoA, ATP, NAD+, thiamine-pyrophosphate and pyridoxal-phosphate, based on direct or indirect data, appeared normal as were tissue plasmalogen and very long chain fatty acid levels. However, upon dietary phytol administration, the knockout mice displayed hepatomegaly, liver inflammation, and an induction of peroxisomal enzymes. This phenotype was partially mediated by PPARα. Hepatic triacylglycerols and cholesterylesters were elevated and both phytanic acid and pristanic acid accumulated in the liver lipids, in females to higher extent than in males. In addition, pristanic acid degradation products were detected, as wells as the CoA-esters of all these branched fatty acids. Hence, PMP34 is important for the degradation of phytanic/pristanic acid and/or export of their metabolites. Whether this is caused by a shortage of peroxisomal CoA affecting the intraperoxisomal formation of pristanoyl-CoA (and perhaps of phytanoyl-CoA), or the SCPx-catalyzed thiolytic cleavage during pristanic acid ß-oxidation, could not be proven in this model, but the phytol-derived acyl-CoA profile is compatible with the latter possibility. On the other hand, the normal functioning of other peroxisomal pathways, and especially bile acid formation, seems to exclude severe transport problems or a shortage of CoA, and other cofactors like FAD, NAD(P)+, TPP. Based on our findings, PMP34 deficiency in humans is unlikely to be a life threatening condition but could cause elevated phytanic/pristanic acid levels in older adults.

Odd Chain Fatty Acids; New Insights of the Relationship Between the Gut Microbiota, Dietary Intake, Biosynthesis and Glucose Intolerance.

Recent findings have shown an inverse association between circulating C15:0/C17:0 fatty acids with disease risk, therefore, their origin needs to be determined to understanding their role in these pathologies. Through combinations of both animal and human intervention studies, we comprehensively investigated all possible contributions of these fatty acids from the gut-microbiota, the diet, and novel endogenous biosynthesis. Investigations included an intestinal germ-free study and a C15:0/C17:0 diet dose response study. Endogenous production was assessed through: a stearic acid infusion, phytol supplementation, and a Hacl1-/- mouse model. Two human dietary intervention studies were used to translate the results. Finally, a study comparing baseline C15:0/C17:0 with the prognosis of glucose intolerance. We found that circulating C15:0/C17:0 levels were not influenced by the gut-microbiota. The dose response study showed C15:0 had a linear response, however C17:0 was not directly correlated. The phytol supplementation only decreased C17:0. Stearic acid infusion only increased C17:0. Hacl1-/- only decreased C17:0. The glucose intolerance study showed only C17:0 correlated with prognosis. To summarise, circulating C15:0 and C17:0 are independently derived; C15:0 correlates directly with dietary intake, while C17:0 is substantially biosynthesized, therefore, they are not homologous in the aetiology of metabolic disease. Our findings emphasize the importance of the biosynthesis of C17:0 and recognizing its link with metabolic disease.