Since activation of PPARγ is the main target for the antidiabetic effect of TZDs, especially when it heterodimerizes with RXR, we aimed to test the potential antidiabetic effect of phytol (250 mg/kg), the natural precursor of phytanic acid, a RXR ligand and/or pioglitazone (5 mg/kg) to diabetic insulin-resistant rats. Regarding the molecular docking simulation on PPARγ, phytanic acid, rather than phytol, showed a binding mode that mimics the crystal orientation of rosiglitazone and pioglitazone, forming H bonds with the same amino acids (S289, H 323, H 449 and Y 473), and the least energy level, which emphasizes their importance for PPARγ molecular recognition, activation, hence antidiabetic activity. In addition, docking on the RXRα/PPARγ heterodimer, revealed that phytanic acid has higher binding affinity and lesser energy score on RXRα, compared to the original ligand, retinoic acid. Phytanic acid binds by 3H bonds and shares retinoic acid in arginine (R 316). These results were further supported biochemically, where oral phytol and/or pioglitazone (5 mg/kg) improved significantly glucose homeostasis, lipid panel, raised serum adiponectin level and lowered TNF-α, reaching in most cases the effect of the 10 mg/kg pioglitazone. The study concluded that the insulin sensitizing/anti-diabetic effect of phytol is mediated by partly from activation of nuclear receptors and heterodimerization of RXR with PPARγ by phytanic acid.

A better understanding of the mechanisms of beige and brown adipogenesis is needed for developing strategies to prevent and treat obesity and associated metabolic disorders. Phytanic acid (PA) exists in a wide range of foods, especially in milk fat and marine foods, but its effects on obesity and beige adipogenesis remain poorly defined. The objective is to investigate the effects and regulatory mechanisms of PA in the beige adipogenesis. In 3T3-L1 preadipocytes, PA elevated the expression of brown adipogenic markers, suggesting that PA promotes beige adipogenic differentiation in committed adipogenic cells. In uncommitted C3H10T1/2 cells, while PA increased PGC1α expression, it did not increase brown adipogenic regulators PRDM16 or UCP1 expression, suggesting that PA had no significant effects on brown adipocyte commitment. PA also enhanced mitochondrial biogenesis and oxygen consumption. Promotion of both mitochondriogenesis and beige adipogenic differentiation were blocked by using PPARα antagonist or with Pparα knockdown, showing that PA-mediated beige/brown adipogenic differentiation is dependent on PPARα. Additionally, the PA-regulated effect is independent on β3-adrenergic receptor. Taken together, PA promotes beige adipogenic differentiation, but not the commitment of progenitor cells to the brown adipocyte lineage. PPARα is a key mediator during PA-induced beige/brown adipogenic differentiation.

We studied the alpha-oxidation of phytanic acid in human fibroblasts of controls and patients affected with classical Refsum disease, rhizomelic chondrodysplasia punctata, generalized peroxisomal disorders and peroxisomal bifunctional protein deficiency. Cultured fibroblasts were incubated with phytanic acid, after which medium and cells were collected separately. 2-Hydroxyphytanic acid and pristanic acid were measured in the medium and cells by stable isotope dilution gas chromatography mass spectrometry. In controls, 2-hydroxyphytanic acid and pristanic acid could be detected in the medium after incubation with phytanic acid, proving that alpha-oxidation of phytanic acid via 2-hydroxyphytanoyl-CoA to pristanic acid was active and intermediates were excreted into the medium. In cells from patients with a defective alpha-oxidation (Refsum disease, rhizomelic chondrodysplasia punctata and generalized peroxisomal disorders) 2-hydroxyphytanic acid and pristanic acid were low or not detectable, showing that in these disorders the hydroxylation of phytanoyl-CoA to 2-hydroxyphytanoyl-CoA is deficient. In cells with a peroxisomal beta-oxidation defect, 2-hydroxyphytanic acid and pristanic acid were formed in amounts comparable to those in the controls.

Phytanic acid produced in ruminants from chlorophyll may have preventive effects on the metabolic syndrome, partly due to its reported RXR and PPAR- α agonist activity. Milk from cows fed increased levels of green plant material, contains increased phytanic acid concentrations, but it is unknown to what extent minor increases in phytanic acid content in dairy fat leads to higher circulating levels of phytanic acid in plasma of the consumers.

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 β-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 β-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.

Phytanic acid (PA) has been implicated in development of cancer and its defective metabolism is known to cause life-threatening conditions, such as Refsum disease, in children. To explore molecular mechanisms of phytanic acid-induced cellular pathology, we investigated its effect on NADPH oxidase (NOX) and epidermal growth factor receptor (EGFR) in rat aortic smooth muscle cells (RASMC).

Among the eight stereoisomers of phytanic acid (PA), the 3RS, 7R, 11R-isomer is naturally occurring and is present in foods and the human body. PA is considered to have possible health benefits in the immune system. However, it remains undetermined whether these effects are elicited by the 3RS, 7R, 11R-PA isomer, because previous studies used a commercially available PA whose isomer configuration is unknown. In this study, we synthesized a preparation of 3RS, 7R, 11R-PA, and investigated its in vitro immunomodulatory effects, especially the T-cell production of interferon (IFN)-γ, which is associated with various autoimmune diseases. This study also investigated the effects of 3RS, 7R, 11R-PA on NF-κB activity in order to address the mechanism of its immunomodulatory effects.

The accumulation of the two branched-chain fatty acids phytanic acid and pristanic acid is known to play an important role in several diseases with peroxisomal impairment, like Refsum disease, Zellweger syndrome and α-methylacyl-CoA racemase deficiency. Recent studies elucidated that the toxic activity of phytanic acid and pristanic acid is mediated by multiple mitochondrial dysfunctions, generation of reactive oxygen species and Ca2+ deregulation via the InsP3-Ca2+ signaling pathway in glial cells. However, the exact signaling mechanism through which both fatty acids mediate toxicity is still under debate. Here, we studied the ability of phytanic acid and pristanic acid to activate the free fatty acid receptor GPR40, a G-protein-coupled receptor, which was described to be involved in the Ca2+ signaling of fatty acids. We treated HEK 293 cells expressing the GPR40 receptor with phytanic acid or pristanic acid. This resulted in a significant increase in the intracellular Ca2+ level, similar to the effect seen after treatment with the synthetic GPR40 agonist GW9508. Furthermore, we demonstrate that the GPR40 activation might be due to an interaction of the carboxylate moiety of fatty acids with the receptor. Our findings indicate that the phytanic acid- and pristanic acid-mediated Ca2+ deregulation can involve the activation of GPR40. Therefore, we suppose that activation of GPR40 might be part of the signaling cascade of the toxicity of phytanic and pristanic acids.

Alzheimer's disease (AD) is the main cause of dementia and cognitive impairment. It has been associated with a significant diminution of omega-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) levels in the brain. Clinical trials with DHA as a treatment in neurological diseases have shown inconsistent results. Previously, we reported that the presence of phytanic acid (PhA) in standard DHA compositions could be blunting DHA's beneficial effects. Therefore, we aimed to analyze the effects of a low PhA-concentrated DHA and a standard PhA-concentrated DHA in Apolipoprotein E knockout (ApoE-/-) mice. Behavioral tests and protein expression of pro-inflammatory, pro-oxidant, antioxidant factors, and AD-related mediators were evaluated. Low PhA-concentrated DHA decreased Aβ, ß-amyloid precursor protein (APP), p-tau, Ca2+/calmodulin-dependent protein kinase II (CAMKII), caspase 3, and catalase, and increased brain derived neurotrophic factor (BDNF) when compared to standard PhA-concentrated DHA. Low PhA-concentrated DHA decreased interleukin (IL)-6 and tumor necrosis factor alpha (TNF-α) protein expression in ApoE-/- mice when compared to standard PhA-concentrated DHA. No significant differences were found in p22phox, inducible nitric oxide synthase (iNOS), glutathione peroxidase (GPx), superoxide dismutase 1 (SOD-1), and tau protein expression. The positive actions of a low PhA-concentrated DHA were functionally reflected by improving the cognitive deficit in the AD experimental model. Therefore, reduction of PhA content in DHA compositions could highlight a novel pathway for the neurodegeneration processes related to AD.

Pristanic acid and phytanic acid are branched-chain fatty acids, which play an important role in diseases with peroxisomal impairment, like Refsum disease (MIM 266500), Zellwegers syndrome and alpha-methylacyl-CoA racemase deficiency (MIM 604489). Several studies revealed that the toxic activity of phytanic acid is mediated by multiple mitochondrial dysfunctions. However, the action of pristanic acid on brain cells is still completely unknown. Here, we exposed astrocytes, oligodendrocytes and neurons in mixed culture to pristanic acid and phytanic acid to analyse cellular consequences. Pristanic acid exerts a strong cytotoxic activity on brain cells, displayed by dramatic Ca2+ deregulation, in situ mitochondrial depolarization and cell death. Interestingly, pristanic acid strongly induced generation of reactive oxygen species (ROS), whereas phytanic acid exerts weaker effects on ROS production. In conclusion, pristanic acid as well as phytanic acid induced a complex array of toxic activities with mitochondrial dysfunction and Ca2+ deregulation.

The acyclic diterpenoid acid geranylgeranoic acid (GGA) has been reported to induce autophagic cell death in several human hepatoma-derived cell lines; however, the molecular mechanism for this remains unknown. In the present study, several diterpenoids were examined for ability to induce XBP1 splicing and/or lipotoxicity for human hepatoma cell lines. Here we show that three groups of diterpenoids emerged: 1) GGA, 2,3-dihydro GGA and 9-cis retinoic acid induce cell death and XBP1 splicing; 2) all-trans retinoic acid induces XBP1 splicing but little cell death; and 3) phytanic acid, phytenic acid and geranylgeraniol induce neither cell death nor XBP1 splicing. GGA-induced ER stress/ unfolded protein response (UPR) and its lipotoxicity were both blocked by co-treatment with oleic acid. The blocking activity of oleic acid for GGA-induced XBP1 splicing was not attenuated by methylation of oleic acid. These findings strongly suggest that GGA at micromolar concentrations induces the so-called lipid-induced ER stress response/UPR, which is oleate-suppressive, and shows its lipotoxicity in human hepatoma cells.

Refsum disease (RD) is an inborn error of metabolism that is characterised by a defect in peroxisomal α-oxidation of the branched-chain fatty acid phytanic acid. The disorder presents with late-onset progressive retinitis pigmentosa and polyneuropathy and can be diagnosed biochemically by elevated levels of phytanate in plasma and tissues of patients. To date, no cure exists for RD, but phytanate levels in patients can be reduced by plasmapheresis and a strict diet. In this study, we reconstructed a fibroblast-specific genome-scale model based on the recently published, FAD-curated model, based on Recon3D reconstruction. We used transcriptomics (available via GEO database with identifier GSE138379), metabolomics and proteomics (available via ProteomeXchange with identifier PXD015518) data, which we obtained from healthy controls and RD patient fibroblasts incubated with phytol, a precursor of phytanic acid. Our model correctly represents the metabolism of phytanate and displays fibroblast-specific metabolic functions. Using this model, we investigated the metabolic phenotype of RD at the genome scale, and we studied the effect of phytanate on cell metabolism. We identified 53 metabolites that were predicted to discriminate between healthy and RD patients, several of which with a link to amino acid metabolism. Ultimately, these insights in metabolic changes may provide leads for pathophysiology and therapy. DATABASES: Transcriptomics data are available via GEO database with identifier GSE138379, and proteomics data are available via ProteomeXchange with identifier PXD015518.

Metabolic changes occur in patients with peroxisomal diseases owing to impairments in the genes involved in peroxisome function. For diagnostic purposes, saturated very-long-chain fatty acids (VLCFAs) such as C24:0 and C26:0, phytanic acid, pristanic acid, and plasmalogens are often measured as metabolic hallmarks. As the direct pathology of peroxisomal disease is yet to be fully elucidated, we sought to explore the fatty acid species that accumulate in patients with peroxisomal diseases. We developed a method for detecting a range of fatty acids implicated in peroxisomal diseases such as Zellweger syndrome (ZS) and X-linked adrenoleukodystrophy (X-ALD). To this end, we employed an ultra-performance liquid chromatography-mass spectrometry (LC-MS) coupled with negatively charged electrospray ionization. Fatty acids from patients and control subjects were extracted from total lipids by acid-hydrolysis and compared. In accordance with previous results, the amounts of VLCFAs, phytanic acid, and pristanic acid differed between the two groups. We identified extremely long and highly polyunsaturated VLCFAs (ultra-VLC-PUFAs) such as C44:12 in ZS samples. Moreover, three unknown molecules were prominent in control samples but scarcely detectable in ZS samples. LC-MS/MS analysis identified these as 1-alkyl-sn-glycerol 3-phosphates derived from ether lipids containing fatty alcohols such as C16:0, C18:0, or C18:1. Our method provides an approach to observing a wide range of lipid-derived fatty acids and related molecules in order to understand the metabolic changes involved in peroxisomal diseases. This technique can therefore be used in identifying metabolic markers and potential clinical targets for future treatment.

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.

The immortalized human hepatocyte (IHH) cell line is increasingly used for studies related to liver metabolism, including hepatic glucose, lipid, lipoprotein and triglyceride metabolism, and the effect of therapeutic interventions. To determine whether the IHH cell line is a good model to investigate hepatic peroxisomal metabolism, we measured several peroxisomal parameters in IHH cells and, for comparison, HepG2 cells and primary skin fibroblasts. This revealed a marked plasmalogen deficiency and a deficient fatty acid α-oxidation in the IHH cells, due to a defect of PEX7, a cytosolic receptor protein required for peroxisomal import of a subset of peroxisomal proteins. These abnormalities have consequences for the lipid homeostasis of these cells and thus should be taken into account for the interpretation of data previously generated by using this cell line and when considering using this cell line for future research.

Peroxisome biogenesis disorders (PBD) are a heterogeneous group of autosomal recessive disorders that affect multiple organ systems. Approximately 80% of PBD patients are classifiedin the Zellweger syndrome spectrum, which is generally caused by mutations in the PEX1, PEX6, PEX10, PEX12, or PEX26 genes.

There is considerable interest in dairy products from low-input systems, such as mountain-pasture grazing cows, because these products are believed to be healthier than products from high-input conventional systems. This may be due to a higher content of bioactive components, such as phytanic acid, a PPAR-agonist derived from chlorophyll. However, the effects of such products on human health have been poorly investigated.

Peroxisomes play an important role in the metabolism of a variety of biomolecules, including lipids and bile acids. Peroxisomal Membrane Protein 4 (PXMP4) is a ubiquitously expressed peroxisomal membrane protein that is transcriptionally regulated by peroxisome proliferator-activated receptor α (PPARα), but its function is still unknown. To investigate the physiological function of PXMP4, we generated a Pxmp4 knockout (Pxmp4-/-) mouse model using CRISPR/Cas9-mediated gene editing. Peroxisome function was studied under standard chow-fed conditions and after stimulation of peroxisomal activity using the PPARα ligand fenofibrate or by using phytol, a metabolite of chlorophyll that undergoes peroxisomal oxidation. Pxmp4-/- mice were viable, fertile, and displayed no changes in peroxisome numbers or morphology under standard conditions. Also, no differences were observed in the plasma levels of products from major peroxisomal pathways, including very long-chain fatty acids (VLCFAs), bile acids (BAs), and BA intermediates di- and trihydroxycholestanoic acid. Although elevated levels of the phytol metabolites phytanic and pristanic acid in Pxmp4-/- mice pointed towards an impairment in peroxisomal α-oxidation capacity, treatment of Pxmp4-/- mice with a phytol-enriched diet did not further increase phytanic/pristanic acid levels. Finally, lipidomic analysis revealed that loss of Pxmp4 decreased hepatic levels of the alkyldiacylglycerol class of neutral ether lipids, particularly those containing polyunsaturated fatty acids. Together, our data show that while PXMP4 is not critical for overall peroxisome function under the conditions tested, it may have a role in the metabolism of (ether)lipids.

Thiamine pyrophosphate (TPP) is a cofactor for 2-hydroxyacyl-CoA lyase 1 (HACL1), a peroxisomal enzyme essential for the alpha-oxidation of phytanic acid and 2-hydroxy straight chain fatty acids. So far, HACL1 is the only known peroxisomal TPP-dependent enzyme in mammals. Little is known about the transport of metabolites and cofactors across the peroxisomal membrane and no peroxisomal thiamine or TPP carrier has been identified in mammals yet. This study was undertaken to get a better insight into these issues and to shed light on the role of TPP in peroxisomal metabolism.

α-Methylacyl-CoA racemase (Amacr) catalyzes the racemization of the 25-methyl group in C27-intermediates in bile acid synthesis and in methyl-branched fatty acids such as pristanic acid, a metabolite derived from phytol. Consequently, patients with Amacr deficiency accumulate C27-bile acid intermediates, pristanic and phytanic acid and display sensorimotor neuropathy, seizures and relapsing encephalopathy. In contrast to humans, Amacr-deficient mice are clinically symptomless on a standard laboratory diet, but failed to thrive when fed phytol-enriched chow. In this study, the effect and the mechanisms behind the development of the phytol-feeding associated disease state in Amacr-deficient mice were investigated. All Amacr-/- mice died within 36weeks on a phytol diet, while wild-type mice survived. Liver failure was the main cause of death accompanied by kidney and brain abnormalities. Histological analysis of liver showed inflammation, fibrotic and necrotic changes, Kupffer cell proliferation and fatty changes in hepatocytes, and serum analysis confirmed the hepatic disease. Pristanic and phytanic acids accumulated in livers of Amacr-/- mice after a phytol diet. Microarray analysis also revealed changes in the expression levels of numerous genes in wild-type mouse livers after two weeks of the phytol diet compared to a control diet. This indicates that detoxification of phytol metabolites in liver is accompanied by activation of multiple pathways at the molecular level and Amacr-/- mice are not able to respond adequately. Phytol causes primary failure in liver leading to death of Amacr-/- mice thus emphasizing the indispensable role of Amacr in detoxification of α-methyl-branched fatty acids.