The importance of choosing the appropriate cholesterol quantification method: enzymatic assay versus gas chromatography.

Cholesterol is a major lipid of the animal realm with many biological roles. It is an important component of cellular membranes and a precursor of steroid hormones and bile acids. It is particularly abundant in nervous tissues, and dysregulation of cholesterol metabolism has been associated with neurodegenerative diseases such as Alzheimer's and Huntington's diseases. Deciphering the pathophysiological mechanisms of these disorders often involves animal models such as mice and Drosophila. Accurate quantification of cholesterol levels in the chosen models is a critical point of these studies. In the present work, we compare two common methods, gas chromatography coupled to flame-ionization detection (GC/FID) and a cholesterol oxidase-based fluorometric assay to measure cholesterol in mouse brains and Drosophila heads. Cholesterol levels measured by the two methods were similar for the mouse brain, which presents a huge majority of cholesterol in its sterol profile. On the contrary, depending on the method, measured cholesterol levels were very different for Drosophila heads, which present a complex sterol profile with a minority of cholesterol. We showed that the enzyme-based assay is not specific for cholesterol and detects other sterols as well. This method is therefore not suited for cholesterol measurement in models such as Drosophila. Alternatively, chromatographic methods, such as GC/FID, offer the required specificity for cholesterol quantification. Understanding the limitations of the quantification techniques is essential for reliable interpretation of the results in cholesterol-related research.

Study protocol of OmegaROP-2 prospective study: expression of placental fatty acid receptors in preterm newborns with retinopathy of prematurity.

BACKGROUND: Incomplete vascularization of the retina in preterm infants carries a risk of retinopathy of prematurity (ROP). Progress in neonatal resuscitation in developing countries has led to the survival of an increasing number of premature infants, resulting in an increased rate of ROP and consequently in visual disability. Strategies to reduce ROP involve optimizing oxygen saturation, nutrition, and normalizing factors such as insulin-like growth factor 1 and n-3 long-chain polyunsaturated fatty acids (LC-PUFA). Our previous study, OmegaROP, showed that there is an accumulation or retention of docosahexaenoic acid (DHA) in mothers of infants developing ROP, suggesting abnormalities in the LC-PUFA placental transfer via fatty acid transporting proteins. The present study aims to better understand the LC-PUFA transport dysfunction in the fetoplacental unit during pregnancy and to find a novel target for the prevention of ROP development. METHODS: The study protocol is designed to evaluate the correlation between the expression level of placental fatty acid receptors and ROP occurrence. This ongoing study will include 100 mother-infant dyads: mother-infant dyads born before 29 weeks of gestational age (GA) and mother-infant dyads with full-term pregnancies. Recruitment is planned over a period of 46 months. Maternal and cord blood samples as well as placental tissue samples will be taken following delivery. ROP screening will be performed using wide-field camera imaging according to the International Classification of ROP consensus statement. DISCUSSION: The results of this study will have a tangible impact on public health. Indeed, if we show a correlation between the expression level of placental omega-3 receptors and the occurrence of ROP, it would be an essential step in discovering novel pathophysiological mechanisms involved in this retinopathy. TRIAL REGISTRATION: NCT04819893.

Bioavailability and spatial distribution of fatty acids in the rat retina after dietary omega-3 supplementation.

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina.

Impact of a high-fat diet on the fatty acid composition of the retina.

Structure and function of the retina mainly rely on its fatty acid (FA) composition. Evidence from epidemiological studies and from animal experiments indicates that FA composition of the retina is influenced by the diet. Mice under chronic high-fat diet (HFD) develop metabolic syndrome, a risk factor for diabetes that is associated with structural and functional alterations of the retina. Here, we studied the impact of chronic exposure of mice to HFD on retinal FA composition. C57BL/6 J male mice were fed either a chow diet or a HFD for 11 weeks. As expected, HFD induced weight gain, adiposity, hyperglycemia and dyslipidemia. The retinal FA composition was determined by gas chromatography coupled to flame ionization detection. No significant change in the relative abundance of total saturated FAs (SFAs), total monounsaturated FAs (MUFAs) or total polyunsaturated FAs (PUFAs) was observed. However, retinas of HFD-fed mice displayed decreased amounts of C24:0 (p = 0.0231), C16:1n-7 (p < 0.0001), C18:1n-7 (p < 0.0001), C20:3n-9 (p = 0.0425) and C20:3n-6 (p = 0.0008), and an increased amount of C20:2n-6 (p < 0.0001). In addition, the ratio of linoleic acid (C18:2n-6) to alpha-linolenic acid (C18:3n-3) was increased in the retinas of HFD-fed mice (15.0 ± 0.8 versus 11.8 ± 0.6 in HFD and CD, respectively, p = 0.0045). No modification in the contents of arachidonic acid (C20:4n-6, AA) and docosahexaenoic acid (C22:6n-3, DHA) were observed. Analysis of dimethylacetals (DMA), which are residues of plasmalogens (Pls), revealed that the amount of Pls containing octadecanal-aldehydes (DMA C18:0) was significantly increased in HFD-fed mice (p = 0.0447). This increase was, at least in part, balanced by a decrease in Pls containing 7-octadecanal-aldehydes (DMA C18:1n-7) (p = 0.0007). In conclusion, HFD had an impact on the relative proportion of essential dietary fatty acids linoleic acid and alpha-linolenic acid that are incorporated in the retina. However, this imbalance in PUFA precursors did not alter the content of the two major retinal long-chain PUFAs, AA and DHA. HFD consumption also led to alterations in the retinal SFAs, MUFAs and Pls profiles.

Is 24(S)-hydroxycholesterol a potent modulator of cholesterol metabolism in Müller cells? An in vitro study about neuron to glia communication in the retina.

Communication between neurons and glia plays a major role in nervous tissue homeostasis. It is thought to participate in tuning cholesterol metabolism to cellular demand, which is a critical issue for neuronal health. Cholesterol is a membrane lipid crucial for nervous tissue functioning, and perturbed regulation of its metabolism has been linked to several neurodegenerative disorders. In the brain, 24(S)-hydroxycholesterol (24S-OHC) is an oxysterol synthesized by neurons to eliminate cholesterol, and 24S-OHC has been shown to regulate cholesterol metabolism in astrocytes, glial cells which provide cholesterol to neurons. In the retina, 24S-OHC is also an elimination product of cholesterol produced by neurons, especially the retinal ganglion cells. However, it is not known whether Müller cells, the major macroglial cells of the retina, play the role of cholesterol provider for retinal neurons and whether they respond to 24S-OHC signaling, similarly to brain glial cells. In the present study, primary cultures of rat Müller cells were treated with 0, 0.5 or 1.5 µM 24S-OHC for 48 hours. The levels of cholesterol, precursors and oxysterols were quantified using gas chromatography coupled to flame-ionization detection or mass spectrometry. In addition, the expression of key genes related to cholesterol metabolism was analyzed using RTq-PCR. Müller cells were shown to express many genes linked to cholesterol metabolism, including genes coding for proteins implicated in cholesterol biosynthesis (HMGCR), cholesterol uptake and export via lipoproteins (LDL-R, SR-BI, ApoE and ABACA1) and regulation of cholesterol metabolism (SREBP2 and LXRß). Cholesterol and several of its precursors and oxidative products were present. CYP27A1, the main retinal enzyme implicated in cholesterol elimination via oxysterol production, was quantified at low transcript levels but neither of its two typical products were detected in Müller cells. Furthermore, our results demonstrate that 24S-OHC has a strong hypocholesterolemic effect in Müller cells, leading to cholesterol depletion (-37 % at 1.5 µM). This was mediated by a decrease in cholesterol synthesis, as illustrated by reduced levels of cholesterol precursors: desmosterol (-38 % at 1.5 µM) and lathosterol (-84 % at 1.5 µM), and strong downregulation of HMGCR gene expression (2.4 fold decrease at 1.5µM). In addition, LDL-R and SR-BI gene expression were reduced in response to 24S-OHC treatment (2 fold and 1.6 fold at 1.5 µM, respectively), suggesting diminished lipoprotein uptake by the cells. On the contrary, there was a dramatic overexpression of ABCA1 transporter (10 fold increase at 1.5 µM), probably mediating an increase in cholesterol efflux. Finally, 24S-OHC induced a small but significant upregulation of the CYP27A1 gene. These data indicate that Müller cells possess the necessary cholesterol metabolism machinery and that they are able to sharply adjust their cholesterol metabolism in response to 24S-OHC, a signal molecule of neuronal cholesterol status. This suggests that Müller cells could be major players of cholesterol homeostasis in the retina via neuron-glia crosstalk.

Argan Oil-Mediated Attenuation of Organelle Dysfunction, Oxidative Stress and Cell Death Induced by 7-Ketocholesterol in Murine Oligodendrocytes 158N.

Argan oil is widely used in Morocco in traditional medicine. Its ability to treat cardiovascular diseases is well-established. However, nothing is known about its effects on neurodegenerative diseases, which are often associated with increased oxidative stress leading to lipid peroxidation and the formation of 7-ketocholesterol (7KC) resulting from cholesterol auto-oxidation. As 7KC induces oxidative stress, inflammation and cell death, it is important to identify compounds able to impair its harmful effects. These compounds may be either natural or synthetic molecules or mixtures of molecules such as oils. In this context: (i) the lipid profiles of dietary argan oils from Berkane and Agadir (Morocco) in fatty acids, phytosterols, tocopherols and polyphenols were determined by different chromatographic techniques; and (ii) their anti-oxidant and cytoprotective effects in 158N murine oligodendrocytes cultured with 7KC (25-50 µM; 24 h) without and with argan oil (0.1% v/v) or α-tocopherol (400 µM, positive control) were evaluated with complementary techniques of cellular and molecular biology. Among the unsaturated fatty acids present in argan oils, oleate (C18:1 n-9) and linoleate (C18:1 n-6) were the most abundant; the highest quantities of saturated fatty acids were palmitate (C16:0) and stearate (C18:0). Several phytosterols were found, mainly schottenol and spinasterol (specific to argan oil), cycloartenol, ß-amyrin and citrostadienol. α- and γ-tocopherols were also present. Tyrosol and protocatechic acid were the only polyphenols detected. Argan and extra virgin olive oils have many compounds in common, principally oleate and linoleate, and tocopherols. Kit Radicaux Libres (KRL) and ferric reducing antioxidant power (FRAP) tests showed that argan and extra virgin olive oils have anti-oxidant properties. Argan oils were able to attenuate the cytotoxic effects of 7KC on 158N cells: loss of cell adhesion, cell growth inhibition, increased plasma membrane permeability, mitochondrial, peroxisomal and lysosomal dysfunction, and the induction of oxiapoptophagy (OXIdation + APOPTOsis + autoPHAGY). Altogether, our data obtained in 158N oligodendrocytes provide evidence that argan oil is able to counteract the toxic effects of 7KC on nerve cells, thus suggesting that some of its compounds could prevent or mitigate neurodegenerative diseases to the extent that they are able to cross the blood-brain barrier.

Plasmatic Ganglioside Profile and Age-Related Macular Degeneration: A Case-Control Study.

PURPOSE: Gangliosides are glycosphingolipids that are particularly abundant in the nervous system, including the retina. However, their precise role in this tissue and its pathologies remain poorly understood. The objective of the present study was to characterize the ganglioside profile of human plasma and to determine whether it is affected in age-related macular degeneration (AMD). METHODS: Eighty-three subjects were included: control subjects (n = 25), atrophic AMD patients (n = 27) and exudative AMD patients (n = 31). For each subject, gangliosides were extracted from plasma and analyzed by liquid chromatography coupled to mass spectrometry. RESULTS: GM3 appeared to be by far the major ganglioside of human plasma, associated with GD3. No specific ganglioside class was detected in the plasma of AMD patients. Fourteen molecular species of GM3 and 9 species of GD3, accounting for the variability of the ceramide moiety of the ganglioside molecule, were identified and characterized. Analyses revealed no significant differences in the proportion of these species between control, atrophic and exudative AMD patient groups. Total GM3 levels did not differ either. CONCLUSION: Although gangliosides are considered important for the retina's structure and function, it seems that circulating gangliosides are not associated with the retinal damage occurring during the course of AMD.

Diversity of Sterol Composition in Tunisian Pistacia lentiscus Seed Oil.

Pistacia lentiscus L. seed oil is used in some Mediterranean forest area for culinary and medicinal purposes. In this study, we aim to examine, for the first time, the effect of growing area on sterol content of Pistacia lentiscus seed oil. Fruits were harvested from 13 different sites located in northern and central Tunisia. Gas chromatography-flame-ionization detection (GC-FID) was used to quantify sterols and gas chromatography/mass spectrometry (GC/MS) was used to identify them. The major sterol identified was ß-sitosterol with a value ranging from 854.12 to 1224.09 mg/kg of oil, thus making up more than 54% of the total sterols. The other two main sterols were cycloartenol (11%) and 24-methylene-cycloartenol (5%). Statistical results revealed that growing location significantly (P < 0.001) affected phytosterol levels in these oils.

Protective Effects of α-Tocopherol, γ-Tocopherol and Oleic Acid, Three Compounds of Olive Oils, and No Effect of Trolox, on 7-Ketocholesterol-Induced Mitochondrial and Peroxisomal Dysfunction in Microglial BV-2 Cells.

Lipid peroxidation products, such as 7-ketocholesterol (7KC), may be increased in the body fluids and tissues of patients with neurodegenerative diseases and trigger microglial dysfunction involved in neurodegeneration. It is therefore important to identify synthetic and natural molecules able to impair the toxic effects of 7KC. We determined the impact of 7KC on murine microglial BV-2 cells, especially its ability to trigger mitochondrial and peroxisomal dysfunction, and evaluated the protective effects of α- and γ-tocopherol, Trolox, and oleic acid (OA). Multiple complementary chemical assays, flow cytometric and biochemical methods were used to evaluate the antioxidant and cytoprotective properties of these molecules. According to various complementary assays to estimate antioxidant activity, only α-, and γ-tocopherol, and Trolox had antioxidant properties. However, only α-tocopherol, γ-tocopherol and OA were able to impair 7KC-induced loss of mitochondrial transmembrane potential, which is associated with increased permeability to propidium iodide, an indicator of cell death. In addition, α-and γ-tocopherol, and OA were able to prevent the decrease in Abcd3 protein levels, which allows the measurement of peroxisomal mass, and in mRNA levels of Abcd1 and Abcd2, which encode for two transporters involved in peroxisomal ß-oxidation. Thus, 7KC-induced side effects are associated with mitochondrial and peroxisomal dysfunction which can be inversed by natural compounds, thus supporting the hypothesis that the composition of the diet can act on the function of organelles involved in neurodegenerative diseases.

Apprehending ganglioside diversity: a comprehensive methodological approach.

Gangliosides (GGs) make a wide family of glycosphingolipids ubiquitously expressed in mammalian tissues and particularly abundant in the brain and nervous system. They exhibit a huge diversity due to structural variations in both their oligosaccharidic chain and ceramide moiety, which represent a real analytical challenge. Since their discovery in the 1940s, methods have persistently improved until the emergence of LC/MS, which offers a high level of specificity and sensitivity and is suitable with high-throughput profiling studies. We describe here a comprehensive approach relying on various techniques and aiming at fully characterizing GGs in biological samples. First, total GG content was determined by a biochemical assay. Second, GG class composition was assessed by high-performance thin-layer chromatography followed by colorimetric revelation. Then, ceramide types of GG classes were identified, and their relative quantification was performed thanks to the development of a powerful and reliable LC/MS method. Finally, ceramides were structurally characterized, and minor and less common GG classes were identified using high-resolution MS. These methods were applied to the rat retina to provide an exhaustive description of its GG composition, giving the base for a better understanding of the precise roles of GGs in this tissue.

In vivo consequences of cholesterol-24S-hydroxylase (CYP46A1) inhibition by voriconazole on cholesterol homeostasis and function in the rat retina.

Cholesterol 24S-hydroxylase (CYP46A1) converts cholesterol into 24S-hydroxycholesterol in neurons and participates in cholesterol homeostasis in the central nervous system, including the retina. We aimed to evaluate the consequences of CYP46A1 inhibition by voriconazole on cholesterol homeostasis and function in the retina. Rats received daily intraperitoneal injections of voriconazole (60mg/kg), minocycline (22mg/kg), voriconazole plus minocycline, or vehicle during five consecutive days. The rats were submitted to electroretinography to monitor retinal functionality. Cholesterol and 24S-hydroxycholesterol were measured in plasma, brain and retina by gas chromatography-mass spectrometry. The expression of CYP46A1, and GFAP as a marker for glial activation was analyzed in the retina and brain. Cytokines and chemokines were measured in plasma, vitreous, retina and brain. Voriconazole significantly impaired the functioning of the retina as exemplified by the reduced amplitude and increased latency of the b-wave of the electroretinogram, and altered oscillary potentials. Voriconazole decreased 24S-hydroxycholesterol levels in the retina. Unexpectedly, CYP46A1 and GFAP expression was increased in the retina of voriconazole-treated rats. ICAM-1 and MCP-1 showed significant increases in the retina and vitreous body. Minocycline did not reverse the effects of voriconazole. Our data highlighted the cross talk between retinal ganglion cells and glial cells in the retina, suggesting that reduced 24S-hydroxycholesterol concentration in the retina may be detected by glial cells, which were consequently activated.

Long-term intake of Lactobacillus helveticus enhances bioavailability of omega-3 fatty acids in the mouse retina.

Omega-3 (n-3) polyunsaturated fatty acids (PUFAs), particularly docosahexaenoic acid (DHA), are required for the structure and function of the retina. Several observational studies indicate that consumption of a diet with relatively high levels of n-3 PUFAs, such as those provided by fish oils, has a protective effect against the development of age-related macular degeneration. Given the accumulating evidence showing the role of gut microbiota in regulating retinal physiology and host lipid metabolism, we evaluated the potential of long-term dietary supplementation with the Gram-positive bacterium Lactobacillus helveticus strain VEL12193 to modulate the retinal n-3 PUFA content. A set of complementary approaches was used to study the impact of such a supplementation on the gut microbiota and host lipid/fatty acid (FA) metabolism. L. helveticus-supplementation was associated with a decrease in retinal saturated FAs (SFAs) and monounsaturated FAs (MUFAs) as well as an increase in retinal n-3 and omega-6 (n-6) PUFAs. Interestingly, supplementation with L. helveticus enriched the retina in C22:5n-3 (docosapentaenoic acid, DPA), C22:6n-3 (DHA), C18:2n-6 (linoleic acid, LA) and C20:3n-6 (dihomo gamma-linolenic acid, DGLA). Long-term consumption of L. helveticus also modulated gut microbiota composition and some changes in OTUs abundance correlated with the retinal FA content. This study provides a proof of concept that targeting the gut microbiota could be an effective strategy to modulate the retinal FA content, including that of protective n-3 PUFAs, thus opening paths for the design of novel preventive and/or therapeutical strategies for retinopathies.

Effects of topical docosahexaenoic acid on postoperative fibrosis in an animal model of glaucoma filtration surgery.

PURPOSE: The aim of this study was to evaluate docosahexaenoic acid (DHA) as a potential antifibrotic agent after glaucoma filtration surgery (GFS) in rats. METHODS: A total of 36 10-week-old Brown Norway rats underwent GFS. Animals were equally divided into three groups: a control group, a DHA group and a mitomycin C (MMC) group. Intraocular pressure (IOP) was measured using a dynamic rebound tonometer, and a photograph of the surgical site was taken on days 1, 3, 7, 10, 14 and 17. The incorporation of DHA into fibroblasts was evaluated by gas chromatography. The expression of alfa-smooth muscle actin (α-SMA) and Smad proteins was assessed by Western blotting. RESULTS: IOP decreased after surgery in animals from the three groups on day 1 after surgery. Over time, IOP remained lower in the DHA and MMC groups than in the control group (median [interquartile range] 8.0 [7.0-8.0] and 8.0 [7.3-8.0] mmHg vs. 9.0 [8.0-9.0] mmHg, respectively; p < 0.001). Bleb area in the DHA and MMC groups remained larger than that of the control group from day 7 to day 14 (3.9 [2.9-5.2] and 3.5 [2.3-4.4] mm2 vs. 2.3 [2.0-2.8] mm2 , respectively; p = 0.0021). We did not observe any change in DHA concentrations in the fibroblasts of the DHA group compared with the other groups. CONCLUSION: The impact of DHA on IOP and bleb area was similar to that of MMC. The mechanisms of action of DHA in rat eye fibroblasts deserve further investigation.

Retinal cholesterol metabolism is perturbated in response to experimental glaucoma in the rat.

Alterations of cholesterol metabolism have been described for many neurodegenerative pathologies, such as Alzheimer's disease in the brain and age-related macular degeneration in the retina. Recent evidence suggests that glaucoma, which is characterized by the progressive death of retinal ganglion cells, could also be associated with disruption of cholesterol homeostasis. In the present study we characterized cholesterol metabolism in a rat model of laser-induced intraocular hypertension, the main risk factor for glaucoma. Sterol levels were measured using gas-chromatography and cholesterol-related gene expression using quantitative RT-PCR at various time-points. As early as 18 hours after the laser procedure, genes implicated in cholesterol biosynthesis and uptake were upregulated (+49% and +100% for HMG-CoA reductase and LDLR genes respectively, vs. naive eyes) while genes involved in efflux were downregulated (-26% and -37% for ApoE and CYP27A1 genes, respectively). Cholesterol and precursor levels were consecutively elevated 3 days post-laser (+14%, +40% and +194% for cholesterol, desmosterol and lathosterol, respectively). Interestingly, counter-regulatory mechanisms were transcriptionally activated following these initial dysregulations, which were associated with the restoration of retinal cholesterol homeostasis, favorable to ganglion cell viability, one month after the laser-induced ocular hypertension. In conclusion, we report here for the first time that ocular hypertension is associated with transient major dynamic changes in retinal cholesterol metabolism.

Profiles of Fatty Acids, Polyphenols, Sterols, and Tocopherols and Scavenging Property of Mediterranean Oils: New Sources of Dietary Nutrients for the Prevention of Age-related Diseases.

The present study provides the fatty acid, tocopherol, phytosterol, and polyphenol profiles of some Mediterranean oils extracted from pumpkin, melon, and black cumin seed oils and those of dietary argan seed oil. Gas chromatography analysis revealed that oleic and linoleic acids were the most abundant fatty acids. Argan and melon seed oils exhibited the highest levels of oleic acid (47.32±0.02%) and linoleic acid (58.35±0.26%), respectively. In terms of tocopherols, melon seed oil showed the highest amount (652.1±3.26 mg/kg) with a predominance of γ-tocopherol (633.1±18.81 mg/kg). The phytosterol content varied between 2237.00±37.55 µg/g for argan oil to 6995.55±224.01 µg/g for melon seed oil. High Performance Liquid Chromatography analysis also revealed the presence of several polyphenols: vanillin (0.59 mg equivalents Quercetin/100 g) for melon seed oil, and p-hydroxycinnamic acid (0.04 mg equivalents Quercetin/100 g), coumarine (0.05 mg equivalents Quercetin/100 g), and thymoquinone (1.2 mg equivalents Quercetin/100 g) for black cumin seed oil. The "Kit Radicaux Libres" (KRL) assay used to evaluate the scavenging properties of the oils showed that black cumin seed oil was the most efficient. On the light of the richness of all Mediterranean oil samples in bioactive compounds, the seed oils studied can be considered as important sources of nutrients endowed with cytoprotective properties which benefits in preventing age-related diseases which are characterized by an enhanced oxidative stress.

Dietary Inulin Supplementation Affects Specific Plasmalogen Species in the Brain.

Plasmalogens (Pls) are glycerophospholipids that play critical roles in the brain. Evidence supports the role of diet and that of the gut microbiota in regulating brain lipids. We investigated the impact of dietary intake of inulin-a soluble fiber used as prebiotic-on the Pl content of the cortex in mice. No global modification in the Pl amounts was observed when evaluated by gas chromatographic analysis of dimethyl acetals (DMAs). However, the analysis of individual molecular species of Pls by liquid chromatography revealed a reduced abundance of major species of ethanolamine Pls (PlsEtn)-PE(P-18:0/22:6) and PE(P-34:1)-in the cortex of mice fed a diet supplemented with inulin. DMA and expression levels of genes (Far-1, Gnpat, Agps, Pla2g6 and Tmem86b) encoding key enzymes of Pl biosynthesis or degradation were not altered in the liver and in the cortex of mice exposed to inulin. In addition, the fatty acid profile and the amount of lyso forms derived from PlsEtn were not modified in the cortex by inulin consumption. To conclude, inulin affects the brain levels of major PlsEtn and further investigation is needed to determine the exact molecular mechanisms involved.

Protective effects of milk thistle (Sylibum marianum) seed oil and α-tocopherol against 7ß-hydroxycholesterol-induced peroxisomal alterations in murine C2C12 myoblasts: Nutritional insights associated with the concept of pexotherapy.

Peroxisomes play an important role in regulating cell metabolism and RedOx homeostasis. Peroxisomal dysfunctions favor oxidative stress and cell death. The ability of 7ß-hydroxycholesterol (7ß-OHC; 50 µM, 24 h), known to be increased in patients with age-related diseases such as sarcopenia, to trigger oxidative stress, mitochondrial and peroxisomal dysfunction was studied in murine C2C12 myoblasts. The capacity of milk thistle seed oil (MTSO, 100 µg/mL) as well as α-tocopherol (400 µM; reference cytoprotective agent) to counteract the toxic effects of 7ß-OHC, mainly at the peroxisomal level were evaluated. The impacts of 7ß-OHC, in the presence or absence of MTSO or α-tocopherol, were studied with complementary methods: measurement of cell density and viability, quantification of reactive oxygen species (ROS) production and transmembrane mitochondrial potential (ΔΨm), evaluation of peroxisomal mass as well as topographic, morphologic and functional peroxisomal changes. Our results indicate that 7ß-OHC induces a loss of cell viability and a decrease of cell adhesion associated with ROS overproduction, alterations of mitochondrial ultrastructure, a drop of ΔΨm, and several peroxisomal modifications. In the presence of 7ß-OHC, comparatively to untreated cells, important quantitative and qualitative peroxisomal modifications were also identified: a) a reduced number of peroxisomes with abnormal sizes and shapes, mainly localized in cytoplasmic vacuoles, were observed; b) the peroxisomal mass was decreased as indicated by lower protein and mRNA levels of the peroxisomal ABCD3 transporter; c) lower mRNA level of Pex5 involved in peroxisomal biogenesis as well as higher mRNA levels of Pex13 and Pex14, involved in peroxisomal biogenesis and/or pexophagy, was found; d) lower levels of ACOX1 and MFP2 enzymes, implicated in peroxisomal ß-oxidation, were detected; e) higher levels of very-long-chain fatty acids, which are substrates of peroxisomal ß-oxidation, were found. These different cytotoxic effects were strongly attenuated by MTSO, in the same range of order as with α-tocopherol. These findings underline the interest of MTSO and α-tocopherol in the prevention of peroxisomal damages (pexotherapy).

Soluble Fiber Inulin Consumption Limits Alterations of the Gut Microbiota and Hepatic Fatty Acid Metabolism Caused by High-Fat Diet.

Diet shapes the gut microbiota which impacts hepatic lipid metabolism. Modifications in liver fat content are associated with metabolic disorders. We investigated the extent of dietary fat and fiber-induced alterations in the composition of gut microbiota and hepatic fatty acids (FAs). Mice were fed a purified low-fat diet (LFD) or high-fat diet (HFD) containing non-soluble fiber cellulose or soluble fiber inulin. HFD induced hepatic decreases in the amounts of C14:0, C16:1n-7, C18:1n-7 and increases in the amounts of C17:0, C20:0, C16:1n-9, C22:5n-3, C20:2n-6, C20:3n-6, and C22:4n-6. When incorporated in a LFD, inulin poorly affected the profile of FAs. However, when incorporated in a HFD, it (i) specifically led to an increase in the amounts of hepatic C18:0, C22:0, total polyunsaturated FAs (PUFAs), total n-6 PUFAs, C18:3n-3, and C18:2n-6, (ii) exacerbated the HFD-induced increase in the amount of C17:0, and (iii) prevented the HFD-induced increases in C16:1n-9 and C20:3n-6. Importantly, the expression/activity of some elongases and desaturases, as well as the gut microbiota composition, were impacted by the dietary fat and fiber content. To conclude, inulin modulated gut microbiota and hepatic fatty acid composition, and further investigations will determine whether a causal relationship exists between these two parameters.

Lipidomic profile of human nasal mucosa and associations with circulating fatty acids and olfactory deficiency.

The nasal mucosa (NM) contains olfactory mucosa which contributes to the detection of odorant molecules and the transmission of olfactory information to the brain. To date, the lipid composition of the human NM has not been adequately characterized. Using gas chromatography, liquid chromatography coupled to mass spectrometry and thin layer chromatography, we analyzed the fatty acids and the phospholipid and ceramide molecular species in adult human nasal and blood biopsies. Saturated and polyunsaturated fatty acids (PUFAs) accounted for 45% and 29% of the nasal total fatty acids, respectively. Fatty acids of the n-6 family were predominant in the PUFA subgroup. Linoleic acid and arachidonic acid (AA) were incorporated in the main nasal phospholipid classes. Correlation analysis revealed that the nasal AA level might be positively associated with olfactory deficiency. In addition, a strong positive association between the AA levels in the NM and in plasma cholesteryl esters suggested that this blood fraction might be used as an indicator of the nasal AA level. The most abundant species of ceramides and their glycosylated derivatives detected in NM contained palmitic acid and long-chain fatty acids. Overall, this study provides new insight into lipid species that potentially contribute to the maintenance of NM homeostasis and demonstrates that circulating biomarkers might be used to predict nasal fatty acid content.

Causal Link between n-3 Polyunsaturated Fatty Acid Deficiency and Motivation Deficits.

Reward-processing impairment is a common symptomatic dimension of several psychiatric disorders. However, whether the underlying pathological mechanisms are common is unknown. Herein, we asked if the decrease in the n-3 polyunsaturated fatty acid (PUFA) lipid species, consistently described in these pathologies, could underlie reward-processing deficits. We show that reduced n-3 PUFA biostatus in mice leads to selective motivational impairments. Electrophysiological recordings revealed increased collateral inhibition of dopamine D2 receptor-expressing medium spiny neurons (D2-MSNs) onto dopamine D1 receptor-expressing MSNs in the nucleus accumbens, a main brain region for the modulation of motivation. Strikingly, transgenically preventing n-3 PUFA deficiency selectively in D2-expressing neurons normalizes MSN collateral inhibition and enhances motivation. These results constitute the first demonstration of a causal link between a behavioral deficit and n-3 PUFA decrease in a discrete neuronal population and suggest that lower n-3 PUFA biostatus in psychopathologies could participate in the etiology of reward-related symptoms.