Major Facilitator Superfamily Domain Research Articles

Direct uptake of sphingosine-1-phosphate independent of phospholipid phosphatases

Sphingosine-1-phosphate (S1P) is a lipid mediator that is relatively abundant in plasma and plays an important role in the vascular and immune systems. To date, the only known mechanism for removing S1P from plasma has been dephosphorylation by phospholipid phosphatases (PLPPs) on the surface of cells in contact with the plasma. However, there remains a possibility that PLPP-independent dephosphorylation or direct S1P uptake into cells could occur. To examine these possibilities, here we generated triple KO (TKO) HAP1 cells that lacked all PLPPs (PLPP1–3) present in mammals. In the TKO cells, the intracellular metabolism of externally added deuterium-labeled S1P to ceramide was reduced to 17% compared with the WT cells, indicating that most extracellular S1P is dephosphorylated by PLPPs and then taken up into cells. However, this result also reveals the existence of a PLPP-independent S1P uptake pathway. Tracer experiments using [32P]S1P showed the existence of a direct S1P uptake pathway that functions without prior dephosphorylation. Overexpression of sphingolipid transporter 2 (SPNS2) or of major facilitator superfamily domain containing 2B (MFSD2B), both known S1P efflux transporters, in TKO cells increased the direct uptake of S1P, whereas KO of MFSD2B in TKO cells reduced this uptake. These results suggest that these are channel-type transporters and capable of not only exporting but also importing S1P. Furthermore, we observed that erythroid cells expressing MFSD2B, exhibited high S1P uptake activity. Our findings describing direct S1P uptake may contribute to the elucidation of the molecular mechanisms that regulate plasma S1P concentration.

GFI1 Is a Downstream Target of EVI1 in Normal Hematopoiesis

Acute myeloid leukemia (AML) with high expression of a transcriptional factor, Ecotropic viral integration site 1 (EVI1), is associated with extremely poor prognosis. EVI1 is, however, also essential for maintaining normal hematopoietic stem cells (HSCs), rendering it potentially difficult to target this molecule. To overcome this therapeutic difficulty, it is important to comprehensively elucidate differentially regulated downstream targets between normal and leukemia cells. In this study, we searched downstream targets of EVI1 in normal hematopoiesis by combining a chromatin immunoprecipitation sequence (ChIP-seq) and RNA-sequence (RNA-seq) analysis using a mouse hematopoietic cell line 32D-cl3 with high EVI1 expression. We deleted Evi1 using CRISPR/Cas9 in 32D-cl3 cells. Evi1 knock-out (KO) 32D-cl3 cells showed comparable cell growth with parental cells in the presence of IL-3, which enables them to proliferate permanently without differentiation. When they are allowed to differentiate by adding G-CSF, the number of KO cells decreased sharply at day 5-6, compared with parental 32D-cl3 cells. Along with the decreased cell number, KO cells also demonstrated higher positive rate of Gr-1 at day 7, a typical marker of differentiation into granulocytes, indicating accelerated differentiation of KO cells. These results indicated that EVI1 is required to maintain undifferentiated status of 32D-cl3 cells in a differentiation-permissive conditions, which can model normal hematopoiesis. We knocked in 3×FLAG tag at the 3' end of the Evi1 gene to perform ChIP-seq using anti-FLAG antibody. By using these knock-in cells, ChIP-seq was performed on day 0 and day 3 of G-CSF treatment, when they had started to differentiate with still maintained EVI1 expression. The peaks observed in undifferentiated day 0 sample were considered to contain a group of genes involved in undifferentiated hematopoietic cells in cooperation with EVI1. Genes associated HDAC class I, RAC1 signaling were enriched in these genes. To investigate the functional implications of the result of ChIP-seq, RNA-seq data using two clones of KO cells and parental cells were combined. We found that 152 genes were significantly up-regulated, and 155 genes were down-regulated in the KO cells, with false discovery rate less than 0.05. Twenty-four genes were identified by extracting common genes between ChIP-seq and RNA-seq; namely, genes which had day 0-specific peaks in ChIP-seq, and whose expression were decreased in the KO cells. In order to further examine the physiological implications of 24 genes in vivo, we referred to the results of RNA-seq using murine bone marrow transplantation model, where murine hematopoietic progenitor cells retrovirally transduced with Evi1 were transplanted into irradiated syngeneic mice, finally leading to AML after a long latency. Samples obtained early after post transplantation and those after AML onset were compared to those of normal hematopoietic progenitor cells. Among the above 24 genes, the expression of 5 genes was increased early after transplantation and decreased after the onset of AML, that is, these genes were up-regulated by EVI1 but don't seem to be involved in AML maintenance. We functionally validated the role of these genes in 32D-cl3 cells. Of the above, CRISPR/Cas9-mediated knock-out of Gfi1(Growth Factor Independent 1 Transcriptional Repressor) and Mfsd2b (Major facilitator superfamily domain containing 2B) in 32D-cl3 cells led to high Gr-1 positivity at day 7 like Evi1-KO cells, suggesting that these genes are involved in the functions of EVI1 in the normal hematopoiesis. The mRNA expression of these genes was compared in LSK (Lineage- Sca1+ c-kit+) cells from the bone marrow of Evi1 conditional knockout (cKO) mice and control mice. The expression of Gfi1 and Mfsd2b was decreased in LSK cells from Evi1 cKO mice. Furthermore, retroviral expression of Gfi1 in LSK cells restored the reduced colony-forming ability of Evi1 cKO cells. These results collectively suggest that GFI1 is regulated by EVI1 and is involved in the function of EVI1 regulating the stemness of hematopoietic stem and progenitor cells in normal hematopoiesis. These findings provide us with the novel insights on EVI1-mediated HSC maintenance as well as on the therapeutic strategy that specifically targets leukemia-specific EVI1 effectors while preserving normal hematopoiesis. Disclosures Kurokawa: Shire Plc: Speakers Bureau; Jansen Pharmaceutical: Speakers Bureau; Ono: Research Funding, Speakers Bureau; Boehringer Ingelheim: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Eisai: Research Funding, Speakers Bureau; Sumitomo Dainippon Pharma: Research Funding, Speakers Bureau; Teijin: Research Funding; Takeda: Research Funding, Speakers Bureau; Kyowa Kirin: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Astellas: Research Funding, Speakers Bureau; Otsuka: Research Funding, Speakers Bureau; Pfizer: Research Funding; Sanwa-Kagaku: Consultancy; MSD: Consultancy, Research Funding, Speakers Bureau; Chugai: Consultancy, Research Funding, Speakers Bureau; Bioverativ Japan: Consultancy; Celgene: Consultancy, Speakers Bureau; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Nippon Shinyaku: Research Funding, Speakers Bureau.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the tunicamycin transporter (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

MFSD7C switches mitochondrial ATP synthesis to thermogenesis in response to heme

ATP synthesis and thermogenesis are two critical outputs of mitochondrial respiration. How these outputs are regulated to balance the cellular requirement for energy and heat is largely unknown. Here we show that major facilitator superfamily domain containing 7C (MFSD7C) uncouples mitochondrial respiration to switch ATP synthesis to thermogenesis in response to heme. When heme levels are low, MSFD7C promotes ATP synthesis by interacting with components of the electron transport chain (ETC) complexes III, IV, and V, and destabilizing sarcoendoplasmic reticulum Ca2+-ATPase 2b (SERCA2b). Upon heme binding to the N-terminal domain, MFSD7C dissociates from ETC components and SERCA2b, resulting in SERCA2b stabilization and thermogenesis. The heme-regulated switch between ATP synthesis and thermogenesis enables cells to match outputs of mitochondrial respiration to their metabolic state and nutrient supply, and represents a cell intrinsic mechanism to regulate mitochondrial energy metabolism.

Thyrocyte cell survival and adaptation to chronic endoplasmic reticulum stress due to misfolded thyroglobulin

The large secretory glycoprotein thyroglobulin is the primary translation product of thyroid follicular cells. This difficult-to-fold protein is susceptible to structural alterations that disable export of the misfolded thyroglobulin from the endoplasmic reticulum (ER), which is a known cause of congenital hypothyroidism characterized by severe chronic thyrocyte ER stress. Nevertheless, individuals with this disease commonly grow a goiter, indicating thyroid cell survival and adaptation. To model these processes, here we continuously exposed rat PCCL3 thyrocytes to tunicamycin, which causes a significant degree of ER stress that is specifically attributable to thyroglobulin misfolding. We found that, in response, PCCL3 cells down-regulate expression of the "tunicamycin transporter" (major facilitator superfamily domain containing-2A, Mfsd2a). Following CRISPR/Cas9-mediated Mfsd2a deletion, PCCL3 cells could no longer escape the chronic effects of high-dose tunicamycin, as demonstrated by persistent accumulation of unglycosylated thyroglobulin; nevertheless, these thyrocytes survived and grew. A proteomic analysis of these cells adapted to chronic ER protein misfolding revealed many hundreds of up-regulated proteins, indicating stimulation of ER chaperones, oxidoreductases, stress responses, and lipid biosynthesis pathways. Further, we noted increased phospho-AMP-kinase, suggesting up-regulated AMP-kinase activity, and decreased phospho-S6-kinase and protein translation, suggesting decreased mTOR activity. These changes are consistent with conserved cell survival/adaptation pathways. We also observed a less-differentiated thyrocyte phenotype with decreased PAX8, FOXE1, and TPO protein levels, along with decreased thyroglobulin mRNA levels. In summary, we have developed a model of thyrocyte survival and growth during chronic continuous ER stress that recapitulates features of congenital hypothyroid goiter caused by mutant thyroglobulin.

Probable role for major facilitator superfamily domain containing 6 (MFSD6) in the brain during variable energy consumption

Purpose: The major facilitator superfamily (MFS) is known as the largest and most diverse superfamily containing human transporters, and these transporters are essential as they sustain the homeostasis within cellular compartments by moving substances over lipid membranes.Methods: We have identified a novel MFS protein, named Major facilitator superfamily domain containing 6 (MFSD6), and confirmed that it is phylogenetically related to the human Solute Carrier (SLC) transporter family. A homology model of MFSD6 revealed 12 predicted transmembrane segments (TMS) with the classical MFS fold between TMS 6 and 7.Results: Immunohistological analyses showed specific MFSD6 staining in neurons of wildtype mouse brain tissue, but no expression in astrocytes. Furthermore, we explored expression and probable function(s) of MFSD6 in relation to its phylogenetically related proteins, major facilitator superfamily domain containing 8 (MFSD8) and 10 (MFSD10), which is of interest as both these proteins are involved in diseases.Conclusions: We showed that expression levels of Mfsd6 and Mfsd10 were decreased with elevated or depleted energy consumption, while that of Mfsd8 remained unaffected.

Decreased Blood Level of MFSD2a as a Potential Biomarker of Alzheimer's Disease.

The protein Major Facilitator Superfamily Domain containing 2A (MFSD2a) was recently described as the primary carrier for docosahexaenoic acid (DHA) into the brain. Alzheimer’s disease (AD) is a progressive neurodegenerative disorder characterized by lower DHA levels in blood lipids. The aim of this study was to investigate the expression of MFSD2a in the whole blood and brain as a potential biomarker of AD. Three groups were established: 38 healthy controls, 48 subjects with moderate AD (GDS4), and 47 with severe AD (GDS6). We analyzed postmortem brain samples from the hippocampus of 11 healthy controls and 11 severe AD patients. Fatty acid (FA) was determined in serum and brain by gas chromatography. Blood and brain MFSD2a protein expression was analyzed by Western blotting. We found a significant and progressive decline of MFSD2a levels in blood of AD patients (Control 0.83 ± 0.13, GDS4 0.72 ± 0.09, GDS6 0.48 ± 0.05*, p ˂ 0.01). We also corroborated a significant reduction of DHA and other n-3 long-chain polyunsaturated FA in serum of AD. No differences were found in MFSD2a expression or FA levels in brain of controls and AD subjects. MFSD2A carrier was analyzed in AD patients for the first time and the level of MFSD2a in the whole blood could be a potential biomarker of this disease.

Frameshift Variant in MFSD12 Explains the Mushroom Coat Color Dilution in Shetland Ponies.

Mushroom is a unique coat color phenotype in Shetland Ponies characterized by the dilution of the chestnut coat color to a sepia tone and is hypothesized to be a recessive trait. A genome wide association study (GWAS), utilizing the Affymetrix 670K array (MNEc670k) and a single locus mixed linear model analysis (EMMAX), identified a locus on ECA7 for further investigation (Pcorrected = 2.08 × 10−10). This locus contained a 3 Mb run of homozygosity in the 12 mushroom ponies tested. Analysis of high throughput Illumina sequencing data from one mushroom Shetland pony compared to 87 genomes from horses of various breeds, uncovered a frameshift variant, p.Asp201fs, in the MFSD12 gene encoding the major facilitator superfamily domain containing 12 protein. This variant was perfectly concordant with phenotype in 96 Shetland Ponies (P = 1.15 × 10−22), was identified in the closely related Miniature Horse for which the mushroom phenotype is suspected to occur (fmu = 0.02), and was absent in 252 individuals from seven additional breeds not reported to have the mushroom phenotype. MFSD12 is highly expressed in melanocytes and variants in this gene in humans, mice, and dogs impact pigmentation. Given the role of MFSD12 in melanogenesis, we propose that p.Asp201fs is causal for the dilution observed in mushroom ponies.

Reproductive state and choline intake influence enrichment of plasma lysophosphatidylcholine-DHA: a post hoc analysis of a controlled feeding trial.

The major facilitator superfamily domain 2a protein was identified recently as a lysophosphatidylcholine (LPC) symporter with high affinity for LPC species enriched with DHA (LPC-DHA). To test the hypothesis that reproductive state and choline intake influence plasma LPC-DHA, we performed a post hoc analysis of samples available through 10weeks of a previously conducted feeding study, which provided two doses of choline (480 and 930mg/d) to non-pregnant (n 21), third-trimester pregnant (n 26), and lactating (n 24) women; all participants consumed 200mg of supplemental DHA and 22% of their daily choline intake as 2H-labelled choline. The effects of reproductive state and choline intake on total LPC-DHA (expressed as a percentage of LPC) and plasma enrichments of labelled LPC and LPC-DHA were assessed using mixed and generalised linear models. Reproductive state interacted with time (P=0·001) to influence total LPC-DHA, which significantly increased by week 10 in non-pregnant women, but not in pregnant or lactating women. Contrary to total LPC-DHA, patterns of labelled LPC-DHA enrichments were discordant between pregnant and lactating women (P<0·05), suggestive of unique, reproductive state-specific mechanisms that result in reduced production and/or enhanced clearance of LPC-DHA during pregnancy and lactation. Regardless of the reproductive state, women consuming 930 v. 480mg choline per d exhibited no change in total LPC-DHA but higher d3-LPC-DHA (P=0·02), indicating that higher choline intakes favour the production of LPC-DHA from the phosphatidylethanolamine N-methyltransferase pathway of phosphatidylcholine biosynthesis. Our results warrant further investigation into the effect of reproductive state and dietary choline on LPC-DHA dynamics and its contribution to DHA status.

Chronic cerebral hypoperfusion alters amyloid-β transport related proteins in the cortical blood vessels of Alzheimer’s disease model mouse

Abnormal accumulation of amyloid-β (Aβ) peptide defines progression of Alzheimer’s disease (AD) pathology in brain. Here, we investigated expressive changes of two main Aβ transport receptors low-density lipoprotein receptor related protein-1 (LRP1) and receptor for advanced glycation end products (RAGE) in a novel AD mice (APP23) with chronic cerebral hypoperfusion (CCH) model, moreover, examined a protective effect of a free radical scavenger edaravone (Eda). In contrast to wild type (WT) and APP23 mice, CCH strongly accelerated abnormal Aβ40 depositions and cerebral amyloid angiopathy (CAA) pathology, increased both LRP1 and RAGE expressions in brain parenchyma, while a decrease of LRP1 and an increase of RAGE were observed in vascular endothelial cells at age 12 months (M) of AD mice. Furthermore, CCH strongly increased expressions of two hypoxia-related proteins hypoxia inducible factor-1α (HIF-1α) and heme oxygenase-1 (HO-1), two oxidative-related proteins 4-hydroxy-2-nonenal (4-HNE) and 8-hydroxy-2′-deoxyguanosine (8-OHdG), and decreased both two vital nutrient transporter proteins major facilitator super family domain containing 2a (Mfsd2a) and glucose transporter 1 (Glut1) expressions. Such the above abnormal pathological changes were significantly ameliorated by edaravone treatment. The present study demonstrated that CCH strongly enhanced primary AD pathology causing double imbalances of Aβ efflux and influx transport related proteins in the cortical blood vessels in AD mice, and that such a neuropathologic abnormality was greatly ameliorated by Eda.

The Lysophosphatidylcholine Transporter MFSD2A Is Essential for CD8+ Memory T Cell Maintenance and Secondary Response to Infection

Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. In this study, we discovered that the sodium-dependent lysophosphatidylcholine (LPC) transporter major facilitator superfamily domain containing 2A (MFSD2A) is upregulated on activated CD8+ T cells and is required for memory T cell maintenance. MFSD2A deficiency in mice resulted in decreased import of LPC esterified to long chain fatty acids into activated CD8+ T cells, and MFSD2A-deficient cells are at a competitive disadvantage resulting in reduced memory T cell formation and maintenance and reduced response to secondary infection. Mechanistically, import of LPCs was required to maintain T cell homeostatic turnover, which when lost resulted in a decreased memory T cell pool and thus a reduced secondary response to repeat infection.

The lysophosphatidylcholine transporter, MFSD2A, is essential for CD8+ memory T cell maintenance and secondary response to infection

Abstract Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous fatty acids has remained enigmatic. Here we discovered the sodium-dependent lysophosphatidylcholine transporter, Major Facilitator Super Family Domain Containing 2a (MFSD2A), is upregulated on activated CD8+ T cells and is required for memory T cell maintenance. MFSD2A deficiency in mice resulted in decreased import of lysophosphatidylcholine (LPC) esterified to long chain fatty acids (LCFAs) into activated CD8+ T cells, and MFSD2A deficient cells are at a competitive disadvantage resulting in reduced memory T cell formation and maintenance and reduced response to secondary infection. Mechanistically, import of LPCs was required to maintain T cell homeostatic turnover, that when lost resulted in a decreased memory T cell pool and thus a reduced secondary response to repeat infection.

Association of MFSD3 promoter methylation level and weight regain after gastric bypass: Assessment for 3 y after surgery.

Genetic predisposition and epigenetic signatures could explain why some individuals regain the weight lost after different obesity treatments. Major facilitator superfamily domain 3 (MFSD3) is a family of membrane-bound solute carriers whose expression has been recently associated with nutrient intake and adipose tissue homeostasis. This study aimed to evaluate a possible association between MFSD3 preoperative methylation pattern and weight regain after bariatric surgery. This is a longitudinal study comprising 24 obese (body mass index > 35 m/kg2) women submitted to gastric bypass. Anthropometric measurements were evaluated at preoperative time and 1, 2, and 3 y after surgery, and then weight regain was calculated. Genomic DNA was extracted from leukocytes and was bisulfite modified by specific kits, according manufacturer's instructions. Methylation analysis was performed with the Infinium Human Methylation 450 K bead chip technology, and methylation level was expressed as a β value ranging from 0 (unmethylated) to 1 (fully methylated). Shapiro-Wilk, repeated-means analysis of variance, Spearman correlation, Mann-Whitney, and independent t tests were used in statistical analysis (P < 0.05). A total of 25% of patients regained significant weight. Weight regain after bariatric surgery was positively correlated with cg00010266 MFSD3 preoperative methylation levels (r = 0.6804, P = 0.0126). Moreover, cg00010266 MFSD3 baseline methylation levels were significantly higher in regainer patients than non-regainers (6.2 ± 1.5 versus 3.9 ± 1.2%, P = 0.026). Patients allocated in the higher cg00010266 MFSD3 preoperative methylation group had higher weight regain (4.1 ± 1.8 versus 6.7 ± 2.2 kg, P = 0.037). Preoperative hypermethylation of MFSD3 gene is significantly associated with weight regain and a worse response to gastric bypass.

MFSD2A expression predicts better prognosis in gastric cancer

Major facilitator superfamily domain containing-2A (MFSD2A) is reported to correlated with some tumors, but its clinical significance in gastric cancer (GC) is still unknown. The expression of MFSD2A and CD34 were examined on tissue microarrays of 170 set of GC and adjacent normal tissue (ANT) by immunohistochemistry. The relationship of MFSD2A with microvessel density (MVD) and clinicopathological characteristics was also investigated. MFSD2A expression is lower in GC tissue (35.3%) than in ANT (78.2%, P < 0.01). Mean MVD was higher in cancer tissue (49.7 ± 5.46) than in ANT (19.3 ± 2.19, P < 0.01), and higher in MFSD2A− GC (56.5 ± 7.27), than in MFSD2A+ GC (24.8 ± 4.31, P < 0.01). MFSD2A expression was significantly higher in moderately/well differentiated GC (47.4%) than in poorly differentiated GC (25.0%, P < 0.01) and in early-stage GC (46.4%) than in advanced GC (27.7%, P = 0.012). Patients with MFSD2A+ specimens (n = 60) had significantly better prognoses than the MFSD2A− group (n = 110; P < 0.0001). These results suggest that MFSD2A might affect angiogenesis and inhibit GC development and progression. MFSD2A may help predict prognosis and could be a therapeutic target in GC.

The lysolipid transporter Mfsd2a regulates lipogenesis in the developing brain

Brain development requires a massive increase in brain lipogenesis and accretion of the essential omega-3 fatty acid docosahexaenoic acid (DHA). Brain acquisition of DHA is primarily mediated by the transporter Major Facilitator Superfamily Domain containing 2a (Mfsd2a) expressed in the endothelium of the blood-brain barrier (BBB) and other abundant cell types within the brain. Mfsd2a transports DHA and other polyunsaturated fatty acids (PUFAs) esterified to lysophosphatidylcholine (LPC-DHA). However, the function of Mfsd2a and DHA in brain development is incompletely understood. Here, we demonstrate, using vascular endothelial-specific and inducible vascular endothelial-specific deletion of Mfsd2a in mice, that Mfsd2a is uniquely required postnatally at the BBB for normal brain growth and DHA accretion, with DHA deficiency preceding the onset of microcephaly. In Mfsd2a-deficient mouse models, a lipidomic signature was identified that is indicative of increased de novo lipogenesis of PUFAs. Gene expression profiling analysis of these DHA-deficient brains indicated that sterol regulatory-element binding protein (Srebp)-1 and Srebp-2 pathways were highly elevated. Mechanistically, LPC-DHA treatment of primary neural stem cells down-regulated Srebp processing and activation in a Mfsd2a-dependent fashion, resulting in profound effects on phospholipid membrane saturation. In addition, Srebp regulated the expression of Mfsd2a. These data identify LPC-DHA transported by Mfsd2a as a physiological regulator of membrane phospholipid saturation acting in a feedback loop on Srebp activity during brain development.

Relation of placental alkaline phosphatase expression in human term placenta with maternal and offspring fat mass.

Alkaline phosphatase is implicated in intestinal lipid transport and in the development of obesity. Placental alkaline phosphatase is localised to the microvillous plasma membrane of the placental syncytiotrophoblast at the maternal-fetal interface, but its role is unclear. We investigated the relations of placental alkaline phosphatase activity and mRNA expression with maternal body composition and offspring fat mass in humans. Term human placentas from the UK Birthright cohort (n = 52) and the Southampton Women's Survey (SWS) (n = 95) were studied. In the Birthright cohort, alkaline phosphatase activity was measured in placental microvillous plasma membrane vesicles. In the SWS, alkaline phosphatase mRNA was measured using Nanostring. Alkaline phosphatase gene expression was compared to other lipid-related genes. In Birthright samples placental microvillous plasma membrane alkaline phosphatase activity was positively associated with maternal triceps skinfold thickness and BMI (β = 0.04 (95% CI: 0.01-0.06) and β = 0.02 (0.00-0.03) µmol/mg protein/min per SD, P = 0.002 and P = 0.05, respectively) after adjusting for potential confounders. In SWS samples placental alkaline phosphatase mRNA expression in term placenta was positively associated with maternal triceps skinfold (β = 0.24 (0.04, 0.44) SD/SD, P = 0.02), had no association with neonatal %fat mass (β = 0.01 (-0.20 to 0.21) SD/SD, P = 0.93) and was negatively correlated with %fat mass at ages 4 (β = -0.28 (-0.52 to -0.04) SD/SD, P = 0.02), 6-7 (β = -0.25 (-0.49 to -0.02) SD/SD, P = 0.03) years. When compared with placental expression of other genes, alkaline phosphatase expression was positively related to genes including the lysophosphatidylcholine transporter MFSD2A (major facilitator superfamily domain containing 2A, P < 0.001) and negatively related to genes including the fatty acid transport proteins 2 and 3 (P = 0.001, P < 0.001). Our findings suggest relationships between placental alkaline phosphatase and both maternal and childhood adiposity. The inverse relationship between placental alkaline phosphatase gene expression and childhood %fat mass suggests that placental alkaline phosphatase may help to protect the foetus from the adverse effects of maternal obesity.

Metastatic Brain Tumors Disrupt the Blood-Brain Barrier and Alter Lipid Metabolism by Inhibiting Expression of the Endothelial Cell Fatty Acid Transporter Mfsd2a

Disruption of the blood-brain barrier (BBB) by cancer cells is linked to metastatic tumor initiation and progression; however, the pathways that drive these events remain poorly understood. Here, we have developed novel patient-derived xenograft (PDX) models of brain metastases that recapitulate pathological growth features found in original patient samples, thus allowing for analysis of BBB disruption by tumor cells. We report that the BBB is selectively disrupted in brain metastases, in part, via inhibition of the endothelial cell-expressed docosahexaenoic acid (DHA) transporter, major facilitator superfamily domain 2a (Mfsd2a). Loss of Mfsd2a expression in the tumor endothelium results in enhanced BBB leakage, but reduced DHA transport and altered lipid metabolism within metastases. Mfsd2a expression in normal cerebral endothelial cells is cooperatively regulated by TGFβ and bFGF signaling pathways, and these pathways are pathologically diminished in the brain metastasis endothelium. These results not only reveal a fundamental pathway underlying BBB disruption by metastatic cancer cells, but also suggest that restoring DHA metabolism in the brain tumor microenvironment may be a novel therapeutic strategy to block metastatic cell growth and survival.

The long chain fatty acid transporter, MFSD2A, is essential for memory CD8+ T cell formation and maintenance

Abstract Access to nutrients is critical for an effective T cell immune response to infection. Although transporters for sugars and amino acids have previously been described in the context of the CD8+ T cell immune response, the active transport of exogenous esterified fatty acids has remained enigmatic. Here we discovered the long chain fatty acid transporter, Major Facilitator Super Family Domain Containing 2a (MFSD2A), is upregulated on activated CD8+ T cells and is essential for their memory cell formation. MFSD2A deficiency resulted in decreased import of long chain fatty acids (LCFAs) esterified to lysophosphatidylcholine (LPC) into activated CD8+ T cells but overall resulted in a normal primary effector T cell response. However, loss of MFSD2A led to reduced memory T cell formation and maintenance. MFSD2A deficient memory CD8+ T cells showed reduced CD127 and CD62L expression and their cell turnover was significantly impaired in contrast to their wildtype counterparts. Moreover, the secondary response to infection was severely diminished in MFSD2A deficient T cells. Mechanistically, import of LCFAs was required to maintain cell energy requirements and ‘fitness’, that when lost resulted in a decreased memory T cell pool and inability to proliferate upon secondary stimulation. Our results show that MFSD2A and LPC may be useful for future small molecule therapy design to generate a more robust T cell response for new vaccines or cancer treatments.

DHA supplementation during pregnancy as phospholipids or TAG produces different placental uptake but similar fetal brain accretion in neonatal piglets.

The great variety of n-3 long-chain PUFA sources raises the question of the most adequate for using as a DHA supplement during pregnancy. Placental and fetal availability of different DHA sources remains unclear. We investigated DHA availability in maternal lipoproteins, placenta and fetal tissues in pregnant sows fed DHA as phospholipid (PL) or TAG to identify the best DHA source during this period. Pregnant Iberian sows were fed diets containing 0·8 % DHA of total fatty acids as PL from egg yolk or TAG from algae oil during the last third of gestation (40 d). Maternal tissues, placentas and fetal tissues were obtained at delivery and DHA quantified by GC. Major Facilitator Superfamily Domain Containing 2a (MFSD2a) carrier expression was analysed in both placenta and fetal brain by Western blotting. Sows fed the DHA-PL diet showed higher DHA incorporation in plasma LDL but not in plasma total lipids. No differences were found in DHA content between groups in maternal liver, adipose tissue or brain. Placental tissue incorporated more DHA in both total lipids and PL fraction in sows fed DHA-PL. However, this did not lead to an enhanced DHA accretion either in fetal plasma, fetal liver or fetal brain. MFSD2a expression was similar between both experimental groups. Maternal DHA supplementation during pregnancy in sow either as PL or TAG produces similar DHA accretion in fetal tissues but not in placenta. Both fat sources are equally available for fetal brain.

Deoxynivalenol- and zearalenone-contaminated feeds alter gene expression profiles in the livers of piglets.

ObjectiveThe Fusarium mycotoxins of deoxynivalenol (DON) and zerolenone (ZEN) cause health hazards for both humans and farm animals. Therefore, the main intention of this study was to reveal DON and ZEN effects on the mRNA expression of pro-inflammatory cytokines and other immune related genes in the liver of piglets.MethodsIn the present study, 15 six-week-old piglets were randomly assigned to the following three different dietary treatments for 4 weeks: control diet, diet containing 8 mg DON/kg feed, and diet containing 0.8 mg ZEN/kg feed. After 4 weeks, liver samples were collected and sequenced using RNA-Seq to investigate the effects of the mycotoxins on genes and gene networks associated with the immune systems of the piglets.ResultsOur analysis identified a total of 249 differentially expressed genes (DEGs), which included 99 upregulated and 150 downregulated genes in both the DON and ZEN dietary treatment groups. After biological pathway analysis, the DEGs were determined to be significantly enriched in gene ontology terms associated with many biological pathways, including immune response and cellular and metabolic processes. Consistent with inflammatory stimulation due to the mycotoxin-contaminated diet, the following Kyoto encyclopedia of genes and genomes pathways, which were related to disease and immune responses, were found to be enriched in the DEGs: allograft rejection pathway, cell adhesion molecules, graft-versus-host disease, autoimmune thyroid disease (AITD), type I diabetes mellitus, human T-cell leukemia lymphoma virus infection, and viral carcinogenesis. Genome-wide expression analysis revealed that DON and ZEN treatments downregulated the expression of the majority of the DEGs that were associated with inflammatory cytokines (interleukin 10 receptor, beta, chemokine [C-X-C motif] ligand 9), proliferation (insulin-like growth factor 1, major facilitator superfamily domain containing 2A, insulin-like growth factor binding protein 2, lipase G, and salt inducible kinase 1), and other immune response networks (paired immunoglobulin-like type 2 receptor beta, Src-like-adaptor-1 [SLA1], SLA3, SLA5, SLA7, claudin 4, nicotinamide N-methyltransferase, thyrotropin-releasing hormone degrading enzyme, ubiquitin D, histone H2B type 1, and serum amyloid A).ConclusionIn summary, our results demonstrated that high concentrations DON and ZEN disrupt immune-related processes in the liver.