Decreased activity of folate transporters in lipid rafts resulted in reduced hepatic folate uptake in chronic alcoholism in rats

Folic acid is an essential nutrient that is required for one-carbon biosynthetic processes and for methylation of biomolecules. Deficiency of this micronutrient leads to disturbances in normal physiology of cell. Chronic alcoholism is well known to be associated with folate deficiency, which is due in part to folate malabsorption. The present study deals with the regulatory mechanisms of folate uptake in liver during chronic alcoholism. Male Wistar rats were fed 1 g/kg body weight/day ethanol (20 % solution) orally for 3 months, and the molecular mechanisms of folate uptake were studied in liver. The characterization of the folate transport system in liver basolateral membrane (BLM) suggested it to be a carrier mediated and acidic pH dependent, with the major involvement of proton coupled folate transporter and folate binding protein in the uptake. The folate transporters were found to be associated with lipid raft microdomain of liver BLM. Moreover, ethanol ingestion decreased the folate transport by altering the Vmax of folate transport process and downregulated the expression of folate transporters in lipid rafts. The decreased transporter levels were associated with reduced protein and mRNA levels of these transporters in liver. The deranged folate uptake together with reduced folate transporter levels in lipid rafts resulted in reduced folate levels in liver and thereby to its reduced levels in serum of ethanol-fed rats. The chronic ethanol ingestion led to decreased folate uptake in liver, which was associated with the decreased number of transporter molecules in the lipid rafts that can be ascribed to the reduced synthesis of these transporters.     

Down-regulation of reduced folate carrier may result in folate malabsorption across intestinal brush border membrane during experimental alcoholism

Folate plays a critical role in maintaining normal metabolic, energy, differentiation and growth status of all mammalian cells. The intestinal folate uptake is tightly and diversely regulated, and disturbances in folate homeostasis are observed in alcoholism, attributable, in part, to intestinal malabsorption of folate. The aim of this study was to delineate the regulatory mechanisms of folate transport in intestinal absorptive epithelia in order to obtain insights into folate malabsorption in a rat model of alcoholism. The rats were fed 1 g.kg(-1) body weight of ethanol daily for 3 months. A reduced uptake of [(3)H]folic acid in intestinal brush border membrane was observed over the course of ethanol administration for 3 months. Folate transport exhibited saturable kinetics and the decreased intestinal brush border membrane folate transport in chronic alcoholism was associated with an increased K(m) value and a low V(max) value. Importantly, the lower intestinal [(3)H]folic acid uptake in ethanol-fed rats was observed in all cell fractions corresponding to villus tip, mid-villus and crypt base. RT-PCR analysis for reduced folate carrier, the major folate transporter, revealed that reduced folate carrier mRNA levels were decreased in jejunal tissue derived from ethanol-fed rats. Parallel changes were observed in reduced folate carrier protein levels in brush border membrane along the entire crypt-villus axis. In addition, immunohistochemical staining for reduced folate carrier protein showed that, in alcoholic conditions, deranged reduced folate carrier localization was observed along the entire crypt-villus axis, with a more prominent effect in differentiating crypt base stem cells. These changes in functional activity of the membrane transport system were not caused by a general loss of intestinal architecture, and hence can be attributed to the specific effect of ethanol ingestion on the folate transport system. The low folate uptake activity observed in ethanol-fed rats was found to be associated with decreased serum and red blood cell folate levels, which might explain the observed jejunal genomic hypomethylation. These findings offer possible mechanistic insights into folate malabsorption during alcoholism.     

A novel deletion mutation in the proton-coupled folate transporter (PCFT; SLC46A1) in a Nicaraguan child with hereditary folate malabsorption

Hereditary folate malabsorption (OMIM 229050) is a rare autosomal recessive disorder caused by loss-of-function mutations in the proton-coupled folate transporter gene (pcft/SLC46A1) resulting in impaired folate transport across the intestine and into the central nervous system. We report a novel, homozygous, deletion mutation in a child of Nicaraguan descent in exon 2 (c.558–588 del, ss778190447) at amino acid position I188 resulting in a frameshift with a premature stop.     

Hereditary folate malabsorption: A positively charged amino acid at position 113 of the proton-coupled folate transporter (PCFT/SLC46A1) is required for folic acid binding

The proton-coupled folate transporter (PCFT/SLC46A1) mediates intestinal folate uptake at acidic pH. Some loss of folic acid (FA) transport mutations in PCFT from hereditary folate malabsorption (HFM) patients cluster in R113, thereby suggesting a functional role for this residue. Herein, unlike non-conservative substitutions, an R113H mutant displayed 80-fold increase in the FA transport Km while retaining parental Vmax, hence indicating a major fall in folate substrate affinity. Furthermore, consistent with the preservation of 9% of parental transport activity, R113H transfectants displayed a substantial decrease in the FA growth requirement relative to mock transfectants. Homology modeling based on the crystal structures of the Escherichia coli transporter homologues EmrD and glycerol-3-phosphate transporter revealed that the R113H rotamer properly protrudes into the cytoplasmic face of the minor cleft normally occupied by R113. These findings constitute the first demonstration that a basic amino acid at position 113 is required for folate substrate binding.     

Roles of lipid rafts in membrane transport.

Cholesterol-sphingolipid microdomains (lipid rafts) are part of the machinery ensuring correct intracellular trafficking of proteins and lipids. The most apparent roles of rafts are in sorting and vesicle formation, although their roles in vesicle movement and cytoskeletal connections as well as in vesicle docking and fusion are coming into focus. New evidence suggests that compositionally distinct lipid microdomains are assembled and may coexist within a given membrane. Important clues have also been uncovered about the mechanisms coupling raft-dependent signaling and endocytic uptake.     

Interaction of phosphoinositolglycan(-peptides) with plasma membrane lipid rafts of rat adipocytes

Insulin receptor-independent activation of the insulin signal transduction cascade in insulin-responsive target cells by phosphoinositolglycans (PIG) and PIG-peptides (PIG-P) is accompanied by redistribution of glycosylphosphatidylinositol (GPI)-anchored plasma membrane proteins (GPI proteins) and dually acylated nonreceptor tyrosine kinases from detergent/carbonate-resistant glycolipid-enriched plasma membrane raft domains of high-cholesterol content (hcDIGs) to rafts of lower cholesterol content (lcDIGs). Here we studied the nature and localization of the primary target of PIG(-P) in isolated rat adipocytes. Radiolabeled PIG-P (Tyr-Cys-Asn-NH-(CH(2))(2)-O-PO(OH)O-6Manalpha1(Manalpha1-2)-2Manalpha1-6Manalpha1-4GluN1-6Ino-1,2-(cyclic)-phosphate) prepared by chemical synthesis or a radiolabeled lipolytically cleaved GPI protein from Saccharomyces cerevisiae, which harbors the PIG-P moiety, bind to isolated hcDIGs but not to lcDIGs. Binding is saturable and abolished by pretreatment of intact adipocytes with trypsin followed by NaCl or with N-ethylmaleimide, indicating specific interaction of PIG-P with a cell surface protein. A 115-kDa polypeptide released from the cell surface by the trypsin/NaCl-treatment is labeled by [(14)C]N-ethylmaleimide. The labeling is diminished upon incubation of adipocytes with PIG-P which can be explained by direct binding of PIG-P to the 115-kDa protein and concomitant loss of its accessibility to N-ethylmaleimide. Binding of PIG-P to hcDIGs is considerably increased after pretreatment of adipocytes with (glycosyl)phosphatidylinositol-specific phospholipases compatible with lipolytic removal of endogenous ligands, such as GPI proteins/lipids. These data demonstrate that in rat adipocytes synthetic PIG(-P) as well as lipolytically cleaved GPI proteins interact specifically with hcDIGs. The interaction depends on the presence of a trypsin/NaCl/NEM-sensitive 115-kDa protein located at hcDIGs which thus represents a candidate for a binding protein for exogenous insulin-mimetic PIG(-P) and possibly endogenous GPI proteins/lipids.     

Ceramide-domain formation and collapse in lipid rafts: membrane reorganization by an apoptotic lipid

The effect of physiologically relevant ceramide concentrations (< or = 4 mol %) in raft model membranes with a lipid composition resembling that of cell membranes, i.e., composed of different molar ratios of an unsaturated glycerophospholipid, sphingomyelin, and cholesterol (Chol) along a liquid-disordered-liquid-ordered tie line was explored. The application of a fluorescence multiprobe and multiparameter approach, together with multiple fluorescence resonance energy transfer (FRET) pairs, in the well-characterized palmitoyl-oleoyl-phosphocholine (POPC)/palmitoyl-sphingomyelin (PSM)/Chol ternary mixture, revealed that low palmitoyl-ceramide (PCer) concentrations strongly changed both the biophysical properties and lipid lateral organization of the ternary mixtures in the low-to-intermediate Chol/PSM-, small raft size range (<25 mol % Chol). For these mixtures, PCer recruited up to three PSM molecules for the formation of very small ( approximately 4 nm) and highly ordered gel domains, which became surrounded by rafts (liquid-ordered phase) when Chol/PSM content increased. However, the size of these rafts did not change, showing that PCer did not induce the formation of large platforms or the coalescence of small rafts. In the high Chol/PSM-, large raft domains range (>33 mol % Chol), Chol completely abolished the effect of PCer by competing for PSM association. Lipid rafts govern the biophysical properties and lateral organization in these last mixtures.     

Interaction of phosphatidylinositolglycan(-peptides) with plasma membrane lipid rafts triggers insulin-mimetic signaling in rat adipocytes

The phosphoinositolglycan(-peptide) (PIG-P) portion of glycosylphosphatidylinositol-anchored plasma membrane (GPI) proteins or synthetic PIG(-P) molecules interact with proteinaceous binding sites which are located in high-cholesterol-containing detergent/carbonate-insoluble glycolipid-enriched raft domains (hcDIGs) of the plasma membrane. In isolated rat adipocytes, PIG(-P) induce the redistribution of GPI proteins from hcDIGs to low-cholesterol-containing DIGs (lcDIGs) and concomitantly provoke insulin-mimetic signaling and metabolic action. Using a set of synthetic PIG(-P) derivatives we demonstrate here that their specific binding to hcDIGs and their insulin-mimetic signaling/metabolic activity strictly correlate with respect to (i) translocation of the GPI proteins, Gce1 and 5(')-nucleotidase, from hcDIGs to lcDIGs, (ii) dissociation of the nonreceptor tyrosine kinase, pp59(Lyn), from caveolin residing at hcDIGs, (iii) translocation of pp59(Lyn) from hcDIGs to lcDIGs, (iv) activation of pp59(Lyn), (v) tyrosine phosphorylation of insulin receptor substrate proteins-1/2, and finally (vi) stimulation of glucose transport. The natural PIG(-P) derived from the carboxy-terminal tripeptide of Gce1, YCN-PIG, exhibits the highest potency followed by a combination of the separate peptidylethanolamidyl and PIG constituents. We conclude that efficient positive cross-talk of PIG(-P) to the insulin signaling cascade requires their interaction with hcDIGs. We suggest that PIG(-P) thereby displace GPI proteins from binding to hcDIGs leading to their release from hcDIGs for lateral movement to lcDIGs which initiates signal transduction from DIGs via caveolin and pp59(Lyn) to the insulin receptor substrate proteins of the insulin signaling pathway.     

The platelet receptor for type III collagen (TIIICBP) is present in platelet membrane lipid microdomains (rafts)

Platelet interactions with collagen are orchestrated by the presence or the migration of platelet receptor(s) for collagen into lipid rafts, which are specialized lipid microdomains from the platelet plasma membrane enriched in signalling proteins. Electron microscopy shows that in resting platelets, TIIICBP, a receptor specific for type III collagen, is present on the platelet membrane and associated with the open canalicular system, and redistributes to the platelet membrane upon platelet activation. After platelet lysis by 1% Triton X-100 and the separation of lipid rafts on a discontinuous sucrose gradient, TIIICBP is recovered in lipid raft-containing fractions and Triton X-100 insoluble fractions enriched in cytoskeleton proteins. Platelet aggregation, induced by type III collagen, was inhibited after disruption of the lipid rafts by cholesterol depletion, whereas platelet adhesion under static conditions did not require lipid raft integrity. These results indicate that TIIICBP, a platelet receptor involved in platelet interaction with type III collagen, is localized within platelet lipid rafts where it could interact with other platelet receptors for collagen (GP VI and α2β1 integrin) for efficient platelet activation. Pascal Maurice and Ludovic Waeckel have contributed equally to this work.     

The isolation and structure of membrane lipid rafts from rat brain

The action of detergents in the isolation of detergent-resistant membrane fractions from rat brain is reported. Triton X-100 treatment of whole rat brain homogenate at 4 degrees C produced detergent-resistant membranes with a density of 1.07g/ml compared with Brij96 where the density of the membrane was only 1.05g/ml. The DRM fractions isolated using Triton X-100 are considerably heavier than those isolated from homogenates treated with Brij96. The major polar lipid composition of DRMs derived from Brij96 treated homogenates have a higher proportion of aminophospholipids compared with choline phospholipids than Triton X-100 derived DRMs; this may indicate that DRMs from Brij96 treated homogenates are more closely related to the parent membrane in lipid composition. Solubilization by Triton X-100 at higher temperatures resulted in the appearance of a second detergent-resistant membrane fraction distinctly lighter in density than the membrane recovered at density 1.07g/ml. Analysis of phospholipid composition of the brain homogenate during detergent treatment for up to 30min at 37 degrees C showed a decreasing proportion of sphingomyelin. Treatment of homogenates at 37 degrees C appears to activate phospholipases/sphingomyelinases that may alter the lipid content of isolated DRMs. The presence of K+/Mg2+ with Brij96 treatment results in DRM fractions with significantly thicker bilayers and of larger vesicle diameter than DRMs isolated from either Triton X-100 or Brij96 treated homogenates in the absence of cations.     

Detergent-insoluble glycosphingolipid/cholesterol-rich membrane domains, lipid rafts and caveolae (review).

Within the cell membrane glycosphingolipids and cholesterol cluster together in distinct domains or lipid rafts, along with glycosyl-phosphatidylinositol (GPI)-anchored proteins in the outer leaflet and acylated proteins in the inner leaflet of the bilayer. These lipid rafts are characterized by insolubility in detergents such as Triton X-100 at 4 degrees C. Studies on model membrane systems have shown that the clustering of glycosphingolipids and GPI-anchored proteins in lipid rafts is an intrinsic property of the acyl chains of these membrane components, and that detergent extraction does not artefactually induce clustering. Cholesterol is not required for clustering in model membranes but does enhance this process. Single particle tracking, chemical cross-linking, fluorescence resonance energy transfer and immunofluorescence microscopy have been used to directly visualize lipid rafts in membranes. The sizes of the rafts observed in these studies range from 70-370 nm, and depletion of cellular cholesterol levels disrupts the rafts. Caveolae, flask-shaped invaginations of the plasma membrane, that contain the coat protein caveolin, are also enriched in cholesterol and glycosphingolipids. Although caveolae are also insoluble in Triton X-100, more selective isolation procedures indicate that caveolae do not equate with detergent-insoluble lipid rafts. Numerous proteins involved in cell signalling have been identified in caveolae, suggesting that these structures may function as signal transduction centres. Depletion of membrane cholesterol with cholesterol binding drugs or by blocking cellular cholesterol biosynthesis disrupts the formation and function of both lipid rafts and caveolae, indicating that these membrane domains are involved in a range of biological processes.     

Spatio-temporal localization of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos

We report for the first time the detection of membrane lipid rafts in mouse oocytes and cleaving preimplantation embryos. Cholera toxin β (CTβ), which binds to the raft-enriched ganglioside GM1, was selected to label rafts. In a novel application a Qdot reagent was used to detect CTβ labeling. This is the first reported use of nanocrystals in mammalian embryo imaging. Comparative membrane labeling with CTβ and lipophilic membrane dyes containing saturated or unsaturated aliphatic tails showed that the detection of GM1 in mouse oocytes and embryo membranes was consistent with the identification of cholesterol- and sphingolipid-enriched rafts in the cell membrane. Distribution of the GM1 was compared with the known distribution of non-raft membrane components, and disruption of membrane rafts with detergents confirmed the cholesterol dependence of GM1 on lipid raft labeling. Complementary functional studies showed that cholesterol depletion using methyl-β-cyclodextrin inhibited preimplantation development in culture. Our results show that the membranes of the mouse oocyte and zygote are rich in lipid rafts, with heterogeneous and stage-dependent distribution. In dividing embryos, the rafts were clearly associated with the cleavage furrow. At the morula stage, rafts were also apically enriched in each blastomere. In blastocysts, rafts were detectable in the trophectoderm layer, but could not be detected in the inner cell mass without prior fixation and permeabilization of the embryo. Lipid rafts and their associated proteins are, therefore, spatio-temporally positioned to a play a critical role in preimplantation developmental events.     

Hyperglycemia-induced membrane lipid peroxidation and elevated homocysteine levels are poorly attenuated by exogenous folate in embryonic chick brains

Injection of L-glucose (9.29 micromol/kg egg) into the air sac of fertile chicken eggs during the first 3 days of embryonic development (E(0-2)) has been reported to cause hyperglycemia and membrane lipid peroxidation in embryonic chick hepatic membranes. These observations have now been extended into embryonic chick brains at 11 days of development (theoretical stage 37). L-glucose caused a 1.7-fold increase in serum D-glucose levels (p< or =0.05), a 1.4-fold decrease in the % living embryos (p< or =0.05), a 1.1-fold decrease in embryonic masses (p< or =0.05), and a 1.4-fold decrease in embryonic brain masses (p< or =0.05) as compared to controls. L-glucose also caused a 3.8-fold increase in brain lipid hydroperoxide (LPO) levels (p< or =0.05) and complex changes in the relative fatty acid composition of brain membranes. Consistent with the hypothesis of hyperglycemia-induced increases in lipid peroxidation were decreased docosahexaenoic acid (DHA: 22: 6, n-3) levels as compared to controls (p< or =0.05). However, hyperglycemia-induced increased docosapentaenoic acid (DPA: 22:5, n-6) levels, decreased arachidonic acid (20; 4, n-6) levels, decreased linoleic acid (18:2, n-6) levels, and increased levels of several saturated short-chain membrane fatty acids were also observed as compared to controls (p< or =0.05). l-glucose caused a 12-fold increase in brain homocysteine levels, a 2.5-fold decrease in S-adenosylmethionine levels, and a 2-fold increase in S-adenosylhomocysteine levels as compared to controls (p< or =0.05). These hyperglycemia-induced alterations were poorly attenuated by exogenous folic acid (181.2 micromol/kg egg).     

CLN3P, the Batten disease protein, localizes to membrane lipid rafts (detergent-resistant membranes)

Juvenile neuronal ceroid lipofuscinosis is an inherited pediatric neurodegenerative disorder, which occurs as a result of mutations in the CLN3 gene that is located on chromosome 16p12.1. The encoded protein, CLN3P, is a putative transmembrane protein with no known function. In this study, we demonstrate that CLN3P resides on membrane lipid raft domains (detergent-resistant membranes) and provide important new data towards possible functions of the protein.     

The epithelial sodium channel (ENaC) traffics to apical membrane in lipid rafts in mouse cortical collecting duct cells

We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.     

Analysis of detergent-resistant membranes in Arabidopsis. Evidence for plasma membrane lipid rafts

The trafficking and function of cell surface proteins in eukaryotic cells may require association with detergent-resistant sphingolipid- and sterol-rich membrane domains. The aim of this work was to obtain evidence for lipid domain phenomena in plant membranes. A protocol to prepare Triton X-100 detergent-resistant membranes (DRMs) was developed using Arabidopsis (Arabidopsis thaliana) callus membranes. A comparative proteomics approach using two-dimensional difference gel electrophoresis and liquid chromatography-tandem mass spectrometry revealed that the DRMs were highly enriched in specific proteins. They included eight glycosylphosphatidylinositol-anchored proteins, several plasma membrane (PM) ATPases, multidrug resistance proteins, and proteins of the stomatin/prohibitin/hypersensitive response family, suggesting that the DRMs originated from PM domains. We also identified a plant homolog of flotillin, a major mammalian DRM protein, suggesting a conserved role for this protein in lipid domain phenomena in eukaryotic cells. Lipid analysis by gas chromatography-mass spectrometry showed that the DRMs had a 4-fold higher sterol-to-protein content than the average for Arabidopsis membranes. The DRMs were also 5-fold increased in sphingolipid-to-protein ratio. Our results indicate that the preparation of DRMs can yield a very specific set of membrane proteins and suggest that the PM contains phytosterol and sphingolipid-rich lipid domains with a specialized protein composition. Our results also suggest a conserved role of lipid modification in targeting proteins to both the intracellular and extracellular leaflet of these domains. The proteins associated with these domains provide important new experimental avenues into understanding plant cell polarity and cell surface processes.