Expression Profiles Of Transporters Research Articles

The WWOX/HIF1A Axis Downregulation Alters Glucose Metabolism and Predispose to Metabolic Disorders.

Recent reports indicate that the hypoxia-induced factor (HIF1α) and the Warburg effect play an initiating role in glucotoxicity, which underlies disorders in metabolic diseases. WWOX has been identified as a HIF1α regulator. WWOX downregulation leads to an increased expression of HIF1α target genes encoding glucose transporters and glycolysis’ enzymes. It has been proven in the normoglycemic mice cells and in gestational diabetes patients. The aim of the study was to determine WWOX’s role in glucose metabolism regulation in hyperglycemia and hypoxia to confirm its importance in the development of metabolic disorders. For this purpose, the WWOX gene was silenced in human normal fibroblasts, and then cells were cultured under different sugar and oxygen levels. Thereafter, it was investigated how WWOX silencing alters the genes and proteins expression profile of glucose transporters and glycolysis pathway enzymes, and their activity. In normoxia normoglycemia, higher glycolysis genes expression, their activity, and the lactate concentration were observed in WWOX KO fibroblasts in comparison to control cells. In normoxia hyperglycemia, it was observed a decrease of insulin-dependent glucose uptake and a further increase of lactate. It likely intensifies hyperglycemia condition, which deepen the glucose toxic effect. Then, in hypoxia hyperglycemia, WWOX KO caused weaker glucose uptake and elevated lactate production. In conclusion, the WWOX/HIF1A axis downregulation alters glucose metabolism and probably predispose to metabolic disorders.

Fe3O4-urea nanocomposites as a novel nitrogen fertilizer for improving nutrient utilization efficiency and reducing environmental pollution

Almost 81% of nitrogen fertilizers are applied in form of urea but most of it is lost due to volatilization and leaching leading to environmental pollution. In this regard, slow-release nano fertilizers can be an effective solution. Here, we have synthesized different Fe3O4-urea nanocomposites with Fe3O4 NPs: urea ratio (1:1, 1:2, 1:3) ie. NC-1, 2, and 3 respectively, and checked their efficacy for growth and yield enhancement. Oryza sativa L. cv. Swarna seedlings were treated with different NCs for 14 days in hydroponic conditions and significant up-regulation of photosynthetic efficiency and nitrogen metabolism were observed due to increased availability of nitrogen and iron. The discriminant functional analysis confirmed that the NC3 treatment yielded the best results so further gene expression studies were performed for NC-3 treated seedlings. Significant changes in expression profiles of ammonia and nitrate transporters indicated that NC-3 treatment enhanced nitrogen utilization efficiency (NUE) due to sustained slow release of urea. From pot experiments, we found significant enhancement of growth, grain nutrient content, and NUE in NC supplemented sets. 1.45 fold increase in crop yield was achieved when 50% N was supplemented in form of NC-3 and the rest in form of ammonium nitrate. NC supplementation can also play a vital role in minimizing the use of bulk N fertilizers because, when 75% of the recommended N dose was supplied in form of NC-3, 1.18 fold yield enhancement was found. Thus our results highlight that, slow-release NC-3 can play a major role in increasing the NUE of rice.

Expression of proposed methionine transporters along the gastrointestinal tract of pigs and their regulation by dietary methionine sources

BackgroundGiven the key role of methionine (Met) in biological processes like protein translation, methylation, and antioxidant defense, inadequate Met supply can limit performance. This study investigated the effect of different dietary Met sources on the expression profile of various Met transporters along the gastrointestinal tract (GIT) of pigs.MethodsA total of 27 pigs received a diet supplemented with 0.21% DL-Met, 0.21% L-Met, or 0.31% DL-2-hydroxy-4-(methylthio)butanoic acid (DL-HMTBA). Changes in mRNA expression of B0AT1, ATB0,+, rBAT, ASCT2, IMINO, LAT4, y+LAT1, LAT2, and SNAT2 were evaluated in the oral mucosa, cardia, fundus, pylorus, duodenum, proximal jejunum, middle jejunum, ileum, cecum, proximal colon, and distal colon, complemented by protein expression analysis of B0AT1, ASCT2, LAT2, and LAT4.ResultsExpression of all investigated transcripts differed significantly along the GIT. B0AT1, rBAT, y+LAT1, LAT2, and LAT4 showed strongest mRNA expression in small intestinal segments. ASCT2, IMINO, and SNAT2 were similarly expressed along the small and large intestines but expression differed in the oral mucosa and stomach. ATB0,+ showed highest mRNA expression in large intestinal tissues, cardia, and pylorus. In pigs fed DL-Met, mRNA expression of ASCT2 was higher than in pigs fed DL-HMTBA in small intestinal tissues and mRNA expression of IMINO was lower than in pigs fed L-Met in large intestinal tissues. Dietary DL-HMTBA induced a stronger mRNA expression of basolateral uptake systems either in the small (LAT2) or large (y+LAT1) intestine. Protein expression of B0AT1 was higher in the middle jejunum and ileum in pigs fed DL-Met when compared with the other Met supplements. LAT4 expression was higher in pigs fed DL-HMTBA when compared with DL-Met (small intestine) and L-Met (small intestine, oral mucosa, and stomach).ConclusionA high expression of several Met transporters in small intestinal segments underlines the primary role of these segments in amino acid absorption; however, some Met transporters show high transcript and protein levels also in large intestine, oral mucosa, and stomach. A diet containing DL-Met has potential to increase apical Met transport in the small intestine, whereas a diet containing DL-HMTBA has potential to increase basolateral Met transport in the small intestine and, partly, other gastrointestinal tissues.

L-amino acid oxidase from snake venom: Biotransformation and induction of apoptosis in human colon cancer cells

This study evaluated the potential of antitumor activity of snake venom from Vipera ammodytes and L-amino acid oxidase from Crotalus adamanteus on different colorectal cancer cell lines through determination of cytotoxic activity by MTT assay, pro-apoptotic activity by acridine orange/ethidium bromide staining, and concentrations of redox status parameters (superoxide, reduced glutathione, lipid peroxidation) by colorimetric methods. The expression of genes involved in the biotransformation process and metabolite efflux was determined by qPCR method, while protein expression of glutathione synthetase and P-glycoprotein were analysed by immunocytochemistry. The analysis of cell death shows that snake venom dominantly leads cells to necrosis. Induction of apoptosis by L-amino acid oxidase was in correlation with oxidative disbalance in cancer cells. Gene expression profile of membrane transporters and CYP genes were different in each cell line and in correlation with their sensitivity of treatment. Our results show that L-amino acid oxidase from snake venom is a potent cytotoxic substance with pronounced pro-apoptotic activity. The inhibition of P-glycoprotein suggests that L-amino acid oxidase is a good substance for furter research of antitumor effect, with unexpressed potential for occurrence of drug resistance in vitro.

Skeletal muscle phenotype and game performance in elite women football players.

We combined game activity analyses with skeletal muscle phenotypes and comprehensive physiological testing to elucidate factors of importance for physical performance in elite women's football. GPS-data from an experimental game, sprint and endurance testing, and muscle tissue analysis of metabolic enzyme activity, protein expression and fiber type composition were completed for international top-level women players (n=20; age; 23±4 yrs, height; 166±10cm, weight; 60±8kg; VO2max ; 51±6ml/min/kg). Muscle monocarboxylate transporter 4 (MCT4) protein expression explained 46% of the variance in total game distance, while the ability to maintain high-intensity running (HIR) during the final 15min of the game correlated to myosin heavy chain 1 (MHCI) and Na+ -K+ ATPase β1, FXYD1 (phospholemman) and superoxide dismutase 2 (SOD2) protein expression (range: r=0.51-0.71; all p<0.05). Total HIR distance correlated with (MHCIIa) protein expression (r=0.51; p<0.05), while muscle Na+ /H+ exchanger 1 (NHE1) protein explained 36% of the variance in game sprint distance (p<0.05). Total game accelerations (actions >4m/s2 ) correlated with platelet endothelial cell adhesion molecule (PECAM-1) protein expression (r=0.51; p<0.05), while concentric knee flexor strength explained 42-62% of the variance in intense decelerations (>4m/s2 ). In conclusion, for elite women players' game endurance performance and resistance to end-game fatigue were affected by monocarboxylate transporter expression and myosin heavy chain profile. HIR was also correlated to ion transporter expression and muscle antioxidative capacity. Finally, the importance of functional strength and measures of muscle vascularization in relation to total game decelerations and accelerations, respectively, illustrates the complex physiological demands in elite women's football.

SERCA Inhibition as a Potential Therapeutic Target for Breast Cancer

Chemotherapy is both a palliative treatment for many cancers and adjuvant treatment before, during, or after local treatment (surgery and/or radiation therapy) in single or combination regimens. Despite current treatments, approximately 1 in 39 women (3%) still die from breast cancer. A major complication with systemic therapy is drug resistance which limits the efficacy of clinical chemotherapy. Hence, current research aims to uncover novel targets for breast cancer therapy. Various studies have shown altered calcium signaling in breast cancer cells, therefore, calcium modulators may be potential therapeutic targets. Studies have also shown that Thapsigargin (TG) and cyclopiazonic acid (CPZ), two potent and specific inhibitors of the sarcoplasmic reticulum (SR) Ca2+-ATPase (SERCA), can impair cancer cell growth. However, TG and CPZ also impair non-malignant cells, which results in significant toxicity, thus preventing use in chemotherapy. Using spectrometry UV-visible measurements to assess calcium loading and ATPase activity in SR microsomes, our lab has found that various drugs which display cell protective effects in ischemia also inhibit SERCA. They include novel benzothiazepines (BZTs; K201, CGP37157, PH00095) and have shown to inhibit SERCA in a calcium dependent mechanism. They decreased SR uptake when cytosolic Ca2+ levels ([Ca2+]c) are low, similar to competitive inhibition of Ca2+ uptake, which produces mild store depletion. Contrarily, TG and CPZ block Ca2+ uptake at all [Ca2+]c and largely deplete SR stores. Using various SERCA inhibitors, we sought out to determine their effects on breast cancer cell lines of different subtypes. MTS assay studies show that TG and CPZ significantly reduce cell viability in MCF7 and MCF10A cell lines while having no significant effects on triple negative breast cancer (TNBC) cell line MDAMB231. Preliminary studies also show no significant effect by TG on BT20 cells, another TNBC cell line. BZT K201 also significantly reduced cell viability in MCF7 cells but not MDAMB231. Immunoblot shows MCF7 cells have significantly higher SERCA2 expression than MDAMB231. We observe no significant differences in plasma membrane calcium channel ATPase (PMCA) protein expression. Preliminary RT-PCR studies estimate MCF7 cells have a higher fold change in SERCA genes. Thus, targeting SERCA may serve as a potential therapeutic target for breast cancer. Here, we find that SERCA inhibitors TG, CPZ, and BZT K201 reduce cell viability in MCF7 cells. Still, these studies indicate that SERCA inhibition may only be efficacious in certain breast cancer cell types which do not express a TNBC phenotype. TNBC cell lines (MDAMB231, BT20) are not significantly affected by SERCA inhibition, which may be due to higher dependence for signaling on plasmalemma calcium channels and transporters. Additional studies are required to confirm the expression profiles of calcium transporters among cell lines, including those in the plasmalemma (PMCA, calcium channels), which may help to understand if alternative sources of Ca2+ for intracellular signaling make TNBC cell lines more resistant to SERCA inhibitors.

Changing expression patterns of TonB-dependent transporters suggest shifts in polysaccharide consumption over the course of a spring phytoplankton bloom

Algal blooms produce large quantities of organic matter that is subsequently remineralised by bacterial heterotrophs. Polysaccharide is a primary component of algal biomass. It has been hypothesised that individual bacterial heterotrophic niches during algal blooms are in part determined by the available polysaccharide substrates present. Measurement of the expression of TonB-dependent transporters, often specific for polysaccharide uptake, might serve as a proxy for assessing bacterial polysaccharide consumption over time. To investigate this, we present here high-resolution metaproteomic and metagenomic datasets from bacterioplankton of the 2016 spring phytoplankton bloom at Helgoland island in the southern North Sea, and expression profiles of TonB-dependent transporters during the bloom, which demonstrate the importance of both the Gammaproteobacteria and the Bacteroidetes as degraders of algal polysaccharide. TonB-dependent transporters were the most highly expressed protein class, split approximately evenly between the Gammaproteobacteria and Bacteroidetes, and totalling on average 16.7% of all detected proteins during the bloom. About 93% of these were predicted to take up organic matter, and for about 12% of the TonB-dependent transporters, we predicted a specific target polysaccharide class. Most significantly, we observed a change in substrate specificities of the expressed transporters over time, which was not reflected in the corresponding metagenomic data. From this, we conclude that algal cell wall-related compounds containing fucose, mannose, and xylose were mostly utilised in later bloom stages, whereas glucose-based algal and bacterial storage molecules including laminarin, glycogen, and starch were used throughout. Quantification of transporters could therefore be key for understanding marine carbon cycling.

Atmospheric reactive oxygen species and some aspects of the antiviral protection of the respiratory epithelium

The review focuses on molecular and biochemical mechanisms of nonspecific protection of respiratory epithelium. The authors provide a comprehensive analysis of up-to-date data on the activity of the lactoperoxidase system expressed on the surface of the respiratory epithelium which provides the generation of hypothiocyanate and hypoiodite in the presence of locally produced or inhaled hydrogen peroxide. Molecular mechanisms of production of active compounds with antiviral and antibacterial effects, expression profiles of enzymes, transporters and ion channels involved in the generation of hypothiocyanite and hypoiodate in the mucous membrane of the respiratory system in physiological and pathological conditions (inflammation) are discussed. In the context of antibacterial and antiviral defense special attention is paid to recent data confirming the effects of atmospheric air composition on the efficiency of hypothiocyanite and hypoiodate synthesis in the respiratory epithelium. The causes and outcomes of lactoperoxidase system impairment due to the action of atmospheric factors are discussed in the context of controlling the sensitivity of the epithelium to the action of bacterial agents and viruses. Restoration of the lactoperoxidase system activity can be achieved by application of pharmacological agents aimed to compensate for the lack of halides in tissues, and by the control of chemical composition of the inhaled air.

Butyrate Rescues Oxidative Stress-Induced Transport Deficits of Tryptophan: Potential Implication in Affective or Gut-Brain Axis Disorders

Introduction: Butyrate is a short-chain fatty acid metabolite produced by microbiota in the colon. With its antioxidant properties, butyrate has also been shown to alter the neurological functions in affective disorder models, suggesting it as a key mediator in gut-brain interactions. Objective: Here, we evaluated the negative effect of oxidative stress on the transport of the serotonin precursor tryptophan as present in affective disorders. Butyrate was hypothesized to be able to rescue these deficits due to its antioxidative capacities and its effect on transmembrane transport of tryptophan. Human skin-derived fibroblasts were used as cellular models to address these objectives. Methods: Human fibroblasts were treated with hydrogen peroxide to induce oxidative stress. Stressed as well as control cells were treated with different concentrations of butyrate. Tryptophan (3H) was used as a tracer to measure the transport of tryptophan across the cell membranes (n = 6). Furthermore, gene expression profiles of different amino acid transporters were analyzed (n = 2). Results: As hypothesized,oxidative stress significantly decreased the uptake of tryptophan in fibroblast cells, while butyrate counteracted this effect. Oxidative stress did not alter the gene expression profile of amino acid transporters. However, treatment of stressed and control cells with different concentrations of butyrate differentially regulated the gene expression of large amino acid transporters 1 and 2, which are the major transporters of tryptophan. Conclusions:Gut microbiota-derived butyrate may have therapeutic potential in affective disorders characterized by either aberrant serotonergic activity or neuroinflammation due to its role in rescuing the oxidative stress-induced perturbations of tryptophan transport.

Arbuscular mycorrhizal symbiosis alters the expression of PHT1 phosphate transporters in roots and nodules of P-starved soybean plants

Roots acquire phosphorus (P) as orthophosphate (Pi) through phosphate transporters of the PHT1 family with different affinities to Pi, a process significantly influenced by arbuscular mycorrhizal (AM) symbiosis. However, scarce P availability may constrain nodulation and performance in legumes. Soybean (Glycine max) is an ideal model to study tripartite symbiosis between roots, AM fungi and rhizobia. To evaluate AM influence on GmPHT1 expression in roots and nodules, Bradyrhizobium elkanii-inoculated soybean plants were exposed to low Pi concentration (50 µM) via nutrient solution and inoculated (+ AM) or not (− AM) with the AM fungus Glomus macrocarpum. Control treatment consisted of non-inoculated plants grown under sufficient P conditions (500 µM; − AM + P). Plants were collected at the flowering and grain filling stages. Under P-starvation, mycorrhizal plants showed low intraradical colonization and did not differ in terms of biomass, nodulation and P content from the non-mycorrhizal plants, indicating strong P-limitation and no AM-related growth promotion. However, the expression profile of PHT1 transporters in roots and nodules was effectively altered by mycorrhization. P-starvation induced the expression of several GmPHT1 genes in roots and nodules, while AM symbiosis repressed GmPHT1;6, 7 and 10 in roots and GmPHT1;3, 5, 7, 8, and 10 in nodules. Therefore, even under low levels of root colonization, AM symbiosis significantly modulated the pattern of PHT1 expression under P-starvation. GmPHT1 expression profile suggests different pathways of Pi acquisition in mycorrhizal and non-mycorrhizal plants during P-starvation, however, the low mycorrhizal colonization was not able to deliver adequate P nutrition to the plant.

Expression profile of stem cell markers and ABC transporters in 5-fluorouracil resistant Hep-2 cells.

Resistance of laryngeal squamous cell carcinoma cells to traditional therapeutic regimens still remains to be a major reason for therapeutic failure in patients. In this study, we aimed at investigating the expression profiles of ATP-binding cassette (ABC) transporters and stem cell markers in 5-fluorouracil (5-FU) resistant laryngeal Hep-2 cells. We treated parental Hep-2 cells, with stepwise increased doses of 5-FU for almost 1year to develop 5-FU resistant sub-lines with resistance against varying levels of 5-FU concentrations (4 sub-lines resistant to 1, 2, 4, and eightfold of 5-FU). Then, we measured the expression levels of 10 genes from ABC transporters family and 4 stem cell associated markers using quantitative reverse transcription polymerase chain reaction (qRT-PCR) to find out a potential relationship between these markers and chemoresistance. We found that stemness-associated markers had elevated expressions from the beginning of 5-FU resistance acquisition. Their expressions elevated stepwise while parental Hep-2 cells got resistance to higher doses of 5-FU. Expressions of tested ABC transporters (ABCA5, ABCB1, ABCB6, ABCC1, ABCC2, ABCC3, ABCC5, ABCC10 and ABCF2, and ABCG2) were also deregulated in 5-FU resistant Hep-2 cells. Although their expressions remained unaltered at the beginning of acquisition of resistance, expressions of ABC transporters except from ABCB6 increased significantly when cells became resistant to higher doses of 5-FU. Our results suggest that enrichment of cells with stemness characteristics and upregulation of ABC transporters might be amongst the crucial contributors of chemoresistance in laryngeal cancer cells.

Polarized Expression of Ion Channels and Solute Carrier Family Transporters on Heterogeneous Cultured Human Corneal Endothelial Cells.

PurposeTo clarify the expression profiles of ion channels and transporters of metabolic substrates among heterogeneous cultured human corneal endothelial cells (cHCECs) distinct in their effectiveness in reconstituting the corneal endothelium.MethodsIntegrated proteomics for cell lysates by liquid chromatography–tandem mass spectrometry was carried out from three aliquots of cHCECs enriched in either cluster of definition (CD)44−/+ (mature) cHCECs or CD44++/+++ cell-state transition (CST) cHCECs. The expression profiles of cations/anions, monocarboxylic acid transporters (MCTs), and solute carrier (SLC) family proteins, as well as carbonic anhydrases (CAs), were investigated.ResultsThe polarized expression of cations/anions, MCTs, and SLC family proteins, as well as CAs, was clarified for mature and CST cHCECs. Most SLC4 family members, including SLC4A11 and SLC4A4 (NBCe1), were upregulated in the CST cHCECs, whereas SLC9A1 (Na+/H+ exchanger isoform one [NHE1]) and CA5B were detected only in the mature cHCECs. In addition, SLC25A42, catalyzing the entry of coenzyme A into the mitochondria, and SLC25A18, functioning as a mitochondrial glutamate carrier 2 (both relevant for providing the substrates for mitochondrial bioenergetics), were selectively expressed in the mature cHCECs.ConclusionsOur findings may suggest the relevance of qualifying the polarized expression of these ion channels and transporter-like proteins to ensure not only the suitability but also the in vivo biological functionality of cHCECs selected for use in a cell-injection therapy.

Expression profile of xenobiotic efflux transporters in mouse neural stem cells

Expression profile of xenobiotic efflux transporters in mouse neural stem cells

Evaluating Metabolic Regulation of Human Erythropoiesis By Profiling Nutrient Transporter Expression

Recent studies have documented that cell metabolism regulates hematopoietic stem cell (HSC) renewal and lineage commitment. However, the detailed metabolic changes that occur during human erythropoiesis remain to be defined. As erythroid cell differentiation is likely to be associated with changes in metabolic requirements, we hypothesized that progenitors adapt to these metabolic modulations by altering their nutrient transporter expression profile. Using an in vitro erythroid-inducing cell culture system employing CD34+ cells from human bone marrow and peripheral blood as well as primary erythroid cells isolated from fresh bone marrow samples, we assessed the cell surface nutrient transporter profiles of progenitors at different stages of erythroid development. Quantification of cell surface nutrient transporter expression was performed using a novel scaffold of retroviral envelope receptor binding domains (RBDs) that function as specific ligands of solute carrier (SLC) nutrient transporters. This bank allowed an evaluation of diverse metabolite transporters including GLUT1/SLC2A1 glucose transporter, the PiT1/SLC20A1 and PiT2/SLC20A2 phosphate importers, the XPR1/SLC53A1 phosphate exporter, the FLVCR1 heme exporter, the RFVT1/2 (SLC52A1/SLC52A2) riboflavin importers, the CAT1/SLC7A1 arginine importer, the ASCT2/SLC1A5 glutamine transporter and the SMVT/SLC5A6 sodium-dependent multivitamin transporter. Notably, the cell surface expression profiles of these nutrient transporters, as evaluated by flow cytometry, revealed marked changes as a function of the stage of erythroid differentiation, as shown in the figure below. Specifically, while FLVCR1, RFVT1/2, and SMVT are highly expressed on erythroid progenitors, the levels of these transporters decrease starting at the proerythroblast stage. In addition, PiT1, PiT2, XPR1, and CAT1 are expressed highly during the erythroid colony forming unit (CFU-E) stage while GLUT1 gradually increases and reaches a peak at late stages of erythroid differentiation, remaining elevated on mature red cells. The noted distinct changes in transporter expression are likely a reflection of the changing demands of various nutrients during human erythropoiesis. In summary, we have established a comprehensive metabolite transporter profile at distinct stages of normal human erythropoiesis using a novel experimental strategy. These original findings form a strong foundation for future studies aiming at elucidating the metabolic requirements of normal erythropoiesis, evaluating diseases affecting red blood cell maturation due to aberrant metabolic regulation, and for identifying new therapeutic targets. Figure Disclosures Bitaudeau: Metafora-biosystems: Employment. Petit:Metafora-biosystems: Equity Ownership, Other: CEO and co-founder. Sitbon:Metafora-biosystems: Membership on an entity's Board of Directors or advisory committees, Other: Co-founder.

Prediction of Drug Permeability Using In Vitro Blood-Brain Barrier Models with Human Induced Pluripotent Stem Cell-Derived Brain Microvascular Endothelial Cells.

The strong barrier function of the blood–brain barrier (BBB) protects the central nervous system (CNS) from xenobiotic substances, while the expression of selective transporters controls the transportation of nutrients between the blood and brain. As a result, the delivery of drugs to the CNS and prediction of the ability of specific drugs to penetrate the BBB can be difficult. Although in vivo pharmacokinetic analysis using rodents is a commonly used method for predicting human BBB permeability, novel in vitro BBB models, such as Transwell models, have been developed recently. Induced pluripotent stem cells (iPSCs) have the potential to differentiate into various types of cells, and protocols for the differentiation of iPSCs to generate brain microvascular endothelial cells (BMECs) have been reported. The use of iPSCs makes it easy to scale-up iPSC-derived BMECs (iBMECs) and enables production of BBB disease models by using iPSCs from multiple donors with disease, which are advantageous properties compared with models that utilize primary BMECs (pBMECs). There has been little research on the value of iBMECs for predicting BBB permeability. This study focused on the similarity of iBMECs to pBMECs and investigated the ability of iPSC-BBB models (monoculture and coculture) to predict in vivo human BBB permeability using iBMECs. iBMECs express BMEC markers (e.g., VE-cadherin and claudin-5) and influx/efflux transporters (e.g., Glut-1, SLC7A5, CD220, P-gp, ABCG2, and MRP-1) and exhibit high barrier function (transendothelial electrical resistance, >1000 Ω × cm2) as well as similar transporter expression profiles to pBMECs. We determined that the efflux activity using P-glycoprotein (P-gp) transporter is not sufficient in iBMECs, while in drug permeability tests, iPSC-derived BBB models showed a higher correlation with in vivo human BBB permeability compared with a rat BBB model and the Caco-2 model. In a comparison between monoculture and coculture models, the coculture BBB model showed higher efflux activity for compounds with low CNS permeability (e.g., verapamil and thioridazine). In conclusion, iPSC-BBB models make it possible to predict BBB permeability, and employing coculturing can improve iPSC-BBB function.

The circadian clock regulates RPE-mediated lactate transport via SLC16A1 (MCT1)

Multiple retinal cells harbor a circadian oscillator, including retinal pigment epithelial cells (RPE). However, little is known about the functions that are regulated by the RPE clock. The aim of this study was to investigate whether the circadian clock in the RPE regulates the transport of glucose and its glycolytic metabolic by-product - lactate. To that end, we first characterized the mRNA expression profile of glucose and monocarboxylate transporters in ARPE-19 cells. We found that SLC2A1 and SLC16A1 were, respectively, the most abundantly expressed glucose and lactate (monocarboxylate) transporters. We further observed that the protein products of SLC2A1 (encoding GLUT1) and SLC16A1 (encoding MCT1) localize on the apical membrane of ARPE-19 monolayers. In a subsequent time-course experiment, we found that SLC2A1 and SLC16A1 mRNA oscillated in ARPE-19 monolayers, but not in dispersed cells, suggesting that monolayer cellular organization is necessary for rhythmic regulation of these transporters. In these monolayers, we found that MCT1 proteins varied over time, in contrast to GLUT1 proteins which did not vary over time. Spectrophotometric measurements of supernatants sampled from ARPE-19 monolayer cultures revealed that glucose concentrations did not significantly differ between apical (Api) supernatants and basolateral (BL) ones. In addition, we did not find rhythms in Api or BL glucose concentrations. Conversely, we found higher lactate concentrations in Api supernatants than BL ones. Further, we found that Api lactate concentrations were rhythmic. Pearson's r revealed that the concentration gradients (Api - BL) of glucose and lactate correlated with the gene expression of respective SLC2A1 and SLC16A1 transporters. Incubation with photoreceptor outer segments (POS) affected the mRNA expression of SLC16A1 and SLC2A1 in ARPE-19 monolayers in a time-dependent manner, thus suggesting that the retina might modulate the RPE clock-controlled expression of transporters via interactions with POS. In conclusion, this work provides evidence that the transport of lactate is regulated by the circadian clock in the RPE.

Tissue and nitrogen-linked expression profiles of ammonium and nitrate transporters in maize

BackgroundIn order to grow, plants rely on soil nutrients which can vary both spatially and temporally depending on the environment, the soil type or the microbial activity. An essential nutrient is nitrogen, which is mainly accessible as nitrate and ammonium. Many studies have investigated transport genes for these ions in Arabidopsis thaliana and recently in crop species, including Maize, Rice and Barley. However, in most crop species, an understanding of the participants in nitrate and ammonium transport across the soil plant continuum remains undefined.ResultsWe have mapped a non-exhaustive set of putative nitrate and ammonium transporters in maize. The selected transporters were defined based on previous studies comparing nitrate transport pathways conserved between Arabidopsis and Zea mays (Plett D et. al, PLOS ONE 5:e15289, 2010). We also selected genes from published studies (Gu R et. al, Plant and Cell Physiology, 54:1515-1524, 2013, Garnett T et. al, New Phytol 198:82-94, 2013, Garnett T et. al, Frontiers in Plant Sci 6, 2015, Dechorgnat J et. al, Front Plant Sci 9:531, 2018). To analyse these genes, the plants were grown in a semi-hydroponic system to carefully control nitrogen delivery and then harvested at both vegetative and reproductive stages. The expression patterns of 26 putative nitrogen transporters were then tested. Six putative genes were found not expressed in our conditions. Transcripts of 20 other genes were detected at both the vegetative and reproductive stages of maize development. We observed the expression of nitrogen transporters in all organs tested: roots, young leaves, old leaves, silks, cobs, tassels and husk leaves. We also followed the gene expression response to nitrogen starvation and resupply and uncovered mainly three expression patterns: (i) genes unresponsiveness to nitrogen supply; (ii) genes showing an increase of expression after nitrogen starvation; (iii) genes showing a decrease of expression after nitrogen starvation.ConclusionsThese data allowed the mapping of putative nitrogen transporters in maize at both the vegetative and reproductive stages of development. No growth-dependent expression was seen in our conditions. We found that nitrogen transporter genes were expressed in all the organs tested and in many cases were regulated by the availability of nitrogen supplied to the plant. The gene expression patterns in relation to organ specificity and nitrogen availability denote a speciality of nitrate and ammonium transporter genes and their probable function depending on the plant organ and the environment.

Diet-induced obese alters the expression and function of hepatic drug-metabolizing enzymes and transporters in rats

Obesity increases the incidences of metabolic syndrome, including type 2 diabete, fatty liver, dyslipidemia, hyperglycemia, heart disease, hypertension and cancer. In particular, pharmacokinetics and pharmacodynamics of many drugs have changed in obese patients. However, little is known about the hepatic drug-metabolizing enzymes and transporters that are influenced by diet-induced obese. In this report, we established obesity and fatty liver models in male rats by high-fat diet. The expression profiles of drug-metabolizing enzymes and transporters were studied by quantitative real-timePCR and Western blotting analysis. The function of these enzymes and transporters were assessed by their substrates and cocktail methods. The expression and activity of phase I enzymes (CYP1A2, CYP2B1, CYP2C11, CYP3A1, CYP4A1 and FMO1) and phase II enzymes (UGT1A1, UGT1A3, UGT1A6, UGT1A9, UGT2B7, NAT1 and GSTT1) were decreased in the liver of obese rats. In addition, the mRNA levels of hepatic transporter Slco1a2, Slco1b2, Slc22a5, Abcc2, Abcc3, Abcb1a and Abcg2 decreased significantly in obese animals, while Abcb1b increased significantly. Furthermore, the decreased expression of hepatic phase I and II enzymes and transporter may be due to changes of Hnf4α, LXRα and FXR. In conclusion, the diet-induced obese altered the expression and function of hepatic drug-metabolizing enzymes and transporters in male rats, thereby impacting drug metabolism and pharmacokinetics.

Comparison of mRNA expression profiles of drug-metabolizing enzymes and transporters in fresh and cryopreserved cynomolgus monkey hepatocytes

In this study, freshly isolated and cryopreserved cynomolgus monkey hepatocytes were seeded on Cell-able® plates with feeder cells to form spheroids and were cultured for 28 days. As a control, hepatocytes were also cultured with or without feeder cells on collagen-coated plates. We verified the mRNA expression levels of drug-metabolizing enzyme-related genes and the leakage of enzymes (AST, ALT, LDH, and γ-GTP) as indicators of cell survival. As a result, the patterns of target mRNA expression in fresh and cryopreserved hepatocytes were very similar during the culture period between culture methods. mRNA expression levels were highly maintained at day 28 using the 3D spheroid and co-culture methods, demonstrating that these methods are useful for maintenance of liver function. Leakage of AST and ALT was higher at day 3 but decreased at day 14. LDH was not detected, suggesting that the cell viability was also maintained during the culture period. Furthermore, the functional differences between fresh and cryopreserved hepatocytes were not clearly detected. The co-culture method was useful for long-term culture not requiring 3D structure, and the 3D spheroid culture method was effective as well. With these techniques, cynomolgus monkey hepatocytes are expected to exhibit smaller individual differences and high reproducibility.

Proteomic Quantification of Human Blood-Brain Barrier SLC and ABC Transporters in Healthy Individuals and Dementia Patients.

The blood-brain barrier (BBB) maintains brain homeostasis by controlling traffic of molecules from the circulation into the brain. This function is predominantly dependent on proteins expressed at the BBB, especially transporters and tight junction proteins. Alterations to the level and function of BBB proteins can impact the susceptibility of the central nervous system to exposure to xenobiotics in the systemic circulation with potential consequent effects on brain function. In this study, expression profiles of drug transporters and solute carriers in the BBB were assessed in tissues from healthy individuals ( n = 12), Alzheimer's patients ( n = 5), and dementia with Lewy bodies patients ( n = 5), using targeted, accurate mass retention time (AMRT) and global proteomic methods. A total of 53 transporters were quantified, 19 for the first time in the BBB. A further 20 novel transporters were identified but not quantified. The global proteomic method identified another 3333 BBB proteins. Transporter abundances, taken together with the scaling factor, microvessel protein content per unit tissue (BMvPGB also measured here), can be used in quantitative systems pharmacology models predicting drug disposition in the brain and permitting dose adjustment (precision dosing) in special populations of patients, such as those with dementia. Even in this small study, we see differences in transporter profile between healthy and diseased brain tissue.