Carrier-mediated Process Research Articles

Bacterial lipopolysaccharide inhibits free thiamin uptake along the intestinal tract via interference with membrane expression of thiamin transporters 1 and 2.

This study examined the effect of exposure of small and large intestinal epithelial cells to the bacterial lipopolysaccharide (LPS) on uptake of free form of vitamin B1, i.e., thiamin. The intestinal tract encounters two sources of thiamin: diet and the gut microbiota. Absorption of thiamin in both the small and large intestine occurs via a carrier-mediated process that involves thiamin transporters 1 and 2 (THTR-1 and -2). Complementary in vitro (human duodenal epithelial HuTu-80 cells and human colonic epithelial NCM460 cells), in vivo (mice), and ex vivo (human primary differentiated enteroid and colonoid monolayers) models were used. The results showed that exposure to LPS causes a significant inhibition in carrier-mediated [3H]-thiamin uptake by small and large intestinal epithelia, with no change in the levels of expression of THTR-1 and -2 mRNAs and their total cellular proteins. However, a significant decrease in the fractions of the THTR-1 and -2 proteins that are expressed at the cell membranes of these epithelial cells was observed. These effects of LPS appeared to involve a protein kinase A (PKA) signaling pathway as activating this pathway caused a reversal in the inhibition of thiamin uptake and level of expression of its transporters at the cell membrane. These findings demonstrate that exposure of gut epithelia to LPS (a situation that occurs under different pathological conditions) leads to inhibition in thiamin uptake due to a decrease in level of expression of its transporters at the cell membrane that is likely mediated via a PKA signaling pathway. NEW & NOTEWORTHY This study shows that the exposure of gut epithelial cells to bacterial LPS negatively impact the uptake process of the free form of vitamin B1 (i.e., thiamin). This appears to be mediated via suppression in the level of thiamin transporters 1 and 2 (THTR-1 and -2) expression at the cell membrane and involves a protein kinase A (PKA) signaling pathway.

Effect of knocking out mouse Slc44a4 on colonic uptake of the microbiota-generated thiamine pyrophosphate and colon physiology.

Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation.NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.

SLC44A4 Is the Predominant Transport System for Uptake of the Microbiota-Generated Phosphorylated Form of Vitamin B1 (Thiamin Pyrophosphate; TPP) in Colonocytes: Role in Colonocyte's Physiology

Background: Humans/mammals obtain vitamin B1 from diet and the gut-microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP. We have previously shown that colonocytes are capable of absorbing intact TPP via a specific and effcient carrier-mediated process ( JBC 289:4405-16, 2014); we have also identified the SLC44A4 as a colonic TPP transporter (cTPPT) in both human and mouse and have characterized different aspects of its regulation and cell biology ( Plos One 14: e0224234, 2019). Little, however, is known about the relative contribution of the cTPPT toward total colonic carrier-mediated TPP uptake. Aims: To determine the relative contribution of cTPPT toward total colonic carrier-mediated TPP uptake, and to investigate the effect of its knockout/down on colonocyte's physiology. Methods: An Slc44a4 knockout (KO) mouse model was generated by crossing homozygous Slc44a4 LoxP (+/+) animals with CMV promoter-driven Cre-transgenic mice. Knocking down the SLC44A4 in human colonic epithelial CCD841 cells and in human primary differentiated colonocyte monolayers (from organ donors) was achieved using gene-specific siRNAs. [3H]-TPP was used in uptake studies. Results: Body weight and colonic length of the Slc44a4 KO mice were significantly lower than those of WT-littermates. Near complete (and specific) inhibition in colonic carrier-mediated [3H]-TPP uptake was observed in Slc44a4 KO mice compared to WT-littermates. Similarly, knocking down SLC44A4 of human CCD841 cells and that of human primary colonocyte monolayers led to significant reduction in [3H]-TPP uptake. Since colonocytes have limited ability to synthesize TPP endogenously [they express a very low level of the enzyme thiamin pyrophosphokinase; BBA 1517:320-2, 2001] and that recent genomic studies have suggested that the SLC44A4 gene could be a potential “ulcerative colitis susceptibility” gene ( Genes Immun 17:105-9, 2016), we also examined the effect of Slc44a4 KO on colonocytes physiology. Results of our RNA-seq and Ingenuity Pathway Analysis showed an increase in several genes related to chronic colitis and intestinal inflammation, and changes in genes involved in energy metabolism. Also, the Slc44a4 KO mice were found to be more susceptible to developing colitis when exposed to a low dose of dextran sodium sulfate compared to their WT-littermates. Conclusion: The SLC44A4 is the predominant/only transport system for uptake of the microbiota-generated TPP and that its loss compromise normal colonocyte's physiology. Supported by NIH grants DK-56061 & AA-018071, and VA grants 101BX001142 & IK6BX006189. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Identification of IQ Motif Containing GTPase Activating Protein 2, IQGAP-2 as an interacting partner with the Human Colonic Thiamin Pyrophosphate Transporter (hcTPPT): Effects on physiology and Cell Biology of the Transporter

Background:Humans/mammals obtain vitamin B1 from two sources: the diet and the gut-microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of thiamin pyrophosphate (TPP). We have previously shown that colonocytes process an effcient and specific carrier-mediated process for uptake of the generated TPP that involves the human colonic thiamine pyrophosphate transporter (hcTPPT, product of the SLC44A4 gene). We have also shown that expression of the cTPPT along the GI tract is restricted to the large intestine and have characterized different aspects of its regulation at both the transcriptional and post-transcriptional levels.Aim:To determine whether the hcTPPT has interacting protein partner(s) in human colonocytes that affect its functionality and/or cell biology.Methods: Yeast Two-Hybrid system (Y2H) was used to screen a random-primed normal human colonic cDNA library using the full-length SLC44A4 as a bait. Effect of knocking-down (with gene-specific siRNA) and over-expressing the putative interactor was examined using human-derived colonic epithelial NCM460 cells and human primary differentiated colonoid monolayers (obtained form organ donors). We also used confocal imaging, RT-qPCR and western blotting in our investigations. Results:Our Y2H screening have identified three putative interactors with the hcTPPT, namely: IQGAP-2, SNX-6, and DMXL-1. While all these putative interactors are expressed in human colonocytes, expression of the IQGAP-2 was the highest, and thus, we focused our investigation on this putative interactor. First, we verified the co-localization of IQGAP-2 with the hcTPPT in human colonic NCM460 cells using confocal imaging. We then examined the effect of expressing IQGAP-2 in NCM460 cells and in differentiated colonoids on carrier-mediated TPP uptake and observed significant induction with both models. Knock-down IQGAP-2 (with gene-specific siRNA), on the other hand, led to a significant decrease in TPP uptake by colonocytes. We also found that expressing the IQGAP-2 in colonocytes lead to a marked enhancement in hTPPT protein stability (increased protein half-life). Interestingly, we also found that expression of IQGAP-2 was suppressed by factors/conditions (e.g. lipopolysaccharide, pro-inflammatory cytokine TNF-α and hypoxia, respectively) that inhibit colonic TPP uptake.Conclusion: The accessory protein IQGAP-2 is an interacting partner with the hcTPPT in human colonocytes and that the interaction has physiological/cell biological consequences. Supported by NIH grants AA-018071 & DK-56061, and VA grants 101BX001142 & IK6BX006189. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Blood-to-Testis Transport of Ribavirin Involves Carrier-Mediated Processes at the Blood–Testis Barrier

Ribavirin, an antiretroviral agent targeting the hepatitis C virus, causes male reproductive toxicity. This study investigated the mechanism of ribavirin transport at the blood–testis barrier (BTB). In vivo mouse integration plot analysis after intravenous administration revealed that the net influx clearance of [3H]ribavirin in the testis was 3.6-fold greater than that of [14C]D-mannitol, a paracellular transport marker, implying transcellular transport of ribavirin across the BTB. Moreover, [3H]ribavirin uptake by TM4 cells, mouse-derived Sertoli cells, was time- and concentration-dependent, with a Km value of 2.49 mM. S-[(4-nitrophenyl)methyl]-6-thioinosine, an inhibitor of Na+-independent equilibrative nucleoside transporters (ENTs), strongly inhibited the [3H]ribavirin uptake by TM4 cells at 100 µM. Compared to the uptake of [3H]adenosine, a typical endogenous nucleoside, [3H]ribavirin uptake was relatively similar to ENT2 transport. [3H]Ribavirin uptake was also observed in mouse ENT2-expressing Xenopus laevis oocytes, and gene silencing via the transfection of ENT2 small interfering RNA significantly reduced the [3H]ribavirin transport into TM4 cells by 13%. Taken together, these results suggest that ENT2 partially contributes to ribavirin transport at the BTB.

Hydrogen evolution with hot electrons on a plasmonic-molecular catalyst hybrid system

Plasmonic systems convert light into electrical charges and heat, mediating catalytic transformations. However, there is ongoing controversy regarding the involvement of hot carriers in the catalytic process. In this study, we demonstrate the direct utilisation of plasmon hot electrons in the hydrogen evolution reaction with visible light. We intentionally assemble a plasmonic nanohybrid system comprising NiO/Au/[Co(1,10-Phenanthrolin-5-amine)2(H2O)2], which is unstable at water thermolysis temperatures. This assembly limits the plasmon thermal contribution while ensuring that hot carriers are the primary contributors to the catalytic process. By combining photoelectrocatalysis with advanced in situ spectroscopies, we can substantiate a reaction mechanism in which plasmon-induced hot electrons play a crucial role. These plasmonic hot electrons are directed into phenanthroline ligands, facilitating the rapid, concerted proton-electron transfer steps essential for hydrogen generation. The catalytic response to light modulation aligns with the distinctive profile of a hot carrier-mediated process, featuring a positive, though non-essential, heat contribution.

MiR-122-5p is involved in posttranscriptional regulation of the mitochondrial thiamin pyrophosphate transporter (SLC25A19) in pancreatic acinar cells.

Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.

Fermi-level dependence of magnetism and magnetotransport in the magnetic topological insulators Bi2Te3 and BiSbTe3 containing self-organized MnBi2Te4 septuple layers

The magnetic coupling mechanisms underlying ferromagnetism and magnetotransport in magnetically doped topological insulators (TIs) have been a central issue to gain controlled access to the magnetotopological phenomena such as the quantum anomalous Hall effect (AHE) and the topological axion insulating state. Here, we focus on the role of bulk carriers in magnetism of the family of magnetic TIs, in which the host material is either ${\mathrm{Bi}}_{2}{\mathrm{Te}}_{3}$ or $\mathrm{BiSb}{\mathrm{Te}}_{3}$, containing Mn self-organized in $\mathrm{Mn}{\mathrm{Bi}}_{2}{\mathrm{Te}}_{4}$ septuple layers. We tune the Fermi level using the electron irradiation technique and study how magnetic properties vary through the change in carrier density; the role of the irradiation defects is also discussed. Ferromagnetic resonance spectroscopy and magnetotransport measurements show no effect of the Fermi-level position on the magnetic anisotropy field and the Curie temperature, respectively, excluding bulk magnetism based on a carrier-mediated process. Furthermore, the magnetotransport measurements show that the AHE is dominated by the intrinsic and dissipationless Berry-phase-driven mechanism, with the Hall resistivity enhanced near the bottom/top of the conduction/valence band, due to the Berry curvature which is concentrated near the avoided band crossings. These results demonstrate that the AHE can be effectively managed, maximized, or turned off by adjusting the Fermi level.

Carrier-Mediated Process of Putrescine Elimination at the Rat Blood-Retinal Barrier.

Putrescine is a bioactive polyamine. Its retinal concentration is strictly controlled to maintain a healthy sense of vision. The present study investigated putrescine transport at the blood-retinal barrier (BRB) to gain a better understanding of the mechanisms of putrescine regulation in the retina. Our microdialysis study showed that the elimination rate constant during the terminal phase was significantly greater (1.90-fold) than that of [14C]D-mannitol, which is a bulk flow marker. The difference in the apparent elimination rate constants of [3H]putrescine and [14C]D-mannitol was significantly decreased by unlabeled putrescine and spermine, suggesting active putrescine transport from the retina to the blood across the BRB. Our study using model cell lines of the inner and outer BRB showed that [3H]putrescine transport was time-, temperature-, and concentration-dependent, suggesting the involvement of carrier-mediated processes in putrescine transport at the inner and outer BRB. [3H]Putrescine transport was significantly reduced under Na+-free, Cl--free, and K+-replacement conditions, and attenuated by polyamines or organic cations such as choline, a choline transporter-like protein (CTL) substrate. Rat CTL1 cRNA-injected oocytes exhibited marked alterations in [3H]putrescine uptake, and CTL1 knockdown significantly reduced [3H]putrescine uptake in model cell lines, suggesting the possible participation of CTL1 in putrescine transport at the BRB.

A Na+-Dependent Mechanism Is Involved in Mucosal Uptake of Cinnamic Acid across the Jejunal Brush Border in Rats

Phenolic acids are present in all plant-derived foods and in most diets. Indirect evidence indicates substantial absorption of phenolic monomers from the gastrointestinal tract. However, the mechanisms involved in the absorptive process are unknown. The present study investigates mucosal uptake of radioactively labeled cinnamic acid as a model substance for monomeric cinnamic acid derivatives (e.g., cinnamic, ferulic or caffeic acid) in the rat jejunum using an in vitro mucosal uptake technique. The results indicate the existence of a Na+-dependent saturable transport mechanism for uptake of cinnamic acid across the jejunal brush border membrane. The observed Na+ dependence of jejunal cinnamate uptake seems not to be related to the activity of the Na+, H+ exchanger. Lowering the pH of the incubation medium resulted in a pronounced increase in mucosal cinnamate uptake that can be only partially explained by an increase in nonionic diffusion of cinnamic acid. Furthermore, jejunal uptake of cinnamate seems to be influenced by intracellular HCO3- and/or pH, since the addition of methazolamide to a HCO3- and CO2-free incubation medium significantly inhibited mucosal cinnamate uptake, whereas methazolamide was without an effect in the presence of HCO3- and CO2 in the incubation medium. Unlabeled cinnamic and ferulic acid as well as short-chain fatty acids (acetic, propionic and butyric acid) significantly inhibited Na+-dependent uptake of radioactively labeled cinnamic acid. Taken together, the results indicate the involvement of a Na+-dependent, carrier-mediated transport process in the uptake of cinnamic acid and structurally related substrates such as ferulic acid across the brush border membrane of rat jejunum.

Functional characteristics of 3′-azido-3′-deoxythymidine transport at the blood-testis barrier

3′-Azido-3′-deoxythymidine (AZT), an antiretroviral drug, is often adopted in the therapy for human immunodeficiency virus (HIV) infection, and the characteristics of AZT transport at the blood-testis barrier (BTB) were investigated in this study. In the integration plot analysis that evaluates the transport activity in vivo, the apparent influx clearance of [3H]AZT was significantly greater than that of [14C]D-mannitol, a non-permeable paracellular transport marker. In the uptake study in vitro with TM4 cells derived from mouse Sertoli cells, [3H]AZT uptake exhibited a time- and concentration-dependent manner, of which Km and Vmax values being 20.3 µM and 102 pmol/(min·mg protein), respectively. In the inhibition analysis, [3H]AZT uptake was not affected by extracellular inorganics and some substrates of transporters putatively involved in AZT transport. In the further inhibition analyses to elucidate the characteristics of AZT transport, [3H]AZT uptake was strongly reduced in the presence of several nucleosides, that are categorized as 2′-deoxynucleosides with pyrimidine, whereas little effect on [3H]AZT uptake was exhibited in the presence of other nucleosides, nucleobases, and antiretrovirals. These results suggest the influx transport of AZT from the circulating blood to the testis, and the involvement of carrier-mediated process at the BTB, which selectively recognizes 2′-deoxynucleosides with a pyrimidine base.

Physiological Characterization of the Transporter-Mediated Uptake of the Reversible Male Contraceptive H2-Gamendazole Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is formed by a tight network of Sertoli cells (SCs) to limit the movement of reproductive toxicants from the blood into the male genital tract. Transporters expressed at the basal membranes of SCs also influence the disposition of drugs across the BTB. The reversible, nonhormonal contraceptive, H2-gamendazole (H2-GMZ), is an indazole carboxylic acid analog that accumulates over 10 times more in the testes compared with other organs. However, the mechanism(s) by which H2-GMZ circumvents the BTB are unknown. This study describes the physiologic characteristics of the carrier-mediated process(es) that permit H2-GMZ and other analogs to penetrate SCs. Uptake studies were performed using an immortalized human SC line (hT-SerC) and liquid chromatography-tandem mass spectrometry (LC-MS/MS). Uptake of H2-GMZ and four analogs followed Michaelis-Menten transport kinetics (one analog exhibited poor penetration). H2-GMZ uptake was strongly inhibited by indomethacin, diclofenac, MK-571, and several analogs. Moreover, H2-GMZ uptake was stimulated by an acidic extracellular pH, reduced at basic pHs, and independent of extracellular Na+, K+, or Cl- levels, which are intrinsic characteristics of OATP-mediated transport. Therefore, the characteristics of H2-GMZ transport suggest that one or more OATPs may be involved. However, endogenous transporter expression in wild-type Chinese hamster ovary (CHO), Madin-Darby canine kidney (MDCK), and human embryonic kidney-293 (HEK-293) cells limited the utility of heterologous transporter expression to identify a specific OATP transporter. Altogether, characterization of the transporters involved in the flux of H2-GMZ provides insight into the selectivity of drug disposition across the human BTB to understand and overcome the pharmacokinetic and pharmacodynamic difficulties presented by this barrier. SIGNIFICANCE STATEMENT: Despite major advancements in female contraceptives, male alternatives, including vasectomy, condom usage, and physical withdrawal, are antiquated and the widespread availability of nonhormonal, reversible chemical contraceptives is nonexistent. Indazole carboxylic acid analogs such as H2-GMZ are promising new reversible, antispermatogenic drugs that are highly effective in rodents. This study characterizes the carrier-mediated processes that permit H2-GMZ and other drugs to enter Sertoli cells and the observations made here will guide the development of drugs that effectively circumvent the BTB.

Involvement of TauT/SLC6A6 in Taurine Transport at the Blood-Testis Barrier.

Taurine transport was investigated at the blood–testis barrier (BTB) formed by Sertoli cells. An integration plot analysis of mice showed the apparent influx permeability clearance of [3H]taurine (27.7 μL/(min·g testis)), which was much higher than that of a non-permeable paracellular marker, suggesting blood-to-testis transport of taurine, which may involve a facilitative taurine transport system at the BTB. A mouse Sertoli cell line, TM4 cells, showed temperature- and concentration-dependent [3H]taurine uptake with a Km of 13.5 μM, suggesting that the influx transport of taurine at the BTB involves a carrier-mediated process. [3H]Taurine uptake by TM4 cells was significantly reduced by the substrates of taurine transporter (TauT/SLC6A6), such as β-alanine, hypotaurine, γ-aminobutyric acid (GABA), and guanidinoacetic acid (GAA), with no significant effect shown by L-alanine, probenecid, and L-leucine. In addition, the concentration-dependent inhibition of [3H]taurine uptake revealed an IC50 of 378 μM for GABA. Protein expression of TauT in the testis, seminiferous tubules, and TM4 cells was confirmed by Western blot analysis and immunohistochemistry by means of anti-TauT antibodies, and knockdown of TauT showed significantly decreased [3H]taurine uptake by TM4 cells. These results suggest the involvement of TauT in the transport of taurine at the BTB.

Redox-Activated Contrast-Enhanced T1-Weighted Imaging Visualizes Glutathione-Mediated Biotransformation Dynamics in the Liver.

GSH-mediated liver biotransformation is a crucial physiological process demanding efficient research tools. Here, we report a type of amorphous FexMnyO nanoparticles (AFMO-ZDS NPs) as redox-activated probes for in vivo visualization of the dynamics of GSH-mediated biotransformation in liver with T1-weighted magnetic resonance imaging (MRI). This imaging technique reveals the periodic variations in GSH concentration during the degradation of AFMO-ZDS NPs due to the limited transportation capacity of GSH carriers in the course of GSH efflux from hepatocytes to perisinusoidal space, providing direct imaging evidence for this important carrier-mediated process during GSH-mediated biotransformation. Therefore, this technique offers an effective method for in-depth investigations of GSH-related biological processes in liver under various conditions as well as a feasible means for the real-time assessment of liver functions, which is highly desirable for early diagnosis of liver diseases and prompt a toxicity evaluation of pharmaceuticals.

Thyroid Disorders.

Thyroid Disorders.

Calsyntenin-3 interacts with the sodium-dependent vitamin C transporter-2 to regulate vitamin C uptake

Ascorbic acid (AA) uptake in neurons occurs via a Na+-dependent carrier-mediated process mediated by the sodium-dependent vitamin C transporter-2 (SVCT2). Relatively little information is available concerning the network of interacting proteins that support human (h)SVCT2 trafficking and cell surface expression in neuronal cells. Here we identified the synaptogenic adhesion protein, calsyntenin-3 (CLSTN3) as an hSVCT2 interacting protein from yeast two-hybrid (Y2H) screening of a human adult brain cDNA library. This interaction was confirmed by co-immunoprecipitation, mammalian two-hybrid (M2H), and co-localization in human cell lines. Co-expression of hCLSTN3 with hSVCT2 in SH-SY5Y cells led to a marked increase in AA uptake. Reciprocally, siRNA targeting hCLSTN3 inhibited AA uptake. In the J20 mouse model of Alzheimer's disease (AD), mouse (m)SVCT2 and mCLSTN3 expression levels in hippocampus were decreased. Similarly, expression levels of hSVCT2 and hCLSTN3 were markedly decreased in hippocampal samples from AD patients. These findings establish CLSTN3 as a novel hSVCT2 interactor in neuronal cells with potential pathophysiological significance.

A Proton-Coupled Transport System for β-Hydroxy-β-Methylbutyrate (HMB) in Blood–Brain Barrier Endothelial Cell Line hCMEC/D3

β-Hydroxy-β-methylbutyrate (HMB), a leucine metabolite, is used as a nutritional ingredient to improve skeletal muscle health. Preclinical studies indicate that this supplement also elicits significant benefits in the brain; it promotes neurite outgrowth and prevents age-related reductions in neuronal dendrites and cognitive performance. As orally administered HMB elicits these effects in the brain, we infer that HMB crosses the blood–brain barrier (BBB). However, there have been no reports detailing the transport mechanism for HMB in BBB. Here we show that HMB is taken up in the human BBB endothelial cell line hCMEC/D3 via H+-coupled monocarboxylate transporters that also transport lactate and β-hydroxybutyrate. MCT1 (monocarboxylate transporter 1) and MCT4 (monocarboxylate transporter 4) belonging to the solute carrier gene family SLC16 (solute carrier, gene family 16) are involved, but additional transporters also contribute to the process. HMB uptake in BBB endothelial cells results in intracellular acidification, demonstrating cotransport with H+. Since HMB is known to activate mTOR with potential to elicit transcriptomic changes, we examined the influence of HMB on the expression of selective transporters. We found no change in MCT1 and MCT4 expression. Interestingly, the expression of LAT1 (system L amino acid transporter 1), a high-affinity transporter for branched-chain amino acids relevant to neurological disorders such as autism, is induced. This effect is dependent on mTOR (mechanistic target of rapamycine) activation by HMB with no involvement of histone deacetylases. These studies show that HMB in systemic circulation can cross the BBB via carrier-mediated processes, and that it also has a positive influence on the expression of LAT1, an important amino acid transporter in the BBB.

Comprehensive Evidence of Carrier-Mediated Distribution of Amantadine to the Retina across the Blood-Retinal Barrier in Rats.

Amantadine, a drug used for the blockage of NMDA receptors, is well-known to exhibit neuroprotective effects. Accordingly, assessment of amantadine transport at retinal barriers could result in the application of amantadine for retinal diseases such as glaucoma. The objective of this study was to elucidate the retinal distribution of amantadine across the inner and outer blood–retinal barrier (BRB). In vivo blood-to-retina [3H]amantadine transport was investigated by using the rat retinal uptake index method, which was significantly reduced by unlabeled amantadine. This result indicated the involvement of carrier-mediated processes in the retinal distribution of amantadine. In addition, in vitro model cells of the inner and outer BRB (TR-iBRB2 and RPE-J cells) exhibited saturable kinetics (Km in TR-iBRB2 cells, 79.4 µM; Km in RPE-J cells, 90.5 and 9830 µM). The inhibition of [3H]amantadine uptake by cationic drugs/compounds indicated a minor contribution of transport systems that accept cationic drugs (e.g., verapamil), as well as solute carrier (SLC) organic cation transporters. Collectively, these outcomes suggest that carrier-mediated transport systems, which differ from reported transporters and mechanisms, play a crucial role in the retinal distribution of amantadine across the inner/outer BRB.

Comparison of 6:2 chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA) and perfluorooctane sulfonate (PFOS) accumulation and toxicity in mung bean

With the regulation of perfluorooctanesulfonate (PFOS), 6:2 chlorinated polyfluoroalkyl ether sulfonate (6:2 Cl-PFESA) has been used as a potential PFOS alternative in electroplating. In this study, the uptake, translocation and phytotoxicity of PFOS and 6:2 Cl-PFESA in mung bean (Vigna radiata (Linn.) Wilczek.) were investigated. The uptake kinetics of PFOS and 6:2 Cl-PFESA fit the Michaelis-Menten equation well, suggesting that the uptake is a carrier-mediated process. The root concentration factor (RCF) of 6:2 Cl-PFESA (34.55 mL g−1 dw) was 1.27 times that of PFOS (27.11 mL g−1 dw), and the translocation factor (TF) of 6:2 Cl-PFESA (0.177) was 1.07 times that of PFOS (0.165). Exposure to 6:2 Cl-PFESA and PFOS both resulted in the inhibition of mung bean seedling development. Treatment with 6:2 Cl-PFESA and PFOS led to the concentration-dependent elevation of malondialdehyde (MDA), carbonyl groups, and phosphorylated histone H2AX (γ-H2AX) levels in mung bean roots. The MDA and carbonyl group contents induced by 6:2 Cl-PFESA were 1.10–1.35 and 1.03–1.14 times, respectively, those of PFOS. The hydroxyl free radical (·OH) levels in mung bean roots after exposure to PFOS and 6:2 Cl-PFESA were elevated significantly, and the ·OH levels induced by 6:2 Cl-PFESA were higher than those induced by PFOS. Hydroxyl free radical levels were positively correlated with the MDA and carbonyl group contents in mung bean roots (p < 0.05). The dynamic changes in some antioxidative enzyme activities in mung bean seedlings were determined, including peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). The results demonstrated the phytotoxicities of 6:2 Cl-PFESA and PFOS to mung bean in the early developmental stage. 6:2 Cl-PFESA is more harmful to mung beans than PFOS. The production of hydroxyl radical is the mechanism that causes the toxicity of PFOS and 6:2 Cl-PFESA toward plants.

Developmental maturation of the colonic uptake process of the microbiota-generated thiamin pyrophosphate.

The water-soluble vitamin B1 is essential for normal human health and physiology. In its main biologically active form, i.e., thiamin pyrophosphate (TPP), the vitamin plays many critical roles in cell metabolism; thus, its deficiency leads to a variety of adverse effects. Humans/mammals obtain vitamin B1 from two exogenous sources: diet and gut microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP, and colonocytes can efficiently absorb this TPP via a high-affinity and specific carrier-mediated mechanism that involves the recently cloned colonic TPP transporter (cTPPT; product of SLC44A4 gene). There is nothing currently known about colonic uptake of TPP during early stages of life and whether the process undergoes developmental regulation. We addressed this issue using the mouse as animal model. Our results showed that colonic uptake of TPP undergoes developmental upregulation as the animal moves from the suckling period to weanling and adulthood. This upregulation in uptake was found to be associated with a parallel induction in level of expression of the cTPPT protein, mRNA, and heterogeneous nuclear RNA, suggesting possible involvement of transcriptional mechanism(s). We also found a parallel upregulation in the level of expression of the two nuclear factors that drive activity of the SLC44A4 promoter (i.e., CREB-1 and Elf-3) with maturation. These results demonstrate, for the first time, to our knowledge, that colonic TPP uptake process and cTPPT expression are developmentally upregulated and that this upregulation is likely driven via transcriptional mechanism(s).NEW & NOTEWORTHY The colonic carrier-mediated uptake process of the microbiota-generated and phosphorylated form of vitamin B1, i.e., thiamin pyrophosphate, undergoes ontogenic changes that parallel the development of the gut microbiota (and their ability to generate vitamins) during early stages of life.