Transepithelial transport of ferulic acid by monocarboxylic acid transporter in Caco-2 cell monolayers

Our previous study (Biosci. Biotechnol. Biochem., 66, 2449-2457 (2002)), suggested that ferulic acid was transported via a monocarboxylic acid transporter (MCT). Transepithelial transport of ferulic acid was examined in this study by directly measuring the rate of its transport across Caco-2 cell monolayers. Ferulic acid transport was dependent on pH, and in a vectorical way in the apical-basolateral direction. The permeation of ferulic acid was concentration-dependent and saturable; the Michaelis constant was 16.2 mM and the maximum velocity was 220.4 nmol min-1 (mg protein)-1. Various substrates for MCTs, such as benzoic acid and acetic acid, strongly inhibited the permeation of ferulic acid, demonstrating that ferulic acid is obviously transported by MCT. Antioxidative phenolic acid compounds from dietary sources like ferulic acid would be recognized and transported by MCT by intestinal absorption.     

Monocarboxylate transporter (MCT)-1 is up-regulated by PPARalpha

Peroxisome proliferator-activated receptor (PPAR)-alpha mediates an adaptive response to fasting by up-regulation of genes involved in fatty acid oxidation and ketone body synthesis. Ketone bodies are transferred in and out of cells by monocarboxylate transporter (MCT)-1. In this study we observed for the first time that activation of PPARalpha in rats by clofibrate treatment or fasting increased hepatic mRNA concentration of MCT1. In Fao rat hepatoma cells, incubation with the PPARalpha agonist WY 14,643 increased mRNA concentration of MCT1 whereas the PPARgamma agonist troglitazone did not. To elucidate whether up-regulation of MCT1 is indeed mediated by PPARalpha we treated wild-type and PPARalpha-null mice with WY 14,643. In wild-type mice, treatment with WY 14,643 increased mRNA concentrations of MCT1 in liver, kidney and small intestine whereas no up-regulation was observed in PPARalpha-null mice.     

Expression of the monocarboxylate transporter 1 (MCT1) in cells of the porcine intestine

Uptake of energy into cells and its allocation to individual cellular compartments by transporters are essential for tissue homeostasis. The present study gives an analysis of MCT1 expression and its cellular occurrence in the porcine intestine. Tissue portions from duodenum, jejunum, ileum, colon ascendens, colon transversum and colon descendens were collected and prepared for immunohistochemistry, Western blot and real time RT-PCR. A 169bp porcine MCT1 cDNA fragment was amplified and published. MCT1 mRNA expression in the large intestine was 20 fold higher compared to the small intestine. Western blot detected a single protein band of 41kDa at a much higher amount of MCT1 protein in the large intestine vs. the small intestine. MCT1 protein was detected in mitochondrial fractions of the large but not the small intestine. Immunohistochemistry in the small intestine showed that immune cells in the lamina propria and in the lymphoid follicles primarily expressed MCT1 while in the colon epithelial cells were the main source of MCT1. In summary, cellular expression of MCT1 differs between epithelial cells in the colon and small intestine. A possible role of MCT1 for uptake of butyrate into immune cells and the overall role of MCT1 for intestinal immune cell function remains elusive.     

Nucleobase transport by human equilibrative nucleoside transporter 1 (hENT1)

The human equilibrative nucleoside transporters hENT1 and hENT2 (each with 456 residues) are 40% identical in amino acid sequence and contain 11 putative transmembrane helices. Both transport purine and pyrimidine nucleosides and are distinguished functionally by a difference in sensitivity to inhibition by nanomolar concentrations of nitrobenzylmercaptopurine ribonucleoside (NBMPR), hENT1 being NBMPR-sensitive. Previously, we used heterologous expression in Xenopus oocytes to demonstrate that recombinant hENT2 and its rat ortholog rENT2 also transport purine and pyrimidine bases, h/rENT2 representing the first identified mammalian nucleobase transporter proteins (Yao, S. Y., Ng, A. M., Vickers, M. F., Sundaram, M., Cass, C. E., Baldwin, S. A., and Young, J. D. (2002) J. Biol. Chem. 277, 24938-24948). The same study also revealed lower, but significant, transport of hypoxanthine by h/rENT1. In the present investigation, we have used the enhanced Xenopus oocyte expression vector pGEMHE to demonstrate that hENT1 additionally transports thymine and adenine and, to a lesser extent, uracil and guanine. Fluxes of hypoxanthine, thymine, and adenine by hENT1 were saturable and inhibited by NBMPR. Ratios of V(max) (pmol/oocyte · min(-1)):K(m) (mm), a measure of transport efficiency, were 86, 177, and 120 for hypoxantine, thymine, and adenine, respectively, compared with 265 for uridine. Hypoxanthine influx was competitively inhibited by uridine, indicating common or overlapping nucleobase and nucleoside permeant binding pockets, and the anticancer nucleobase drugs 5-fluorouracil and 6-mercaptopurine were also transported. Nucleobase transport activity was absent from an engineered cysteine-less version hENT1 (hENT1C-) in which all 10 endogenous cysteine residues were mutated to serine. Site-directed mutagenesis identified Cys-414 in transmembrane helix 10 of hENT1 as the residue conferring nucleobase transport activity to the wild-type transporter.     

Lactate transport in rat adipocytes: identification of monocarboxylate transporter 1 (MCT1) and its modulation during streptozotocin-induced diabetes

We have characterised L-lactate transport in rat adipocytes and determined whether these cells express a carrier belonging to the monocarboxylate transporter family. L-Lactate was taken up by adipocytes in a time-dependent, non-saturable manner and was inhibited (by approximately 90%) by alpha-cyano-4-hydroxycinnamate. Lactate transport was stimulated by 3.7-fold upon lowering extracellular pH from 7.5 to 6.5 suggesting the presence of a lactate/proton-cotransporter. Antibodies against mono carboxylate transporter 1 (MCT1) reacted positively with plasma membranes (PM), but not with intracellular membranes, prepared from adipocytes. MCTI expression was down-regulated in PM of adipocytes from diabetic rats, which also displayed a corresponding loss (approximately 64%) in their capacity to transport lactate. The data support a role for MCT1 in lactate transport and suggest that changes in MCT1 expression are likely to have important implications for adipocyte lactate metabolism.     

Diet-induced ketosis increases monocarboxylate transporter (MCT1) levels in rat brain

Monocarboxylate transporter (MCT1) levels in brains of adult Long-Evans rats on a high-fat (ketogenic) diet were investigated using light and electron microscopic immunocytochemical methods. Rats given the ketogenic diet (91% fat and 9% protein) for up to 6 weeks had increased levels of the monocarboxylate transporter MCT1 (and of the glucose transporter GLUT1) in brain endothelial cells and neuropil compared to rats on a standard diet. In ketonemic rats, electron microscopic immunogold methods revealed an 8-fold greater MCT1 labeling in the brain endothelial cells at 4 weeks. Abluminal endothelial membranes were twice as heavily labeled as luminal membranes. In controls, luminal and abluminal labeling was not significantly different. The endothelial cytoplasmic compartment was sparsely labeled (<8% of total endothelial labeling) in all brains. Neuropil MCT1 staining was more intense throughout the brain in ketonemic rats, especially in neuropil of the molecular layer of the cerebellum, as revealed by avidin-biotin immunocytochemistry. This study demonstrates that adult rats retain the capacity to upregulate brain MCT1 levels. Furthermore, their brains react to a diet that increases monocarboxylate levels in the blood by enhancing their capability to take up both monocarboxylates (MCT1 upregulation) and glucose (GLUT1 upregulation). This may have important implications for delivery of fuel to the brain under stressful and pathological conditions, such as epilepsy and GLUT1 deficiency syndrome.     

IGF-I receptor gene activation enhanced the expression of monocarboxylic acid transporter 1 in hepatocarcinoma cells

The present study aims to investigate the effect of IGF-I receptor (IGF-IR) gene activation on the expression of monocarboxylic acid transporters (MCTs) in hepatocarcinoma cells. In order to reflect malignant hepatoma, H4IIE cells (a rat hepatoma cell line) stably expressing IGF-IR (IGF-IR-H4IIE cells) have been established by retroviral infection and then the effect of IGF-IR gene up-regulation on the modulation of MCT expression was determined in IGF-IR-H4IIE cells. Immunoblot assay indicated that the expression level of MCT1 was 3.3-fold higher in IGF-IR-H4IIE cells compared to that in control cells, implying that IGF-IR signaling is coupled with the process of MCT1 expression. In contrast, the expression level of MCT2 was not affected by the IGF-IR activation, suggesting that MCT1 and MCT2 are regulated by the distinct type of signals. Furthermore, the cellular uptake of benzoic acid, a representative substrate of MCT1, was significantly (p<0.05) enhanced following the activation of IGF-IR via the pre-incubation with IGF-I (10 ng/ml). In conclusion, MCT1 expression was up-regulated in hepatocarcinoma cells and the IGF-IR signaling appeared to be coupled with the modulation of MCT1 expression.     

Allan-Herndon-Dudley syndrome and the monocarboxylate transporter 8 (MCT8) gene

Allan-Herndon-Dudley syndrome was among the first of the X-linked mental retardation syndromes to be described (in 1944) and among the first to be regionally mapped on the X chromosome (in 1990). Six large families with the syndrome have been identified, and linkage studies have placed the gene locus in Xq13.2. Mutations in the monocarboxylate transporter 8 gene (MCT8) have been found in each of the six families. One essential function of the protein encoded by this gene appears to be the transport of triiodothyronine into neurons. Abnormal transporter function is reflected in elevated free triiodothyronine and lowered free thyroxine levels in the blood. Infancy and childhood in the Allan-Herndon-Dudley syndrome are marked by hypotonia, weakness, reduced muscle mass, and delay of developmental milestones. Facial manifestations are not distinctive, but the face tends to be elongated with bifrontal narrowing, and the ears are often simply formed or cupped. Some patients have myopathic facies. Generalized weakness is manifested by excessive drooling, forward positioning of the head and neck, failure to ambulate independently, or ataxia in those who do ambulate. Speech is dysarthric or absent altogether. Hypotonia gives way in adult life to spasticity. The hands exhibit dystonic and athetoid posturing and fisting. Cognitive development is severely impaired. No major malformations occur, intrauterine growth is not impaired, and head circumference and genital development are usually normal. Behavior tends to be passive, with little evidence of aggressive or disruptive behavior. Although clinical signs of thyroid dysfunction are usually absent in affected males, the disturbances in blood levels of thyroid hormones suggest the possibility of systematic detection through screening of high-risk populations.     

Comparison between copper and cisplatin transport mediated by human copper transporter 1 (hCTR1)

Copper transporter 1 (CTR1) is a transmembrane protein that imports copper(i) into yeast and mammalian cells. Surprisingly, the protein also mediates the uptake of platinum anticancer drugs, e.g. cisplatin and carboplatin. To study the effects of several metal-binding residues/motifs of hCTR1 on the transport of both Cu(+) and cisplatin, we have constructed Hela cells that stably express a series of hCTR1 variant proteins including H22-24A, NHA, C189S, hCTR1ΔC, H139R and Y156A, and compared their abilities to regulate the accumulation and cytotoxicity of these metal compounds. Our results demonstrated that the cells expressing the hCTR1 mutants of histidine-rich motifs in the N-terminus (H22-24A, NHA) resulted in a higher basal copper level in the steady state compared to those expressing wild-type protein. However, the cellular accumulation of both copper and cisplatin in these variants was found at a similar level to that of wild type when incubated with an excess of metal compounds (100 μM). The cells expressing hCTR1 variants of H139R and Y156A exhibit lower capacities towards accumulation of copper but not cisplatin. Significantly, cells with the C189S variant partially retained the ability of the wild-type hCTR1 protein to accumulate both copper and cisplatin, while for cells expressing the C-terminus truncated variant of hCTR1 (hCTR1ΔC) this ability was absolutely abolished, suggesting that this motif is crucial for the function of the transporter.     

Potent blockers of the monocarboxylate transporter MCT1: novel immunomodulatory compounds

A novel series of potent blockers of the monocarboxylate transporter, MCT1, is disclosed. From very potent but lipophilic lead compounds, systematic changes to all parts of the molecule, targeting reduction in log D, afforded compounds with significantly improved overall properties. These compounds show potent immunomodulatory activity.     

p-aminohippuric acid transport at renal apical membrane mediated by human inorganic phosphate transporter NPT1

Organic anions are secreted into urine via organic anion transporters across the renal basolateral and apical membranes. However, no apical membrane transporter for organic anions such as p-aminohippuric acid (PAH) has yet been identified. In the present study, we showed that human NPT1, which is present in renal apical membrane, mediates the transport of PAH. The K(m) value for PAH uptake was 2.66 mM and the uptake was chloride ion sensitive. These results are compatible with those reported for the classical organic anion transport system at the renal apical membrane. PAH transport was inhibited by various anionic compounds. Human NPT1 also accepted uric acid, benzylpenicillin, faropenem, and estradiol-17beta-glucuronide as substrates. Considering its chloride ion sensitivity, Npt1 is expected to function for secretion of PAH from renal proximal tubular cells. This is the first molecular demonstration of an organic anion transport function for PAH at the renal apical membrane.     

Transport model of the human Na+-coupled L-ascorbic acid (vitamin C) transporter SVCT1

Vitamin C (L-ascorbic acid) is an essential micronutrient that serves as an antioxidant and as a cofactor in many enzymatic reactions. Intestinal absorption and renal reabsorption of the vitamin is mediated by the epithelial apical L-ascorbic acid cotransporter SVCT1 (SLC23A1). We explored the molecular mechanisms of SVCT1-mediated L-ascorbic acid transport using radiotracer and voltage-clamp techniques in RNA-injected Xenopus oocytes. L-ascorbic acid transport was saturable (K(0.5) approximately 70 microM), temperature dependent (Q(10) approximately 5), and energized by the Na(+) electrochemical potential gradient. We obtained a Na(+)-L-ascorbic acid coupling ratio of 2:1 from simultaneous measurement of currents and fluxes. L-ascorbic acid and Na(+) saturation kinetics as a function of cosubstrate concentrations revealed a simultaneous transport mechanism in which binding is ordered Na(+), L-ascorbic acid, Na(+). In the absence of L-ascorbic acid, SVCT1 mediated pre-steady-state currents that decayed with time constants 3-15 ms. Transients were described by single Boltzmann distributions. At 100 mM Na(+), maximal charge translocation (Q(max)) was approximately 25 nC, around a midpoint (V(0.5)) at -9 mV, and with apparent valence approximately -1. Q(max) was conserved upon progressive removal of Na(+), whereas V(0.5) shifted to more hyperpolarized potentials. Model simulation predicted that the pre-steady-state current predominantly results from an ion-well effect on binding of the first Na(+) partway within the membrane electric field. We present a transport model for SVCT1 that will provide a framework for investigating the impact of specific mutations and polymorphisms in SLC23A1 and help us better understand the contribution of SVCT1 to vitamin C metabolism in health and disease.     

Role of monocarboxylic acid transport in intracellular pH regulation of isolated proximal cells

Isolated proximal cells were prepared from rabbit kidney cortex by mechanical dissociation. The intracytoplasmic pH (pHi) was measured in HCO3(-)-free media (external pH (pHe), 7.3) using the fluorescent dye 2,7-biscarboxyethyl-5,6-carboxyfluorescein (BCECF). Cells were acid-loaded by the nigericin technique. Addition of 70 mM Na+ to the cells caused a rapid pHi recovery, which was blocked by 0.5 mM amiloride. When the cells were exposed to 5 mM sodium butyrate in the presence of 1 mM amiloride, the H+ efflux was significantly increased and followed Michaelis-Menten kinetics. Increasing pHe from 6.4 to 7.6 at a constant pHi of 6.4 enhanced the butyrate activation of the H+ efflux. Increasing pHi from 6.5 to 7.2 at a constant pHe of 7.2 reduced the butyrate effect. 22Na uptake experiments in the presence of 1 mM amiloride showed that 1.5 mM butyrate increased the Na+ flux in the proximal cells (pHi 7.10). The efficiency of monocarboxylic anions in promoting a pHi recovery increased with the length of their straight chain (acetate less than propionate less than butyrate less than valerate). The data show that when the Na+/H+ antiporter is blocked, the proximal cells can regulate their pHi by a Na+-coupled absorption of butyrate followed by non-ionic diffusion of butyric acid out of the cell and probably also by OH- influx by means of the OH-/anion exchanger.