Thyroid Hormone Transporters MCT8 and OATP1C1 Are Expressed in Projection Neurons and Interneurons of Basal Ganglia and Motor Thalamus in the Adult Human and Macaque Brains.

Monocarboxylate transporter 8 (MCT8) and organic anion-transporting polypeptide 1C1 (OATP1C1) are thyroid hormone (TH) transmembrane transporters relevant for the availability of TH in neural cells, crucial for their proper development and function. Mutations in MCT8 or OATP1C1 result in severe disorders with dramatic movement disability related to alterations in basal ganglia motor circuits. Mapping the expression of MCT8/OATP1C1 in those circuits is necessary to explain their involvement in motor control. We studied the distribution of both transporters in the neuronal subpopulations that configure the direct and indirect basal ganglia motor circuits using immunohistochemistry and double/multiple labeling immunofluorescence for TH transporters and neuronal biomarkers. We found their expression in the medium-sized spiny neurons of the striatum (the receptor neurons of the corticostriatal pathway) and in various types of its local microcircuitry interneurons, including the cholinergic. We also demonstrate the presence of both transporters in projection neurons of intrinsic and output nuclei of the basal ganglia, motor thalamus and nucleus basalis of Meynert, suggesting an important role of MCT8/OATP1C1 for modulating the motor system. Our findings suggest that a lack of function of these transporters in the basal ganglia circuits would significantly impact motor system modulation, leading to clinically severe movement impairment.

Possible mechanism of bamboo shoots (Bambusa balcooa) induced thyroid disruption - An in vitro study.

Endemic goitre and associated iodine deficiency disorders (IDDs) are a major concern in public health even in the period of post salt iodization in many regions. Among others the consumption of cyanogenic plants found responsible for the persistence of such diseases. Bamboo shoots (BS) is one such cyanogenic plant food that caused disruption of certain thyroid hormone synthesizing regulatory element as has already been reported in our earlier study. In this investigation the possible mechanism of thyrocytes disruption along with interruption of thyroid hormone biosynthesis by BS has been worked out. Commonly consumed BS, Bambusa Balcooa Roxb (BBR) water extract was analysed by GC MS; three doses below IC50 were administered to thyrocytes in culture with and without iodine. Expressions of thyroglobulin (Tg), pendrin (PDS) and monocarboxylate transporter 8 (MCT8) were evaluated in thyrocytes with cell cycle analysis, reactive oxygen species (ROS) generation, DNA oxidation and apoptotic regulation through Bax, Bcl-2 and p53. Phytochemical analysis of BBR extract revealed the presence of precursors and metabolic end products of cyanogenic glycosides. Dose dependent decrease in expression of Tg and PDS with concomitant decrease in gene expression of these with MCT8 were observed. Increased ROS, DNA oxidation and associated imbalance were found through increased Bax and p53 with decreased Bcl-2 that perturbed thyrocytes cell cycle. Cyanogenic constituents of BBR generates ROS associated oxidative changes in thyrocytes with DNA damage and oxidation and cell cycle disruption followed by inhibition of thyroid hormone synthesizing regulatory elements; addition of extra iodine showed partial prevention.

Contribution of Monocarboxylate Transporter 6 to the Pharmacokinetics and Pharmacodynamics of Bumetanide in Mice.

Bumetanide, a sulfamyl loop diuretic, is used for the treatment of edema in association with congestive heart failure. Being a polar, anionic compound at physiologic pH, bumetanide uptake and efflux into different tissues is largely transporter-mediated. Of note, organic anion transporters (SLC22A) have been extensively studied in terms of their importance in transporting bumetanide to its primary site of action in the kidney. The contribution of one of the less-studied bumetanide transporters, monocarboxylate transporter 6 (MCT6; SLC16A5), to bumetanide pharmacokinetics (PK) and pharmacodynamics (PD) has yet to be characterized. The affinity of bumetanide for murine Mct6 was evaluated using Mct6-transfected Xenopus laevis oocytes. Furthermore, bumetanide was intravenously and orally administered to wild-type mice (Mct6+/+) and homozygous Mct6 knockout mice (Mct6-/-) to elucidate the contribution of Mct6 to bumetanide PK/PD in vivo. We demonstrated that murine Mct6 transports bumetanide at a similar affinity compared with human MCT6 (78 and 84 µM, respectively, at pH 7.4). After bumetanide administration, there were no significant differences in plasma PK. Additionally, diuresis was significantly decreased by ∼55% after intravenous bumetanide administration in Mct6-/- mice. Kidney cortex concentrations of bumetanide were decreased, suggesting decreased Mct6-mediated bumetanide transport to its site of action in the kidney. Overall, these results suggest that Mct6 does not play a major role in the plasma PK of bumetanide in mice; however, it significantly contributes to bumetanide's pharmacodynamics due to changes in kidney concentrations. SIGNIFICANCE STATEMENT: Previous evidence suggested that MCT6 transports bumetanide in vitro; however, no studies to date have evaluated the in vivo contribution of this transporter. In vitro studies indicated that mouse and human MCT6 transport bumetanide with similar affinities. Using Mct6 knockout mice, we demonstrated that murine Mct6 does not play a major role in the plasma pharmacokinetics of bumetanide; however, the pharmacodynamic effect of diuresis was attenuated in the knockout mice, likely because of the decreased bumetanide concentrations in the kidney.

Oil Body Formation in Marchantia polymorpha Is Controlled by MpC1HDZ and Serves as a Defense against Arthropod Herbivores.

The origin of a terrestrial flora in the Ordovician required adaptation to novel biotic and abiotic stressors. Oil bodies, a synapomorphy of liverworts, accumulate secondary metabolites, but their function and development are poorly understood. Oil bodies of Marchantia polymorpha develop within specialized cells as one single large organelle. Here, we show that a class I homeodomain leucine-zipper (C1HDZ) transcription factor controls the differentiation of oil body cells in two different ecotypes of the liverwort M. polymorpha, a model genetic system for early divergent land plants. In flowering plants, these transcription factors primarily modulate responses to abiotic stress, including drought. However, loss-of-function alleles of the single ortholog gene, MpC1HDZ, in M. polymorpha did not exhibit phenotypes associated with abiotic stress. Rather, Mpc1hdz mutant plants were more susceptible to herbivory, and total plant extracts of the mutant exhibited reduced antibacterial activity. Transcriptomic analysis of the mutant revealed a reduction in expression of genes related to secondary metabolism that was accompanied by a specific depletion of oil body terpenoid compounds. Through time-lapse imaging, we observed that MpC1HDZ expression maxima precede oil body formation, indicating that MpC1HDZ mediates differentiation of oil body cells. Our results indicate that M. polymorpha oil bodies, and MpC1HDZ, are critical for defense against herbivory, but not for abiotic stress tolerance. Thus, C1HDZ genes were co-opted to regulate separate responses to biotic and abiotic stressors in two distinct land plant lineages.

Effects of postbiotic supplementation on growth performance, ruminal fermentation and microbial profile, blood metabolite and GHR, IGF-1 and MCT-1 gene expression in post-weaning lambs.

BACKGROUND: Postbiotics have been established as potential feed additive to be used in monogastric such as poultry and swine to enhance health and growth performance. However, information on the postbiotics as feed additive in ruminants is very limited. The aim of this study was to elucidate the effects of supplementation of postbiotics in newly-weaned lambs on growth performance, digestibility, rumen fermentation characteristics and microbial population, blood metabolite and expression of genes related to growth and volatile fatty acid transport across the rumen epithelium. RESULTS: Postbiotic supplementation increased weight gain, feed intake, nutrient intake and nutrient digestibility of the lambs. No effect on ruminal pH and total VFA, whereas butyrate and ruminal ammonia-N concentration were improved. The lambs fed with postbiotics had higher blood total protein, urea nitrogen and glucose. However, no difference was observed in blood triglycerides and cholesterol levels. Postbiotics increased the population of fibre degrading bacteria but decreased total protozoa and methanogens in rumen. Postbiotics increased the mRNA expression of hepatic IGF-1 and ruminal MCT-1. CONCLUSIONS: The inclusion of postbiotics from L. plantarum RG14 in newly-weaned lambs improved growth performance, nutrient intake and nutrient digestibility reflected from better rumen fermentation and microbial parameters, blood metabolites and upregulation of growth and nutrient intake genes in the post-weaning lambs.

Mitochondrial Pyruvate Carriers Prevent Cadmium Toxicity by Sustaining the TCA Cycle and Glutathione Synthesis.

Cadmium (Cd) is a major heavy metal pollutant, and Cd toxicity is a serious cause of abiotic stress in the environment. Plants protect themselves against Cd stress through a variety of pathways. In a recent study, we found that mitochondrial pyruvate carriers (MPCs) are involved in Cd tolerance in Arabidopsis (Arabidopsis thaliana). Following the identification of MPCs in yeast (Saccharomyces cerevisiae) in 2012, most studies have focused on the function of MPCs in animals, as a possible approach to reduce the risk of cancer developing. The results of this study show that AtMPC protein complexes are required for Cd tolerance and prevention of Cd accumulation in Arabidopsis. AtMPC complexes are composed of two elements, AtMPC1 and AtMPC2 (AtNRGA1 or AtMPC3). When the formation of AtMPCs was interrupted by the loss of AtMPC1, glutamate could supplement the synthesis of acetyl-coenzyme A and sustain the TCA cycle. With the up-regulation of glutathione synthesis following exposure to Cd stress, the supplementary pathway could not efficiently drive the tricarboxylic acid cycle without AtMPC. The ATP content decreased concomitantly with the deletion of tricarboxylic acid activity, which led to Cd accumulation in Arabidopsis. More importantly, ScMPCs were also required for Cd tolerance in yeast. Our results suggest that the mechanism of Cd tolerance may be similar in other species.

Brain and Hepatic Mt mRNA Is Reduced in Response to Mild Energy Restriction and n-3 Polyunsaturated Fatty Acid Deficiency in Juvenile Rats.

Metallothioneins (MTs) perform important regulatory and cytoprotective functions in tissues including the brain. While it is known that energy restriction (ER) and dietary n-3 polyunsaturated fatty acid (PUFA) deficiency impact postnatal brain growth and development, little data exist regarding the impact of undernutrition upon MT expression in growing animals. We tested the hypothesis that ER with and without dietary n-3 PUFA deficiency reduces MT expression in juvenile rats. ER rats were individually pair-fed at 75% of the ad libitum (AL) intake of control rats provided diets consisting of either soybean oil (SO) that is α-linolenic acid (ALA; 18:3n-3) sufficient or corn oil (CO; ALA-deficient). Fatty acids (FA) and metal concentrations of liver and brain regions were analyzed. Tissue expression of MTs (Mt1-3) and modulators of MT expression including glucocorticoid receptors (Nr3c1 and Nr3c2) and several mediators of thyroid hormone regulation (Dio1-3, Mct8, Oatp1c1, Thra, and Thrb) were measured. Plasma corticosterone and triiodothyronine levels were also evaluated. ER, but not metal deficiency, reduced Mt2 expression in the cerebellum (50%) and cerebral cortex (23%). In liver, a reduction in dietary n-3 PUFA reduced Mt1, Mt2, Nr3c1, Mct8, and Thrb. ER elevated Nr3c1, Dio1, and Thrb and reduced Thra in the liver. Given MT's role in cellular protection, further studies are needed to evaluate whether ER or n-3 PUFA deficiency may leave the juvenile brain and/or liver more susceptible to endogenous or environmental stressors.

A perillyl alcohol-conjugated analog of 3-bromopyruvate without cellular uptake dependency on monocarboxylate transporter 1 and with activity in 3-BP-resistant tumor cells.

The anticancer agent 3-bromopyruvate (3-BP) is viewed as a glycolytic inhibitor that preferentially kills glycolytic cancer cells through energy depletion. However, its cytotoxic activity is dependent on cellular drug import through transmembrane monocarboxylate transporter 1 (MCT-1), which restricts its anticancer potential to MCT-1-positive tumor cells. We created and characterized an MCT-1-independent analog of 3-BP, called NEO218. NEO218 was synthesized by covalently conjugating 3-BP to perillyl alcohol (POH), a natural monoterpene. The responses of various tumor cell lines to treatment with either compound were characterized in the presence or absence of supplemental pyruvate or antioxidants N-acetyl-cysteine (NAC) and glutathione (GSH). Drug effects on glyceraldehyde 3-phosphate dehydrogenase (GAPDH) enzyme activity were investigated by mass spectrometric analysis. The development of 3-BP resistance was investigated in MCT-1-positive HCT116 colon carcinoma cells in vitro. Our results show that NEO218: (i) pyruvylated GAPDH on all 4 of its cysteine residues and shut down enzymatic activity; (ii) severely lowered cellular ATP content below life-sustaining levels, and (iii) triggered rapid necrosis. Intriguingly, supplemental antioxidants effectively prevented cytotoxic activity of NEO218 as well as 3-BP, but supplemental pyruvate powerfully protected cells only from 3-BP, not from NEO218. Unlike 3-BP, NEO218 exerted its potent cytotoxic activity irrespective of cellular MCT-1 status. Treatment of HCT116 cells with 3-BP resulted in prompt development of resistance, based on the emergence of MCT-1-negative cells. This was not the case with NEO218, and highly 3-BP-resistant cells remained exquisitely sensitive to NEO218. Thus, our study identifies a mechanism by which tumor cells develop rapid resistance to 3-BP, and presents NEO218 as a superior agent not subject to this cellular defense. Furthermore, our results offer alternative interpretations of previously published models on the role of supplemental antioxidants: Rather than quenching reactive oxygen species (ROS), supplemental NAC or GSH directly interact with 3-BP, thereby neutralizing the drug's cytotoxic potential before it can trigger ROS production. Altogether, our study introduces new aspects of the cytotoxic mechanism of 3-BP, and characterizes NEO218 as an analog able to overcome a key cellular defense mechanism towards this drug.

The Chemical Chaperone Phenylbutyrate Rescues MCT8 Mutations Associated With Milder Phenotypes in Patients With Allan-Herndon-Dudley Syndrome.

Mutations in the thyroid hormone transporter monocarboxylate transporter 8 (MCT8) prevent appropriate entry of thyroid hormones into brain cells during development and cause severe mental retardation in affected patients. The current treatment options are thyromimetic compounds that enter the brain independently of MCT8. Some MCT8-deficient patients (e.g., those carrying MCT8delF501) will not be as severely affected as most others. We have shown that the MCT8delF501 protein has decreased protein stability but important residual function once it reaches the plasma membrane. We were able to rescue protein expression and the function of MCT8delF501 in a Madin-Darby canine kidney cell model by application of the chemical chaperone sodium phenylbutyrate (NaPB), a drug that has been used to treat patients with cystic fibrosis and urea cycle defects for extended periods of time. In the present study, we have extended our previous study and report on the NaPB-dependent rescue of a series of other pathogenic MCT8 mutants associated with milder patient phenotypes. We show that NaPB can functionally rescue the expression and activities of Ser194Phe, Ser290Phe, Leu434Trp, Arg445Cys, Leu492Pro, and Leu568Pro mutations in MCT8 in a dose-dependent manner. The soy isoflavone genistein, a dietary supplement, which was effective in MCT8delF501, was also effective in increasing the expression and transport of these MCT8 mutants; however, the effect size differed among mutants. Kinetic analyses revealed that the Michaelis constants of the mutants toward the primary substrate 3,3',5-triiodothyronine were not much different from the wild-type value, suggesting that these mutants are not impaired in their interaction with substrate but rather destabilized by the mutation and degraded.

Maternal plasma folate impacts differential DNA methylation in an epigenome-wide meta-analysis of newborns.

Folate is vital for fetal development. Periconceptional folic acid supplementation and food fortification are recommended to prevent neural tube defects. Mechanisms whereby periconceptional folate influences normal development and disease are poorly understood: epigenetics may be involved. We examine the association between maternal plasma folate during pregnancy and epigenome-wide DNA methylation using Illumina's HumanMethyl450 Beadchip in 1,988 newborns from two European cohorts. Here we report the combined covariate-adjusted results using meta-analysis and employ pathway and gene expression analyses. Four-hundred forty-three CpGs (320 genes) are significantly associated with maternal plasma folate levels during pregnancy (false discovery rate 5%); 48 are significant after Bonferroni correction. Most genes are not known for folate biology, including APC2, GRM8, SLC16A12, OPCML, PRPH, LHX1, KLK4 and PRSS21. Some relate to birth defects other than neural tube defects, neurological functions or varied aspects of embryonic development. These findings may inform how maternal folate impacts the developing epigenome and health outcomes in offspring.

Efficient Activation of Pathogenic ΔPhe501 Mutation in Monocarboxylate Transporter 8 by Chemical and Pharmacological Chaperones.

Monocarboxylate transporter 8 (MCT8) is a thyroid hormone transmembrane transporter expressed in many cell types, including neurons. Mutations that inactivate transport activity of MCT8 cause severe X-linked psychomotor retardation in male patients, a syndrome originally described as the Allan-Herndon-Dudley syndrome. Treatment options currently explored the focus on finding thyroid hormone-like compounds that bypass MCT8 and enter cells through different transporters. Because MCT8 is a multipass transmembrane protein, some pathogenic mutations affect membrane trafficking while potentially retaining some transporter activity. We explore here the effects of chemical and pharmacological chaperones on the expression and transport activity of the MCT8 mutant ΔPhe501. Dimethylsulfoxide, 4-phenylbutyric acid as well as its sodium salt, and the isoflavone genistein increase T3 uptake into MDCK1 cells stably transfected with mutant MCT8-ΔPhe501. We show that ΔPhe501 represents a temperature-sensitive mutant protein that is stabilized by the proteasome inhibitor MG132. 4-Phenylbutyrate has been used to stabilize ΔPhe508 mutant cystic fibrosis transmembrane conductance regulator protein and is in clinical use in patients with urea cycle defects. Genistein is enriched in soy and available as a nutritional supplement. It is effective in stabilizing MCT8-ΔPhe501 at 100 nM concentration. Expression of the L471P mutant is increased in response to phenylbutyrate, but T3 uptake activity is not induced, supporting the notion that the chaperone specifically increases membrane expression. Our findings suggest that certain pathogenic MCT8 mutants may be responsive to (co-)treatment with readily available compounds, which increase endogenous protein function.

Monocarboxylate transporter 1 is up-regulated in Caco-2 cells by the methionine precursor DL-2-hydroxy-(4-methylthio)butanoic acid.

The methionine precursor, DL-2-hydroxy-(4-methylthio)butanoic acid (HMTBA), is a synthetic source of dietary methionine, which is widely used as a poultry nutritional supplement. In the intestinal epithelium, HMTBA transport across the apical membrane is mediated by monocarboxylate transporter 1 (MCT1). The first step in biological utilisation of this methionine precursor is the stereospecific conversion of HMTBA to the corresponding keto acid. In the present study, the regulation of trans-epithelial HMTBA transport was investigated in Caco-2 cell monolayers. Differentiated Caco-2 cells were maintained under control conditions (apical compartment: 0.2 mmol/L L-methionine) or in a HMTBA-enriched medium (2 mmol/L HMTBA). The effect of culture on HMTBA transport was evaluated from apical and basolateral kinetic parameters. MCT1 and MCT4 immuno-localisation and gene expression were investigated by confocal microscopy and real-time quantitative RT-PCR, respectively. The results indicated that apical MCT1 was up-regulated by exposure to HMTBA (1.4-fold increase in Vmax without changes in Km). Moreover, total monolayer MCT1 immunoreactivity increased 1.8-fold in HMTBA-supplemented cultures, this effect mainly being localised at the apical membrane. Functional and immuno-localisation data suggest involvement of MCT1 and MCT4 in basolateral HMTBA transport, although, in this case, no effect was observed for HMTBA-enrichment. Molecular analysis confirmed MCT1 mRNA up-regulation (1.8-fold), with no effect on MCT4 mRNA expression. Thus, exposure to HMTBA up-regulates the trans-epithelial transport of this methionine precursor by increasing the expression and the transport capacity of apical MCT1.

Tissue-specific expression of monocarboxylate transporters during fasting in mice.

Monocarboxylates such as pyruvate, lactate and ketone bodies are crucial for energy supply of all tissues, especially during energy restriction. The transport of monocarboxylates across the plasma membrane of cells is mediated by monocarboxylate transporters (MCTs). Out of 14 known mammalian MCTs, six isoforms have been functionally characterized to transport monocarboxylates and short chain fatty acids (MCT1-4), thyroid hormones (MCT8, -10) and aromatic amino acids (MCT10). Knowledge on the regulation of the different MCT isoforms is rare. In an attempt to get more insights in regulation of MCT expression upon energy deprivation, we carried out a comprehensive analysis of tissue specific expression of five MCT isoforms upon 48 h of fasting in mice. Due to the crucial role of peroxisome proliferator-activated receptor (PPAR)-α as a central regulator of energy metabolism and as known regulator of MCT1 expression, we included both wildtype (WT) and PPARα knockout (KO) mice in our study. Liver, kidney, heart, small intestine, hypothalamus, pituitary gland and thyroid gland of the mice were analyzed. Here we show that the expression of all examined MCT isoforms was markedly altered by fasting compared to feeding. Expression of MCT1, MCT2 and MCT10 was either increased or decreased by fasting dependent on the analyzed tissue. MCT4 and MCT8 were down-regulated by fasting in all examined tissues. However, PPARα appeared to have a minor impact on MCT isoform regulation. Due to the fundamental role of MCTs in transport of energy providing metabolites and hormones involved in the regulation of energy homeostasis, we assumed that the observed fasting-induced adaptations of MCT expression seem to ensure an adequate energy supply of tissues during the fasting state. Since, MCT isoforms 1-4 are also necessary for the cellular uptake of drugs, the fasting-induced modifications of MCT expression have to be considered in future clinical care algorithms.

Post-natal changes in MCT1 expression in the forestomach of calves.

The monocarboxylate transporter 1 (MCT1) has been demonstrated to be involved in the transfer of short-chain fatty acids (SCFA) and/or their intraepithelial metabolites from the rumen to the blood. As MCT1 plays a role in SCFA transfer, it is assumed that SCFA are the main substrates influencing its expression. However, there are hints that MCT1 may also be expressed during the early life of the animal when SCFA are not released in the forestomach. To figure out whether MCT1 expression in the forestomach is influenced independently of SCFA during that period, we studied post-natal MCT1 expression immunohistochemically in the epithelia of omasum, atrium ruminis, saccus dorsalis ruminis, saccus ventralis ruminis and reticulum of calves born preterm and at term. The calves were nourished by colostrum or by milk-based formula diet. MCT1 could be found in all the forestomach compartments tested, even in preterm calves. The protein was mainly oriented to the luminal side in the immature epithelium 24 h after birth. Orientation to the blood side of the cells developed during the first 4 days after birth. In the rumen epithelia (but not in the other forestomach compartments tested), orientation of MCT1 to the blood side of the cells was paralleled by an increase in the overall expression rate during the first 4 days after birth. As lactate levels were very high directly after birth, a lactate-dependent substrate induction may have been the underlying mechanism. However, non-specific changes due to general differential processes might also be the cause. Both early upregulation of MCT1 and high blood lactate levels may provide the epithelia with lactate as energy source.

Effect of white tea (Camellia sinensis (L.)) extract in the glycolytic profile of Sertoli cell.

PURPOSE: Many health benefits have been attributed to tea (Camellia sinensis (L.)), and tea infusions are used as dietary agent and included in food supplements. Herein, we report the effect of a white tea (WTEA) extract in Sertoli cell (SC) metabolism. The SC is responsible for the nutritional support of the developing germ cells. METHODS: An aqueous WTEA extract was prepared and analyzed by (1)H-NMR. Rat SCs were cultured with or without the WTEA extract. mRNA and protein levels of glucose transporters (GLUT1 and GLUT3), phosphofructokinase, lactate dehydrogenase (LDH) and monocarboxylate transporter 4 were determined by qPCR and western blot. LDH activity was assessed and metabolite production/consumption determined by (1)H-NMR. RESULTS: WTEA-exposed SCs presented decreased protein and mRNA levels of GLUT1 and decreased glucose uptake. However, intracellular LDH activity was increased and SC lactate production was stimulated by the presence of the WTEA extract. Interestingly, alanine production was also found to be stimulated in WTEA extract-exposed SCs. CONCLUSION: WTEA extract altered the glycolytic profile of cultured SCs, stimulating lactate production. Since lactate is used as metabolic substrate and has an anti-apoptotic effect in the developing germ cells, the supplementation with WTEA extract may be advantageous to improve male reproductive health.

Crucial residue involved in L-lactate recognition by human monocarboxylate transporter 4 (hMCT4).

BACKGROUND: Monocarboxylate transporters (MCTs) transport monocarboxylates such as lactate, pyruvate and ketone bodies. These transporters are very attractive therapeutic targets in cancer. Elucidations of the functions and structures of MCTs is necessary for the development of effective medicine which targeting these proteins. However, in comparison with MCT1, there is little information on location of the function moiety of MCT4 and which constituent amino acids govern the transport function of MCT4. The aim of the present work was to determine the molecular mechanism of L-lactate transport via hMCT4. EXPERIMENTAL APPROACH: Transport of L-lactate via hMCT4 was determined by using hMCT4 cRNA-injected Xenopus laevis oocytes. hMCT4 mediated L-lactate uptake in oocytes was measured in the absence and presence of chemical modification agents and 4,4'-diisothiocyanostilbene-2,2'-disulphonate (DIDS). In addition, L-lactate uptake was measured by hMCT4 arginine mutants. Immunohistochemistry studies revealed the localization of hMCT4. RESULTS: In hMCT4-expressing oocytes, treatment with phenylglyoxal (PGO), a compound specific for arginine residues, completely abolished the transport activity of hMCT4, although this abolishment was prevented by the presence of L-lactate. On the other hand, chemical modifications except for PGO treatment had no effect on the transport activity of hMCT4. The transporter has six conserved arginine residues, two in the transmembrane-spanning domains (TMDs) and four in the intracellular loops. In hMCT4-R278 mutants, the uptake of L-lactate is void of any transport activity without the alteration of hMCT4 localization. CONCLUSIONS: Our results suggest that Arg-278 in TMD8 is a critical residue involved in substrate, L-lactate recognition by hMCT4.

Oat ß-glucan and dietary calcium and phosphorus differentially modify intestinal expression of proinflammatory cytokines and monocarboxylate transporter 1 and cecal morphology in weaned pigs.

Physiologic effects of dietary oat ß-glucan and low and high dietary calcium-phosphorus (CaP) on intestinal morphology and gene expression related to SCFA absorption, mucus production, inflammation, and peptide digestion have not been established in weaned mammals. We therefore randomized 32 weaned pigs into 4 equal groups that received a cornstarch-casein-based diet with low (65% of the Ca and P requirement) and high (125 and 115% of the Ca and P requirement, respectively) CaP levels and low- and high-CaP diets supplemented with 8.95% oat ß-glucan concentrate for 14 d. High-CaP diets downregulated duodenal expression of IL-1ß (P < 0.05) by 30% compared with low-CaP diets. Furthermore, high-CaP diets reduced (P < 0.05) cecal crypt depth by 14% compared with low-CaP diets. Dietary ß-glucan upregulated the expression of cecal MCT1 (P < 0.05) by 40% and that of colonic IL-6 (P < 0.05) by 142% compared with the control diet. Correlation analysis indicated that cecal MCT1 (r = 0.99, P < 0.001) and colonic IL-6 (r = 0.84, P < 0.05) expression was positively related to luminal butyrate and total SCFA, respectively, indicating that ß-glucan may partly modify gene expression via increased SCFA generation. In conclusion, ß-glucan and CaP levels modulated the expression of selected genes and morphology in the postweaning period, but effects were specific to intestinal segment. The present results further indicate that, in addition to being essential nutrients for bone accretion, dietary CaP level may modify the intestinal tissue response in young pigs.

Tyrosine kinase inhibitors noncompetitively inhibit MCT8-mediated iodothyronine transport.

CONTEXT: Tyrosine kinase inhibitors (TKI) are used for the treatment of various cancers. Case reports and clinical trials have reported abnormal thyroid function tests (TFT) after treatment with sunitinib, imatinib, sorafenib, dasatinib, and nilotinib. An increased requirement for levothyroxine was reported in thyroidectomized patients during TKI treatment. OBJECTIVE: We hypothesized that abnormal TFT are compatible with inhibition of thyroid hormone (TH) transporters and subsequently reduced pituitary-TH feedback. Monocarboxylate transporter 8 (MCT8) is a TH transmembrane transporter in brain, pituitary, and other organs. MCT8 mutation leads to abnormal TFT in patients and respective mouse models. We tested whether TKI are able to inhibit MCT8-mediated TH uptake into cells. DESIGN: Madin-Darby-canine kidney (MDCK1) cells stably expressing human MCT8 were exposed in vitro to TKI at increasing concentrations, and MCT8-mediated [(125)I]T(3) uptake and efflux were measured. The mode of inhibition was determined. RESULTS: TKI exposure dose-dependently inhibited MCT8-dependent T(3) and T(4) uptake. IC(50) values for sunitinib, imatinib, dasatinib, and bosutinib ranged from 13-38 µm, i.e. similar to the Michaelis-Menten constant K(m) for T(3) and T(4), 4 and 8 µm, respectively. Kinetic experiments revealed a noncompetitive mode of inhibition for all TKI tested. CONCLUSIONS: Partial inhibition by TKI of pituitary or hypothalamic TH feedback may increase TSH or increase the levothyroxine requirement of thyroidectomized patients. It is still possible that other mechanisms contribute to TKI-mediated impairments of TFT, e.g. altered metabolism of TH. Bosutinib was not previously reported to alter TFT.

[Effect of brazilein on energy metabolism of cerebral ischemia-reperfusion in mice].

OBJECTIVE: To investigate brazilein's role in energy metabolism of cerebral ischemia-reperfusion in mice. METHOD: Fourty mice were randomly divided into the sham group, ischemia group, brazilein 5 mg x kg(-1) group and brazilein 10 mg x kg(-1) group, each with ten cases. Cerebral ischemia model was the built. Mice were injected with brazilein three days before the operation, then they were killed. Cerebrum homogenate was prepared for the detecting of ATP, ADP, AMP and lactic acid by HPLC, expressions of MCT1 and MCT2 in mRNA level by RT-PCR. RESULT: The lactic acid in cerebrum increased sharply 20 minutes after cerebral ischemia and decreased 1 hour after reperfusion, then returned to the normal level 24 hours after reperfusion. The charge of energy decreased significantly at the beginning of the ischemia-reperfusion, and the charge restored 1 hour after reperfusion though it was still much lower than the normal level at the time point of 24 hours. Moreover, MCT1 and MCT2 upregulated accompanied with the increase of lactate, MCT2 mRNA enhanced in brazilein 5 mg x kg(-1) group (P < 0.05) while both the two factors increased in brazilein 10 mg x kg(-1) group (P < 0.01). CONCLUSION: Brazilein might protect neurons by changing the charge of energy.

[Disorders in thyroid hormone metabolism].

The levels of the serum thyroid hormone (free T4 and free T3) are determined not only by thyroid hormone synthesis/secretion but also by their metabolism. Thyroid hormone metabolism is mediated by three selenoproteins, selenodeiodinase type 1, 2, and 3 (D1, D2, and D3), the expression and function of which are tightly regulated in a tissue-specific manner. Among them, D2 increases and D3 decreases the intracellular thyroid hormone levels, whereas D1 seems to play a role as a housekeeping/scavenger enzyme in general thyroid hormone metabolism. Although no mutation in either of the deiodinase enzyme genes has been reported, some related genes (SECISBP2, DEHAL1, and MCT8) can cause thyroid hormone-related inherited disorders. In addition, a variety of hormones, cytokines, and drugs can influence thyroid function through altered thyroid hormone metabolism.