Thiamine is a substrate of organic cation transporters in Caco-2 cells

doi:10.1016/j.ejphar.2012.02.028

European Journal of Pharmacology

Volume 682, Issues 1–3, 5 May 2012, Pages 37-42

https://doi.org/10.1016/j.ejphar.2012.02.028 Get rights and content

Abstract

The aim of this study was to characterize the intestinal absorption of thiamine, by investigating the hypothesis of an involvement of Organic Cation Transporter (OCT) family members in this process. [³H]-T⁺ uptake was found to be: 1) time-dependent, 2) Na⁺- and Cl⁻-dependent, 3) pH-dependent, with uptake increasing with a decrease in extracellular pH and decreasing with a decrease in intracellular pH, 4) inhibited by amiloride, 5) inhibited by the thiamine structural analogues oxythiamine and amprolium, 6) inhibited by the unrelated organic cations MPP⁺, clonidine, dopamine, serotonin, 7) inhibited by the OCT inhibitors decynium22 and progesterone. Moreover, the dependence of [³H]-T⁺ uptake on phosphorylation/dephosphorylation mechanisms was also investigated and [³H]-T⁺ uptake was found to be reduced by PKA activation and protein tyrosine phosphatase and alkaline phosphatase inhibition. In conclusion, our results are compatible with the possibility of thiamine being transported not only by ThTr1 and/or ThTr2, but also by members of the OCT family of transporters (most probably OCT1 and/or OCT3), thus sharing the same transporters with several other organic cations at the small intestinal level.

Introduction

Thiamine (vitamin B₁), a water-soluble micronutrient, is essential for normal cellular functions and growth. It is a precursor of thiamine pyrophosphate which plays a critical role in normal carbohydrate metabolism where it participates in the decarboxylation of pyruvic and α-ketoglutamic acids, and in the utilization of pentose in the hexose monophosphate shunt (Rindi and Laforenza, 2000).

Thiamine deficiency in humans leads to a variety of clinical abnormalities including neurological and cardiovascular disorders (Kumar, 2010, Morley, 2010, Soukoulis et al., 2009, Wooley, 2008). Thiamine deficiency represents a significant nutritional problem in developing countries. This is not an exclusive problem in these countries, as it is also found in affluent countries as a clinically significant problem in individuals with chronic alcoholism (Koike and Sobue, 2006, Subramanya, n.d, Tallaksen et al., 1992), diabetes (Saito et al., 1987, Thornalley et al., 2007), in patients with renal and intestinal diseases (Frank et al., 2000), and in the elderly (Fabian and Elmadfa, 2008, Lengyel et al., 2008), despite an average daily intake of this vitamin that exceeds the recommended requirements (Olsen et al., 2009).

Humans and other mammals cannot synthesize thiamine and thus must obtain this vitamin from exogenous sources via intestinal absorption. Thus, the intestine plays a critical role in regulating body thiamine homeostasis and understanding the mechanism of intestinal thiamine absorption process is of significant nutritional importance.

Chemically, thiamine is an organic cation (quaternary ammonium compound) with a high molecular weight. Biological membranes prevent transmembrane diffusion of the majority of organic molecules that bear net charges at physiological pH, and so membrane-bound transport systems are generally involved in the absorption, distribution, and elimination of these compounds. The first thiamine transporter (ThTr1, SLC19A2) was cloned in 1999 in yeast. Mutations in the human SLC19A2 gene are responsible for thiamine-responsive megaloblastic anemia (Rogers Syndrome) (Ganapathy et al., 2004). However, these patients do not present the characteristic cardiovascular and/or neurological symptoms (known as beriberi) seen in nutritional thiamine deficiency (Ganapathy et al., 2004), and have normal thiamine plasmatic levels, which suggest that, besides ThTr1, other transporter(s) are also involved in the intestinal transport of thiamine. Accordingly, a second high-affinity transporter was described for thiamine (ThTr2; SLC19A3). This transporter is expressed in different tissues, including intestine (Said et al., 2004).

Since thiamine is an organic cation, organic cation transporters (OCTs; members of the SLC22 family), which are polyspecific transporters of organic cations, may also contribute for global thiamine transport. At the intestinal level, two distinct OCTs are known to be functionally present: OCT1 (Martel et al., 2000) and OCT3 (also known as the extraneuronal monoamine transporter, EMT) (Martel et al., 2000, Martel et al., 2001).

For studies on the intestinal uptake of thiamine, Caco-2 cells, an enterocyte-like cell line derived from a human colonic adenocarcinoma, were used as an intestinal epithelial model. This human intestinal epithelial cell line forms confluent monolayers of well-differentiated enterocyte-like cells with the functional properties of transporting epithelia (Delie and Rubas, 1997, Yee, 1997).

Section snippets

Materials

[³H]-thiamine (specific activity 10 Ci mmol^− 1; American Radiolabeled Chemicals Inc., St. Louis, MO, USA); DMSO, Triton X-100 (Merck, Darmstadt, Germany); tetraethylammonium bromide (TEA), cimetidine, choline chloride, N-methylnicotinamide chloride (NMN), corticosterone, decynium22 (1,1′-diethyl-2,2′-cyanine iodide), dibutyryl cAMP sodium salt, histamine dihydrochloride, β-estradiol, pyridoxal 5′-phosphate, HEPES (N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid), amiloride hydrochloride,

Characterization of thiamine ([³H]-T⁺) uptake by Caco-2 cells

Cellular uptake of [³H]-T⁺ was determined using Caco-2 cells as a model of intestinal epithelia. In the first series of experiments, we verified that cells took up [³H]-T⁺ in a time-dependent way, and that uptake of [³H]-T⁺ was linear with time for up to 5 min of incubation. So, all the subsequent experiments were performed using a 3 min incubation period, in the presence or absence of compounds, in order to determine initial rates of uptake (Fig. 1).

pH-dependence and amiloride effect

The effect of extracellular medium pH on [³H]-T

Discussion

The intestinal transport of thiamine is pharmacologically poorly characterized. The absorption of this vitamin is made primarily in intestine, and due to the fact that thiamine is an organic cation, it needs a transporter in the plasma membrane.

Previous studies with brush border membrane vesicles (BBMVs) revealed that thiamine uptake by enterocytes is a Na⁺-independent, pH-dependent, amiloride-sensitive, electroneutral carrier-mediated mechanism inhibited by thiamine structural analogs such as

Conclusion

Our results are compatible with the possibility of thiamine being transported not only by ThTr1 and/or ThTr2, but also by members of the OCT family of transporters (most probably OCT1 and/or OCT3), thus sharing the same transporters with several other organic cations at the small intestinal level. The hypothesis of thiamine being transported by OCT family members is supported by the following data: i) pH-dependence characteristics of [³H]-T⁺ uptake by Caco-2 cells are consistent with a T⁺/H⁺

Acknowledgements

This work was supported by FCT, COMPETE, QREN and FEDER (PTDC/SAU-OSM/102239/2008 and PTDC/QUI/65501/2006), two post-doctoral research fellows (SFRH/BPD/75294/2010, SFRH/BPD/40110/2007) and a PhD fellow (SFRH/BD/78367/2011).

Clara Lemos has an Humboldt Research Fellowship for Postdoctoral Researchers from the Alexander von Humboldt Foundation.

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Thiamin (vitamin B1) is a precursor of thiamin diphosphate (ThDP), which is a known coenzyme of central metabolism. However, data on the noncoenzyme action of thiamin and/or its derivatives have been accumulating since the observation of synaptic corelease of thiamin with acetylcholine. In thiamin-synthesizing organisms, the noncoenzyme action involves the ThDP riboswitch. The energy-dependent biosynthesis of thiamin triphosphate and its adenylated derivatives in all clades provides further evidence for the noncoenzyme action of thiamin and/or derivatives. In the mammalian brain, thiamin is linked to protein acetylation. Thiamin and/or its derivatives are allosteric regulators of the malate and glutamate dehydrogenases, aminotransferases and pyridoxal kinase. Noncanonical thiamin-binding proteins of systemic significance include p53, PARP1, signal-related phosphatases, and kinases. Coupled to coenzyme actions of thiamin in central metabolism, the thiamin binding to noncanonical proteins may well provide for systemic regulation by thiamin-dependent signaling cascades, similar to those involving essential metabolites, such as ATP and NAD⁺. In particular the site-specific competition between thiamin and (di)nucleotides may attenuate purinergic signaling and NAD⁺-dependent polyADP-ribosylation or deacetylation. Elucidation of protein targets of the noncoenzyme thiamin action is advanced through structure-based identification of thiamin- or derivatives-binding protein patterns, bioinformatics analysis of their distribution among proteins and pathways, and experimental verification of the pattern-based predictions. As a result, noncanonical thiamin-binding proteins emerge as important players in the thiamin bioactivity essential for human health, including pain relief, immunity, neurodegeneration, and cancer.
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Decreased thiamine and reduced activity of thiamine diphosphate (ThDP)-dependent 2-oxoglutarate dehydrogenase (OGDH) cause neurodegeneration. We hypothesized on concerted cell-specific regulation of the thiamine metabolism and ThDP-dependent reactions. We identified a smaller thiamine pool, a lower expression of the mitochondrial ThDP transporter, and a higher expression of OGDH in rat astrocytes versus neuroblastoma N2A. According to the data, the astrocytic OGDH may be up-regulated by an increase in intracellular ThDP, while the neuroblastomal OGDH functions at full ThDP saturation. Indeed, in rat astrocytes and brain cortex, OGDH inhibition by succinyl phosphonate (SP) enlarged the pool of thiamine compounds. Increased ThDP level in response to the OGDH inhibition presumably up-regulated the enzyme to compensate for a decrease in reducing power which occurred in SP-treated astrocytes. Under the same SP treatment of N2A cells, their thiamine pool and reducing power were unchanged, although SP action was evident from accumulation of glutamate. The presented data indicate that functional interplay between OGDH, other proteins of the tricarbocylic acid cycle and proteins of thiamine metabolism is an important determinant of physiology-specific networks and their homeostatic mechanisms.
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An increased carbon flux and exploitation of metabolic pathways for the rapid generation of biosynthetic precursors is a common phenotype observed in breast cancer. To support this metabolic phenotype, cancer cells adaptively regulate the expression of glycolytic enzymes and nutrient transporters. However, activity of several enzymes involved in glucose metabolism requires an adequate supply of cofactors. In particular, vitamin B1 (thiamine) is utilized as an essential cofactor for metabolic enzymes that intersect at critical junctions within the glycolytic network. Intracellular availability of thiamine is facilitated by the activity of thiamine transporters and thiamine pyrophosphokinase-1 (TPK-1). Therefore, the objective of this study was to establish if the cellular determinants regulating thiamine homeostasis differ between breast cancer and normal breast epithelia. Employing cDNA arrays of breast cancer and normal breast epithelial tissues, SLC19A2, SLC25A19 and TPK-1 were found to be significantly up-regulated. Similarly, up-regulation was also observed in breast cancer cell lines compared to human mammary epithelial cells. Thiamine transport assays and quantitation of intracellular thiamine and thiamine pyrophosphate established a significantly greater extent of thiamine transport and free thiamine levels in breast cancer cell lines compared to human mammary epithelial cells. Overall, these findings demonstrate an adaptive response by breast cancer cells to increase cellular availability of thiamine.

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Molecular and Cellular PharmacologyThiamine is a substrate of organic cation transporters in Caco-2 cells