Correction: Mitochondrial Uptake of Thiamin Pyrophosphate: Physiological and Cell Biological Aspects.

[This corrects the article DOI: 10.1371/journal.pone.0073503.].

Correction: Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter.

[This corrects the article DOI: 10.1371/journal.pone.0149255.].

Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter.

Microbiota of the large intestine synthesizes considerable amount of vitamin B1 (thiamine) in the form of thiamine pyrophosphate (TPP). We have recently demonstrated the existence of an efficient and specific carrier-mediated uptake process for TPP in human colonocytes, identified the TPP transporter (TPPT) involved (product of the SLC44A4 gene), and shown that expression of TPPT along the gastrointestinal (GI) tract is restricted to the colon. Our aim in this study was to determine the molecular basis of the colon-specific expression of TPPT focusing on a possible epigenetic mechanism. Our results showed that the CpG island predicted in the SLC44A4 promoter is non-methylated in the human colonic epithelial NCM460 cells, but is hyper-methylated in the human duodenal epithelial HuTu80 cells (as well as in the human retinal pigment epithelial ARPE19 cells). In the mouse (where TPPT expression in the GI tract is also restricted to the colon), the CpG island predicted in the Slc44a4 promoter is non-methylated in both the jejunum and colon, thus arguing against possible contribution of DNA methylation in the colon-specific expression of TPPT. A role for histone modifications in the tissue-specific pattern of Slc44a4 expression, however, was suggested by the findings that in mouse colon, histone H3 in the 5'-regulatory region of Slc44a4 is tri-methylated at lysine 4 and acetylated at lysine 9, whereas the tri-methylation at lysine 27 modification was negligible. In contrast, in the mouse jejunum, histone H3 is hyper-trimethylated at lysine 27 (repressor mark). Similarly, possible involvement of miRNA(s) in the tissue-specific expression of TPPT was also suggested by the findings that the 3'-UTR of SLC44A4 is targeted by specific miRNAs/RNA binding proteins in non-colonic, but not in colonic, epithelial cells. These studies show, for the first time, epigenetic mechanisms (histone modifications) play a role in determining the tissue-specific pattern of expression of TPPT in the GI tract.

The human colonic thiamine pyrophosphate transporter (hTPPT) is a glycoprotein and N-linked glycosylation is important for its function.

The recently identified human thiamine pyrophosphate transporter (hTPPT; product of the SLC44A4 gene) is responsible for absorption of the microbiota-generated TPP in the large intestine. The hTPPT is highly expressed in the colon, but not in other regions of the intestinal tract and is localized exclusively at the apical membrane domain of epithelia. The hTPPT protein is predicted to have multiple TM domains with a number of putative N-glycosylation sites, but it is not known if the protein is actually glycosylated, and if so at which site, and their role in the functionality of the transporter. Using several approaches including inhibiting de novo N-glycosylation in human colonic epithelial NCM460 cells with tunicamycin as well as enzymatic de-glycosylation, we show that the hTPPT protein is, indeed, a glycoprotein. Glycosylation of hTPPT was shown, by mean of site-directed mutagenesis, to occur at Asn(69), Asn(155), Asn(197), Asn(393), and Asn(416). However, only N-glycosylation at Asn(69), Asn(155), and Asn(393) appeared to be important for transporter functionality possibly through an effect on protein conformation and/or interaction with its ligand (but not through changes in expression at the cell membrane as determined by live cell confocal imaging). Results of this study showed, for the first time, that the hTPPT is glycosylated and that N-linked glycosylation occurs at multiple sites with some of them being important for function. The results also provide an indirect support for a membrane topology for hTPPT with 10 transmembrane domains as predicted by the TMHMM transmembrane helixes prediction program.

Molecular Mechanisms Mediating the Adaptive Regulation of Intestinal Riboflavin Uptake Process.

The intestinal absorption process of vitamin B2 (riboflavin, RF) is carrier-mediated, and all three known human RF transporters, i.e., hRFVT-1, -2, and -3 (products of the SLC52A1, 2 & 3 genes, respectively) are expressed in the gut. We have previously shown that the intestinal RF uptake process is adaptively regulated by substrate level, but little is known about the molecular mechanism(s) involved. Using human intestinal epithelial NCM460 cells maintained under RF deficient and over-supplemented (OS) conditions, we now show that the induction in RF uptake in RF deficiency is associated with an increase in expression of the hRFVT-2 & -3 (but not hRFVT-1) at the protein and mRNA levels. Focusing on hRFVT-3, the predominant transporter in the intestine, we also observed an increase in the level of expression of its hnRNA and activity of its promoter in the RF deficiency state. An increase in the level of expression of the nuclear factor Sp1 (which is important for activity of the SLC52A3 promoter) was observed in RF deficiency, while mutating the Sp1/GC site in the SLC52A3 promoter drastically decreased the level of induction in SLC52A3 promoter activity in RF deficiency. We also observed specific epigenetic changes in the SLC52A3 promoter in RF deficiency. Finally, an increase in hRFVT-3 protein expression at the cell surface was observed in RF deficiency. Results of these investigations show, for the first time, that transcriptional and post-transcriptional mechanisms are involved in the adaptive regulation of intestinal RF uptake by the prevailing substrate level.

Regulation of basal promoter activity of the human thiamine pyrophosphate transporter SLC44A4 in human intestinal epithelial cells.

Microbiota of the large intestine synthesize considerable amount of vitamin B1 in the form of thiamine pyrophosphate (TPP). There is a specific high-affinity regulated carrier-mediated uptake system for TPP in human colonocytes (product of the SLC44A4 gene). The mechanisms of regulation of SLC44A4 gene expression are currently unknown. In this study, we characterized the SLC44A4 minimal promoter region and identified transcription factors important for basal promoter activity in colonic epithelial cells. The 5'-regulatory region of the SLC44A4 gene (1,022 bp) was cloned and showed promoter activity upon transient transfection into human colonic epithelial NCM460 cells. With the use of a series of 5'- and 3'-deletion luciferase reporter constructs, the minimal genomic region that required basal transcription of the SLC44A4 gene expression was mapped between nucleotides -178 and +88 (using the distal transcriptional start site as +1). Mutational analysis performed on putative cis-regulatory elements established the involvement of ETS/ELF3 [E26 transformation-specific sequence (ETS) proteins], cAMP-responsive element (CRE), and SP1/GC-box sequence motifs in basal SLC44A4 promoter activity. By means of EMSA, binding of ELF3 and CRE-binding protein-1 (CREB-1) transcription factors to the SLC44A4 minimal promoter was shown. Contribution of CREB into SLC44A4 promoter activity was confirmed using NCM460 cells overexpressing CREB. We also found high expression of ELF3 and CREB-1 in colonic (NCM460) compared with noncolonic (ARPE19) cells, suggesting their possible contribution to colon-specific pattern of SLC44A4 expression. This study represents the first characterization of the SLC44A4 promoter and reports the importance of both ELF3 and CREB-1 transcription factors in the maintenance of basal promoter activity in colonic epithelial cells.

Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter.

Colonic microbiota synthesize a considerable amount of thiamine in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes. Nothing, however, is known about the molecular identity of this system. Here we report on the molecular identification of the colonic TPP uptake system as the product of the SLC44A4 gene. We cloned the cDNA of SLC44A4 from human colonic epithelial NCM460 cells, which, upon expression in ARPE19 cells, led to a significant (p < 0.01, >5-fold) induction in [(3)H]TPP uptake. Uptake by the induced system was also found to be temperature- and energy-dependent; Na(+)-independent, slightly higher at acidic buffer pH, and highly sensitive to protonophores; saturable as a function of TPP concentration, with an apparent Km of 0.17 ± 0.064 µM; and highly specific for TPP and not affected by free thiamine, thiamine monophosphate, or choline. Expression of the human TPP transporter was found to be high in the colon and negligible in the small intestine. A cell surface biotinylation assay and live cell confocal imaging studies showed the human TPP transporter protein to be expressed at the apical membrane domain of polarized epithelia. These results show, for the first time, the molecular identification and characterization of a specific and high-affinity TPP uptake system in human colonocytes. The findings further support the hypothesis that the microbiota-generated TPP is absorbable and could contribute toward host thiamine homeostasis, especially toward cellular nutrition of colonocytes.

Association of TM4SF4 with the human thiamine transporter-2 in intestinal epithelial cells.

BACKGROUND: The human thiamine transporter-2 (hTHTR-2) is involved in the intestinal absorption of thiamine. Recent studies with membrane transporters of other nutrients/substrates have shown that they have associated proteins that affect different aspects of their physiology and cell biology. Nothing is known about protein(s) that interact with hTHTR-2 in intestinal epithelial cells and influence its physiological function and/or its cell biology. AIMS: The aim of this study was to identify protein partner(s) that interact with hTHTR-2 in human intestinal cells and determine the physiological/biological consequence of that interaction. METHODS: The yeast split-ubiquitin two-hybrid approach was used to screen a human intestinal cDNA library. GST-pull-down and cellular co-localization approaches were used to confirm the interaction between hTHTR-2 and the associated protein(s). The effect of such an interaction on hTHTR-2 function was examined by (3)H-thiamine uptake assays. RESULTS: Our screening results identified the human TransMembrane 4 SuperFamily 4 (TM4SF4) as a potential interactor with hTHTR-2. This interaction was confirmed by an in vitro GST-pull-down assay, and by live-cell confocal imaging of HuTu-80 cells co-expressing hTHTR-2-GFP and mCherry-TM4SF4 (the latter displayed a significant overlap of these two proteins in intracellular vesicles and at the cell membrane). Co-expression of hTHTR-2 with TM4SF4 in HuTu-80 cells led to a significant induction in thiamine uptake. In contrast, silencing TM4SF4 with gene-specific siRNA led to a significant decrease in thiamine uptake. CONCLUSIONS: These results show for the first time that the accessory protein TM4SF4 interacts with hTHTR-2 and influences the physiological function of the thiamine transporter.

Mitochondrial uptake of thiamin pyrophosphate: physiological and cell biological aspects.

Mammalian cells obtain vitamin B1 (thiamin) from their surrounding environment and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. Most of TPP is then transported into the mitochondria via a carrier-mediated process that involves the mitochondrial thiamin pyrophosphate transporter (MTPPT). Knowledge about the physiological parameters of the MTPP-mediated uptake process, MTPPT targeting and the impact of clinical mutations in MTPPT in patients with Amish lethal microcephaly and neuropathy and bilateral striatal necrosis are not fully elucidated, and thus, were addressed in this study using custom-made (3)H-TPP as a substrate and mitochondria isolated from mouse liver and human-derived liver HepG2 cells. Results showed (3)H-TPP uptake by mouse liver mitochondria to be pH-independent, saturable (Km = 6.79±0.53 µM), and specific for TPP. MTPPT protein was expressed in mouse liver and HepG2 cells, and confocal images showed a human (h)MTPPT-GFP construct to be targeted to mitochondria of HepG2 cells. A serial truncation analysis revealed that all three modules of hMTPPT protein cooperated (although at different levels of efficiency) in mitochondrial targeting rather than acting autonomously as independent targeting module. Finally, the hMTPPT clinical mutants (G125S and G177A) showed proper mitochondrial targeting but displayed significant inhibition in (3)H-TPP uptake and a decrease in level of expression of the MTPPT protein. These findings advance our knowledge of the physiology and cell biology of the mitochondrial TPP uptake process. The results also show that clinical mutations in the hMTPPT system impair its functionality via affecting its level of expression with no effect on its targeting to mitochondria.

Adaptive regulation of human intestinal thiamine uptake by extracellular substrate level: a role for THTR-2 transcriptional regulation.

The intestinal thiamine uptake process is adaptively regulated by the level of vitamin in the diet, but the molecular mechanism involved is not fully understood. Here we used the human intestinal epithelial Caco-2 cells exposed to different levels of extracellular thiamine to delineate the molecular mechanism involved. Our results showed that maintaining Caco-2 cells in a thiamine-deficient medium resulted in a specific and significant increase of [3H]thiamine uptake compared with cell exposure to a high level of thiamine (1 mM). This adaptive regulation was also associated with a higher level of mRNA expression of thiamine transporter-2 (THTR-2), but not thiamine transporter-1 (THTR-1), in the deficient condition and a higher level of promoter activity of gene encoding THTR-2 (SLC19A3). Using 5'-truncated promoter-luciferase constructs, we identified the thiamine level-responsive region in the SLC19A3 promoter to be between -77 and -29 (using transcriptional start site as +1). By means of mutational analysis, a key role for a stimulating protein-1 (SP1)/guanosine cytidine box in mediating the effect of extracellular thiamine level on SLC19A3 promoter was established. Furthermore, extracellular level of thiamine was found to affect SP1 protein expression and binding pattern to the thiamine level-responsive region of SLC19A3 promoter in Caco-2 cells as shown by Western blotting and electrophoretic mobility shift assay analysis, respectively. These studies demonstrate that the human intestinal thiamine uptake is adaptively regulated by the extracellular substrate level via transcriptional regulation of the THTR-2 system, and report that SP1 transcriptional factor is involved in this regulation.

Characterization of the human mitochondrial thiamine pyrophosphate transporter SLC25A19 minimal promoter: a role for NF-Y in regulating basal transcription.

Transcriptional regulation of expression of the human mitochondrial thiamine pyrophosphate transporter (the product of the SLC25A19 gene) is unknown. To understand this regulation, we cloned and characterized the 5'-regulatory region of the SLC25A19 gene (1,080 bp). The cloned fragment was found to possess promoter activity in transiently transfected human-derived liver HepG2 cells. 5'- and 3'-deletion analysis has identified the minimal region required for basal SLC25A19 promoter activity to be between -131 and +20 (using the distal transcriptional start site as +1). The minimal promoter lacks typical TATA motif and contains two inverted CCAAT boxes (binding sites for NF-Y transcriptional factor). By means of mutational analysis, the critical role of both the upstream and downstream CCAAT boxes in basal SLC25A19 promoter activity was established; however, each of these boxes alone was found to be unable to support promoter activity. EMSA and supershift EMSA (with the use of specific antibodies against NF-Y subunits) studies, as well as chromatin immunoprecipitation assay, demonstrated the binding of NF-Y to both CCAAT boxes in vitro and in vivo, respectively. The requirement for NF-Y in SLC25A19 promoter activity in vivo was directly confirmed by the use of a dominant negative NF-YA mutant in transiently transfected HepG2 cells. These studies report for the first time the characterization of the SLC25A19 promoter and demonstrate an essential role for NF-Y in its basal activity.

Glyoxalate reductase/hydroxypyruvate reductase interacts with the sodium-dependent vitamin C transporter-1 to regulate cellular vitamin C homeostasis.

The human sodium-dependent vitamin C transporter 1 (hSVCT1) contributes to cellular uptake of ascorbic acid (AA). Although different aspects of hSVCT1 cell biology have been extensively studied, nothing is currently known about the broader hSVCT1 interactome that modulates its role in cellular physiology. Here, we identify the enzyme human glyoxalate reductase/hydroxypyruvate reductase (hGR/HPR) as an hSVCT1 associated protein by yeast two-hybrid (Y2H) screening of a human liver cDNA library. The interaction between hSVCT1 and hGR/HPR was further confirmed by in vitro GST pull-down assay, in vivo coimmunoprecipitation and mammalian two-hybrid firefly luciferase assays. This interaction had functional significance as coexpression of hGR/HPR with hSVCT1 led to an increase in AA uptake. Reciprocally, siRNA-mediated knockdown of endogenous hGR/HPR led to an inhibition of AA uptake. Given that oxalate is a degradation product of vitamin C and hGR/HPR acts to limit cellular oxalate levels, this association physically couples two independent regulators of cellular oxalate production. Furthermore, confocal imaging of human liver HepG2 cells coexpressing GFP-hSVCT1 and hGR/HPR-mCherry demonstrated that these two proteins colocalize within a subpopulation of intracellular organelles. This provides a possible molecular basis for organellar AA transport and regulation of local glyoxylate/glycolate concentration in the vicinity of organelle membranes.

A high-affinity and specific carrier-mediated mechanism for uptake of thiamine pyrophosphate by human colonic epithelial cells.

All mammals require exogenous sources of thiamine (vitamin B1), as they lack the ability to synthesize the vitamin. These sources are dietary and bacterial (the latter is in reference to the vitamin, which is synthesized by the normal microflora of the large intestine). Bacterially generated thiamine exists in the free, as well as the pyrophosphorylated [thiamine pyrophosphate (TPP)], form. With no (or very little) phosphatase activity in the colon, we hypothesized that the bacterially generated TPP can also be taken up by colonocytes. To test this hypothesis, we examined [(3)H]TPP uptake in the human-derived, nontransformed colonic epithelial NCM460 cells and purified apical membrane vesicles isolated from the colon of human organ donors. Uptake of TPP by NCM460 cells occurred without metabolic alterations in the transported substrate and 1) was pH- and Na(+)-independent, but energy-dependent, 2) was saturable as a function of concentration (apparent K(m) = 0.157 ± 0.028 µM), 3) was highly specific for TPP and not affected by free thiamine (or its analogs) or by thiamine monophosphate and unrelated folate derivatives, 4) was adaptively regulated by extracellular substrate (TPP) level via what appears to be a transcriptionally mediated mechanism(s), and 5) appeared to be influenced by an intracellular Ca(2+)/calmodulin-mediated regulatory pathway. These findings suggest the involvement of a carrier-mediated mechanism for TPP uptake by colonic NCM460 cells, which was further confirmed by results from studies of native human colonic apical membrane vesicles. The results also suggest that the bacterially synthesized TPP in the large intestine is bioavailable and may contribute to overall body homeostasis of vitamin B1 and, especially, to the cellular nutrition of the local colonocytes.

Effect of clinical mutations on functionality of the human riboflavin transporter-2 (hRFT-2).

The Brown-Vialetto-Van Laere syndrome (BVVLS) is a rare neurological disease characterized by ponto-bulbar palsy, bilateral sensorineural deafness, and respiratory insufficiency. Recent genetic studies have identified mutations in the C20orf54 gene, which encodes the human riboflavin (RF) transporter -2 (hRFT-2) and suggested their link to the manifestation of BVVLS. However, there is nothing currently known about the effect of these mutations on functionality of hRFT-2, a protein that is expressed in a variety of tissues with high expression in the intestine. We addressed this issue using the human-derived intestinal epithelial Caco-2 cells. Our results showed significant (P<0.01) impairment in RF uptake by Caco-2 cells transiently expressing W17R, P28T, E36K, E71K, and R132W (but not L350M) hRFT-2 mutants. This impairment in RF transport was not due to a decrease in transcription and/or translation of hRFT-2, since mRNA and protein levels of the carrier were similar in cells expressing the mutants and wild-type hRFT-2. Confocal images of live Caco-2 cells transiently transfected with hRFT-2 mutants (fused with green fluorescent protein) showed the P28T, E36K, E71K, and R132W mutants were retained within the endoplasmic reticulum, while the W17R and L350M mutants were expressed at the cell membrane; cell surface expression of the W17R mutant was further confirmed by direct determination of cell surface transporter density. These results show for the first time that some of the BVVLS associated mutations in hRFT-2 affect the transporter functionality and that this effect is mediated via alterations in membrane targeting and/or activity of the transporter.

Tspan-1 interacts with the thiamine transporter-1 in human intestinal epithelial cells and modulates its stability.

The human thiamine transporter-1 (hTHTR-1) contributes to intestinal thiamine uptake, and its function is regulated at both the transcriptional and posttranscriptional levels. Nothing, however, is known about the protein(s) that may interact with hTHTR-1 and affects its cell biology and physiology. We addressed this issue in the present investigation using a bacterial two-hybrid system to screen a human intestinal cDNA library with the complete coding sequence of hTHTR-1 as a bait. Our results showed that a member of the tetraspanin family of proteins, Tspan-1, interacts with hTHTR-1. Coimmunoprecipitation and glutathione S-transferase (GST)-pulldown assays confirmed the existence of such an interaction between hTspan-1 and hTHTR-1 in human intestinal epithelial Caco-2 cells. Furthermore, live cell confocal imaging demonstrated that hTspan-1 and hTHTR-1 colocalize in human intestinal epithelial HuTu-80 cells. The importance of the interaction between hTspan-1 and hTHTR-1 for cell biology of the thiamine transporter was examined in HuTu-80 cells stably expressing hTHTR-1. Coexpression of hTspan-1 in these cells led to a significant decrease in the rate of degradation of hTHTR-1 compared with cells expressing the hTHTR-1 alone; in fact the half-life of the hTHTR-1 protein was twice longer in the former cell type compared with the latter cell type (12 h vs. 6 h, respectively). This finding was also confirmed at the functional level when a significantly higher thiamine uptake was observed in cycloheximide-treated (6 h) cells expressing hTHTR-1 together with hTspan-1 compared with those expressing hTHTR-1 alone. These studies demonstrate for the first time that Tspan-1 is an interacting partner with hTHTR-1 and that this interaction affects hTHTR-1 stability.

Association of PDZ-containing protein PDZD11 with the human sodium-dependent multivitamin transporter.

Intestinal absorption of biotin is mediated via the sodium-dependent multivitamin transporter (SMVT). Studies from our laboratory and others have characterized different aspects of the human SMVT (hSMVT), but nothing is currently known about protein(s) that may interact with hSMVT and affect its physiology/biology. In this study, a PDZ-containing protein PDZD11 was identified as an interacting partner with hSMVT using yeast two-hybrid screen of a human intestinal cDNA library. The interaction between hSMVT and PDZD11 was confirmed by in vitro GST-pull-down assay and in vivo in a mammalian cell environment by a two-hybrid luciferase and coimmunoprecipitation assays. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells expressing hSMVT-GFP and DsRed-PDZD11 demonstrated colocalization of these two proteins. We also examined the functional consequence of the interaction between hSMVT and PDZD11 in HuTu-80 cells and observed significant induction in [(3)H]biotin uptake upon coexpression of hSMVT and PDZD11. In contrast, knocking down of PDZD11 with gene-specific small interfering RNA led to a significant decrease in biotin uptake; biotinylation assay showed this to be associated with a marked decrease in level of expression of hSMVT at the cell membrane. By truncation approach, we also demonstrated that the PDZ binding domain that is located in the COOH-terminal tail of hSMVT polypeptide is involved in the interaction with PDZD11. These results demonstrate for the first time that PDZD11 is an interacting partner with hSMVT in intestinal epithelial cells and that this interaction affects hSMVT function and cell biology.

Identification of dynein light chain road block-1 as a novel interaction partner with the human reduced folate carrier.

The reduced folate carrier (RFC) is a major folate transport system in mammalian cells. RFC is highly expressed in the intestine and believed to play a role in folate absorption. Studies from our laboratory and others have characterized different aspects of the intestinal folate absorption process, but little is known about possible existence of accessory protein(s) that interacts with RFC and influences its physiology and/or cell biology. We investigated this issue by employing a bacterial two-hybrid system to screen a BacterioMatch II human intestinal cDNA library using the large intracellular loop between transmembrane domains 6 and 7 of the human RFC (hRFC) as bait. Our screening has resulted in the identification of dynein light chain road block-1 (DYNLRB1) as an interacting partner with hRFC. Existence of a direct protein-protein interaction between hRFC and DYNLRB1 was confirmed by in vitro pull-down assay and in vivo mammalian two-hybrid luciferase assay and coimmunoprecipitation analysis. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells demonstrated colocalization of DYNLRB1 with hRFC. Coexpression of DYNLRB1 with hRFC led to a significant (P < 0.05) increase in folate uptake. On the other hand, inhibiting the endogenous DYNLRB1 with gene-specific small interfering RNA or pharmacologically with a specific inhibitor (vanadate) led to a significant (P < 0.05) decrease in folate uptake. This study demonstrates for the first time the identification of DYNLRB1 as an interacting protein partner with hRFC. Furthermore, DYNLRB1 appears to influence the function and cell biology of hRFC.

Pancreatic beta cells and islets take up thiamin by a regulated carrier-mediated process: studies using mice and human pancreatic preparations.

Thiamin is essential for the normal function of the endocrine pancreas, but very little is known about uptake mechanism(s) and regulation by beta cells. We addressed these issues using mouse-derived pancreatic beta-TC-6 cells, and freshly isolated primary mouse and human pancreatic islets. Results showed that thiamin uptake by beta-TC-6 cells involves a pH (but not Na+)-dependent carrier-mediated process that is saturable at both the nanomolar (apparent K(m) = 37.17 +/- 9.9 nM) and micromolar (apparent K(m) = 3.26 +/- 0.86 microM) ranges, cis-inhibited by thiamin structural analogs, and trans-stimulated by unlabeled thiamin. Involvement of carrier-mediated process was also confirmed in primary mouse and human pancreatic islets. Both THTR-1 and THTR-2 were found to be expressed in these mouse and human pancreatic preparations. Maintaining beta-TC-6 cells in the presence of a high level of thiamin led to a significant (P < 0.01) decrease in thiamin uptake, which was associated with a significant downregulation in level of expression of THTR-1 and THTR-2 at the protein and mRNA levels and a decrease in transcriptional (promoter) activity. Modulators of intracellular Ca2+/calmodulin- and protein-tyrosine kinase-mediated pathways also altered thiamin uptake. Finally, confocal imaging of live beta-TC-6 cells showed that clinical mutants of THTR-1 have mixed expression phenotypes and all led to impairment in thiamin uptake. These studies demonstrate for the first time that thiamin uptake by the endocrine pancreas is carrier mediated and is adaptively regulated by the prevailing vitamin level via transcriptional mechanisms. Furthermore, clinical mutants of THTR-1 impair thiamin uptake via different mechanisms.

Mechanism of nicotinic acid transport in human liver cells: experiments with HepG2 cells and primary hepatocytes.

This study reports on the functional expression of a specific, high-affinity carrier-mediated mechanism for the transport of niacin (nicotinic acid) in human liver cells. Both human-derived liver HepG2 cells and human primary hepatocytes were used as models in these investigations. The initial rate of transport of nicotinic acid into HepG2 cells was found to be acidic pH, temperature, and energy dependent; it was, however, Na(+) independent in nature. Evidence for the existence of a carrier-mediated system that is specific for [(3)H]nicotinic acid transport was found and included the following: 1) saturability as a function of concentration with an apparent K(m) of 0.73 +/- 0.16 microM and V(max) of 25.02 +/- 1.45 pmol.mg protein(-1).3 min(-1), 2) cis-inhibition by unlabeled nicotinic acid and nicotinamide but not by unrelated organic anions (lactate, acetate, butyrate, succinate, citrate, and valproate), and 3) trans-stimulation of [(3)H]nicotinic acid efflux by unlabeled nicotinic acid. Transport of the vitamin into human primary hepatocytes occurs similarly via an acidic pH-dependent and specific carrier-mediated process. Inhibitors of the Ca(2+)-calmodulin-mediated pathway (but not modulators of the PKC-, PKA-, and protein tyrosine kinase-mediated pathways) inhibited nicotinic acid transport into both HepG2 cells and human primary hepatocytes. Maintenance of HepG2 cells (for 48 h) in growth medium oversupplemented with nicotinic acid (or nicotinamide) did not affect the subsequent transport of [(3)H]nicotinic acid into HepG2 cells. These results show, for the first time, the existence of a specific and regulated membrane carrier-mediated system for nicotinic acid transport in human liver cells.