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

Svetlana M Nabokina; Hamid M Said

doi:10.1152/ajpgi.00151.2012

. 2012 May 24;303(3):G389–G395. doi: 10.1152/ajpgi.00151.2012

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

Svetlana M Nabokina ¹, Hamid M Said ^1,^✉

PMCID: PMC3423106 PMID: 22628036

Abstract

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 [³H]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 Ca²⁺/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.

Keywords: thiamine pyrophosphate uptake, colonocytes

thiamine (vitamin B1), a water-soluble micronutrient, is absolutely required for normal functions of all living cells. Thiamine in its diphosphorylated form [i.e., thiamine pyrophosphate (TPP)] is an indispensable cofactor for oxidative metabolism, as it acts as a cofactor for several enzymes involved in carbohydrate and energy metabolism, including pyruvate dehydrogenase, α-ketoglutarate dehydrogenase, branched-chain α-ketoacid dehydrogenase, and transketolase (reviewed in Refs. 3, 5, and 28). Also, because thiamine bridges the glycolytic and pentose phosphate pathways (which generate chemical reducing power in the cell), it plays a role in reducing cellular oxidative stress (8, 15). Furthermore, it appears to play an important role in maintaining normal mitochondrial function and organization (4). Thus, deficiency of this micronutrient at the cellular level leads to impairment of energy metabolism, oxidative stress, and mitochondrial abnormalities. Thiamine deficiency in humans, which leads to serious clinical abnormalities that include cardiovascular and neurological disorders, occurs in a high percentage of individuals with chronic alcoholism and in a number of other conditions, such as diabetes mellitus and gastrointestinal disorders (3, 28–30).

Humans (and other mammals) cannot synthesize thiamine and, thus, must obtain this vitamin from exogenous sources: the diet and the normal microflora of the large intestine. Thiamine in the diet exists mainly in the TPP form. It is generally assumed that dietary TPP is first hydrolyzed to free thiamine (by the abundant small intestinal phosphatases) primarily in the proximal part of the small gut (24). Free thiamine is then absorbed via a specific carrier-mediated mechanism (reviewed in Refs. 24 and 28) that involves high-affinity thiamine transporter-1 and -2 (THTR-1 and THTR-2; for humans these systems are referred to as hTHTR-1 and hTHTR-2).

It has been long recognized that the normal microflora of the large intestine synthesizes a considerable amount of thiamine and that this thiamine exists in the free and phosphorylated forms (1, 17, 18, 22). More recent studies have shown that the human gut microbiome can be classified into three functionally different enterotypes (enterotypes 1, 2, and 3), with enterotype 2 (which is enriched in Prevotella and Desulfovibrio) being enriched with enzymes that are involved in the biosynthesis of TPP (1). Despite clear evidence showing the production and availability of thiamine and its derivatives by gut microbiota, which clearly points to its importance in host nutrition, our understanding of the ability of the human colon to absorb these forms of vitamins is still emerging. Thus we previously showed that human colonocytes possess a highly efficient and specific uptake mechanism for free thiamine (27). However, it is not known if human colonocytes can also absorb the bacterially generated TPP. Knowing that colonocytes possess no or little alkaline phosphatase activity compared with epithelial cells of the small intestine (2, 9, 11), we hypothesized that these cells may be able to take up this form of the vitamin. There is no evidence showing such an uptake system for TPP in the human colon. Studies in the nematode Caenorhabditis elegans tpk-1 (qm162) mutant carrying a partial loss-of-function mutation in a thiamine pyrophosphokinase gene (thiamine pyrophosphokinase enzymatically converts thiamine to TPP) showed that C. elegans can be rescued by supplementation with TPP but not with thiamine (12). The same study (12) also showed that expression of wild-type thiamine pyrophosphokinase in one tissue of C. elegans can rescue the function of other tissues that express only the mutant thiamine pyrophosphokinase, suggesting that TPP is being transported from one tissue to another. Evidence for efflux of TPP from mammalian (murine leukemia) cells via the reduced folate carrier RFC1 has also been reported (32).

Thus our aim in this study was to determine whether human colonic epithelial cells can take up TPP directly from the intestinal lumen and, if so, to determine the mechanism involved in this uptake and its regulation. We used as models nontransformed human-derived colonic epithelial NCM460 cells [a well-established model for studying colonocyte physiology (20, 21, 25–27)] and native purified apical membrane vesicles isolated from the mucosa of the proximal colon of human organ donors (7, 19). Our data demonstrate, for the first time, that human colonocytes possess a specific, high-affinity, and regulated carrier-mediated system for TPP uptake.

MATERIALS AND METHODS

Chemicals

Custom-made ³H-labeled TPP ([³H]TPP, specific activity 1.3 Ci/mmol, radiochemical purity 99.9%) was obtained from Moravek Biochemicals (Brea, CA). ³H-labeled biotin ([³H]biotin, specific activity 60 Ci/mmol, radiochemical purity 99.0%) and ³H-labeled riboflavin ([³H]RF, specific activity 21.2 Ci/mmol, radiochemical purity 99.9%) were obtained from American Radiolabeled Chemicals (St. Louis, MO) and Moravek Biochemicals, respectively. Unlabeled TPP and thiamine analogs were purchased from Sigma. All other chemicals and reagents were purchased from commercial sources and were of analytical grade.

Cell Culture and Uptake Studies

The human-derived nontransformed colonic epithelial cell line NCM460 (INCELL, San Antonio, TX) was maintained and subcultured as described by us previously (26, 27). For uptake studies, NCM460 cells were trypsinized and plated onto 12-well plates (∼2 × 10⁵ cells/well) in Ham's F-12 culture medium (Invitrogen) supplemented with 20% (vol/vol) FBS and antibiotics. For assays of TPP oversupplementation effect, cells were grown for 4 days in Ham's F-12 medium including 1 mM unlabeled TPP, with medium changed every day. Uptake studies were performed on confluent cell monolayers (between passages 29 and 44) 2–4 days following confluence. Uptake was examined in cells incubated at 37°C in Krebs-Ringer (KR) buffer containing (in mM) 123 NaCl, 4.93 KCl, 1.23 MgSO₄, 0.85 CaCl₂, 5 glucose, 5 glutamine, 10 HEPES, and 10 MES (pH 7.4). Although colonocytes are known to express no (or little) alkaline phosphatases (2, 9, 11), which we also confirmed by demonstration of no metabolism in the TPP taken up and the TPP left in the incubation medium (see results), we added the alkaline phosphatase inhibitor as an additional layer of precaution [phenylalanine (10 mM) did not affect TPP uptake; data not shown]. [³H]TPP was added to the incubation medium at the onset of the uptake experiment. The reaction was terminated after 3 min (unless otherwise stated) by the addition of 2 ml of ice-cold KR buffer, and the cells were immediately aspirated and washed with ice-cold KR buffer. Cells were lysed with 1 ml of 1 N NaOH, lysates were neutralized with HCl, and radioactivity was assessed in a liquid scintillation counter. The protein content of cell digests was measured using the DC protein assay kit (Bio-Rad, Hercules, CA).

Human Colonic Apical Membrane Vesicles and Uptake Studies

Human colonic apical membrane vesicles were kindly provided by Dr. P. K. Dudeja (University of Illinois at Chicago) and isolated from epithelial cells of proximal colon of human organ donors by a well-established and validated procedure (7) and in accordance with the institutional protocols approved by the Institutional Review Board of the University of Illinois at Chicago. Uptake of [³H]TPP into colonic apical membrane vesicles was measured using a rapid-filtration technique, as described by us previously (14). Briefly, vesicles were preloaded with a buffer containing (in mM) 280 mannitol and 20 HEPES-Tris, pH 7.4, and then incubated in a buffer containing (in mM) 140 mannitol, 100 NaCl, 10 HEPES, 10 MES, and 10 phenylalanine (pH 7.4) in the presence of [³H]TPP (0.38 μM). Uptake studies were performed for 15 s at 37°C.

Thin-Layer Chromatography

Chemical stability of [³H]TPP in the incubation buffer and the metabolic forms of the intracellular radioactivity following uptake were examined under the same conditions used for the uptake experiments. After incubation of NCM460 cells in 1 ml of KR buffer containing [³H]TPP (0.23 μM) for 3 min (unless otherwise indicated) at 37°C, the forms of extracellular or intracellular ³H radioactivity were identified by thin-layer chromatography (TLC), as described previously (33). For assessment of chemical stability of TPP, 100% trichloroacetic acid (15 μl) was added to the aliquot (250 μl) of extracellular medium or cells, and the cells were subjected to centrifugation and several extractions with water-saturated ethyl ether. For determination of the metabolic forms of the intracellular radioactivity, cells were processed as described above (see Cell Culture and Uptake Studies). The sample (∼20 μl) was mixed with the unlabeled thiamine, thiamine monophosphate (TMP), and TPP (to check the separation of thiamine derivatives under UV light), applied to the TLC plate (20 × 20 cm plate coated with a 250-μm layer of silica gel; Whatman), and run using a solvent system of diethanolamine-methanol-formic acid-67 mM dibasic sodium phosphate (1:15:1.5:5). Silica gel was cut into equal-sized (∼1-cm) pieces, and distribution of radioactivity was assessed in a liquid scintillation counter.

Data Presentation and Statistical Analysis

Values are means ± SE of multiple individual uptake determinations and are expressed in picomoles or femtomoles per milligram of protein per unit of time. Student's t-test was used in statistical analysis, with statistical significance set at 0.01 (P < 0.01). Kinetic parameters of the saturable TPP uptake process [i.e., maximal velocity (V_max) and apparent Michaelis-Menten constant (K_m)] were calculated as described by Wilkinson (31). Uptake by the saturable component was calculated at each concentration by subtraction of uptake by simple diffusion [determined from the slope of the line between the point of origin and uptake at high pharmacological concentration of TPP (1 mM)] from total uptake.

RESULTS

Uptake of TPP by NCM460 Cells: General Characteristics of the Process

Uptake of TPP (0.23 μM) by NCM460 cells as a function of time was studied and found to be linear (r = 0.99) for up to 10 min of incubation and occurred at 8.57 ± 1.17 pmol/mg protein (Fig. 1). A 3-min incubation time (initial rate) was used in all subsequent experiments.

Fig. 1. — Uptake of thiamine pyrophosphate (TPP) by NCM460 cells as a function of time. Confluent monolayers of NCM460 cells were incubated in Krebs-Ringer buffer, pH 7.4, at 37°C. [³H]TPP (0.23 μM) was added to the incubation medium at the start of uptake. Values are means ± SE of 3–4 separate uptake determinations. SEs are not visible on the scale of the graph.

The effect of uptake buffer pH on the initial rate of TPP uptake by NCM460 cells was examined. The results showed TPP (0.23 μM) uptake to be pH-independent over the pH range 5.5–8.0 (Fig. 2). All subsequent studies were performed at buffer pH 7.4.

Fig. 2. — Effect of incubation buffer pH on initial rate of TPP uptake by NCM460 cells. Confluent monolayers of NCM460 cells were incubated at 37°C in Krebs-Ringer buffer at pH 5.5–8.0. [³H]TPP (0.23 μM) was added to the incubation medium at the onset of a 3-min incubation (i.e., initial rate). Values are means ± SE of 3–4 separate uptake determinations. Some SEs are not visible on the scale of the graph.

The role of Na⁺ in TPP uptake by NCM460 cells was studied by replacement of Na⁺ in the uptake buffer with an equimolar concentration (123 mM) of the monovalent cations K⁺ and choline or with inert mannitol. No inhibition of the initial rate of TPP (0.23 μM) uptake was found upon Na⁺ replacement (22.04 ± 1.68, 21.43 ± 0.76, 22.35 ± 1.9, and 22.86 ± 1.65 pmol·mg protein⁻¹·3 min⁻¹ in the presence of Na⁺, K⁺, choline, and mannitol, respectively). We also measured the initial rate of TPP (0.23 μM) uptake by NCM460 cells that were pretreated for 30 min with the Na⁺-K⁺-ATPase inhibitor ouabain (1 mM). Similar uptake was observed for control and ouabain-pretreated cells (23.94 ± 0.66 and 22.39 ± 0.68 pmol·mg protein⁻¹·3 min⁻¹, respectively).

The energy dependence of TPP uptake was examined by measurement of the initial rate of TPP (0.23 μM) uptake by NCM460 cells that were pretreated for 30 min with the metabolic inhibitor 2,4-dinitrophenol (DNP, 1 mM). The results show significant (P < 0.01) inhibition of TPP uptake by DNP pretreatment (23.94 ± 0.66 and 14.22 ± 0.71 pmol·mg protein⁻¹·3 min⁻¹ for control and DNP-pretreated cells, respectively).

The effect of the membrane transport inhibitors probenecid and amiloride (each at 1 mM) on the initial rate of TPP (0.23 μM) uptake by NCM460 cells was examined. TPP uptake was significantly (P < 0.01) inhibited by both compounds, with probenecid showing a more prominent inhibitory effect (24.44 ± 0.29, 21.68 ± 0.33, and 10.81 ± 0.29 pmol·mg protein⁻¹·3 min⁻¹ for control, amiloride, and probenecid, respectively).

Although colonocytes are known to express no (or little) alkaline phosphatase activity (2, 9, 11), we estimated chemical stability of TPP in the uptake buffer (and its possible metabolism after exposure to cells) under the same conditions used in the uptake experiments. NCM460 cells were incubated in KR buffer containing [³H]TPP (0.23 μM) for 3 min (initial rate) at 37°C; then the forms of extracellular ³H radioactivity were identified by TLC (see materials and methods). Cells incubated in the presence of inhibitor of intestinal alkaline phosphatases (phenylalanine, 10 mM) served as a control. The results showed that ∼97% and ∼98% of the extracellular ³H radioactivity was in the form of intact TPP after 3 min of exposure to cells in the presence and absence of phenylalanine, respectively. We also estimated the effect of prolonged (60 min) exposure of [³H]TPP (0.23 μM) to NCM460 cells in the absence of phenylalanine. The results showed that ∼95% of the extracellular radioactivity existed in the form of intact TPP. These data clearly point to the absence of detectable degradation of [³H]TPP as a result of contact with NCM460 cells.

In a related study, we also determined the metabolic forms of the transported radioactivity following a 3-min (initial rate) incubation of NCM460 cells with [³H]TPP (0.23 μM). The results showed that the majority (∼96%) of the transported substrate was in the form of intact TPP.

Involvement of a Carrier-Mediated System for TPP Transport in NCM460 Cells and Native Human Colon

We examined the effect of unlabeled TPP (1 mM) on the initial rate of [³H]TPP (0.23 μM) uptake by NCM460 cells. The results showed significant (P < 0.01) reduction of TPP uptake in the presence of unlabeled TPP (22.23 ± 0.37 and 0.08 ± 0.01 pmol·mg protein⁻¹·3 min⁻¹ for control and 1 mM TPP, respectively).

To establish the physiological relevance of these findings to the native human intestine, we examined the effect of unlabeled TPP (1 mM) on the initial rate of TPP (0.38 μM) uptake by purified apical membrane vesicles isolated from epithelial cells of proximal human colon. The results showed significant (P < 0.01) inhibition of TPP transport by unlabeled TPP (2.71 ± 0.08 and 0.65 ± 0.01 pmol·mg protein⁻¹·15 s⁻¹ for control and unlabeled TPP, respectively). These results provide the physiological validation for our observations with NCM460 cells with regard to the involvement of a carrier-mediated system for TPP transport.

To further confirm the existence of a carrier-mediated system for TPP uptake in colonocytes, we looked for the possible existence of saturability in the initial rate (3 min) of TPP uptake by NCM460 cells as a function of substrate concentration. The results (Fig. 3) showed the uptake to indeed include a saturable component with kinetic parameters of 0.157 ± 0.028 μM for apparent K_m and 22.31 ± 0.98 pmol·mg protein⁻¹·3 min⁻¹ for V_max. These data further confirm the existence of a carrier-mediated mechanism for TPP uptake by human colonocytes.

Fig. 3. — Uptake of TPP by NCM460 cells as a function of substrate concentration. Confluent monolayers of NCM460 cells were incubated at 37°C in Krebs-Ringer buffer, pH 7.4, in the presence of different concentrations of TPP. Uptake was measured after 3 min of incubation (i.e., initial rate). Uptake by the carrier-mediated system was calculated as described in materials and methods. Values are means ± SE of 6–9 separate uptake determinations. Some SEs are not visible on the scale of the graph.

Specificity of the TPP Uptake System of NCM460 Cells

To determine the specificity of the TPP uptake system, we examined the effect of unlabeled TPP and a wide spectrum of its structural analogs, including TMP, thiamine, pyrithiamine, benfotiamine, oxythiamine, amprolium, and thiamine disulfide (each at 200 μM), on the initial rate of TPP (0.23 μM) uptake by NCM460 cells. The results (Table 1) showed that none of the compounds, other than unlabeled TPP, significantly affected uptake.

Table 1.

Effect of TPP structural analogs and related compounds on the initial rate of [³H]TPP uptake by NCM460 cells

Compound (200 μM)	Uptake, pmol·mg protein⁻¹·3 min⁻¹	P Value
Control	22.81 ± 0.16
TPP	0.18 ± 0.01	<0.01
TMP	20.95 ± 1.04	NS
Thiamine	23.66 ± 1.31	NS
Pyrithiamine	21.38 ± 0.31	NS
Benfotiamine	22.13 ± 0.42	NS
Oxythiamine	22.61 ± 0.39	NS
Amprolium	22.98 ± 0.37	NS
Thiamine disulfide	23.23 ± 0.35	NS
ADP	22.54 ± 1.38	NS
ATP	19.31 ± 0.76	NS

Open in a new tab

Values are means ± SE of 3–6 separate uptake determinations. Monolayers of NCM460 cells were incubated in Krebs-Ringer buffer, pH 7.4, at 37°C. ³H-labeled thiamine pyrophosphate ([³H]TPP, 0.23 μM) and the compound under investigation were added to the incubation medium at onset of 3-min incubation. TMP, thiamine monophosphate; NS, not significant.

We also examined the effect of the organic phosphates, ADP and ATP (200 μM each), on the initial rate of TPP (0.23 μM) uptake by NCM460 cells. No significant inhibition in TPP uptake was observed for these compounds (Table 1).

Because the RFC has been reported to induce efflux of TPP in murine leukemia cells (32), we next determined the possibility of involvement of the RFC system in TPP uptake by NCM460 cells. To do this, we examined the effect of adding high concentrations of the naturally occurring folate derivative 5-formyltetrahydrofolate, the anti-folate methotrexate, and folic acid (each at 200 μM) to the incubation medium on the initial rate of TPP (0.23 μM) uptake by NCM460 cells. The results (Fig. 4) showed that none of these compounds inhibits TPP uptake by NCM460 cells, ruling out the possibility that RFC is involved in TPP uptake by human colonocytes.

Fig. 4. — Effect of structural analogs of folic acid on the initial rate of TPP uptake by NCM460 cells. Confluent monolayers of NCM460 cells were incubated at 37°C in Krebs-Ringer buffer, pH 7.4, in the presence of [³H]TPP (0.23 μM) and folic acid (FA), folic acid (FA), unlabeled 5-formyltetrahydrofolate (5-FTHF), the anti-folate methotrexate (MTX), or TPP (each at 200 μM). Uptake was measured after 3 min of incubation (i.e., initial rate). Values are means ± SE of 3–6 separate uptake determinations. *P < 0.01.

In a related study, we examined the effect of TPP structural analogs, including TMP and thiamine (each at 120 μM) on the initial rate of [³H]TPP (0.38 μM) uptake by apical membrane vesicles isolated from proximal human colon (the effect of unlabeled TPP was also simultaneously tested as a positive control). The results showed no significant inhibition of TPP uptake by unlabeled TMP and thiamine but the expected significant (P < 0.01) inhibition by unlabeled TPP (2.44 ± 0.09, 2.56 ± 0.2, 2.75 ± 0.18, and 0.46 ± 0.01 pmol·mg protein⁻¹·15 s⁻¹ for control, TMP, thiamine, and unlabeled TPP, respectively). These data provide the physiological validation for our observations with NCM460 cells with regard to specificity of the TPP carrier system.

Regulation of the TPP Uptake Process in NCM460 Cells by Extracellular and Intracellular Factors

Effect of extracellular substrate level.

We examined the effect of substrate (TPP) level in the extracellular environment on initial rate of [³H]TPP uptake. Figure 5 depicts the effect of maintaining NCM460 cells in TPP-oversupplemented (1 mM TPP) growth medium for 4 days on the initial rate of TPP (0.23 μM) uptake. Uptake by cells maintained in a medium containing a sufficient amount of thiamine (0.9 μM), but lacking TPP, served as a control in these experiments. As shown in Fig. 5, [³H]TPP uptake by cells maintained in TPP-oversupplemented conditions was significantly (P < 0.01) lower than uptake by cells maintained in control medium. Uptake of unrelated vitamins [³H]RF and [³H]biotin was not affected by the TPP level in cell growth medium (Fig. 5), confirming the specificity of the observation.

Fig. 5. — Effect of TPP level in the extracellular environment on initial rate of [³H]TPP uptake by NCM460 cells. After NCM460 cells were maintained in TPP-oversupplemented (1 mM TPP) or control (no TPP) growth medium for 4 days, uptake of TPP and unrelated vitamins, biotin and riboflavin (RF), was measured. A: initial rate of [³H]TPP (0.23 μM) uptake. B: initial rate of [³H]biotin (5 nM) uptake. C: initial rate of [³H]RF (14 nM) uptake. Values are means ± SE of 3–6 separate uptake determinations. *P < 0.01.

Since adaptive regulation of a number of vitamin transporters by substrate level has been shown to include transcriptional mechanisms (reviewed in Ref. 28), we aimed to examine the possibility of involvement of transcriptional mechanisms in TPP uptake regulation by substrate level. NCM460 cells grown in TPP-oversupplemented conditions for 4 days (control) were transferred to the medium lacking TPP and treated with the transcriptional inhibitor actinomycin D (0.3 μg/ml) for 24 h. Significant (P < 0.01) induction of TPP (0.23 μM) uptake was observed for cells in TPP-deficient medium compared with those continuously kept in TPP-oversupplemented medium, with the induction being significantly (P < 0.01) inhibited by actinomycin D (Fig. 6). These data suggest that the adaptive regulation of the TPP transporter system by the substrate level occurs at the transcriptional level.

Fig. 6. — TPP uptake by NCM460 cells grown under TPP-deficient and control conditions: effect of actinomycin D. NCM460 cells grown in TPP-oversupplemented medium (1 mM) for 4 days (control) were transferred to TPP-deficient medium and treated with actinomycin D (0.3 μg/ml) for 24 h. Uptake (for 3 min) was measured in Krebs-Ringer buffer, pH 7.4, in the presence of 0.23 μM [³H]TPP. Values are means ± SE of 3–6 separate uptake determinations. Significant induction of TPP uptake was observed for cells in TPP-deficient medium compared with those continuously kept in TPP-oversupplemented medium, with induction being significantly inhibited by actinomycin D: *P < 0.01.

Role of the Ca²⁺/calmodulin-mediated intracellular regulatory pathway.

Pathways known to affect the nutrient uptake regulation in epithelial cells, including the Ca²⁺/calmodulin-mediated, PKC-mediated, cAMP (or PKA)-mediated, nitric oxide (NO)-mediated, and protein tyrosine kinase (PTK)-mediated pathways (6, 10, 13, 16, 23), were chosen for our studies. Possible regulation of TPP uptake in colonocytes by these intracellular pathways was studied by examining the effect of a modulator of a specific pathway on TPP uptake. The modulators of the PKC-mediated (phorbol 12-myristate 13-acetate, 10 μM), cAMP (or PKA)-mediated (dibutyryl cAMP, 5 mM), NO-mediated (S-nitroso-N-acetylpenicillamine, 1 mM), and PTK-mediated (genistein and tyrphostin A-1, 50 μM each) pathways do not affect TPP uptake by NCM460 cells (data not shown). In contrast, the Ca²⁺/calmodulin-mediated intracellular regulatory pathway plays a role in regulation of TPP uptake in NCM460 cells. Pretreating NCM460 cells with calmidazolium (1, 10, and 50 μM), an inhibitor of the Ca²⁺/calmodulin-mediated pathway, for 1 h caused a significant (P < 0.01) concentration-dependent decrease in TPP uptake (Table 2). Another inhibitor of the Ca²⁺/calmodulin-mediated pathway, KN62 (50 μM), was also found to significantly (P < 0.05) inhibit TPP uptake (Table 2). We determined the effect of calmidazolium on the kinetic parameters of TPP uptake. Figure 7 depicts the results of the initial rate of TPP uptake as a function of substrate concentration by NCM460 cells pretreated for 1 h with 25 μM calmidazolium. As shown in Fig. 7, calmidazolium causes a significant (P < 0.01) inhibition of V_max (18.96 ± 1.21 and 5.79 ± 0.20 pmol·mg protein⁻¹·3 min⁻¹ for control and calmidazolium, respectively), as well as a significant (P < 0.01) inhibition of apparent K_m (0.14 ± 0.03 and 0.05 ± 0.01 μM for control and calmidazolium, respectively) of the TPP uptake process in NCM460 cells.

Table 2.

Effect of modulators of the Ca²⁺/calmodulin-mediated intracellular regulatory pathway on [³H]TPP uptake by NCM460 cells

	Uptake, pmol·mg protein⁻¹·3 min⁻¹	P Value
Control	23.37 ± 0.89
Calmidazolium
1 μM	22.84 ± 0.03	NS
10 μM	14.55 ± 0.16	<0.01
50 μM	6.09 ± 0.11	<0.01
KN62 (50 μM)	17.77 ± 0.32	<0.05

Open in a new tab

Values are means ± SE of 3–4 separate uptake determinations. Monolayers of NCM460 cells were incubated in Krebs-Ringer buffer, pH 7.4, at 37°C. [³H]TPP (0.23 μM) and the compound under investigation were added to the incubation medium at onset of 3-min incubation.

Fig. 7. — Effect of calmidazolium on kinetic parameters of TPP uptake by NCM460 cells. Confluent monolayers of NCM460 cells were pretreated (for 1 h) with 25 μM calmidazolium or kept in Krebs-Ringer buffer (control). Initial rate of uptake of different concentrations of TPP was then examined in Krebs-Ringer buffer, pH 7.4. Values are means ± SE of 6–9 separate uptake determinations. Some SEs are not visible on the scale of the graph.

DISCUSSION

In contrast to the situation in the small intestine, there is no (or very little) alkaline phosphatase activity in the colon (2, 9, 11). This raises the possibility that the bacterially synthesized TPP in the colonic lumen remains (for the most part) unchanged and, thus, is available for uptake by the colonic epithelial cells. The current study was designed to directly test whether colonic epithelial cells are capable of taking up TPP and, if so, to determine the mechanism involved and how it is regulated. We used nontransformed human-derived colonic epithelial NCM460 cells and native human colonic apical membrane vesicles as models in our investigations (19–21, 25–27).

Our studies with NCM460 cells showed no degradation of TPP in the incubation medium whether phenylalanine (an inhibitor of alkaline phosphatases) was present or absent. Even prolonged (60 min) incubation of the cells with [³H]TPP in the absence of phenylalanine was not associated with detectable hydrolysis of TPP. Furthermore, the metabolic form of the transported TPP following 3 min of incubation of cells with [³H]TPP was mainly intact TPP. These findings clearly show that little, if any, degradation occurs in TPP prior its absorption, as well as after its internalization in colonic epithelial cells. In addition, the data support the belief that no (or little) alkaline phosphatase activity exists in colonocytes.

Uptake of TPP by NCM460 cells was found to be energy-dependent, but it was Na⁺- and pH-independent. We also obtained evidence indicating the involvement of a carrier-mediated mechanism for TPP uptake by NCM460 cells. This was shown by the significant inhibition of the initial rate of uptake of [³H]TPP by unlabeled TPP and further supported by the observation of saturation in the uptake of TPP as a function of substrate concentration in the incubation medium. An apparent K_m of the saturable TPP uptake system was determined to be 0.157 ± 0.028 μM, suggesting high affinity for the system involved. The physiological relevance of our findings with NCM460 cells with regard to involvement of a carrier-mediated system for TPP uptake was confirmed by the findings with purified native human colonic apical membrane vesicles, which again showed significant inhibition of [³H]TPP uptake by unlabeled TPP.

The functionally identified TPP uptake system in human colonocytes appears to be highly specific for TPP, as neither free thiamine (including a host of its analogs), TMP, the adenine nucleotides (ADP and ATP), nor folate derivatives were able to inhibit the initial rate of [³H]TPP uptake by NCM460 cells. The inability of TMP to inhibit [³H]TPP uptake by NCM460 cells indicates that the TPP uptake system can discriminate between mono- and diphosphorylated thiamine. Also, the inability of 5-methyltetrahydrofolate and methotrexate to inhibit TPP uptake by NCM460 cells indicates that the system involved is not related to the RFC (32). Further studies are required to determine the molecular identity of the colonic TPP uptake system.

We also examined the possible regulation of the TPP uptake process by extracellular and intracellular factors. Our results showed that maintaining NCM460 cells in TPP-oversupplemented growth medium leads to a specific and significant downregulation of the initial rate of [³H]TPP uptake by these cells. This adaptive regulation of the TPP uptake process by extracellular substrate level seems to be mediated, at least in part, at the transcriptional level, as indicated in the study involving the transcriptional inhibitor actinomycin D. We also examined possible regulation of the TPP uptake process of NCM460 cells by intracellular regulatory pathways. Pathways known to play a role in nutrient uptake regulation in epithelial cells, such as the Ca²⁺/calmodulin-mediated, PKC-mediated, cAMP (or PKA)-mediated, NO-mediated, and PTK-mediated pathways (6, 10, 13, 16, 23), were studied. Our results show that modulators of the PKC-, PKA-, NO-, and PTK-mediated signaling pathways were unable to affect TPP uptake by NCM460 cells, suggesting that these pathways do not play an important role in TPP uptake by NCM460 cells. In contrast, a role for the Ca²⁺/calmodulin-mediated intracellular regulatory pathway was evident. Calmidazolium, a widely used modulator of this pathway, caused a significant and concentration-dependent inhibition of TPP uptake, and its effect appeared to be mainly mediated via a significant inhibition of the V_max of the TPP uptake process of NCM460 cells. This observation suggests that the effect of calmidazolium is a result of alterations in the activity (and/or number) of the TPP carrier. The exact mechanism (direct, through an effect on the uptake system, or indirect, through an effect of another entity that then affects the TPP uptake system) through which the Ca²⁺/calmodulin-mediated pathway affects the TPP uptake process in NCM460 cells is not clear, and further studies are required to address this issue.

Finally, it is worth mentioning that recent studies have shown that human thiamine pyrophosphokinase (the cytoplasmic enzyme that converts transported free thiamine to TPP) is barely expressed in the human colon, while it is abundantly expressed in the human small intestine (34). This is most likely because colonic epithelial cells do not need this enzyme, as they import an already made TPP from the lumen. In summary, results of this study demonstrate, for the first time, the existence of a specific and high-affinity carrier-mediated mechanism for TPP uptake by human colonocytes. The results also show that the TPP uptake process is adaptively regulated by the extracellular substrate level and appears to be under the regulation of an intracellular Ca²⁺/calmodulin-mediated pathway.

GRANTS

This study was supported by grants from the National Institutes of Health (DK-56061 and AA-018071) and the Department of Veterans Affairs.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

S.M.N. and H.M.S. are responsible for conception and design of the research; S.M.N. performed the experiments; S.M.N. and H.M.S. analyzed the data; S.M.N. and H.M.S. interpreted the results of the experiments; S.M.N. prepared the figures; S.M.N. and H.M.S. drafted the manuscript; S.M.N. and H.M.S. edited and revised the manuscript; H.M.S. approved the final version of the manuscript.

REFERENCES

1. Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto JM, Bertalan M, Borruel N, Casellas F, Fernandez L, Gautier L, Hansen T, Hattori M, Hayashi T, Kleerebezem M, Kurokawa K, Leclerc M, Levenez F, Manichanh C, Nielsen HB, Nielsen T, Pons N, Poulain J, Qin J, Sicheritz-Ponten T, Tims S, Torrents D, Ugarte E, Zoetendal EG, Wang J, Guarner F, Pedersen O, de Vos WM, Brunak S, Doré J; MetaHIT Consortium, Antolín M, Artiguenave F, Blottiere HM, Almeida M, Brechot C, Cara C, Chervaux C, Cultrone A, Delorme C, Denariaz G, Dervyn R, Foerstner KU, Friss C, van de Guchte M, Guedon E, Haimet F, Huber W, van Hylckama-Vlieg J, Jamet A, Juste C, Kaci G, Knol J, Lakhdari O, Layec S, Le Roux K, Maguin E, Mérieux A, Melo Minardi R, M'rini C, Muller J, Oozeer R, Parkhill J, Renault P, Rescigno M, Sanchez N, Sunagawa S, Torrejon A, Turner K, Vandemeulebrouck G, Varela E, Winogradsky Y, Zeller G, Weissenbach J, Ehrlich SD, Bork P. Enterotypes of the human gut microbiome. Nature 473: 174–180, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
2. Barrow BJ, Ortiz-Reyes R, O'Riordan MA, Pretlow TP. In situ localization of enzymes and mucin in normal rat colon embedded in plastic. Histochem J 21: 289–295, 1989 [DOI] [PubMed] [Google Scholar]
3. Berdanier CD. Advanced Nutrition: Micronutrients. Boca Raton, FL: CRC, 1998, p. 80–88 [Google Scholar]
4. Bettendorff L, Goessens G, Sluse F, Wins P, Bureau M, Laschet J, Grisar T. Thiamine deficiency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors. J Neurochem 64: 2013–2021, 1995 [DOI] [PubMed] [Google Scholar]
5. Bettendorff L, Wins P. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors. FEBS J 276: 2917–2925, 2009 [DOI] [PubMed] [Google Scholar]
6. Brandsch M, Miyamoto Y, Ganapathy V, Leibach FH. Regulation of taurine transport in human colon carcinoma cell line (HT-29 and Caco-2) by protein kinase C. Am J Physiol Gastrointest Liver Physiol 264: G939–G946, 1993 [DOI] [PubMed] [Google Scholar]
7. Brasitus TA, Keresztes RS. Protein-lipid interactions in antipodal plasma membranes of rat colonocytes. Biochim Biophys Acta 773: 290–300, 1984 [DOI] [PubMed] [Google Scholar]
8. Calingasan NY, Gandy SE, Baker H, Sheu KF, Smith JD, Lamb BT, Gearhart JD, Buxbaum JD, Harper C, Selkoe DJ, Price DL, Sisodia SS, Gibson GE. Novel neuritic clusters with accumulations of amyloid precursor protein and amyloid precursor-like protein 2 immunoreactivity in brain regions damaged by thiamine deficiency. Am J Pathol 149: 1063–1071, 1996 [PMC free article] [PubMed] [Google Scholar]
9. Chernobilsky B, Tsou KC. Adenocarcinoma, adenomas and polyps of the colon. Histochem Study Cancer 21: 165–177, 1968 [DOI] [PubMed] [Google Scholar]
10. Cohen ME, Reinlib L, Watson AJM, Gorelick F, Sikora KR, Tse M, Rood RP, Czernik AJ, Sharp GW, Donowitz M. Rabbit ileal villus cell brush border Na⁺/H⁺ exchanger is regulated by Ca²⁺/calmodulin-dependent protein kinase II, a brush border membrane protein. Proc Natl Acad Sci USA 87: 8990–8994, 1990 [DOI] [PMC free article] [PubMed] [Google Scholar]
11. Dawson I, Pryse-Davies J. The distribution of certain enzyme systems in the normal human gastrointestinal tract. Gastroenterology 44: 745–760, 1963 [PubMed] [Google Scholar]
12. de Jong L, Meng Y, Dent J, Hekimi S. Thiamine pyrophosphate biosynthesis and transport in the nematode Caenorhabditis elegans. Genetics 168: 845–854, 2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
13. Donowitz M, Montgomery JLM, Walker MS, Cohen ME. Brush border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na⁺/H⁺ exchange. Am J Physiol Gastrointest Liver Physiol 266: G647–G656, 1994 [DOI] [PubMed] [Google Scholar]
14. Dudeja PK, Tyagi S, Kavilaveettil RJ, Gill R, Said HM. Mechanism of thiamin uptake by human jejunal brush-border membrane vesicles. Am J Physiol Cell Physiol 281: C786–C792, 2001 [DOI] [PubMed] [Google Scholar]
15. Frederikse PH, Farnsworth P, Zigler JS., Jr Thiamine deficiency in vivo produces fiber cell degeneration in mouse lenses. Biochem Biophys Res Commun 258: 703–707, 1999 [DOI] [PubMed] [Google Scholar]
16. Gill RK, Saksena S, Syed IA, Tyagi S, Alrefui WA, Malakooti J, Ramasmamy K, Dudeja PK. Regulation of NHE-3 by nitric oxide in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 283: G747–G756, 2002 [DOI] [PubMed] [Google Scholar]
17. Gurerrant NB, Dutcher RA. Assay of vitamins B and G as influenced by coprophagy. J Biol Chem 98: 225–235, 1932 [Google Scholar]
18. Gurerrant NB, Dutcher RA, Brown RA. Further studies concerning formation of B vitamins in digestive tract of rat. J Nutr 13: 305–315, 1937 [Google Scholar]
19. Mahajan RJ, Baldwin ML, Harig JM, Ramaswamy K, Dudeja PK. Chloride transport in human proximal colonic apical membrane vesicles. Biochim Biophys Acta 1280: 12–18, 1996 [DOI] [PubMed] [Google Scholar]
20. Moyer MP, Stauffer JS, Manzano LA, Tanzer LL, Merriman RL. NCM460: a normal colon mucosal epithelial cell line. In Vitro Cell Dev Biol Anim 32: 315–317, 1996 [DOI] [PubMed] [Google Scholar]
21. Na X, Zhao D, Koon HW, Kim H, Husmark J, Moyer MP, Pothoulakis C, LaMont JT. Clostridium difficile toxin B activates the EGF receptor and the ERK/MAP kinase pathway in human colonocytes. Gastroenterology 128: 1002–1011, 2005 [DOI] [PubMed] [Google Scholar]
22. Najjar VA, Holt LE. The biosynthesis of thiamin in man and its implication in human nutrition. JAMA 123: 683–684, 1943 [Google Scholar]
23. Piper RC, James DE, Slot JW, Puri C, Lawrence JC., Jr GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP. J Biol Chem 268: 16557–16563, 1993 [PubMed] [Google Scholar]
24. Rindi G, Laforenza U. Thiamine intestinal transport and related issues: recent aspects. Proc Soc Exp Biol Med 224: 246–255, 2000 [DOI] [PubMed] [Google Scholar]
25. Sahi J, Nataraja SG, Layden TJ, Goldstein JL, Moyer MP, Rao MC. Cl⁻ transport in an immortalized human epithelial cell line (NCM460) derived from the normal transverse colon. Am J Physiol Cell Physiol 275: C1048–C1057, 1998 [DOI] [PubMed] [Google Scholar]
26. Said HM, Ortiz A, Moyer MP, Yanagawa N. Riboflavin uptake by human-derived colonic epithelial NCM460 cells. Am J Physiol Cell Physiol 278: C270–C276, 2000 [DOI] [PubMed] [Google Scholar]
27. Said HM, Ortiz A, Subramanian VS, Neufeld EJ, Moyer MP, Dudeja PK. Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460. Am J Physiol Gastrointest Liver Physiol 281: G144–G150, 2001 [DOI] [PubMed] [Google Scholar]
28. Said HM. Intestinal absorption of water-soluble vitamins in health and disease. Biochem J 437: 357–372, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]
29. Tanphaichirt V. Modern Nutrition in Health and Disease. New York: Lea and Febiger, 1994, p. 359–375 [Google Scholar]
30. Victor M, Adams RD, Collins GH. The Wernicke-Korsakoff Syndrome and Related Neurological Disorders Due to Alcoholism and Malnutrition. Philadelphia, PA: Davis, 1989 [Google Scholar]
31. Wilkinson GN. Statistical estimation in enzyme kinetics. Biochem J 80: 324–332, 1961 [DOI] [PMC free article] [PubMed] [Google Scholar]
32. Zhao R, Gao F, Wang Y, Diaz GA, Gelb BD, Goldman ID. Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells. J Biol Chem 276: 1114–1118, 2001 [DOI] [PubMed] [Google Scholar]
33. Zhao R, Gao F, Goldman ID. Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells. Am J Physiol Cell Physiol 282: C1512–C1517, 2002 [DOI] [PubMed] [Google Scholar]
34. Zhao R, Gao F, Goldman ID. Molecular cloning of human thiamin pyrophosphokinase. Biochim Biophys Acta 1517: 320–322, 2001 [DOI] [PubMed] [Google Scholar]

[B1] 1. Arumugam M, Raes J, Pelletier E, Le Paslier D, Yamada T, Mende DR, Fernandes GR, Tap J, Bruls T, Batto JM, Bertalan M, Borruel N, Casellas F, Fernandez L, Gautier L, Hansen T, Hattori M, Hayashi T, Kleerebezem M, Kurokawa K, Leclerc M, Levenez F, Manichanh C, Nielsen HB, Nielsen T, Pons N, Poulain J, Qin J, Sicheritz-Ponten T, Tims S, Torrents D, Ugarte E, Zoetendal EG, Wang J, Guarner F, Pedersen O, de Vos WM, Brunak S, Doré J; MetaHIT Consortium, Antolín M, Artiguenave F, Blottiere HM, Almeida M, Brechot C, Cara C, Chervaux C, Cultrone A, Delorme C, Denariaz G, Dervyn R, Foerstner KU, Friss C, van de Guchte M, Guedon E, Haimet F, Huber W, van Hylckama-Vlieg J, Jamet A, Juste C, Kaci G, Knol J, Lakhdari O, Layec S, Le Roux K, Maguin E, Mérieux A, Melo Minardi R, M'rini C, Muller J, Oozeer R, Parkhill J, Renault P, Rescigno M, Sanchez N, Sunagawa S, Torrejon A, Turner K, Vandemeulebrouck G, Varela E, Winogradsky Y, Zeller G, Weissenbach J, Ehrlich SD, Bork P. Enterotypes of the human gut microbiome. Nature 473: 174–180, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B2] 2. Barrow BJ, Ortiz-Reyes R, O'Riordan MA, Pretlow TP. In situ localization of enzymes and mucin in normal rat colon embedded in plastic. Histochem J 21: 289–295, 1989 [DOI] [PubMed] [Google Scholar]

[B3] 3. Berdanier CD. Advanced Nutrition: Micronutrients. Boca Raton, FL: CRC, 1998, p. 80–88 [Google Scholar]

[B4] 4. Bettendorff L, Goessens G, Sluse F, Wins P, Bureau M, Laschet J, Grisar T. Thiamine deficiency in cultured neuroblastoma cells: effect on mitochondrial function and peripheral benzodiazepine receptors. J Neurochem 64: 2013–2021, 1995 [DOI] [PubMed] [Google Scholar]

[B5] 5. Bettendorff L, Wins P. Thiamin diphosphate in biological chemistry: new aspects of thiamin metabolism, especially triphosphate derivatives acting other than as cofactors. FEBS J 276: 2917–2925, 2009 [DOI] [PubMed] [Google Scholar]

[B6] 6. Brandsch M, Miyamoto Y, Ganapathy V, Leibach FH. Regulation of taurine transport in human colon carcinoma cell line (HT-29 and Caco-2) by protein kinase C. Am J Physiol Gastrointest Liver Physiol 264: G939–G946, 1993 [DOI] [PubMed] [Google Scholar]

[B7] 7. Brasitus TA, Keresztes RS. Protein-lipid interactions in antipodal plasma membranes of rat colonocytes. Biochim Biophys Acta 773: 290–300, 1984 [DOI] [PubMed] [Google Scholar]

[B8] 8. Calingasan NY, Gandy SE, Baker H, Sheu KF, Smith JD, Lamb BT, Gearhart JD, Buxbaum JD, Harper C, Selkoe DJ, Price DL, Sisodia SS, Gibson GE. Novel neuritic clusters with accumulations of amyloid precursor protein and amyloid precursor-like protein 2 immunoreactivity in brain regions damaged by thiamine deficiency. Am J Pathol 149: 1063–1071, 1996 [PMC free article] [PubMed] [Google Scholar]

[B9] 9. Chernobilsky B, Tsou KC. Adenocarcinoma, adenomas and polyps of the colon. Histochem Study Cancer 21: 165–177, 1968 [DOI] [PubMed] [Google Scholar]

[B10] 10. Cohen ME, Reinlib L, Watson AJM, Gorelick F, Sikora KR, Tse M, Rood RP, Czernik AJ, Sharp GW, Donowitz M. Rabbit ileal villus cell brush border Na⁺/H⁺ exchanger is regulated by Ca²⁺/calmodulin-dependent protein kinase II, a brush border membrane protein. Proc Natl Acad Sci USA 87: 8990–8994, 1990 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B11] 11. Dawson I, Pryse-Davies J. The distribution of certain enzyme systems in the normal human gastrointestinal tract. Gastroenterology 44: 745–760, 1963 [PubMed] [Google Scholar]

[B12] 12. de Jong L, Meng Y, Dent J, Hekimi S. Thiamine pyrophosphate biosynthesis and transport in the nematode Caenorhabditis elegans. Genetics 168: 845–854, 2004 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B13] 13. Donowitz M, Montgomery JLM, Walker MS, Cohen ME. Brush border tyrosine phosphorylation stimulates ileal neutral NaCl absorption and brush-border Na⁺/H⁺ exchange. Am J Physiol Gastrointest Liver Physiol 266: G647–G656, 1994 [DOI] [PubMed] [Google Scholar]

[B14] 14. Dudeja PK, Tyagi S, Kavilaveettil RJ, Gill R, Said HM. Mechanism of thiamin uptake by human jejunal brush-border membrane vesicles. Am J Physiol Cell Physiol 281: C786–C792, 2001 [DOI] [PubMed] [Google Scholar]

[B15] 15. Frederikse PH, Farnsworth P, Zigler JS., Jr Thiamine deficiency in vivo produces fiber cell degeneration in mouse lenses. Biochem Biophys Res Commun 258: 703–707, 1999 [DOI] [PubMed] [Google Scholar]

[B16] 16. Gill RK, Saksena S, Syed IA, Tyagi S, Alrefui WA, Malakooti J, Ramasmamy K, Dudeja PK. Regulation of NHE-3 by nitric oxide in Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 283: G747–G756, 2002 [DOI] [PubMed] [Google Scholar]

[B17] 17. Gurerrant NB, Dutcher RA. Assay of vitamins B and G as influenced by coprophagy. J Biol Chem 98: 225–235, 1932 [Google Scholar]

[B18] 18. Gurerrant NB, Dutcher RA, Brown RA. Further studies concerning formation of B vitamins in digestive tract of rat. J Nutr 13: 305–315, 1937 [Google Scholar]

[B19] 19. Mahajan RJ, Baldwin ML, Harig JM, Ramaswamy K, Dudeja PK. Chloride transport in human proximal colonic apical membrane vesicles. Biochim Biophys Acta 1280: 12–18, 1996 [DOI] [PubMed] [Google Scholar]

[B20] 20. Moyer MP, Stauffer JS, Manzano LA, Tanzer LL, Merriman RL. NCM460: a normal colon mucosal epithelial cell line. In Vitro Cell Dev Biol Anim 32: 315–317, 1996 [DOI] [PubMed] [Google Scholar]

[B21] 21. Na X, Zhao D, Koon HW, Kim H, Husmark J, Moyer MP, Pothoulakis C, LaMont JT. Clostridium difficile toxin B activates the EGF receptor and the ERK/MAP kinase pathway in human colonocytes. Gastroenterology 128: 1002–1011, 2005 [DOI] [PubMed] [Google Scholar]

[B22] 22. Najjar VA, Holt LE. The biosynthesis of thiamin in man and its implication in human nutrition. JAMA 123: 683–684, 1943 [Google Scholar]

[B23] 23. Piper RC, James DE, Slot JW, Puri C, Lawrence JC., Jr GLUT4 phosphorylation and inhibition of glucose transport by dibutyryl cAMP. J Biol Chem 268: 16557–16563, 1993 [PubMed] [Google Scholar]

[B24] 24. Rindi G, Laforenza U. Thiamine intestinal transport and related issues: recent aspects. Proc Soc Exp Biol Med 224: 246–255, 2000 [DOI] [PubMed] [Google Scholar]

[B25] 25. Sahi J, Nataraja SG, Layden TJ, Goldstein JL, Moyer MP, Rao MC. Cl⁻ transport in an immortalized human epithelial cell line (NCM460) derived from the normal transverse colon. Am J Physiol Cell Physiol 275: C1048–C1057, 1998 [DOI] [PubMed] [Google Scholar]

[B26] 26. Said HM, Ortiz A, Moyer MP, Yanagawa N. Riboflavin uptake by human-derived colonic epithelial NCM460 cells. Am J Physiol Cell Physiol 278: C270–C276, 2000 [DOI] [PubMed] [Google Scholar]

[B27] 27. Said HM, Ortiz A, Subramanian VS, Neufeld EJ, Moyer MP, Dudeja PK. Mechanism of thiamine uptake by human colonocytes: studies with cultured colonic epithelial cell line NCM460. Am J Physiol Gastrointest Liver Physiol 281: G144–G150, 2001 [DOI] [PubMed] [Google Scholar]

[B28] 28. Said HM. Intestinal absorption of water-soluble vitamins in health and disease. Biochem J 437: 357–372, 2011 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B29] 29. Tanphaichirt V. Modern Nutrition in Health and Disease. New York: Lea and Febiger, 1994, p. 359–375 [Google Scholar]

[B30] 30. Victor M, Adams RD, Collins GH. The Wernicke-Korsakoff Syndrome and Related Neurological Disorders Due to Alcoholism and Malnutrition. Philadelphia, PA: Davis, 1989 [Google Scholar]

[B31] 31. Wilkinson GN. Statistical estimation in enzyme kinetics. Biochem J 80: 324–332, 1961 [DOI] [PMC free article] [PubMed] [Google Scholar]

[B32] 32. Zhao R, Gao F, Wang Y, Diaz GA, Gelb BD, Goldman ID. Impact of the reduced folate carrier on the accumulation of active thiamin metabolites in murine leukemia cells. J Biol Chem 276: 1114–1118, 2001 [DOI] [PubMed] [Google Scholar]

[B33] 33. Zhao R, Gao F, Goldman ID. Reduced folate carrier transports thiamine monophosphate: an alternative route for thiamine delivery into mammalian cells. Am J Physiol Cell Physiol 282: C1512–C1517, 2002 [DOI] [PubMed] [Google Scholar]

[B34] 34. Zhao R, Gao F, Goldman ID. Molecular cloning of human thiamin pyrophosphokinase. Biochim Biophys Acta 1517: 320–322, 2001 [DOI] [PubMed] [Google Scholar]

PERMALINK

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

Svetlana M Nabokina

Hamid M Said

Abstract