Effect of chronic alcohol exposure on gut vitamin B7 uptake: involvement of epigenetic mechanisms and effect of alcohol metabolites.

Vitamin B7 (biotin) is essential for normal health and its deficiency/suboptimal levels occur in a variety of conditions including chronic alcoholism. Mammals, including humans, obtain biotin from diet and gut-microbiota via absorption along the intestinal tract. The absorption process is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; SLC5A6). We have previously shown that chronic alcohol exposure significantly inhibits intestinal/colonic biotin uptake via suppression of Slc5a6 transcription in animal and cell line models. However, little is known about the transcriptional/epigenetic factors that mediate this suppression. In addition, the effect of alcohol metabolites (generated via alcohol metabolism by gut microbiota and host tissues) on biotin uptake is still unknown. To address these questions, we first demonstrated that chronic alcohol exposure inhibits small intestinal and colonic biotin uptake and SMVT expression in human differentiated enteroid and colonoid monolayers. We then showed that chronic alcohol exposures of both, Caco-2 cells and mice, are associated with a significant suppression in expression of the nuclear factor KLF-4 (needed for Slc5a6 promoter activity), as well as with epigenetic alterations (histone modifications). We also found that chronic exposure of NCM460 human colonic epithelial cells as well as human differentiated colonoid monolayers, to alcohol metabolites (acetaldehyde, ethyl palmitate, ethyl oleate) significantly inhibited biotin uptake and SMVT expression. These findings shed light onto the molecular/epigenetic mechanisms that mediate the inhibitory effect of chronic alcohol exposure on intestinal biotin uptake. They further show that alcohol metabolites are also capable of inhibiting biotin uptake in the gut.NEW & NOTEWORTHY Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.

Proinflammatory cytokines inhibit thiamin uptake by human and mouse pancreatic acinar cells: involvement of transcriptional mechanism(s).

Thiamin (vitamin B1) plays critical roles in normal metabolism and function of all mammalian cells. Pancreatic acinar cells (PACs) import thiamin from circulation via specific carrier-mediated uptake that involves thiamin transporter-1 and -2 (THTR-1 and -2; products of SLC19A2 and SLC19A3, respectively). Our aim in this study was to investigate the effect(s) of proinflammatory cytokines on thiamin uptake by PACs. We used human primary (h)PACs, PAC 266-6 cells, and mice in vivo as models in the investigations. First, we examined the level of expression of THTR-1 and -2 mRNA in pancreatic tissues of patients with chronic pancreatitis and observed severe reduction in their expression compared with normal control subjects. Exposing hPACs and PAC 266-6 to proinflammatory cytokines (hyper IL-6, TNF-α, and IL-1ß) was found to lead to a significant inhibition in thiamin uptake. Focusing on hyper-IL-6 (which also inhibited thiamin uptake by primary mouse PACs), the inhibition in thiamin uptake was found to be associated with significant reduction in THTR-1 and -2 proteins and mRNA expression as well as in activity of the SLC19A2 and SLC19A3 promoters; it was also associated with reduction in level of expression of the transcription factor Sp1 (which is required for activity of these promoters). Finally, blocking the intracellular Stat3 signaling pathway was found to lead to a significant reversal in the inhibitory effect of hyper IL-6 on thiamin uptake by PAC 266-6. These results show that exposure of PACs to proinflammatory cytokines negatively impacts thiamin uptake via (at least in part) transcriptional mechanism(s).NEW & NOTEWORTHY Findings of the current study demonstrate, for the first time, that exposure of pancreatic acinar cells to proinflammatory cytokines (including hyper IL-6) cause significant inhibition in vitamin B1 (thiamin; a micronutrient that is essential for normal cellular energy metabolism) and that this effect is mediated at the level of transcription of the thiamin transporter genes SLC19A2 and SLC19A3.

Posttranscriptional regulation of thiamin transporter-1 expression by microRNA-200a-3p in pancreatic acinar cells.

The water-soluble vitamin B1 (thiamin) plays essential roles in normal metabolism and function of all human/mammalian cells, including the pancreatic acinar cells (PACs). PACs obtain thiamin from their surrounding circulation via transport across the plasma membrane, a process that is mediated by thiamin transporter (THTR)-1 and THTR-2. We have previously characterized different aspects of thiamin uptake by mouse and human primary PACs, but little is known about posttranscriptional regulation of the uptake event. We addressed this by focusing on the predominant thiamin transporter THTR-1 (encoded by SLC19A2 gene) in PACs. Transfecting pmirGLO-SLC19A2 3'-untranslated region (UTR) into mouse-derived PAC 266-6 cells leads to a significant reduction in luciferase activity compared with cells transfected with empty vector. Subjecting the SLC19A2 3'-UTR to different in silico algorithms identified multiple putative microRNA binding sites in this region. Focusing on miR-200a-3p (since it is highly expressed in mouse and human pancreas), we found that transfecting PAC 266-6 and human primary PACs (hPACs) with mimic miR-200a-3p leads to a significant inhibition of THTR-1 expression (both protein and mRNA levels) and in thiamin uptake. In contrast, transfection by miR-200a-3p inhibitor leads to an increase in THTR-1 expression and thiamin uptake. Additionally, truncating the region carrying miR-200a-3p binding site in SLC19A2 3'-UTR and mutating the binding site lead to abrogation in the inhibitory effect of this microRNA on luciferase activity in PAC 266-6. These results demonstrate that expression of THTR-1 and thiamin uptake in PACs is subject to posttranscriptional regulation by microRNAs.NEW & NOTEWORTHY The findings of this study show, for the first time, that the membrane transporter of vitamin B1, i.e., thiamin transporter-1 (THTR-1), is subject to regulation by microRNAs (specifically miR-200a-3p) in mouse and human primary pancreatic acinar cells (PACs). The results also show that this posttranscriptional regulation has functional consequences on the ability of PACs to take in the essential micronutrient thiamin.

Pyridoxine and pancreatic acinar cells: transport physiology and effect on gene expression profile.

Pyridoxine (vitamin B6), an essential micronutrient for normal cell physiology, plays an important role in the function of the exocrine pancreas. Pancreatic acinar cells (PACs) obtain vitamin B6 from circulation, but little is known about the mechanism involved in the uptake process; limited information also exists on the effect of pyridoxine availability on the gene expression profile in these cells. We addressed both these issues in the current investigation using mouse-derived pancreatic acinar 266-6 cells (PAC 266-6) and human primary PACs (hPACs; obtained from organ donors), together with appropriate physiological and molecular (RNA-Seq) approaches. The results showed [3H]pyridoxine uptake to be 1) pH and temperature (but not Na+) dependent, 2) saturable as a function of concentration, 3) cis-inhibited by unlabeled pyridoxine and its close structural analogs, 4) trans-stimulated by unlabeled pyridoxine, 5) regulated by an intracellular Ca2+/calmodulin-mediated pathway, 6) adaptively-regulated by extracellular substrate (pyridoxine) availability, and 7) negatively impacted by exposure to cigarette smoke extract. Vitamin B6 availability was found (by means of RNA-Seq) to significantly (FDR < 0.05) modulate the expression profile of many genes in PAC 266-6 cells (including those that are relevant to pancreatic health and development). These studies demonstrate, for the first time, the involvement of a regulatable and specific carrier-mediated mechanism for pyridoxine uptake by PACs; the results also show that pyridoxine availability exerts profound effects on the gene expression profile in mammalian PACs.

Effect of bacterial flagellin on thiamin uptake by human and mouse pancreatic acinar cells: inhibition mediated at the level of transcription of thiamin transporters 1 and 2.

Thiamin (vitamin B1) is essential for normal cellular metabolism and function. Pancreatic acinar cells (PACs) obtain thiamin from the circulation via a specific carrier-mediated process that involves the plasma membrane thiamin transporters 1 and 2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3 genes, respectively). There is nothing known about the effect of bacterial products/toxins on thiamin uptake by PACs. We addressed this issue in the present investigation by examining the effect of bacterial flagellin on physiological and molecular parameters of thiamin uptake by PACs. We used human primary PACs, mice in vivo, and cultured mouse-derived pancreatic acinar 266-6 cells in our investigation. The results showed that exposure of human primary PACs to flagellin led to a significant inhibition in thiamin uptake; this inhibition was associated with a significant decrease in expression of THTR-1 and -2 at the protein and mRNA levels. These findings were confirmed in mice in vivo as well as in cultured 266-6 cells. Subsequent studies showed that flagellin exposure markedly suppressed the activity of the SLC19A2 and SLC19A3 promoters and that this effect involved the Sp1 regulatory factor. Finally, knocking down Toll-like receptor 5 by use of gene-specific siRNA was found to lead to abrogation in the inhibitory effect of flagellin on PAC thiamin uptake. These results show, for the first time, that exposure of PACs to flagellin negatively impacts the physiological and molecular parameters of thiamin uptake and that this effect is mediated at the level of transcription of the SLC19A2 and SLC19A3 genes. NEW & NOTEWORTHY The present study demonstrates, for the first time, that prolonged exposure of pancreatic acinar cells to flagellin inhibits uptake of vitamin B1, a micronutrient that is essential for energy metabolism and ATP production. This effect is mediated at the level of transcription of the SLC19A2 and SLC19A3 genes and involves the Sp1 transcription factor.

Effect of the proinflammatory cytokine TNF-α on intestinal riboflavin uptake: inhibition mediated via transcriptional mechanism(s).

Riboflavin (RF), is essential for normal cellular metabolism/function. Intestinal RF absorption occurs via a specific carrier-mediated process that involves the apical transporter RFVT-3 ( SLC52A3) and the basolateral RFVT-1 (SLC52A1). Previously, we characterized different cellular/molecular aspects of the intestinal RF uptake process, but nothing is known about the effect of proinflammatory cytokines on the uptake event. We addressed this issue using in vitro, ex vivo, and in vivo models. First, we determined the level of mRNA expression of the human (h)RFVT-3 and hRFVT-1 in intestinal tissue of patients with inflammatory bowel disease (IBD) and observed a markedly lower level compared with controls. In the in vitro model, exposing Caco-2 cells to tumor necrosis factor-α (TNF-α) led to a significant inhibition in RF uptake, an effect that was abrogated upon knocking down TNF receptor 1 (TNFR1). The inhibition in RF uptake was associated with a significant reduction in the expression of hRFVT-3 and -1 protein and mRNA levels, as well as in the activity of the SLC52A3 and SLC52A1 promoters. The latter effects appear to involve Sp1 and NF-κB sites in these promoters. Similarly, exposure of mouse small intestinal enteroids and wild-type mice to TNF-α led to a significant inhibition in physiological and molecular parameters of intestinal RF uptake. Collectively, these findings demonstrate that exposure of intestinal epithelial cells to TNF-α leads to inhibition in RF uptake and that this effect is mediated, at least in part, via transcriptional mechanism(s). These findings may explain the significantly low RF levels observed in patients with IBD.

Colorimetric Detection of Perfluorinated Compounds by All-Polymer Photonic Transducers.

We report on the highly sensitive optical and colorimetric detection of perfluorinated compounds in the vapor phase achieved by all-polymer dielectric mirrors. High optical quality and uniformly distributed Bragg reflectors were fabricated by alternating thin films of poly(N-vinylcarbazole) and Hyflon AD polymers as high and low refractive index medium, respectively. A new processing procedure has been developed to compatibilize the deposition of poly(N-vinylcarbazole) with the highly solvophobic Hyflon AD polymer layers to achieve mutual processability between the two polymers and fabricate the devices. As a proof of principle, sensing measurements were performed using the Galden HT55 polymer as a prototype of the perfluorinated compound. The Bragg stacks show a strong chromatic response upon exposure to this compound, clearly detectable as both spectral and intensity variations. Conversely, Bragg mirrors fabricated without fluorinated polymers do not show any detectable response, demonstrating that the Hyflon AD polymer acts as the active and selective medium for sensing perfluorinated species. These results demonstrate that organic dielectric mirrors containing perfluorinated polymers can represent an innovative colorimetric monitoring system for fluorinated compounds, suitable to improve both environmental safety and quality of life.

Molecular mechanisms involved in the adaptive regulation of the colonic thiamin pyrophosphate uptake process.

A considerable amount of the thiamin generated by gut microbiota exists in the form of thiamin pyrophosphate (TPP). We have previously shown that human colonocytes possess an efficient carrier-mediated uptake process for TPP that involves the SLC44A4 system and this uptake process is adaptively regulated by prevailing extracellular TPP level. Little is known about the molecular mechanisms that mediate this adaptive regulation. We addressed this issue using human-derived colonic epithelial NCM460 cells and mouse colonoids as models. Maintaining NCM460 cells in the presence of a high level of TPP (1 mM) for short (2 days)- and long-term (9 days) periods was found to lead to a significant reduction in [3H] TPP uptake compared with cells maintained in its absence. Short-term exposure showed no changes in level of expression of SLC44A4 protein in total cell homogenate (although there was a decreased expression in the membrane fraction), mRNA, and promoter activity. However, a significant reduction in the level of expression of the SLC44A4 protein, mRNA, and promoter activity was observed upon long-term maintenance with the substrate. Similar changes in Slc44a4 mRNA expression were observed when mouse colonoids were maintained with TPP for short- and long-term periods. Expression of the transcription factors ELF3 and CREB-1 (which drive the SLC44A4 promoter) following long-term exposure was unchanged, but their binding affinity to the promoter was decreased and specific histone modifications were also observed. These studies demonstrate that, depending on the period of exposure, different mechanisms are involved in the adaptive regulation of colonic TPP uptake by extracellular substrate level.

DnrI of Streptomyces peucetius binds to the resistance genes, drrAB and drrC but is activated by daunorubicin.

The master regulator, DnrI of Streptomyces peucetius is a member of the family of transcriptional activator, Streptomyces antibiotic regulatory proteins (SARP), which controls the biosynthesis of antitumor anthracycline, daunorubicin (DNR) and doxorubicin (DXR). The binding of DnrI to the heptameric repeat sequence found within the -35 promoter region of biosynthetic gene, dpsE activates it. To combat the increased level of intracellular DNR, the cell has developed self resistance mechanism mediated by drrAB and drrC genes which are regulated by regulatory genes. We find that a drug non-producing mutant, ΔdpsA, showed sensitive phenotype in plate assay along with an increased level of dnrI transcript. Whereas the mutant grown in the presence of DNR showed a resistant phenotype with a six and eight folds increase in drrAB and drrC transcripts respectively. Computational studies followed by molecular docking showed that DnrI bound as a monomer to a slightly modified heptameric DNA motif, 5'-ACACGCA in drrA and 5'-ACAACCT in drrC which was also proved by electrophoretic mobility shift assay. These findings confirm that DnrI belongs to winged helix-turn-helix DNA-binding protein with Tetratricopeptide Repeat domain. The transcriptional regulator DnrI binds to the resistance genes at specific sites but they are activated only when an increased load of intracellular DNR is sensed.

Molecular mechanism(s) involved in differential expression of vitamin C transporters along the intestinal tract.

Mammalian cells utilize two transporters for the uptake of ascorbic acid (AA), Na+-dependent vitamin C transporter SVCT-1 and SVCT-2. In the intestine, these transporters are involved in AA absorption and are expressed at the apical and basolateral membrane domains of the polarized epithelia, respectively. Little is known about the differential expression of these two transporters along the anterior-posterior axis of the intestinal tract and the molecular mechanism(s) that dictate this pattern of expression. We used mouse and human intestinal cDNAs to address these issues. The results showed a significantly lower rate of carrier-mediated AA uptake by mouse colon than jejunum. This was associated with a significantly lower level of expression of SVCT-1 and SVCT-2 at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels in the colon than the jejunum, implying the involvement of transcriptional mechanism(s). Similarly, expression levels of SVCT-1 and SVCT-2 mRNA and hnRNA were significantly lower in human colon. We also examined the levels of expression of hepatocyte nuclear factor 1α and specificity protein 1, which drive transcription of the Slc23a1 and Slc23a2 promoters, respectively, and found them to be markedly lower in the colon. Furthermore, significantly lower levels of the activating markers for histone (H3) modifications [H3 trimethylation of lysine 4 (H3K4me3) and H3 triacetylation of lysine 9 (H3K9ac)] were observed in the Slc23a1 and Slc23a2 promoters in the colon. These findings show, for the first time, that SVCT-1 and SVCT-2 are differentially expressed along the intestinal tract and that this pattern of expression is, at least in part, mediated via transcriptional/epigenetic mechanisms.NEW & NOTEWORTHY Our findings show, for the first time, that transporters of the water-soluble vitamin ascorbic acid (i.e., the vitamin C transporters SVCT-1 and SVCT-2) are differentially expressed along the length of the intestinal tract and that the pattern of expression is mediated, at least in part, by transcriptional and epigenetic mechanism(s) affecting both Slc23a1 and Slc23a2 genes.

Uptake of ascorbic acid by pancreatic acinar cells is negatively impacted by chronic alcohol exposure.

Vitamin C (ascorbic acid, AA) is indispensable for normal metabolism of all mammalian cells including pancreatic acinar cells (PACs). PACs obtain AA from their surroundings via transport across the cell membrane. Chronic alcohol exposure negatively affects body AA homeostasis; it also inhibits uptake of other micronutrients into PACs, but its effect on AA uptake is not clear. We examined this issue using both in vitro (266-6 cells) and in vivo (mice) models of chronic alcohol exposure. First, we determined the relative expression of the AA transporters 1 and 2 [i.e., sodium-dependent vitamin C transporter-1 (SVCT-1) and SVCT-2] in mouse and human PACs and found SVCT-2 to be the predominant transporter. Chronic exposure of 266-6 cells to alcohol significantly inhibited AA uptake and caused a marked reduction in SVCT-2 expression at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Similarly, chronic alcohol feeding of mice significantly inhibited AA uptake and caused a marked reduction in level of expression of the SVCT-2 protein, mRNA, and hnRNA. These findings suggest possible involvement of transcriptional mechanism(s) in mediating chronic alcohol effect on AA uptake by PACs. We also observed significant epigenetic changes (histone modifications) in the Slc23a2 gene (reduction in H3K4me3 level and an increase in H3K27me3 level) in the alcohol-exposed 266-6 cells. These findings show that chronic alcohol exposure inhibits PAC AA uptake and that the effect is mediated, in part, at the level of transcription of the Slc23a2 gene and may involve epigenetic mechanism(s).

Inhibition of pancreatic acinar mitochondrial thiamin pyrophosphate uptake by the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

Thiamin is essential for normal metabolism in pancreatic acinar cells (PAC) and is obtained from their microenvironment through specific plasma-membrane transporters, converted to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria through the mitochondrial TPP (MTPP) transporter (MTPPT; product of SLC25A19 gene). TPP is essential for normal mitochondrial function. We examined the effect of long-term/chronic exposure of PAC in vitro (pancreatic acinar 266-6 cells) and in vivo (wild-type or transgenic mice carrying the SLC25A19 promoter) of the cigarette smoke toxin, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), on the MTPP uptake process. Our in vitro and in vivo findings demonstrate that NNK negatively affects MTPP uptake and reduced expression of MTPPT protein, MTPPT mRNA, and heterogenous nuclear RNA, as well as SLC25A19 promoter activity. The effect of NNK on Slc25a19 transcription was neither mediated by changes in expression of transcriptional factor NFY-1 (known to drive SLC25A19 transcription), nor due to changes in methylation profile of the Slc25a19 promoter. Rather, it appears to be due to changes in histone modifications that involve significant decreases in histone H3K4-trimethylation and H3K9-acetylation (activation markers). The effect of NNK on MTPPT function is mediated through the nonneuronal α7-nicotinic acetylcholine receptor (α7-nAChR), as indicated by both in vitro (using the nAChR antagonist mecamylamine) and in vivo (using an α7-nAchR(-/-) mouse model) studies. These findings demonstrate that chronic exposure of PAC to NNK negatively impacts PAC MTPP uptake. This effect appears to be exerted at the level of Slc25a19 transcription, involve epigenetic mechanism(s), and is mediated through the α7-nAchR.

Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.

Thiamin (vitamin B1), a member of the water-soluble family of vitamins, is essential for normal cellular functions; its deficiency results in oxidative stress and mitochondrial dysfunction. Pancreatic acinar cells (PAC) obtain thiamin from the circulation using a specific carrier-mediated process mediated by both thiamin transporters -1 and -2 (THTR-1 and THTR-2; encoded by the SLC19A2 and SLC19A3 genes, respectively). The aim of the current study was to examine the effect of chronic exposure of mouse PAC in vivo and human PAC in vitro to nicotine (a major component of cigarette smoke that has been implicated in pancreatic diseases) on thiamin uptake and to delineate the mechanism involved. The results showed that chronic exposure of mice to nicotine significantly inhibits thiamin uptake in murine PAC, and that this inhibition is associated with a marked decrease in expression of THTR-1 and THTR-2 at the protein, mRNA and hnRNAs level. Furthermore, expression of the important thiamin-metabolizing enzyme, thiamin pyrophosphokinase (TPKase), was significantly reduced in PAC of mice exposed to nicotine. Similarly, chronic exposure of cultured human PAC to nicotine (0.5 µM, 48 h) significantly inhibited thiamin uptake, which was also associated with a decrease in expression of THTR-1 and THTR-2 proteins and mRNAs. This study demonstrates that chronic exposure of PAC to nicotine impairs the physiology and the molecular biology of the thiamin uptake process. Furthermore, the study suggests that the effect is, in part, mediated through transcriptional mechanism(s) affecting the SLC19A2 and SLC19A3 genes.

Chronic alcohol exposure affects pancreatic acinar mitochondrial thiamin pyrophosphate uptake: studies with mouse 266-6 cell line and primary cells.

Thiamin is essential for normal metabolic activity of all mammalian cells, including those of the pancreas. Cells obtain thiamin from their surroundings and enzymatically convert it into thiamin pyrophosphate (TPP) in the cytoplasm; TPP is then taken up by mitochondria via a specific carrier the mitochondrial TPP transporter (MTPPT; product of the SLC25A19 gene). Chronic alcohol exposure negatively impacts the health of pancreatic acinar cells (PAC), but its effect on physiological/molecular parameters of MTPPT is not known. We addressed this issue using mouse pancreatic acinar tumor cell line 266-6 and primary PAC of wild-type and transgenic mice carrying the SLC25A19 promoter that were fed alcohol chronically. Chronic alcohol exposure of 266-6 cells (but not to its nonoxidative metabolites ethyl palmitate and ethyl oleate) led to a significant inhibition in mitochondrial TPP uptake, which was associated with a decreased expression of MTPPT protein, mRNA, and activity of the SLC25A19 promoter. Similarly, chronic alcohol feeding of mice led to a significant inhibition in expression of MTPPT protein, mRNA, heterogeneous nuclear RNA, as well as in activity of SLC25A19 promoter in PAC. While chronic alcohol exposure did not affect DNA methylation of the Slc25a19 promoter, a significant decrease in histone H3 euchromatin markers and an increase in H3 heterochromatin marker were observed. These findings show, for the first time, that chronic alcohol exposure negatively impacts pancreatic MTPPT, and that this effect is exerted, at least in part, at the level of Slc25a19 transcription and appears to involve epigenetic mechanism(s).

Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms.

Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5'-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na(+) dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms.

Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake.

Pancreatic acinar cells (PAC) obtain thiamin from the circulation via a carrier-mediated process that involves thiamin transporters 1 and 2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3, respectively). Chronic alcohol exposure of PAC inhibits thiamin uptake, and, on the basis of in vitro studies, this inhibition appears to be transcriptionally mediated. The aim of this study was to confirm the involvement of a transcriptional mechanism in mediating the chronic alcohol effect in in vivo settings and to delineate the molecular mechanisms involved. Using transgenic mice carrying full-length SLC19A2 and SLC19A3 promoters, we found that chronic alcohol feeding led to a significant reduction in the activity of SLC19A2 and SLC19A3 promoters (as well as in thiamin uptake and expression of THTR-1 and -2). Similar findings were seen in 266-6 cells chronically exposed to alcohol in vitro. In the latter studies, the alcohol inhibitory effect was found to be mediated via the minimal SLC19A2 and SLC19A3 promoters and involved the cis-regulatory elements stimulating protein 1 (SP1)/gut-enriched Kruppel-like factor and SP1-GG-box and SP1/GC, respectively. Chronic alcohol exposure of PAC also led to a significant reduction in the expression of the SP1 transcription factor, which upon correction (via expression) led to the prevention of alcohol inhibitory effects on not only the activity of SLC19A2 and SLC19A3 promoters but also on the expression of THTR-1 and -2 mRNA and thiamin uptake. These results demonstrate that the inhibitory effect of chronic alcohol exposure on physiological/molecular parameters of thiamin uptake by PAC is mediated via specific cis-regulatory elements in SLC19A2 and SLC19A3 minimal promoters.

Effect of the cigarette smoke component, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK), on physiological and molecular parameters of thiamin uptake by pancreatic acinar cells.

Thiamin is indispensable for the normal function of pancreatic acinar cells. These cells take up thiamin via specific carrier-mediated process that involves thiamin transporter-1 and -2 (THTR-1 and THTR-2; products of SLC19A2 and SLC19A3 genes, respectively). In this study we examined the effect of chronic exposure of pancreatic acinar cells in vitro (pancreatic acinar 266-6 cells) and in vivo (wild-type and transgenic mice carrying the SLC19A2 and SLC19A3 promoters) to the cigarette smoke component 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) on physiological and molecular parameters of the thiamin uptake process. The results show that chronic exposure of 266-6 cells to NNK (3 µM, 24 h) leads to a significant inhibition in thiamin uptake. The inhibition was associated with a significant decrease in the level of expression of THTR-1 and -2 at the protein and mRNA levels as well as in the activity of SLC19A2 and SLC19A3 promoters. Similarly chronic exposure of mice to NNK (IP 10 mg/100 g body weight, three times/week for 2 weeks) leads to a significant inhibition in thiamin uptake by freshly isolated pancreatic acinar cells, as well as in the level of expression of THTR-1 and -2 protein and mRNA. Furthermore, activity of the SLC19A2 and SLC19A3 promoters expressed in transgenic mice were significantly suppressed by chronic exposure to NNK. The effect of NNK on the activity of the SLC19A2 and SLC19A3 promoters was not mediated via changes in their methylation profile, rather it appears to be exerted via an SP1/GG and SP1/GC cis-regulatory elements in these promoters, respectively. These results demonstrate, for the first time, that chronic exposure of pancreatic acinar cells to NNK negatively impacts the physiological and molecular parameters of thiamin uptake by pancreatic acinar cells and that this effect is exerted, at least in part, at the level of transcription of the SLC19A2 and SLC19A3 genes.

Daunorubicin efflux in Streptomyces peucetius modulates biosynthesis by feedback regulation.

Streptomyces peucetius self-resistance genes drrA and drrB encode membrane-associated proteins that function like an ABC transporter for the efflux of daunorubicin and to maintain a constant subinhibitory physiological concentration of the drug within the cell. In this study, the drrA and drrB operons were disrupted for investigating drug production, self-resistance and regulation. The drrA-drrB null mutant was highly sensitive to daunorubicin. A 10-fold decrease in drug production was observed in the null mutant compared with the wild-type strain. We propose that the absence of a drug-specific efflux pump increases the intracellular concentration of daunorubicin, which is sensed by the organism to turn down drug production. Quantitative real-time PCR analysis of the mutant showed a drastic reduction in the expression of the key regulator dnrI and polyketide synthase gene dpsA. However, the expression of regulatory genes dnrO and dnrN was increased. Feedback regulation based on the intracellular daunorubicin concentration is discussed.