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Hamid M Said - One of the best experts on this subject based on the ideXlab platform.
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---Pro-inflammatory cytokines inhibit thiamin uptake by human and mouse pancreatic acinar cells: Involvement of transcriptional mechanism(s).
American journal of physiology. Gastrointestinal and liver physiology, 2020Co-Authors: Kasin Yadunandam Anandam, Padmanabhan Srinivasan, Tomoya Yasujima, Saleh Al-juburi, Hamid M SaidAbstract: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 transporters-1 & -2 (THTR-1 & -2; products of SLC19A2 and SLC19A3, respectively). Our aim in this study was to investigate the effect(s) of pro-inflammatory 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 & -2 mRNA in pancreatic tissues of patients with chronic pancreatitis and observed severe reduction in their expression compared to normal control subjects. Exposing hPACs and PAC 266-6 to pro-inflammatory cytokines (hyper IL-6, TNF-α, and IL-1β) 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 & -2 proteins and mRNAs 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 pro-inflammatory cytokines negatively impacts thiamin uptake via (at least in part) transcriptional mechanism(s).
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pH-dependent pyridoxine transport by SLC19A2 and SLC19A3: Implications for absorption in acidic microclimates
The Journal of biological chemistry, 2020Co-Authors: Takahiro Yamashiro, Hamid M Said, Tomoya Yasujima, Hiroaki YuasaAbstract:SLC19A2 and SLC19A3, also known as thiamine transporters (THTR) 1 and 2, respectively, transport the positively charged thiamine (vitamin B1) into cells to enable its efficient utilization. SLC19A2 and SLC19A3 are also known to transport structurally unrelated cationic drugs, such as metformin, but whether this charge selectivity extends to other molecules, such as pyridoxine (vitamin B6), is unknown. We tested this possibility using Madin-Darby canine kidney II (MDCKII) cells and human embryonic kidney 293 (HEK293) cells for transfection experiments, and also using Caco-2 cells as human intestinal epithelial model cells. The stable expression of SLC19A2 and SLC19A3 in MDCKII cells (as well as their transient expression in HEK293 cells) led to a significant induction in pyridoxine uptake at pH 5.5 compared with control cells. The induced uptake was pH-dependent, favoring acidic conditions over neutral to basic conditions, and protonophore-sensitive. It was saturable as a function of pyridoxine concentration, with an apparent Km of 37.8 and 18.5 μm, for SLC19A2 and SLC19A3, respectively, and inhibited by the pyridoxine analogs pyridoxal and pyridoxamine as well as thiamine. We also found that silencing the endogenous SLC19A3, but not SLC19A2, of Caco-2 cells with gene-specific siRNAs lead to a significant reduction in carrier-mediated pyridoxine uptake. These results show that SLC19A2 and SLC19A3 are capable of recognizing/transporting pyridoxine, favoring acidic conditions for operation, and suggest a possible role for these transporters in pyridoxine transport mainly in tissues with an acidic environment like the small intestine, which has an acidic surface microclimate.
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Posttranscriptional regulation of thiamin transporter-1 expression by microRNA-200a-3p in pancreatic acinar cells
American journal of physiology. Gastrointestinal and liver physiology, 2020Co-Authors: Kalidas Ramamoorthy, Padmanabhan Srinivasan, Tomoya Yasujima, Kasin Yadunandam Anandam, Hamid M SaidAbstract: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.
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Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.
PloS one, 2015Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Edwin C. Thrower, Gopalakrishnan Loganathan, Appakalai N. Balamurugan, Fred S. Gorelick, Hamid M SaidAbstract: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.
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Effect of nicotine exposure on cellular and molecular parameters of vitamin B1 (thiamin) uptake by pancreatic acinar cells (896.3)
The FASEB Journal, 2014Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Sameer Kapadia, Hamid M SaidAbstract:Pancreatic acinar cells take in thiamin by a specific carrier-mediated process that involve both THTR-1 and THTR-2 (encoded by SLC19A2 and SLC19A3, respectively). In this study we examined the effe...
Veedamali S. Subramanian - One of the best experts on this subject based on the ideXlab platform.
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Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.
PloS one, 2015Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Edwin C. Thrower, Gopalakrishnan Loganathan, Appakalai N. Balamurugan, Fred S. Gorelick, Hamid M SaidAbstract: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.
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Effect of nicotine exposure on cellular and molecular parameters of vitamin B1 (thiamin) uptake by pancreatic acinar cells (896.3)
The FASEB Journal, 2014Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Sameer Kapadia, Hamid M SaidAbstract:Pancreatic acinar cells take in thiamin by a specific carrier-mediated process that involve both THTR-1 and THTR-2 (encoded by SLC19A2 and SLC19A3, respectively). In this study we examined the effe...
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Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake.
American journal of physiology. Gastrointestinal and liver physiology, 2014Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Hamid M SaidAbstract: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, resp...
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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
PLOS ONE, 2013Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Hamid M SaidAbstract: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.
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Relative contribution of THTR-1 and THTR-2 in thiamin uptake by pancreatic acinar cells: Studies utilizing SLC19A2 and Slc19a3 knockout mouse models
American journal of physiology. Gastrointestinal and liver physiology, 2011Co-Authors: Veedamali S. Subramanian, Sandeep B. Subramanya, Hamid M SaidAbstract:Thiamin is essential for normal function of pancreatic acinar cells, and its deficiency leads to a reduction in pancreatic digestive enzymes. We have recently shown that thiamin uptake by rat pancreatic acinar cells is carrier-mediated and that both thiamin transporter (THTR)-1 and THTR-2 are expressed in these cells; little, however, is known about the relative contribution of these transporters toward total carrier-mediated thiamin uptake by these cells. We addressed this issue using a gene-specific silencing approach (siRNA) in mouse-derived pancreatic acinar 266–6 cells and SLC19A2 and Slc19a3 knockout mouse models. First we established that thiamin uptake by mouse pancreatic acinar cells is via a carrier-mediated process. We also established that these cells as well as native human pancreas express THTR-1 and THTR-2, with expression of the former (and activity of its promoter) being significantly higher than that of the latter. Using gene-specific siRNA against mouse THTR-1 and THTR-2, we observed a significant inhibition in carrier-mediated thiamin uptake by 266–6 cells in both cases. Similarly, thiamin uptake by freshly isolated primary pancreatic acinar cells of the SLC19A2 and Slc19a3 knockout mice was significantly lower than uptake by acinar cells of the respective littermates; the degree of inhibition observed in the former knockout model was greater than that of the latter. These findings demonstrate, for the first time, that both mTHTR-1 and mTHTR-2 are involved in carrier-mediated thiamin uptake by pancreatic acinar cells.
Padmanabhan Srinivasan - One of the best experts on this subject based on the ideXlab platform.
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---Pro-inflammatory cytokines inhibit thiamin uptake by human and mouse pancreatic acinar cells: Involvement of transcriptional mechanism(s).
American journal of physiology. Gastrointestinal and liver physiology, 2020Co-Authors: Kasin Yadunandam Anandam, Padmanabhan Srinivasan, Tomoya Yasujima, Saleh Al-juburi, Hamid M SaidAbstract: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 transporters-1 & -2 (THTR-1 & -2; products of SLC19A2 and SLC19A3, respectively). Our aim in this study was to investigate the effect(s) of pro-inflammatory 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 & -2 mRNA in pancreatic tissues of patients with chronic pancreatitis and observed severe reduction in their expression compared to normal control subjects. Exposing hPACs and PAC 266-6 to pro-inflammatory cytokines (hyper IL-6, TNF-α, and IL-1β) 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 & -2 proteins and mRNAs 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 pro-inflammatory cytokines negatively impacts thiamin uptake via (at least in part) transcriptional mechanism(s).
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Posttranscriptional regulation of thiamin transporter-1 expression by microRNA-200a-3p in pancreatic acinar cells
American journal of physiology. Gastrointestinal and liver physiology, 2020Co-Authors: Kalidas Ramamoorthy, Padmanabhan Srinivasan, Tomoya Yasujima, Kasin Yadunandam Anandam, Hamid M SaidAbstract: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.
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Chronic Nicotine Exposure In Vivo and In Vitro Inhibits Vitamin B1 (Thiamin) Uptake by Pancreatic Acinar Cells.
PloS one, 2015Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Edwin C. Thrower, Gopalakrishnan Loganathan, Appakalai N. Balamurugan, Fred S. Gorelick, Hamid M SaidAbstract: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.
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Effect of nicotine exposure on cellular and molecular parameters of vitamin B1 (thiamin) uptake by pancreatic acinar cells (896.3)
The FASEB Journal, 2014Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Sameer Kapadia, Hamid M SaidAbstract:Pancreatic acinar cells take in thiamin by a specific carrier-mediated process that involve both THTR-1 and THTR-2 (encoded by SLC19A2 and SLC19A3, respectively). In this study we examined the effe...
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Mechanisms involved in the inhibitory effect of chronic alcohol exposure on pancreatic acinar thiamin uptake.
American journal of physiology. Gastrointestinal and liver physiology, 2014Co-Authors: Padmanabhan Srinivasan, Veedamali S. Subramanian, Hamid M SaidAbstract: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, resp...
Ellis J Neufeld - One of the best experts on this subject based on the ideXlab platform.
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role of defective high affinity thiamine transporter SLC19A2 in marrow from a mouse model of thiamine responsive anemia syndrome evidence for defective deoxyribose and heme synthesis
Blood, 2005Co-Authors: Elena Tartaglini, Inderneel Sahai, Judidth C Fleming, Gretchen Chick, Claudia M Montefusco, Laszlo G Boros, Ellis J NeufeldAbstract:The rare autosomal recessive human disorder, TRMA syndrome (thiamine-responsive anemia with diabetes and deafness, OMIM 249270), is caused by mutations in the high affinity vitamin B1 transporter, SLC19A2. In human TRMA, ringed sideroblasts and megaloblastic changes are found in the marrow, suggesting potential defects in heme synthesis or mitochondrial function, and DNA synthesis, respectively. The mechanism whereby defective marrow thiamine transport causes these findings in the marrow was examined in a mouse model for TRMA. We previously demonstrated in fibroblasts from humans with TRMA a defect in the rate and pathway used for de novo ribose synthesis using Stable Isotope Dynamic Metabolic Profiling (SIDMaP analysis - Boros et al, Blood102: 3556, 2003). To examine whether this fibroblast defect accurately defined the marrow pathophysiology, we have now examined this pathway in detail in mice defective for the orthologous SLC19A2 gene by targeted gene disruption (Fleming et al, Mol Genet Metab80:234, 2003). SLC19A2 mutant (−/−) or wildtype (+/+) mice were subjected to normal mouse chow (22 mg/kg thiamine), or thiamine deficient chow diet. Diets were confirmed thiamine deficient by chemical analysis. To examine heme biosynthesis, we used 55 Fe-transferrin, ex vivo , to label marrow from +/+ or −/− mice maintained for 10 days in thiamine replete or deficient chow. Mutant mice had cell surface labeling indistinguishable from +/+ at 0°C (P>0.3), reflecting available transferrin receptors. Total cellular 55 Fe uptake at 37 C was equal to or slightly greater than that of +/+ marrow in thiamine depleted state. However, incorporation of 55 Fe into heme lagged in −/− cells (7.3 +/− 0.4 pmol/10 6 cells/7 min) compared to +/+ (8.6 +/− 0.5 pmol/10 6 cells, P =0.018 in triplicate experiments). To examine deoxyribose (dRibose) synthesis, mice were maintained on thiamine replete or depleted chow for 10 days, then [1,2 13 C] glucose was administered intraperitoneally overnight and 3 hours before sacrifice and marrow harvest. DNA from whole marrow and from sorted basophilic erythroblasts (CD71+/Ter119+) was isolated, hydrolized, derivatized, and dRibose analyzed by GC-MS. The initial rate of incorporation of 13 C into DNA dRibose, reflecting de novo synthetic rate, was reduced 34% in basophilic erythroblasts of −/− mice after thiamine depletion (n=3 in duplicate experiments, P slc9a2 leads to two distinct erythropoietic defects. De novo DNA dRibose synthesis via transketolase is markedly impaired. Further, while iron uptake by marrow cells is maintained at normal levels, the heme biosynthetic rate is decreased in −/− mice in thiamine-depleted status. This is the first direct demonstration of a role for thiamine in normal erythrocyte heme and DNA metabolism. Together, these defects can account directly for the meglaoblastosis and sideroblasts of human SLC19A2 defects in TRMA.
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Thiamine Deprivation Induces Reticulocytopenic Anemia in SLC19A2 Knockout Marrow: Evidence for Cell-Intrinsic Defect.
Blood, 2005Co-Authors: Maria Claudia Montefusco, Gretchen Chick, Judith C Fleming, Ellis J NeufeldAbstract:Megaloblastic anemia, diabetes, and sensorineural deafness characterize the autosomal recessive human disorder, Thiamine-Responsive Megaloblastic Anemia syndrome (TRMA). TRMA is due to mutations of the human SLC19A2 gene, which encodes a high-affinity thiamine membrane transporter. The murine SLC19A2 knock-out (-/-) model (Fleming et al, Mol Gen Metab; 80 (2003), 234–241) develops thiamine-dependent reticulocytopenia without megaloblastic anemia or sideroblasts, but the whole animal knockouts become ill in thiamine-depleted settings, which limit investigation of erythropoiesis. Here we investigate, by bone marrow transplantation experiments, whether the erythropoietic defect is cell-intrinsic and correlated with the correct function of SLC19A2. We conducted this study by transplanting C57BL/6J-GPIb SLC19A2 −/− (−/−) or wild type (+/+) bone marrow into C57BL/6J-GPIa wild type (+/+) mice. These congenic strains were chosen to distinguish host and donor cells by GPI isozymes. After host irradiation (525 cG for 2 doses), we injected 4 X106 donor marrow cells retro-orbitally. After engraftment, confirmed by GPIa/b assay (>60 days), we switched animals to either 2mg/kg chow thiamine diet (control) or 0 mg/kg thiamine (normal mouse chow is 22 mg/kg thiamine). The transplant experiments were carried in duplicate and included a total of 53 transplant recipient mice of both sexes. After 24-day diet both +/+ and −/− groups on 0 mg/kg thiamine diet lost weight (~28%). Blood parameters changed unequally. After 24 days on 0 mg/kg thiamine diet mice transplanted with −/− bone marrow reveled a significant increment of reticulocyte mean cell volume (MCV) (23% with P< 0.01) together with a relevant decrease of red blood cell count (46% and P< 0.01). A severe leukopenia diet correlated was also observed in both groups of mice on thiamine-depleted diet. The blood parameters of mice on 2-mg/kg diet did not reveal significant changes along the entire diet period. These results confirm the involvement of SLC19A2 membrane transporter in the impaired thiamine cell uptake and allow us to say that the erythropoietic defect we first observed in SLC19A2 KO mice is not only diet related, but due to a cell-intrinsic marrow defect. Tables. Peripheral blood parameters of bone marrow transplanted mice at baseline and after 24 days on depleted thiamine diet. | Donor | | WBC | RBC | MCV Retic | |:-----:| ------------ | ------------ | ----------- | ------------ | | −/− | mean (StDev) | 20.41 (4.23) | 10.5 (3.46) | 61.83 (2.38) | | +/+ | mean (StDev) | 20.63 (2.36) | 9.24 (1.45) | 67.4 (3.03) | | | P | 0.9140 | 0.4257 | 0.0136 | BASELINE | Donor | | WBC | RBC | MCV retic | |:-------------------------------------------:| ------------ | ----------- | ----------- | ------------ | | St Dev = Standard Deviation P = probability | | −/− | mean (StDev) | 8.53 (1.75) | 6.79 (1.65) | 76.18 (1.24) | | +/+ | mean (StDev) | 6.51 (3.16) | 6.09 (1.37) | 65.23 (5.04) | | | P | 0.3232 | 0.0321 | 0.0011 | 24 days on 0 mg/kg thiamine diet
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male infertility and thiamine dependent erythroid hypoplasia in mice lacking thiamine transporter SLC19A2
Molecular Genetics and Metabolism, 2003Co-Authors: Judith C Fleming, Ryosuke Kawatsuji, Elena Tartaglini, Jeffrey J. Bednarski, Mark D Fleming, Yuko Fujiwara, Ellis J NeufeldAbstract:Abstract Thiamine-responsive megaloblastic anemia with diabetes and deafness (TRMA) is an autosomal recessive disease caused by mutations in the high-affinity thiamine transporter gene SLC19A2. To study the role of thiamine transport in the pathophysiology of TRMA syndrome and of each of the component disorders, we created a targeted disruption of the SLC19A2 gene in mice. SLC19A2 −/− mice are viable and females are fertile. Male −/− mice on a pure 129/Sv background are infertile with small testes (testis/body weight=0.13 ± 0.04 knockout vs. 0.35 ± 0.05 wild type, P
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characterization of a murine high affinity thiamine transporter SLC19A2
Molecular Genetics and Metabolism, 2001Co-Authors: Judith C Fleming, Ryosuke Kawatsuji, Mara P Steinkamp, Elena Tartaglini, Jack L Pinkus, Geraldine S Pinkus, Mark D Fleming, Ellis J NeufeldAbstract:Abstract Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine SLC19A2 locus and characterized the resulting protein. Murine SLC19A2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans ∼13kb with 6 exons, structurally identical to that of the human homolog. We localized the SLC19A2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of SLC19A2 in HEK293T cells resulted in specific uptake of [ 3 H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of SLC19A2 protein. Immunohistochemistry studies indicate that SLC19A2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.
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thiamine responsive megaloblastic anemia syndrome a disorder of high affinity thiamine transport
Blood Cells Molecules and Diseases, 2001Co-Authors: Ellis J Neufeld, Judith C Fleming, Elena Tartaglini, Mara P SteinkampAbstract:Abstract ABSTRACT Thiamine-responsive megaloblastic anemia (TRMA) syndrome (OMIM No. 249270) comprises a distinctive triad of clinical features: megaloblastic anemia with ringed sideroblasts, diabetes mellitus, and progressive sensorineural deafness. The TRMA gene has been mapped and cloned. Designated "SLC19A2" as a member of the solute carrier gene superfamily, this gene is mutated in all TRMA kindreds studied to date. The product of the SLC19A2 gene is a membrane protein which transports thiamine (vitamin B1) with sub-micromolar affinity. Cells from TRMA patients are uniquely sensitive to thiamine depletion to the nanomolar range, while pharmacologic doses of vitamin B1 ameliorate the anemia and diabetes. Here we review the current status of studies aimed at understanding the pathophysiology of this unique transport defect.
Mara P Steinkamp - One of the best experts on this subject based on the ideXlab platform.
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characterization of a murine high affinity thiamine transporter SLC19A2
Molecular Genetics and Metabolism, 2001Co-Authors: Judith C Fleming, Ryosuke Kawatsuji, Mara P Steinkamp, Elena Tartaglini, Jack L Pinkus, Geraldine S Pinkus, Mark D Fleming, Ellis J NeufeldAbstract:Abstract Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine SLC19A2 locus and characterized the resulting protein. Murine SLC19A2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans ∼13kb with 6 exons, structurally identical to that of the human homolog. We localized the SLC19A2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of SLC19A2 in HEK293T cells resulted in specific uptake of [ 3 H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of SLC19A2 protein. Immunohistochemistry studies indicate that SLC19A2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.
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A novel mutation in the SLC19A2 gene in a Tunisian family with thiamine-responsive megaloblastic anaemia, diabetes and deafness syndrome
British Journal of Haematology, 2001Co-Authors: Sami Gritli, Mara P Steinkamp, Elena Tartaglini, Souheil Omar, Souha Guannouni, Sarrah Baltagi-ben Jilani, Raouf Hafsia, Judith C Fleming, Charles I. Berul, Ali BelhaniAbstract:Thiamine-responsive megaloblastic anaemia (TRMA) syndrome with diabetes and deafness was found in two patients from a Tunisian kindred. The proband was homozygous for a novel mutation, 287delG, in the high-affinity thiamine transporter gene, SLC19A2. We demonstrated that fibroblasts from this patient exhibited defective thiamine transport. These data confirm that the SLC19A2 gene is the high-affinity thiamine carrier and that this novel mutation is responsible for TRMA syndrome.
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thiamine responsive megaloblastic anemia syndrome a disorder of high affinity thiamine transport
Blood Cells Molecules and Diseases, 2001Co-Authors: Ellis J Neufeld, Judith C Fleming, Elena Tartaglini, Mara P SteinkampAbstract:Abstract ABSTRACT Thiamine-responsive megaloblastic anemia (TRMA) syndrome (OMIM No. 249270) comprises a distinctive triad of clinical features: megaloblastic anemia with ringed sideroblasts, diabetes mellitus, and progressive sensorineural deafness. The TRMA gene has been mapped and cloned. Designated "SLC19A2" as a member of the solute carrier gene superfamily, this gene is mutated in all TRMA kindreds studied to date. The product of the SLC19A2 gene is a membrane protein which transports thiamine (vitamin B1) with sub-micromolar affinity. Cells from TRMA patients are uniquely sensitive to thiamine depletion to the nanomolar range, while pharmacologic doses of vitamin B1 ameliorate the anemia and diabetes. Here we review the current status of studies aimed at understanding the pathophysiology of this unique transport defect.
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Characterization of a murine high-affinity thiamine transporter, SLC19A2.
Molecular genetics and metabolism, 2001Co-Authors: Judith C Fleming, Ryosuke Kawatsuji, Mara P Steinkamp, Elena Tartaglini, Jack L Pinkus, Geraldine S Pinkus, Mark D Fleming, Ellis J NeufeldAbstract:Thiamine-responsive megaloblastic anemia with deafness and diabetes (TRMA) is a rare autosomal recessive disorder of thiamine transport. Previous studies have demonstrated that the disease is caused by mutations in the SLC19A2 gene encoding a high-affinity thiamine transporter. We hypothesize that thiamine transport, mediated by SLC19A2, plays a role in the development and or maintenance of several organ systems, in particular the erythropoietic, auditory, and glucose homeostasis systems. To investigate the transporter further, we cloned the murine SLC19A2 locus and characterized the resulting protein. Murine SLC19A2 is a 498 amino acid protein, with 12 predicted transmembrane domains. The gene spans approximately 13kb with 6 exons, structurally identical to that of the human homolog. We localized the SLC19A2 gene to mouse chromosome 1, a region syntenic to human chromosome 1q23 that contains the TRMA locus. Transient expression of SLC19A2 in HEK293T cells resulted in specific uptake of [3H] thiamine, confirming a thiamine transporter function. Western blot analysis of mouse tissues reveals a wide distribution of SLC19A2 protein. Immunohistochemistry studies indicate that SLC19A2 is expressed on the cell surface and intracellularly, and is specifically localized to a subpopulation of cells in cochlea, small intestine, and pancreas.