Publications by authors named "Veedamali S Subramanian"

73 Publications

Thymoquinone, a Dietary Bioactive Compound, Exerts Anti-Inflammatory Effects in Colitis by Stimulating Expression of the Colonic Epithelial PPAR-γ Transcription Factor.

Nutrients 2021 Apr 17;13(4). Epub 2021 Apr 17.

Department of Physiology, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain PO Box 17666, United Arab Emirates.

Inflammatory bowel diseases (IBD) are chronic inflammatory disorders with increasing incidence and prevalence worldwide. Here, we investigated thymoquinone (TQ), a naturally occurring phytochemical present in , for anti-inflammatory effects in colonic inflammation. To address this, we used in vivo (mice) and in vitro (HT-29 cells) models in this investigation. Our results showed that TQ treatment significantly reduced the disease activity index (DAI), myeloperoxidase (MPO) activity, and protected colon microscopic architecture. In addition, TQ also reduced the expression of proinflammatory cytokines and mediators at both the mRNA and protein levels. Further, TQ decreased phosphorylation of the activated mitogen-activated protein kinase (MAPK) signaling pathway and nuclear factor kappa B (NF-κB) proteins and enhanced colon epithelial PPAR-γ transcription factor expression. TQ significantly decreased proinflammatory chemokines (CXCL-1 and IL-8), and mediator (COX-2) mRNA expression in HT-29 cells treated with TNF-α. TQ also increased HT-29 PPAR-γ mRNA, PPAR-γ protein expression, and PPAR-γ promoter activity. These results indicate that TQ inhibits MAPK and NF-κB signaling pathways and transcriptionally regulates PPAR-γ expression to induce potent anti-inflammatory activity in vivo and in vitro models of colon inflammation.
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http://dx.doi.org/10.3390/nu13041343DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8073634PMC
April 2021

Upregulation of Vitamin C Transporter Functional Expression in 5xFAD Mouse Intestine.

Nutrients 2021 Feb 14;13(2). Epub 2021 Feb 14.

Department of Medicine, University of California, Irvine, CA 92697, USA.

The process of obtaining ascorbic acid (AA) via intestinal absorption and blood circulation is carrier-mediated utilizing the AA transporters SVCT1 and SVCT2, which are expressed in the intestine and brain (SVCT2 in abundance). AA concentration is decreased in Alzheimer's disease (AD), but information regarding the status of intestinal AA uptake in the AD is still lacking. We aimed here to understand how AA homeostasis is modulated in a transgenic mouse model (5xFAD) of AD. AA levels in serum from 5xFAD mice were markedly lower than controls. Expression of oxidative stress response genes (glutathione peroxidase 1 (GPX1) and superoxide dismutase 1 (SOD1)) were significantly increased in AD mice jejunum, and this increase was mitigated by AA supplementation. Uptake of AA in the jejunum was upregulated. This increased AA transport was caused by a marked increase in SVCT1 and SVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression. A significant increase in the expression of HNF1α and specific protein 1 (Sp1), which drive SLC23A1 and SLC23A2 promoter activity, respectively, was observed. Expression of hSVCT interacting proteins GRHPR and CLSTN3 were also increased. SVCT2 protein and mRNA expression in the hippocampus of 5xFAD mice was not altered. Together, these investigations reveal adaptive up-regulation of intestinal AA uptake in the 5xFAD mouse model.
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http://dx.doi.org/10.3390/nu13020617DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7918291PMC
February 2021

Enteropathogenic Escherichia coli Infection Inhibits Intestinal Ascorbic Acid Uptake via Dysregulation of Its Transporter Expression.

Dig Dis Sci 2020 Jun 17. Epub 2020 Jun 17.

Department of Medicine, University of California Irvine, Irvine, CA, 92697, USA.

Background: Enteropathogenic Escherichia coli (EPEC) infection causes prolonged, watery diarrhea leading to morbidity and mortality. Although EPEC infection impacts nutrient transporter function and expression in intestinal epithelial cells, the effects of EPEC infection on intestinal absorption of ascorbic acid (AA) have not yet been investigated.

Aims: To investigate the effect of EPEC infection on intestinal AA uptake process and expression of both AA transporters.

Methods: We used two experimental models: human-derived intestinal epithelial Caco-2 cells and mice. C-AA uptake assay, Western blot, RT-qPCR, and promoter assay were performed.

Results: EPEC (WT) as well as ΔespF and ΔespG/G2 mutant-infected Caco-2 cells showed markedly inhibited AA uptake, while other mutants (ΔescN, ΔespA, ΔespB, and ΔespD) did not affect AA uptake. Infection also reduced protein and mRNA expression levels for both hSVCT1 and hSVCT2. EPEC-infected mice showed marked inhibitory effect on AA uptake and decreased protein and mRNA expression levels for both mSVCT1 and mSVCT2 in jejunum and colon. MicroRNA regulators of SVCT1 and SVCT2 (miR103a, miR141, and miR200a) were upregulated significantly upon EPEC infection in both Caco-2 and mouse jejunum and colon. In addition, expression of the accessory protein glyoxalate reductase/hydroxypyruvate reductase (GRHPR), which regulates SVCT1 function, was markedly decreased by EPEC infection in both models.

Conclusions: These findings suggest that EPEC infection causes inhibition in AA uptake through a multifactorial dysregulation of SVCT1 and SVCT2 expression in intestinal epithelial cells.
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http://dx.doi.org/10.1007/s10620-020-06389-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7744340PMC
June 2020

Identification of transmembrane protein 237 as a novel interactor with the intestinal riboflavin transporter-3 (RFVT-3): role in functionality and cell biology.

Am J Physiol Cell Physiol 2019 06 20;316(6):C805-C814. Epub 2019 Mar 20.

Department of Physiology and Biophysics, School of Medicine, University of California , Irvine, California.

The apically localized riboflavin (RF) transporter-3 (RFVT-3) is involved in intestinal absorption of vitamin B2. Previous studies have characterized different physiological/biological aspects of the RFVT-3, but there is a lack of knowledge regarding possible existence of interacting partner(s) and consequence of interaction(s) on its function/cell biology. To address the latter, we performed yeast two-hybrid (Y2H) screening of a human colonic cDNA library and have identified transmembrane protein 237 (TMEM237) as a putative interactor with the human (h)RFVT-3; the interaction was further confirmed via "1-by-1" Y2H assay that involved appropriate positive and negative controls. TMEM237 was found to be highly expressed in human native intestine and in human intestinal epithelial cell lines; further, confocal images showed colocalization of the protein with hRFVT-3. The interaction between TMEM237 with hRFVT-3 in human intestinal epithelial HuTu-80 cells was established by coimmunoprecipitation. Expressing TMEM237 in HuTu-80 cells led to a significant induction in RF uptake, while its knockdown (with the use of gene-specific siRNA) led to a significant reduction in uptake. Transfecting TMEM237 into HuTu-80 cells also led to a marked enhancement in hRFVT-3 protein stability (reflected by an increase in the protein half-life). Interestingly, the level of expression of TMEM237 was found to be markedly reduced following treatment with TNF-α (a proinflammatory cytokine that inhibits intestinal RF uptake), while its expression was significantly upregulated following treatment with butyrate (an inducer of intestinal RF uptake). These findings identify TMEM237 as an interactor with the intestinal hRFVT-3 and show that the interaction has physiological/biological significance.
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http://dx.doi.org/10.1152/ajpcell.00029.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6620576PMC
June 2019

MicroRNA-103a regulates sodium-dependent vitamin C transporter-1 expression in intestinal epithelial cells.

J Nutr Biochem 2019 03 7;65:46-53. Epub 2018 Dec 7.

Department of Medicine, University of California, Irvine, CA 92697; Department of Physiology/Biophysics, University of California, Irvine, CA 92697; VA Medical Center, Long Beach, CA 90822.

Intestinal absorption of ascorbic acid (AA) occurs via a Na-dependent carrier-mediated process facilitated through the human sodium-dependent vitamin C transporters-1 &-2 (hSVCT1 and hSVCT2). Many studies have shown that hSVCT1 (product of the SLC23A1 gene) is expressed on the apical membrane of polarized enterocytes where it mediates AA absorption. hSVCT1 expression levels are therefore an important determinant of physiological vitamin C homeostasis. However, little is known about posttranscriptional mechanisms that regulate hSVCT1 expression in intestinal epithelia. In this study, we investigated regulation of hSVCT1 by microRNA (miRNA). A pmirGLO-SLC23A1-3'-UTR construct transfected into human intestinal cell lines (Caco-2 and NCM460 cells) showed markedly reduced luciferase activity. Bioinformatic analysis of the SLC23A1-3'-UTR predicted five miRNA binding sites (miR-103a, miR-107, miR-328, miR-384, and miR-499-5p) in the 3'-UTR. Expression of mature miR-103a was markedly higher compared to the other four putative miRNA regulators in both intestinal cell lines and mouse jejunal mucosa. Addition of a miR-103a mimic, but not a miR-103a mutant construct, markedly reduced the luminescence of the pmirGLO-SLC23A1-3'-UTR reporter. Reciprocally, addition of a miR-103a inhibitor significantly increased luciferase reporter activity. Addition of the miR-103a mimic led to a significant inhibition in AA uptake, associated with decreased hSVCT1 mRNA and protein expression in Caco-2 cells. In contrast, the miR-103a inhibitor increased AA uptake, associated with increased levels of hSVCT1 mRNA and protein. These findings provide the first evidence for posttranscriptional regulation of hSVCT1 by miRNA in intestinal epithelial cells.
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http://dx.doi.org/10.1016/j.jnutbio.2018.12.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6420349PMC
March 2019

Enterotoxigenic Escherichia coli heat labile enterotoxin inhibits intestinal ascorbic acid uptake via a cAMP-dependent NF-κB-mediated pathway.

Am J Physiol Gastrointest Liver Physiol 2019 01 4;316(1):G55-G63. Epub 2018 Oct 4.

Department of Medicine, University of California , Irvine, California.

Vitamin C is an antioxidant and acts as a cofactor for many enzymatic reactions. Humans obtain vitamin C from dietary sources via intestinal absorption, a process that involves the sodium-dependent vitamin C transporters-1 and -2 (SVCT1 and SVCT2). Enterotoxigenic Escherichia coli (ETEC) infection impacts intestinal absorption/secretory functions, but nothing is known about its effect on ascorbic acid (AA) uptake. Here we demonstrate that infection of Caco-2 cells with ETEC led to a significant inhibition in intestinal AA uptake. This inhibition was associated with a marked reduction in hSVCT1 and hSVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression levels as well as significant inhibition in the activity of both the SLC23A1 and SLC23A2 promoters. Similarly, exposure of mice to ETEC led to a significant inhibition in intestinal AA uptake and reduction in mSVCT1 and mSVCT2 protein, mRNA, and hnRNA expression levels. Inhibition was caused by the action of heat labile enterotoxin (LT), since infecting Caco-2 cells with LT-deficient ETEC (ΔLT) failed to impact AA uptake. Because LT activates adenylate cyclase, we also examined the effect of dibutyryl-cAMP in AA uptake by Caco-2 cells and observed a significant inhibition. Furthermore, treating the cells with celastrol, a specific NF-κB inhibitor, significantly blocked the inhibition of AA uptake caused by ETEC infection. Together, these data demonstrate that ETEC infection impairs intestinal AA uptake through a cAMP-dependent NF-κB-mediated pathway that regulates both SLC23A1 and SLC23A2 transcription. NEW & NOTEWORTHY Our findings demonstrate that heat-labile enterotoxin produced by enterotoxigenic Escherichia coli inhibits AA uptake in intestinal epithelial cells and mouse intestine. This effect is mediated through transcriptional repression of SLC23A1 (SVCT1) and SLC23A2 (SVCT2) via a cAMP-dependent NF-κB signaling pathway.
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http://dx.doi.org/10.1152/ajpgi.00259.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6383388PMC
January 2019

Sodium Butyrate Enhances Intestinal Riboflavin Uptake via Induction of Expression of Riboflavin Transporter-3 (RFVT3).

Dig Dis Sci 2019 01 1;64(1):84-92. Epub 2018 Oct 1.

Departments of Medicine and Physiology/Biophysics, University of California, Irvine, CA, 92697, USA.

Background: Uptake of riboflavin (RF) by intestinal epithelial cells occurs via a specific carrier-mediated process that involves the apically localized RF transporter-3 (RFVT3). Previous studies have shown that sodium butyrate (NaB) affects intestinal uptake of other substrates and expression of their membrane transporters, but its effect on intestinal uptake of RF and expression of RFVT3 has not been examined.

Aims: To investigate the effect of NaB on intestinal RF uptake process and expression of the RFVT3.

Methods: Two experimental models were used in this study: Human-derived intestinal epithelial Caco-2 cells and ex vivo mouse colonoids. H-RF uptake assay, Western blot, RT-qPCR, and chromatin immunoprecipitation assay were performed.

Results: Treating Caco-2 cells with NaB led to a significant increase in carrier-mediated RF uptake. This increase was associated with a significant induction in the level of expression of the hRFVT3 protein, mRNA, and heterogenous nuclear RNA (hnRNA). Similarly, treating mouse colonoids with NaB led to a marked increase in the level of expression of the mRFVT3 protein, mRNA, and hnRNA. NaB did not affect hRFVT3 mRNA stability, rather it caused significant epigenetic changes (histone modifications) in the SLC52A3 gene where an increase in H3Ac and a reduction in H3K27me3 levels were observed in the NaB-treated Caco-2 cells compared to untreated controls.

Conclusion: These findings demonstrate that NaB up-regulates intestinal RF uptake and that the effect appears to be mediated, at least in part, at the level of transcription of the SLC52A3 gene and may involve epigenetic mechanism(s).
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http://dx.doi.org/10.1007/s10620-018-5305-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6320279PMC
January 2019

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

Am J Physiol Cell Physiol 2018 11 29;315(5):C653-C663. Epub 2018 Aug 29.

Department of Medicine, University of California , Irvine, California.

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.
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http://dx.doi.org/10.1152/ajpcell.00295.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6293048PMC
November 2018

Tumor necrosis factor alpha reduces intestinal vitamin C uptake: a role for NF-κB-mediated signaling.

Am J Physiol Gastrointest Liver Physiol 2018 08 6;315(2):G241-G248. Epub 2018 Apr 6.

Department of Medicine, University of California , Irvine, California.

Sodium-dependent vitamin C transporter-1 (SVCT-1) is the major transporter mediating intestinal vitamin C uptake. Intestinal inflammation and prolonged infection are associated with increased serum and intestinal mucosa levels of tumor necrosis factor-α (TNF-α), which also exerts profound effects on the intestinal absorption process. Elevated levels of TNF-α have been linked to the pathogenesis of inflammatory bowel disease (IBD) and malabsorption of nutrients, and patients with this condition have low levels of vitamin C. To date, little is known about the effect of TNF-α on intestinal absorption of vitamin C. We studied the impact of TNF-α on ascorbic acid (AA) transport using a variety of intestinal preparations. The expression level of human SVCT-1 mRNA is significantly lower in patients with IBD. TNF-α treated Caco-2 cells and mice showed a significant inhibition of intestinal C-AA uptake. This inhibition was associated with significant decreases in SVCT-1 protein, mRNA, and heterogeneous nuclear RNA levels in TNF-α treated Caco-2 cells, mouse jejunum, and enteroids. Also, TNF-α caused a significant inhibition in the SLC23A1 promoter activity. Furthermore, treatment of Caco-2 cells with celastrol (NF-κB inhibitor) blocked the inhibitory effect caused by TNF-α on AA uptake, SVCT-1 protein, and mRNA expression, as well as the activity of SLC23A1 promoter. Treatment of TNF-α also led to a significant decrease in the expression of hepatocyte nuclear factor-1-α, which drives the basal activity of SLC23A1 promoter, and this effect was reversed by celastrol. Together, these findings show that TNF-α inhibits intestinal AA uptake, and this effect is mediated, at least in part, at the level of transcription of the SLC23A1 gene via the NF-κB pathway. NEW & NOTEWORTHY Our findings show that tumor necrosis factor-α inhibits intestinal ascorbic acid uptake in both in vitro and in vivo systems, and this inhibitory effect is mediated, at least in part, at the level of transcription of the SLC23A1 (sodium-dependent vitamin C transporter-1) gene via the NF-κB pathway.
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http://dx.doi.org/10.1152/ajpgi.00071.2018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6139644PMC
August 2018

Inhibition of intestinal ascorbic acid uptake by lipopolysaccharide is mediated via transcriptional mechanisms.

Biochim Biophys Acta Biomembr 2018 Feb 10;1860(2):556-565. Epub 2017 Oct 10.

Departments of Medicine, Physiology and Biophysics, University of California, Irvine, CA 92697, United States; Department of Veterans Affairs Medical Center, Long Beach, CA 90822, United States.

Ascorbic acid (AA) accumulation in intestinal epithelial cells is an active transport process mainly mediated by two sodium-dependent vitamin C transporters (SVCT-1 and SVCT-2). To date, little is known about the effect of gut microbiota generated lipopolysaccharide (LPS) on intestinal absorption of water-soluble vitamins. Therefore, the objective of this study was to investigate the effects of bacterially-derived LPS on AA homeostasis in enterocytes using Caco-2 cells, mouse intestine and intestinal enteroids models. Pre-treating Caco-2 cells and mice with LPS led to a significant decrease in carrier-mediated AA uptake. This inhibition was associated with a significant reduction in SVCT-1 and SVCT-2 protein, mRNA, and hnRNA expression. Furthermore, pre-treating enteroids with LPS also led to a marked decrease in SVCT-1 and SVCT-2 protein and mRNA expression. Inhibition of SVCT-1 and SVCT-2 occurred at least in part at the transcriptional level as promoter activity of SLC23A1 and SLC23A2 was attenuated following LPS treatment. Subsequently, we examined the protein and mRNA expression levels of HNF1α and Sp1 transcription factors, which are needed for basal SLC23A1 and SLC23A2 promoter activity, and found that they were significantly decreased in the LPS treated Caco-2 cells and mouse jejunum; this was reflected on level of the observed reduction in the interaction of these transcription factors with their respective promoters in Caco-2 cells treated with LPS. Our findings indicate that LPS inhibits intestinal carrier- mediated AA uptake by down regulating the expression of both vitamin C transporters and transcriptional regulation of SLC23A1 and SLC23A2 genes.
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http://dx.doi.org/10.1016/j.bbamem.2017.10.010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5732882PMC
February 2018

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

Am J Physiol Cell Physiol 2017 12 20;313(6):C655-C663. Epub 2017 Sep 20.

Department of Medical Research, VA Medical Center , Long Beach, California.

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 [H] 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.
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http://dx.doi.org/10.1152/ajpcell.00169.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814587PMC
December 2017

Role of MicroRNA-423-5p in posttranscriptional regulation of the intestinal riboflavin transporter-3.

Am J Physiol Gastrointest Liver Physiol 2017 Dec 14;313(6):G589-G598. Epub 2017 Sep 14.

Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California

Riboflavin (RF) is essential for normal cellular functions and health. Humans obtain RF from exogenous sources via intestinal absorption that involves a highly specific carrier-mediated process. We have recently established that the riboflavin transporter-3 (RFVT3) is vital for the normal intestinal RF uptake process and have characterized certain aspects of its transcriptional regulation. Little is known, however, about how this transporter is regulated at the posttranscriptional level. We address this issue by focusing on the role of microRNAs. Using bioinformatics, we identified two potential interacting miRNAs with the human (h) RFVT3-3'-UTR, and showed (using pmirGLO-hRFVT3-3'-UTR) that the hRFVT3-3'-UTR is, indeed, a target for miRNA effect. Of the two putative miRNAs identified, miR-423-5p was found to be highly expressed in intestinal epithelial cells and that its mimic affected luciferase reporter activity of the pmirGLO-hRFVT3-3'-UTR construct, and also led to inhibition in RF uptake by intestinal epithelial Caco-2 and HuTu-80 cells. Furthermore, cells transfected with mutated seed sequences for miR-423-5p showed an abrogation in inhibitory effect of the miR-423-5p mimic on luciferase activity. While miR-423-5p did not affect the level of expression of the hRFVT3 mRNA, it did lead to a significant inhibition in the level of expression of its protein. Similarly, miR-423-5p was found to affect the level of expression of the mouse RFVT3 in cultured intestinal enteroids. These findings demonstrate, for the first time, that the RFVT3 is a target for posttranscriptional regulation by miRNAs in intestinal epithelial cells and that this regulation has functional consequences on intestinal RF uptake. Our findings show for the first time that RFVT3 is a target for posttranscriptional regulation by miR-423-5p in intestinal epithelial cells, and this regulation has functional consequences on intestinal riboflavin (RF) uptake process.
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http://dx.doi.org/10.1152/ajpgi.00238.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5814671PMC
December 2017

Adaptive regulation of pancreatic acinar mitochondrial thiamin pyrophosphate uptake process: possible involvement of epigenetic mechanism(s).

Am J Physiol Gastrointest Liver Physiol 2017 Nov 20;313(5):G448-G455. Epub 2017 Jul 20.

Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California

The essentiality of thiamin stems from its roles as a cofactor [mainly in the form of thiamin pyrophosphate (TPP)] in critical metabolic reactions including oxidative energy metabolism and reduction of cellular oxidative stress. Like other mammalian cells, pancreatic acinar cells (PAC) obtain thiamin from their surroundings and convert it to TPP; mitochondria then take up TPP by a carrier-mediated process that involves the mitochondrial TPP (MTPP) transporter (MTPPT; product of gene). Previous studies have characterized different physiological/biological aspects of the MTPP uptake process, but little is known about its possible adaptive regulation. We addressed this issue using pancreatic acinar 266-6 cells (PAC 266-6) maintained under thiamin-deficient (DEF) and oversupplemented (OS) conditions, as well as thiamin-DEF and -OS transgenic mice carrying the promoter. We found that maintaining PAC 266-6 under the thiamin-DEF condition leads to a significant induction in mitochondrial [H]TPP uptake, as well as in the level of expression of the MTPPT protein and mRNA compared with thiamin-OS cells. Similar findings were observed in mitochondria from thiamin-DEF mice compared with thiamin-OS. Subsequently, we demonstrated that adaptive regulation of MTTP protein was partly mediated via transcriptional mechanism(s) via studies with PAC 266-6 transfected with the promoter and transgenic mice carrying the promoter. This transcriptional regulation appeared to be, at least in part, mediated via epigenetic mechanism(s) involving histone modifications. These studies report, for the first time, that the PAC mitochondrial TPP uptake process is adaptively regulated by the prevailing thiamin level and that this regulation is transcriptionally mediated and involves epigenetic mechanism(s). Our findings show, for the first time, that the mitochondrial thiamin pyrophosphate (MTPP) uptake process is adaptively regulated by the prevailing thiamin level in pancreatic acinar cells and this regulation is mediated, at least in part, by transcriptional and epigenetic mechanism(s) affecting the promoter.
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http://dx.doi.org/10.1152/ajpgi.00192.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5792211PMC
November 2017

Structure/functional aspects of the human riboflavin transporter-3 (): role of the predicted glycosylation and substrate-interacting sites.

Am J Physiol Cell Physiol 2017 Aug 21;313(2):C228-C238. Epub 2017 Jun 21.

Departments of Medicine, Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California

The human riboflavin (RF) transporter-3 (hRFVT-3; product of the gene) plays an essential role in the intestinal RF absorption process and is expressed exclusively at the apical membrane domain of polarized enterocytes. Previous studies have characterized different physiological/biological aspects of this transporter, but nothing is known about the glycosylation status of the hRFVT-3 protein and role of this modification in its physiology/biology. Additionally, little is known about the residues in the hRFVT-3 protein that interact with the ligand, RF. We addressed these issues using appropriate biochemical/molecular approaches, a protein-docking model, and established intestinal/renal epithelial cells. Our results showed that the hRFVT-3 protein is glycosylated and that glycosylation is important for its function. Mutating the predicted -glycosylation sites at Asn and Asn led to a significant decrease in RF uptake; it also led to a marked intracellular (in the endoplasmic reticulum, ER) retention of the mutated proteins as shown by live-cell confocal imaging studies. The protein-docking model used in this study has identified a number of putative substrate-interacting sites: Ser, Ile, Trp, Phe, Thr, and Asn Mutating these potential interacting sites was indeed found to lead to a significant inhibition in RF uptake and to intracellular (ER) retention of the mutated proteins (except for the Phe mutant). These results demonstrate that the hRFVT-3 protein is glycosylated and this glycosylation is important for its function and cell surface expression. This study also identified a number of residues in the hRFVT-3 polypeptide that are important for its function/cell surface expression.
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http://dx.doi.org/10.1152/ajpcell.00101.2017DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5582875PMC
August 2017

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

Am J Physiol Gastrointest Liver Physiol 2017 Apr 8;312(4):G340-G347. Epub 2016 Dec 8.

Departments of Medicine, Physiology, and Biophysics, University of California, Irvine, California.

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 and 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 and 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. 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 and genes.
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http://dx.doi.org/10.1152/ajpgi.00369.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5407060PMC
April 2017

Mutations in SLC5A6 associated with brain, immune, bone, and intestinal dysfunction in a young child.

Hum Genet 2017 02 30;136(2):253-261. Epub 2016 Nov 30.

Departments of Medicine and Physiology/Biophysics, University of California, Irvine, CA 92697.

The human sodium-dependent multivitamin transporter (hSMVT) is a product of the SLC5A6 gene and mediates biotin, pantothenic acid, and lipoate uptake in a variety of cellular systems. We report here the identification of mutations R94X, a premature termination, and R123L, a dysfunctional amino acid change, both in exon 3 of the SLC5A6 gene in a child using whole genome-scanning. At 15 months of age, the child showed failure to thrive, microcephaly and brain changes on MRI, cerebral palsy and developmental delay, variable immunodeficiency, and severe gastro-esophageal reflux requiring a gastrostomy tube/fundoplication, osteoporosis, and pathologic bone fractures. After identification of the SLC5A6 mutations, he responded clinically to supplemental administration of excess biotin, pantothenic acid, and lipoate with improvement in clinical findings. Functionality of the two mutants was examined by H-biotin uptake assay following expression of the mutants in human-derived intestinal HuTu-80 and brain U87 cells. The results showed severe impairment in biotin uptake in cells expressing the mutants compared to those expressing wild-type hSMVT. Live cell confocal imaging of cells expressing the mutants showed the R94X mutant to be poorly tolerated and localized in the cytoplasm, while the R123L mutant was predominantly retained in the endoplasmic reticulum. This is the first reporting of mutations in the SLC5A6 gene in man, and suggests that this gene is important for brain development and a wide variety of clinical functions.
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http://dx.doi.org/10.1007/s00439-016-1751-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5263180PMC
February 2017

SLC52A2 [p.P141T] and SLC52A3 [p.N21S] causing Brown-Vialetto-Van Laere Syndrome in an Indian patient: First genetically proven case with mutations in two riboflavin transporters.

Clin Chim Acta 2016 Nov 1;462:210-214. Epub 2016 Oct 1.

School of Biotechnology, Madurai Kamaraj University, Madurai 625021, India. Electronic address:

Background: Brown-Vialetto-Van Laere Syndrome (BVVLS), a rare neurological disorder characterized by bulbar palsies and sensorineural deafness, is mainly associated with defective riboflavin transporters encoded by the SLC52A2 and SLC52A3 genes.

Methods: Here we present a 16-year-old BVVLS patient belonging to a five generation consanguineous family from Indian ethnicity with two homozygous missense mutations viz., c.421C>A [p.P141T] in SLC52A2 and c.62A>G [p.N21S] in SLC52A3.

Results: Functional characterization based on H-riboflavin uptake assay and live-cell confocal imaging revealed that the effect of mutation c.421C>A [p.P141T] identified in SLC52A2 had a slight reduction in riboflavin uptake; on the other hand, the c.62A>G [p.N21S] identified in SLC52A3 showed a drastic reduction in riboflavin uptake, which appeared to be due to impaired trafficking and membrane targeting of the hRFVT-3 protein.

Conclusions: This is the first report presenting mutations in both riboflavin transporters hRFVT-2 and hRFVT-3 in the same BVVLS patient. Also, c.62A>G [p.N21S] in SLC52A3 appears to contribute more to the disease phenotype in this patient than c.421C>A [p.P141T] in SLC52A2.
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http://dx.doi.org/10.1016/j.cca.2016.09.022DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5521005PMC
November 2016

Structure-function characterization of the human mitochondrial thiamin pyrophosphate transporter (hMTPPT; SLC25A19): Important roles for Ile(33), Ser(34), Asp(37), His(137) and Lys(291).

Biochim Biophys Acta 2016 08 14;1858(8):1883-90. Epub 2016 May 14.

Department of Medical Research, VA Medical Center, Long Beach, CA 90822, United States; Departments of Medicine and Physiology/Biophysics, University of California, Irvine, CA 92697, United States. Electronic address:

Thiamin plays a critical role in cellular energy metabolism. Mammalian cells obtain the vitamin from their surroundings, converted it to thiamin pyrophosphate (TPP) in the cytoplasm, followed by uptake of TPP by mitochondria via a carrier-mediated process that involves the MTPPT (product of the SLC25A19 gene). Previous studies have characterized different physiological/biological aspects of the human MTPPT (hMTPPT), but less is known about structural features that are important for its function. Here, we used a protein-docking model ("Phyre2" and "DockingServer") to predict residues that may be important for function (substrate recognition) of the hMTPPT; we also examined the role of conserved positively-charged residues predicted ("PRALINE") to be in the trans-membrane domains (TMDs) in uptake of the negatively-charged TPP. Among the six residues predicted by the docking model (i.e., Thr(29), Arg(30), Ile(33), Ser(34), Asp(37) and Phe(298)), only Ile(33), Ser(34) and Asp(37) were found to be critical for function. While no change in translational efficiency/protein stability of the Ser(34) mutant was observed, both the Ile(33) and Asp(37) mutants showed a decrease in this parameter(s); there was also a decrease in the expression of the latter two mutants in mitochondria. A need for a polar residue at position 34 of the hMTPPT was evident. Our findings with the positively-charged residues (i.e., His(82), His(137), Lys(231) and Lys(291)) predicted in the TMD showed that His(137) and Lys(291) are important for function (via a role in proper delivery of the protein to mitochondria). These investigations provide important information about the structure-function relationship of the hMTPPT.
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http://dx.doi.org/10.1016/j.bbamem.2016.05.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4900926PMC
August 2016

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

Am J Physiol Cell Physiol 2016 07 27;311(1):C129-35. Epub 2016 Apr 27.

Department of Medicine, University of California, Irvine, California; Department of Physiology, University of California, Irvine, California; Department of Biophysics, University of California, Irvine, California; Department of Veterans Affairs Medical Center, Long Beach, California

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).
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http://dx.doi.org/10.1152/ajpcell.00042.2016DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4967129PMC
July 2016

Inhibition of Intestinal Thiamin Transport in Rat Model of Sepsis.

Crit Care Med 2016 09;44(9):e875-81

1Department of Medicine, VA Long Beach Healthcare System, Long Beach, CA.2Department of Medicine, University of California, Irvine, CA.3Department of Physiology and Biophysics, University of California, Irvine, CA.

Objectives: Thiamin deficiency is highly prevalent in patients with sepsis, but the mechanism by which sepsis induces thiamin deficiency is unknown. This study aimed to determine the influence of various severity of sepsis on carrier-mediated intestinal thiamin uptake, level of expressions of thiamin transporters (thiamin transporter-1 and thiamin transporter-2), and mitochondrial thiamin pyrophosphate transporter.

Design: Randomized controlled study.

Setting: Research laboratory at a Veterans Affairs Medical Center.

Subjects: Twenty-four Sprague-Dawley rats were randomized into controls, mild, moderate, and severe sepsis with equal number of animals in each group.

Interventions: Sepsis was induced by cecal ligation and puncture with the cecum ligated below the cecal valve at 25%, 50%, and 75% of cecal length, defined as severe, moderate, and mild sepsis, respectively. Control animals underwent laparotomy only.

Measurements And Main Results: After 2 days of induced sepsis, carrier-mediated intestinal thiamin uptake was measured using [H]thiamin. Expressions of thiamin transporter-1, thiamin transporter-2, and mitochondrial thiamin pyrophosphate transporter proteins and messenger RNA were measured. Proinflammatory cytokines (interleukin-1β and interleukin-6) and adenosine triphosphate were also measured. Sepsis inhibited [H]thiamin uptake, and the inhibition was a function of sepsis severity. Both cell membrane thiamin transporters and mitochondrial thiamin pyrophosphate transporter expression levels were suppressed; also levels of adenosine triphosphate in the intestine of animals with moderate and severe sepsis were significantly lower than that of sham-operated controls.

Conclusions: For the first time, we demonstrated that sepsis inhibited carrier-mediated intestinal thiamin uptake as a function of sepsis severity, suppressed thiamin transporters and mitochondrial thiamin pyrophosphate transporter, leading to adenosine triphosphate depletion.
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http://dx.doi.org/10.1097/CCM.0000000000001745DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4987227PMC
September 2016

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

Biochim Biophys Acta 2016 Apr 30;1858(4):866-71. Epub 2016 Jan 30.

Department of Medicine, University of California, Irvine, CA 92697, United States; Department of Physiology/Biophysics, University of California, Irvine, CA 92697, United States; Department of Veterans Affairs Medical Center, Long Beach, CA 90822, United States. Electronic address:

The recently identified human thiamine pyrophosphate transporter (hTPPT; product of the SLC44A4 gene) is responsible for absorption of the microbiota-generated TPP in the large intestine. The hTPPT is highly expressed in the colon, but not in other regions of the intestinal tract and is localized exclusively at the apical membrane domain of epithelia. The hTPPT protein is predicted to have multiple TM domains with a number of putative N-glycosylation sites, but it is not known if the protein is actually glycosylated, and if so at which site, and their role in the functionality of the transporter. Using several approaches including inhibiting de novo N-glycosylation in human colonic epithelial NCM460 cells with tunicamycin as well as enzymatic de-glycosylation, we show that the hTPPT protein is, indeed, a glycoprotein. Glycosylation of hTPPT was shown, by mean of site-directed mutagenesis, to occur at Asn(69), Asn(155), Asn(197), Asn(393), and Asn(416). However, only N-glycosylation at Asn(69), Asn(155), and Asn(393) appeared to be important for transporter functionality possibly through an effect on protein conformation and/or interaction with its ligand (but not through changes in expression at the cell membrane as determined by live cell confocal imaging). Results of this study showed, for the first time, that the hTPPT is glycosylated and that N-linked glycosylation occurs at multiple sites with some of them being important for function. The results also provide an indirect support for a membrane topology for hTPPT with 10 transmembrane domains as predicted by the TMHMM transmembrane helixes prediction program.
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http://dx.doi.org/10.1016/j.bbamem.2016.01.028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4779673PMC
April 2016

Conditional (intestinal-specific) knockout of the riboflavin transporter-3 (RFVT-3) impairs riboflavin absorption.

Am J Physiol Gastrointest Liver Physiol 2016 Feb 10;310(4):G285-93. Epub 2015 Dec 10.

Departments of Medicine, Physiology and Biophysics, University of California, Irvine, California; Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and

Riboflavin (RF) is indispensable for normal cell metabolism, proliferation, and growth. The RFVT-3 protein (product of the Slc52a3 gene) is expressed in the gut with the expression being restricted to the apical membrane domain of the polarized intestinal epithelial cells. The relative contribution of RFVT-3 to total carrier-mediated RF uptake in the native intestine, however, is not clear. We addressed this issue in the current investigation using a conditional (intestinal-specific) RFVT-3 knockout (cKO) mouse model developed by the Cre/Lox approach. All RFVT-3 cKO mice were found to be RF deficient and showed a significant growth and development retardation; also, nearly two-thirds of them died prematurely between the age of 6 and 12 wk. In vivo (intestinal and colonic loops) and in vitro (native isolated intestinal epithelial cells) uptake studies showed a severe inhibition in carrier-mediated RF uptake in the cKO mice compared with control littermates. We also observed a significant increase in the level of expression of oxidative stress-responsive genes in the intestine of the cKO mice compared with control littermates. Supplementation of the RFVT-3 cKO mice with pharmacological doses of RF led to a complete correction of the growth retardation and to normalization in the level of expression of the oxidative stress-responsive genes in the gut. These results show, for the first time, that the RFVT-3 system is the main transporter involved in carrier-mediated RF uptake in the native mouse small and large intestine, and that its dysfunction impairs normal RF body homeostasis.
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http://dx.doi.org/10.1152/ajpgi.00340.2015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4754740PMC
February 2016

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

PLoS One 2015 3;10(12):e0143575. Epub 2015 Dec 3.

Department of Medical Research, VA Medical Center, Long Beach, California, United States of America.

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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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0143575PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4669105PMC
June 2016

Novel nonsense mutation (p.Ile411Metfs*12) in the SLC19A2 gene causing Thiamine Responsive Megaloblastic Anemia in an Indian patient.

Clin Chim Acta 2016 Jan 5;452:44-9. Epub 2015 Nov 5.

School of Biotechnology, Madurai Kamaraj University, Madurai 625 021, India. Electronic address:

Thiamine-responsive megaloblastic anemia (TRMA), an autosomal recessive disorder, is caused by mutations in SLC19A2 gene encodes a high affinity thiamine transporter (THTR-1). The occurrence of TRMA is diagnosed by megaloblastic anemia, diabetes mellitus, and sensorineural deafness. Here, we report a female TRMA patient of Indian descent born to 4th degree consanguineous parents presented with retinitis pigmentosa and vision impairment, who had a novel homozygous mutation (c.1232delT/ter422; p.Ile411Metfs*12) in 5th exon of SLC19A2 gene that causes premature termination of hTHTR-1. PROSITE analysis predicted to abrogate GPCRs family-1 signature motif in the variant by this mutation c.1232delT/ter422, suggesting uncharacteristic rhodopsin function leading to cause RP clinically. Thiamine transport activity by the clinical variant was severely inhibited than wild-type THTR-1. Confocal imaging had shown that the variant p.I411Mfs*12 is targeted to the cell membrane and showed no discrepancy in membrane expression than wild-type. Our findings are the first report, to the best of our knowledge, on this novel nonsense mutation of hTHTR-1 causing TRMA in an Indian patient through functionally impaired thiamine transporter activity.
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http://dx.doi.org/10.1016/j.cca.2015.11.002DOI Listing
January 2016

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

PLoS One 2015 29;10(6):e0131698. Epub 2015 Jun 29.

Department of Medicine, University of California, Irvine, California, United States of America; Department of Physiology/Biophysics, University of California, Irvine, California, United States of America; VAMC, Long Beach, California, United States of America.

The intestinal absorption process of vitamin B2 (riboflavin, RF) is carrier-mediated, and all three known human RF transporters, i.e., hRFVT-1, -2, and -3 (products of the SLC52A1, 2 & 3 genes, respectively) are expressed in the gut. We have previously shown that the intestinal RF uptake process is adaptively regulated by substrate level, but little is known about the molecular mechanism(s) involved. Using human intestinal epithelial NCM460 cells maintained under RF deficient and over-supplemented (OS) conditions, we now show that the induction in RF uptake in RF deficiency is associated with an increase in expression of the hRFVT-2 & -3 (but not hRFVT-1) at the protein and mRNA levels. Focusing on hRFVT-3, the predominant transporter in the intestine, we also observed an increase in the level of expression of its hnRNA and activity of its promoter in the RF deficiency state. An increase in the level of expression of the nuclear factor Sp1 (which is important for activity of the SLC52A3 promoter) was observed in RF deficiency, while mutating the Sp1/GC site in the SLC52A3 promoter drastically decreased the level of induction in SLC52A3 promoter activity in RF deficiency. We also observed specific epigenetic changes in the SLC52A3 promoter in RF deficiency. Finally, an increase in hRFVT-3 protein expression at the cell surface was observed in RF deficiency. Results of these investigations show, for the first time, that transcriptional and post-transcriptional mechanisms are involved in the adaptive regulation of intestinal RF uptake by the prevailing substrate level.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0131698PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4484800PMC
March 2016

Identification of residues/sequences in the human riboflavin transporter-2 that is important for function and cell biology.

Nutr Metab (Lond) 2015 14;12:13. Epub 2015 Mar 14.

Departments of Medicine, Physiology/Biophysics, University of California, Irvine, CA 92697 USA ; Department of Veterans Affairs Medical Center, Long Beach, CA 90822 USA.

Background: Riboflavin (RF) is essential for normal cellular metabolic activities. Human cells obtain RF from their surroundings via a carrier-mediated process that involves RF transporters -1, -2 & -3 (hRFVT -1, -2 & -3; products of SLC52A1, -A2 and -A3 genes, respectively). Little is known about the structural features of these transporters that are important for their function/cell biology. Our aim in this study was to address these issues for the hRFVT-2, a transporter linked to the neurodegenerative disorder Brown-Vialetto-Van Laere Syndrome (BVVLS).

Methods: We used comparative protein-structure modelling to predict residues that interact with two amino acids known to be critical for hRFVT-2 function (the clinical mutants L123 and L339), site-directed mutagenesis, and truncation approach in the human-derived brain U87 cell model.

Results: First we showed that the defect in the function of the L123 and L339 hRFVT-2 clinical mutants is related to a reduction in protein stability/translation efficiency and to retention of the protein in the ER. Mutating V120 and L121 (residues predicted to interact with L123) and L342 (a residue predicted to interact with L339) also led to a significant inhibition in hRFVT-2 function (with no change in membrane expression); this inhibition was associated with changes in protein stability/translation efficiency (in the case of V120A and L342A) and an impairment in transport function (in the case of L121). Truncating the N- and C- terminals of hRFVT-2 led to significant inhibition in RF uptake, which was associated with changes in protein stability/translation efficiency (it was also associated with a partial impairment in membrane targeting in the case of the N-terminal truncation).

Conclusion: These investigations report on identification of residues/sequences in the hRFVT-2 protein that is important for its physiological function and cell biology.
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http://dx.doi.org/10.1186/s12986-015-0008-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4367879PMC
March 2015

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

Am J Physiol Gastrointest Liver Physiol 2014 Apr 13;306(7):G631-9. Epub 2014 Feb 13.

Department of Medical Research, VA Medical Center, Long Beach; Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California.

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.
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http://dx.doi.org/10.1152/ajpgi.00420.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3962591PMC
April 2014

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

J Biol Chem 2014 Feb 30;289(7):4405-16. Epub 2013 Dec 30.

From the Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California 92697.

Colonic microbiota synthesize a considerable amount of thiamine in the form of thiamine pyrophosphate (TPP). Recent functional studies from our laboratory have shown the existence of a specific, high-affinity, and regulated carrier-mediated uptake system for TPP in human colonocytes. Nothing, however, is known about the molecular identity of this system. Here we report on the molecular identification of the colonic TPP uptake system as the product of the SLC44A4 gene. We cloned the cDNA of SLC44A4 from human colonic epithelial NCM460 cells, which, upon expression in ARPE19 cells, led to a significant (p < 0.01, >5-fold) induction in [(3)H]TPP uptake. Uptake by the induced system was also found to be temperature- and energy-dependent; Na(+)-independent, slightly higher at acidic buffer pH, and highly sensitive to protonophores; saturable as a function of TPP concentration, with an apparent Km of 0.17 ± 0.064 μM; and highly specific for TPP and not affected by free thiamine, thiamine monophosphate, or choline. Expression of the human TPP transporter was found to be high in the colon and negligible in the small intestine. A cell surface biotinylation assay and live cell confocal imaging studies showed the human TPP transporter protein to be expressed at the apical membrane domain of polarized epithelia. These results show, for the first time, the molecular identification and characterization of a specific and high-affinity TPP uptake system in human colonocytes. The findings further support the hypothesis that the microbiota-generated TPP is absorbable and could contribute toward host thiamine homeostasis, especially toward cellular nutrition of colonocytes.
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http://dx.doi.org/10.1074/jbc.M113.528257DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3924303PMC
February 2014