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Moaness M, Kamel AM, Salama A, Kamel R, Beherei HH, Mabrouk M. Fast skin healing chitosan/PEO hydrogels: In vitro and in vivo studies. Int J Biol Macromol 2024; 265:130950. [PMID: 38513911 DOI: 10.1016/j.ijbiomac.2024.130950] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/25/2023] [Revised: 03/04/2024] [Accepted: 03/15/2024] [Indexed: 03/23/2024]
Abstract
Due to its outstanding qualities, particularly when it takes the shape of hydrogels, chitosan is a well-known biological macromolecule with many applications. When chitosan hydrogels are modified with other polymers, the desirable function as skin regeneration hydrogels is compromised; nevertheless, the mechanical properties can be improved, which is crucial for commercialization. In this study, for the first time, bimetallic zinc silver metal-organic frameworks (ZAg MOF) loaded with ascorbic acid were added to chitosan/polyethylene oxide (PEO) based interpenetrating polymer network (IPN) hydrogels that were crosslinked with biotin to improve their antimicrobial activity, mechanical characteristics, and sustainable treatment of wounds. Significant changes in the microstructure, hydrophilicity level, and mechanical properties were noticed. Ascorbic acid release patterns were upregulated in an acidic environment pH (5.5) that mimics the initial wound pH. Impressive cell viability (98 %), antimicrobial properties, and almost full skin healing in a short time were achieved for the non-replaceable chitosan/PEO developed hydrogels. Enhancing the wound healing of the treated animals using the prepared CS/PEO hydrogel dressing was found to be a result of the inhibition of dermal inflammation via decreasing IL-1β, suppressing ECM degradation (MMP9), stimulating proliferation through upregulation of TGF-β and increasing ECM synthesis as it elevates collagen 1 and α-SMA contents. The findings support the implementation of developed hydrogels as antimicrobial hydrogels dressing for fast skin regeneration.
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Affiliation(s)
- Mona Moaness
- Refractories, Ceramics and Building Materials Department, Advanced Materials Technology and Mineral Resources Research Institute, National Research Centre, 33 El Bohouth St., Dokki, PO Box 12622, Cairo, Egypt.
| | - Amira M Kamel
- Polymers and Pigments Department, National Research Centre, 33El Bohouth St., Dokki, PO Box12622, Cairo, Egypt
| | - Abeer Salama
- Pharmacology Department National Research Centre, 33 El Bohouth St., Dokki, PO Box 12622, Cairo, Egypt
| | - Rabab Kamel
- Pharmaceutical Technology Department, National Research Centre, Dokki, 12622 Cairo, Egypt
| | - Hanan H Beherei
- Refractories, Ceramics and Building Materials Department, Advanced Materials Technology and Mineral Resources Research Institute, National Research Centre, 33 El Bohouth St., Dokki, PO Box 12622, Cairo, Egypt
| | - Mostafa Mabrouk
- Refractories, Ceramics and Building Materials Department, Advanced Materials Technology and Mineral Resources Research Institute, National Research Centre, 33 El Bohouth St., Dokki, PO Box 12622, Cairo, Egypt
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Ramamoorthy K, Sabui S, Srinivasan P, Al-Juburi S, Pham Q, Chu BD, Simoes RD, Fleckenstein JM, Said HM. Effect of chronic alcohol exposure on gut vitamin B7 uptake: involvement of epigenetic mechanisms and effect of alcohol metabolites. Am J Physiol Gastrointest Liver Physiol 2021; 321:G123-G133. [PMID: 34077272 PMCID: PMC8410103 DOI: 10.1152/ajpgi.00144.2021] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Vitamin B7 (biotin) is essential for normal health and its deficiency/suboptimal levels occur in a variety of conditions including chronic alcoholism. Mammals, including humans, obtain biotin from diet and gut-microbiota via absorption along the intestinal tract. The absorption process is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; SLC5A6). We have previously shown that chronic alcohol exposure significantly inhibits intestinal/colonic biotin uptake via suppression of Slc5a6 transcription in animal and cell line models. However, little is known about the transcriptional/epigenetic factors that mediate this suppression. In addition, the effect of alcohol metabolites (generated via alcohol metabolism by gut microbiota and host tissues) on biotin uptake is still unknown. To address these questions, we first demonstrated that chronic alcohol exposure inhibits small intestinal and colonic biotin uptake and SMVT expression in human differentiated enteroid and colonoid monolayers. We then showed that chronic alcohol exposures of both, Caco-2 cells and mice, are associated with a significant suppression in expression of the nuclear factor KLF-4 (needed for Slc5a6 promoter activity), as well as with epigenetic alterations (histone modifications). We also found that chronic exposure of NCM460 human colonic epithelial cells as well as human differentiated colonoid monolayers, to alcohol metabolites (acetaldehyde, ethyl palmitate, ethyl oleate) significantly inhibited biotin uptake and SMVT expression. These findings shed light onto the molecular/epigenetic mechanisms that mediate the inhibitory effect of chronic alcohol exposure on intestinal biotin uptake. They further show that alcohol metabolites are also capable of inhibiting biotin uptake in the gut.NEW & NOTEWORTHY Using complementary models, including human differentiated enteroid and colonoid monolayers, this study shows the involvement of molecular and epigenetic mechanisms in mediating the inhibitory effect of chronic alcohol exposure on biotin uptake along the intestinal tract. The study also shows that alcohol metabolites (generated by gut microbiota and host tissues) cause inhibition in gut biotin uptake.
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Affiliation(s)
- Kalidas Ramamoorthy
- 1Department of Physiology/Biophysics, University of California, Irvine, California
| | - Subrata Sabui
- 1Department of Physiology/Biophysics, University of California, Irvine, California,5Veterans Affairs Medical Center, Long Beach, California
| | - Padmanabhan Srinivasan
- 1Department of Physiology/Biophysics, University of California, Irvine, California,5Veterans Affairs Medical Center, Long Beach, California
| | - Saleh Al-Juburi
- 1Department of Physiology/Biophysics, University of California, Irvine, California
| | - Quang Pham
- 1Department of Physiology/Biophysics, University of California, Irvine, California
| | - Brian D. Chu
- 1Department of Physiology/Biophysics, University of California, Irvine, California,5Veterans Affairs Medical Center, Long Beach, California
| | - Rita D. Simoes
- 3Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, Missouri
| | - James M. Fleckenstein
- 3Department of Medicine, Division of Infectious Diseases, Washington University School of Medicine, St. Louis, Missouri,4Veterans Affairs Medical Center, St. Louis Missouri
| | - Hamid M. Said
- 1Department of Physiology/Biophysics, University of California, Irvine, California,2Department of Medicine, University of California, Irvine, California,5Veterans Affairs Medical Center, Long Beach, California
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Teafatiller T, Heskett CW, Agrawal A, Marchant JS, Baulch JE, Acharya MM, Subramanian VS. Upregulation of Vitamin C Transporter Functional Expression in 5xFAD Mouse Intestine. Nutrients 2021; 13:nu13020617. [PMID: 33672967 PMCID: PMC7918291 DOI: 10.3390/nu13020617] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/14/2020] [Revised: 02/05/2021] [Accepted: 02/11/2021] [Indexed: 01/14/2023] Open
Abstract
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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Affiliation(s)
- Trevor Teafatiller
- Department of Medicine, University of California, Irvine, CA 92697, USA; (T.T.); (C.W.H.); (A.A.)
| | - Christopher W. Heskett
- Department of Medicine, University of California, Irvine, CA 92697, USA; (T.T.); (C.W.H.); (A.A.)
| | - Anshu Agrawal
- Department of Medicine, University of California, Irvine, CA 92697, USA; (T.T.); (C.W.H.); (A.A.)
| | - Jonathan S. Marchant
- Department of Cell Biology, Neurobiology, and Anatomy, Medical College of Wisconsin, Milwaukee, WI 53226, USA;
| | - Janet E. Baulch
- Department of Radiation Oncology, University of California, Irvine, CA 92697, USA; (J.E.B.); (M.M.A.)
| | - Munjal M. Acharya
- Department of Radiation Oncology, University of California, Irvine, CA 92697, USA; (J.E.B.); (M.M.A.)
| | - Veedamali S. Subramanian
- Department of Medicine, University of California, Irvine, CA 92697, USA; (T.T.); (C.W.H.); (A.A.)
- Correspondence: ; Tel.: +1-949-824-3084
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Yan C, Liang N, Li Q, Yan P, Sun S. Biotin and arginine modified hydroxypropyl-β-cyclodextrin nanoparticles as novel drug delivery systems for paclitaxel. Carbohydr Polym 2019; 216:129-139. [DOI: 10.1016/j.carbpol.2019.04.024] [Citation(s) in RCA: 36] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2018] [Revised: 03/27/2019] [Accepted: 04/05/2019] [Indexed: 12/16/2022]
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Sodium Butyrate Enhances Intestinal Riboflavin Uptake via Induction of Expression of Riboflavin Transporter-3 (RFVT3). Dig Dis Sci 2019; 64:84-92. [PMID: 30276569 PMCID: PMC6320279 DOI: 10.1007/s10620-018-5305-z] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/06/2018] [Accepted: 09/24/2018] [Indexed: 02/06/2023]
Abstract
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. 3H-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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Abstract
Nine compounds are classified as water-soluble vitamins, eight B vitamins and one vitamin C. The vitamins are mandatory for the function of numerous enzymes and lack of one or more of the vitamins may lead to severe medical conditions. All the vitamins are supplied by food in microgram to milligram quantities and in addition some of the vitamins are synthesized by the intestinal microbiota. In the gastrointestinal tract, the vitamins are liberated from binding proteins and for some of the vitamins modified prior to absorption. Due to their solubility in water, they all require specific carriers to be absorbed. Our current knowledge concerning each of the vitamins differs in depth and focus and is influenced by the prevalence of conditions and diseases related to lack of the individual vitamin. Because of that we have chosen to cover slightly different aspects for the individual vitamins. For each of the vitamins, we summarize the physiological role, the steps involved in the absorption, and the factors influencing the absorption. In addition, for some of the vitamins, the molecular base for absorption is described in details, while for others new aspects of relevance for human deficiency are included. © 2018 American Physiological Society. Compr Physiol 8:1291-1311, 2018.
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Affiliation(s)
- Hamid M Said
- University of California-School of Medicine, Irvine, California, USA.,VA Medical Center, Long Beach, California, USA
| | - Ebba Nexo
- Department of Clinical Medicine, Clinical Biochemistry, Aarhus University Hospital, Aarhus, Denmark
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Sabui S, Subramanian VS, Kapadia R, Said HM. Adaptive regulation of pancreatic acinar mitochondrial thiamin pyrophosphate uptake process: possible involvement of epigenetic mechanism(s). Am J Physiol Gastrointest Liver Physiol 2017; 313:G448-G455. [PMID: 28729247 PMCID: PMC5792211 DOI: 10.1152/ajpgi.00192.2017] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/19/2017] [Revised: 07/14/2017] [Accepted: 07/14/2017] [Indexed: 01/31/2023]
Abstract
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 SLC25A19 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 SLC25A19 promoter. We found that maintaining PAC 266-6 under the thiamin-DEF condition leads to a significant induction in mitochondrial [3H]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 SLC25A19 promoter and transgenic mice carrying the SLC25A19 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).NEW & NOTEWORTHY 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 SLC25A19 promoter.
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Affiliation(s)
- Subrata Sabui
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
| | - Veedamali S. Subramanian
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
| | - Rubina Kapadia
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
| | - Hamid M. Said
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
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8
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Sabui S, Bohl JA, Kapadia R, Cogburn K, Ghosal A, Lambrecht NW, Said HM. Role of the sodium-dependent multivitamin transporter (SMVT) in the maintenance of intestinal mucosal integrity. Am J Physiol Gastrointest Liver Physiol 2016; 311:G561-70. [PMID: 27492331 PMCID: PMC5076003 DOI: 10.1152/ajpgi.00240.2016] [Citation(s) in RCA: 21] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/30/2016] [Accepted: 07/25/2016] [Indexed: 02/07/2023]
Abstract
Utilizing a conditional (intestinal-specific) knockout (cKO) mouse model, we have recently shown that the sodium-dependent multivitamin transporter (SMVT) (SLC5A6) is the only biotin uptake system that operates in the gut and that its deletion leads to biotin deficiency. Unexpectedly, we also observed that all SMVT-cKO mice develop chronic active inflammation, especially in the cecum. Our aim here was to examine the role of SMVT in the maintenance of intestinal mucosal integrity [permeability and expression of tight junction (TJ) proteins]. Our results showed that knocking out the mouse intestinal SMVT is associated with a significant increase in gut permeability and with changes in the level of expression of TJ proteins. To determine whether these changes are related to the state of biotin deficiency that develops in SMVT-cKO mice, we induced (by dietary means) biotin deficiency in wild-type mice and examined its effect on the above-mentioned parameters. The results showed that dietary-induced biotin deficiency leads to a similar development of chronic active inflammation in the cecum with an increase in the level of expression of proinflammatory cytokines, as well as an increase in intestinal permeability and changes in the level of expression of TJ proteins. We also examined the effect of chronic biotin deficiency on permeability and expression of TJ proteins in confluent intestinal epithelial Caco-2 monolayers but observed no changes in these parameters. These results show that the intestinal SMVT plays an important role in the maintenance of normal mucosal integrity, most likely via its role in providing biotin to different cells of the gut mucosa.
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Affiliation(s)
- Subrata Sabui
- 1Department of Medical Research, VA Medical Center, Long Beach, California; ,2Departments of Medicine, University of California, Irvine, California; ,3Department of Physiology/Biophysics, University of California, Irvine, California
| | - Jennifer Ann Bohl
- 1Department of Medical Research, VA Medical Center, Long Beach, California; ,2Departments of Medicine, University of California, Irvine, California; ,3Department of Physiology/Biophysics, University of California, Irvine, California
| | - Rubina Kapadia
- 1Department of Medical Research, VA Medical Center, Long Beach, California; ,2Departments of Medicine, University of California, Irvine, California; ,3Department of Physiology/Biophysics, University of California, Irvine, California
| | - Kyle Cogburn
- 1Department of Medical Research, VA Medical Center, Long Beach, California; ,2Departments of Medicine, University of California, Irvine, California; ,3Department of Physiology/Biophysics, University of California, Irvine, California
| | - Abhisek Ghosal
- 1Department of Medical Research, VA Medical Center, Long Beach, California; ,2Departments of Medicine, University of California, Irvine, California; ,3Department of Physiology/Biophysics, University of California, Irvine, California
| | - Nils W. Lambrecht
- 1Department of Medical Research, VA Medical Center, Long Beach, California; ,2Departments of Medicine, University of California, Irvine, California; ,3Department of Physiology/Biophysics, University of California, Irvine, California
| | - Hamid M. Said
- 1Department of Medical Research, VA Medical Center, Long Beach, California; ,2Departments of Medicine, University of California, Irvine, California; ,3Department of Physiology/Biophysics, University of California, Irvine, California
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Cheng Y, El-Kattan A, Zhang Y, Ray AS, Lai Y. Involvement of Drug Transporters in Organ Toxicity: The Fundamental Basis of Drug Discovery and Development. Chem Res Toxicol 2016; 29:545-63. [DOI: 10.1021/acs.chemrestox.5b00511] [Citation(s) in RCA: 16] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/27/2022]
Affiliation(s)
- Yaofeng Cheng
- Pharmaceutical
Candidate Optimization, Bristol-Myers Squibb Company, 3551 Lawrenceville
Road, Princeton, New Jersey 08540, United States
| | - Ayman El-Kattan
- Department
of Pharmacokinetics, Dynamics and Metabolism, Pfizer Inc., 610 Main
Street, Cambridge, Massachusetts 02139, United States
| | - Yan Zhang
- Drug
Metabolism and Biopharmaceutics, Incyte Corporation, 1801 Augustine
Cutoff, Wilmington, Delaware 19803, United States
| | - Adrian S. Ray
- Department
of Drug Metabolism, Gilead Sciences, Inc., 333 Lakeside Drive, Foster City, California 94404, United States
| | - Yurong Lai
- Pharmaceutical
Candidate Optimization, Bristol-Myers Squibb Company, 3551 Lawrenceville
Road, Princeton, New Jersey 08540, United States
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Nabokina SM, Ramos MB, Said HM. Mechanism(S) Involved in the Colon-Specific Expression of the Thiamine Pyrophosphate (Tpp) Transporter. PLoS One 2016; 11:e0149255. [PMID: 26901654 PMCID: PMC4764741 DOI: 10.1371/journal.pone.0149255] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/25/2015] [Accepted: 01/30/2016] [Indexed: 12/16/2022] Open
Abstract
Microbiota of the large intestine synthesizes considerable amount of vitamin B1 (thiamine) in the form of thiamine pyrophosphate (TPP). We have recently demonstrated the existence of an efficient and specific carrier-mediated uptake process for TPP in human colonocytes, identified the TPP transporter (TPPT) involved (product of the SLC44A4 gene), and shown that expression of TPPT along the gastrointestinal (GI) tract is restricted to the colon. Our aim in this study was to determine the molecular basis of the colon-specific expression of TPPT focusing on a possible epigenetic mechanism. Our results showed that the CpG island predicted in the SLC44A4 promoter is non-methylated in the human colonic epithelial NCM460 cells, but is hyper-methylated in the human duodenal epithelial HuTu80 cells (as well as in the human retinal pigment epithelial ARPE19 cells). In the mouse (where TPPT expression in the GI tract is also restricted to the colon), the CpG island predicted in the Slc44a4 promoter is non-methylated in both the jejunum and colon, thus arguing against possible contribution of DNA methylation in the colon-specific expression of TPPT. A role for histone modifications in the tissue-specific pattern of Slc44a4 expression, however, was suggested by the findings that in mouse colon, histone H3 in the 5’-regulatory region of Slc44a4 is tri-methylated at lysine 4 and acetylated at lysine 9, whereas the tri-methylation at lysine 27 modification was negligible. In contrast, in the mouse jejunum, histone H3 is hyper-trimethylated at lysine 27 (repressor mark). Similarly, possible involvement of miRNA(s) in the tissue-specific expression of TPPT was also suggested by the findings that the 3’-UTR of SLC44A4 is targeted by specific miRNAs/RNA binding proteins in non-colonic, but not in colonic, epithelial cells. These studies show, for the first time, epigenetic mechanisms (histone modifications) play a role in determining the tissue-specific pattern of expression of TPPT in the GI tract.
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Affiliation(s)
- Svetlana M. Nabokina
- Departments of Medicine, Physiology/Biophysics, University of California Irvine, Irvine, CA, 92697, United States of America
| | - Mel Brendan Ramos
- Departments of Medicine, Physiology/Biophysics, University of California Irvine, Irvine, CA, 92697, United States of America
| | - Hamid M. Said
- Departments of Medicine, Physiology/Biophysics, University of California Irvine, Irvine, CA, 92697, United States of America
- Department of Veterans Affairs Medical Center, Long Beach, CA, 90822, United States of America
- * E-mail:
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11
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Quick M, Shi L. The sodium/multivitamin transporter: a multipotent system with therapeutic implications. VITAMINS AND HORMONES 2015; 98:63-100. [PMID: 25817866 PMCID: PMC5530880 DOI: 10.1016/bs.vh.2014.12.003] [Citation(s) in RCA: 34] [Impact Index Per Article: 3.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/11/2023]
Abstract
The Na(+)/multivitamin transporter (SMVT) is a member of the solute:sodium symporter family that catalyzes the Na(+)-dependent uptake of the structurally diverse water-soluble vitamins pantothenic acid (vitamin B5) and biotin (vitamin H), α-lipoic acid-a vitamin-like substance with strong antioxidant properties-and iodide. The organic substrates of SMVT play central roles in the cellular metabolism and are, therefore, essential for normal human health and development. For example, biotin deficiency leads to growth retardation, dermatological disorders, and neurological disorders. Animal studies have shown that biotin deficiency during pregnancy is directly correlated to embryonic growth retardation, congenital malformation, and death of the embryo. This chapter focuses on the structural and functional features of the human isoform of SMVT (hSMVT); the discovery of which was greatly facilitated by the cloning and expression of hSMVT in tractable expression systems. Special emphasis will be given to mechanistic implications of the transport process of hSMVT that will inform our understanding of the molecular determinants of hSMVT-mediated transport in dynamic context to alleviate the development and optimization of hSMVT as a multipotent platform for drug delivery.
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Affiliation(s)
- Matthias Quick
- Department of Psychiatry, Division of Molecular Therapeutics, Columbia University College of Physicians and Surgeons, New York State Psychiatric Institute, New York, USA.
| | - Lei Shi
- Department of Physiology and Biophysics, Institute for Computational Biomedicine, Weill Medical College of Cornell University, New York, USA
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12
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Srinivasan P, Kapadia R, Biswas A, Said HM. Chronic alcohol exposure inhibits biotin uptake by pancreatic acinar cells: possible involvement of epigenetic mechanisms. Am J Physiol Gastrointest Liver Physiol 2014; 307:G941-9. [PMID: 25214397 PMCID: PMC4250263 DOI: 10.1152/ajpgi.00278.2014] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/30/2014] [Accepted: 09/04/2014] [Indexed: 01/31/2023]
Abstract
Chronic exposure to alcohol affects different physiological aspects of pancreatic acinar cells (PAC), but its effect on the uptake process of biotin is not known. We addressed this issue using mouse-derived pancreatic acinar 266-6 cells chronically exposed to alcohol and wild-type and transgenic mice (carrying the human SLC5A6 5'-promoter) fed alcohol chronically. First we established that biotin uptake by PAC is Na(+) dependent and carrier mediated and involves sodium-dependent multivitamin transporter (SMVT). Chronic exposure of 266-6 cells to alcohol led to a significant inhibition in biotin uptake, expression of SMVT protein, and mRNA as well as in the activity of the SLC5A6 promoter. Similarly, chronic alcohol feeding of wild-type and transgenic mice carrying the SLC5A6 promoter led to a significant inhibition in biotin uptake by PAC, as well as in the expression of SMVT protein and mRNA and the activity of the SLC5A6 promoters expressed in the transgenic mice. We also found that chronic alcohol feeding of mice is associated with a significant increase in the methylation status of CpG islands predicted to be in the mouse Slc5a6 promoters and a decrease in the level of expression of transcription factor KLF-4, which plays an important role in regulating SLC5A6 promoter activity. These results demonstrate, for the first time, that chronic alcohol exposure negatively impacts biotin uptake in PAC and that this effect is exerted (at least in part) at the level of transcription of the SLC5A6 gene and may involve epigenetic/molecular mechanisms.
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Affiliation(s)
- Padmanabhan Srinivasan
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
| | - Rubina Kapadia
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
| | - Arundhati Biswas
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
| | - Hamid M Said
- Department of Medical Research, Veterans Affairs Medical Center, Long Beach, California; and Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California
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Ghosal A, Sekar TV, Said HM. Biotin uptake by mouse and human pancreatic beta cells/islets: a regulated, lipopolysaccharide-sensitive carrier-mediated process. Am J Physiol Gastrointest Liver Physiol 2014; 307:G365-73. [PMID: 24904078 PMCID: PMC4121639 DOI: 10.1152/ajpgi.00157.2014] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Biotin is essential for the normal function of pancreatic beta cells. These cells obtain biotin from their surroundings via transport across their cell membrane. Little is known about the uptake mechanism involved, how it is regulated, and how it is affected by internal and external factors. We addressed these issues using the mouse-derived pancreatic beta-TC-6 cells and freshly isolated mouse and human primary pancreatic beta cells as models. The results showed biotin uptake by pancreatic beta-TC-6 cells occurs via a Na(+)-dependent, carrier-mediated process, that is sensitive to desthiobiotin, as well as to pantothenic acid and lipoate; the process is also saturable as a function of concentration (apparent Km = 22.24 ± 5.5 μM). These cells express the sodium-dependent multivitamin transporter (SMVT), whose knockdown (with doxycycline-inducible shRNA) led to a sever inhibition in biotin uptake. Similarly, uptake of biotin by mouse and human primary pancreatic islets is Na(+)-dependent and carrier-mediated, and both cell types express SMVT. Biotin uptake by pancreatic beta-TC-6 cells is also adaptively regulated (via transcriptional mechanism) by extracellular substrate level. Chronic treatment of pancreatic beta-TC-6 cells with bacterial lipopolysaccharides (LPS) leads to inhibition in biotin uptake. This inhibition is mediated via a Toll-Like receptor 4-mediated process and involves a decrease in membrane expression of SMVT. These findings show, for the first time, that pancreatic beta cells/islets take up biotin via a specific and regulated carrier-mediated process, and that the process is sensitive to the effect of LPS.
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Affiliation(s)
- Abhisek Ghosal
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California
| | - Thillai V. Sekar
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California
| | - Hamid M. Said
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California; and Department of Veterans Affairs Medical Center, Long Beach, California
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14
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Nabokina SM, Inoue K, Subramanian VS, Valle JE, Yuasa H, Said HM. Molecular identification and functional characterization of the human colonic thiamine pyrophosphate transporter. J Biol Chem 2013; 289:4405-16. [PMID: 24379411 DOI: 10.1074/jbc.m113.528257] [Citation(s) in RCA: 54] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023] Open
Abstract
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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Affiliation(s)
- Svetlana M Nabokina
- From the Departments of Medicine and Physiology/Biophysics, University of California, Irvine, California 92697
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15
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Moradi H, Said HM, Vaziri ND. Post-transcriptional nature of uremia-induced downregulation of hepatic apolipoprotein A-I production. Transl Res 2013; 161:477-85. [PMID: 23219399 PMCID: PMC3609941 DOI: 10.1016/j.trsl.2012.11.001] [Citation(s) in RCA: 12] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 07/25/2012] [Revised: 11/06/2012] [Accepted: 11/08/2012] [Indexed: 01/17/2023]
Abstract
Chronic kidney disease is associated with premature death from cardiovascular disease, which is, in part, driven by high density lipoprotein deficiency and dysfunction. One of the main causes of high density lipoprotein deficiency in chronic kidney disease is diminished plasma apolipoprotein (Apo)A-I level. Plasma ApoA-I is reduced in dialysis patients and hepatic ApoA-I messenger RNA (mRNA) is decreased in the uremic rats. This study explored the mechanism of uremia-induced downregulation of ApoA-I. Human hepatoma derived cells were incubated in media containing whole plasma or plasma subfractionation from normal subjects and patients with end stage renal disease pre- and posthemodialysis. Cells and culture media were isolated to measure ApoA-I protein and mRNA. ApoA-I promoter activity was measured using transfection with a luciferase promoter construct containing the -2096 to +293 segment of ApoA-I gene. Finally, effect of uremic and control plasma was assessed on ApoA-I RNA stability. Exposure to uremic plasma significantly reduced ApoA-I mRNA expression and ApoA-I protein production. These effects were reversed by replacing uremic plasma with normal plasma. Although no difference in ApoA-I promoter activity was found between cells exposed to uremic and normal plasma, uremic plasma significantly reduced ApoA-I RNA stability. Experiments using plasma subfractions revealed that the inhibitory effect of uremic plasma on ApoA-I mRNA expression resides in fractions containing molecules larger but not smaller than 30 kd. The pre- and postdialysis plasma exerted an equally potent inhibitory effect on ApoA-I mRNA abundance. Uremia lowers ApoA-I production by reducing its RNA stability. The inhibitory effect of uremic milieu on ApoA-I mRNA expression is mediated by non-dialyzable molecule(s) larger than 30 kd.
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Affiliation(s)
- Hamid Moradi
- Division of Nephrology and Hypertension, University of California, Irvine, Irvine, CA 92697, USA
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16
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Ghosal A, Lambrecht N, Subramanya SB, Kapadia R, Said HM. Conditional knockout of the Slc5a6 gene in mouse intestine impairs biotin absorption. Am J Physiol Gastrointest Liver Physiol 2013; 304:G64-71. [PMID: 23104561 PMCID: PMC3543636 DOI: 10.1152/ajpgi.00379.2012] [Citation(s) in RCA: 49] [Impact Index Per Article: 4.1] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 09/26/2012] [Accepted: 10/19/2012] [Indexed: 01/31/2023]
Abstract
The Slc5a6 gene expresses a plasma membrane protein involved in the transport of the water-soluble vitamin biotin; the transporter is commonly referred to as the sodium-dependent multivitamin transporter (SMVT) because it also transports pantothenic acid and lipoic acid. The relative contribution of the SMVT system toward carrier-mediated biotin uptake in the native intestine in vivo has not been established. We used a Cre/lox technology to generate an intestine-specific (conditional) SMVT knockout (KO) mouse model to address this issue. The KO mice exhibited absence of expression of SMVT in the intestine compared with sex-matched littermates as well as the expected normal SMVT expression in other tissues. About two-thirds of the KO mice died prematurely between the age of 6 and 10 wk. Growth retardation, decreased bone density, decreased bone length, and decreased biotin status were observed in the KO mice. Microscopic analysis showed histological abnormalities in the small bowel (shortened villi, dysplasia) and cecum (chronic active inflammation, dysplasia) of the KO mice. In vivo (and in vitro) transport studies showed complete inhibition in carrier-mediated biotin uptake in the intestine of the KO mice compared with their control littermates. These studies provide the first in vivo confirmation in native intestine that SMVT is solely responsible for intestinal biotin uptake. These studies also provide evidence for a casual association between SMVT function and normal intestinal health.
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Affiliation(s)
- Abhisek Ghosal
- Department of Medicine, University of California, School of Medicine, Irvine, CA, USA
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17
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Araújo F, Fonte P, Santos HA, Sarmento B. Oral delivery of glucagon-like peptide-1 and analogs: alternatives for diabetes control? J Diabetes Sci Technol 2012; 6:1486-97. [PMID: 23294796 PMCID: PMC3570891 DOI: 10.1177/193229681200600630] [Citation(s) in RCA: 38] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 12/16/2022]
Abstract
Type 2 diabetes mellitus (T2DM) is one of the most prevalent diseases worldwide. Current treatments are often associated with off-target effects and do not significantly impact disease progression. New therapies are therefore urgently needed to overcome this social burden. Glucagon-like peptide-1 (GLP-1), an incretin hormone, has been used to control T2DM symptomatology. However, the administration of peptide or proteins drugs is still a huge challenge in the pharmaceutical field, requiring administration by parenteral routes. This article reviews the main hurdles in oral administration of GLP-1 and focuses on the strategies utilized to overcome them.
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Affiliation(s)
- Francisca Araújo
- Department of Pharmaceutical Sciences, Centro de Investigação em Ciências da Saúde, Health Sciences Research Center,Instituto Superior de Ciências da Saúde, CESPU, Gandra, Portugal
- Instituto de Engenharia Biomédica, University of Porto, Porto, Portugal
| | - Pedro Fonte
- Department of Pharmaceutical Sciences, Centro de Investigação em Ciências da Saúde, Health Sciences Research Center,Instituto Superior de Ciências da Saúde, CESPU, Gandra, Portugal
- Department of Chemistry, Faculty of Pharmacy, REQUIMTE, University of Porto, Porto, Portugal
| | - Hélder A. Santos
- Division of Pharmaceutical Technology, Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
| | - Bruno Sarmento
- Department of Pharmaceutical Sciences, Centro de Investigação em Ciências da Saúde, Health Sciences Research Center,Instituto Superior de Ciências da Saúde, CESPU, Gandra, Portugal
- Instituto de Engenharia Biomédica, University of Porto, Porto, Portugal
- Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Porto, Porto, Portugal
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18
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Thomson ABR, Chopra A, Clandinin MT, Freeman H. Recent advances in small bowel diseases: Part II. World J Gastroenterol 2012; 18:3353-74. [PMID: 22807605 PMCID: PMC3396188 DOI: 10.3748/wjg.v18.i26.3353] [Citation(s) in RCA: 12] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/06/2011] [Revised: 04/05/2012] [Accepted: 04/13/2012] [Indexed: 02/06/2023] Open
Abstract
As is the case in all areas of gastroenterology and hepatology, in 2009 and 2010 there were many advances in our knowledge and understanding of small intestinal diseases. Over 1000 publications were reviewed, and the important advances in basic science as well as clinical applications were considered. In Part II we review six topics: absorption, short bowel syndrome, smooth muscle function and intestinal motility, tumors, diagnostic imaging, and cystic fibrosis.
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Nabokina SM, Subramanian VS, Said HM. Effect of clinical mutations on functionality of the human riboflavin transporter-2 (hRFT-2). Mol Genet Metab 2012; 105:652-7. [PMID: 22273710 PMCID: PMC3309148 DOI: 10.1016/j.ymgme.2011.12.021] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/01/2011] [Revised: 12/27/2011] [Accepted: 12/27/2011] [Indexed: 10/14/2022]
Abstract
The Brown-Vialetto-Van Laere syndrome (BVVLS) is a rare neurological disease characterized by ponto-bulbar palsy, bilateral sensorineural deafness, and respiratory insufficiency. Recent genetic studies have identified mutations in the C20orf54 gene, which encodes the human riboflavin (RF) transporter -2 (hRFT-2) and suggested their link to the manifestation of BVVLS. However, there is nothing currently known about the effect of these mutations on functionality of hRFT-2, a protein that is expressed in a variety of tissues with high expression in the intestine. We addressed this issue using the human-derived intestinal epithelial Caco-2 cells. Our results showed significant (P<0.01) impairment in RF uptake by Caco-2 cells transiently expressing W17R, P28T, E36K, E71K, and R132W (but not L350M) hRFT-2 mutants. This impairment in RF transport was not due to a decrease in transcription and/or translation of hRFT-2, since mRNA and protein levels of the carrier were similar in cells expressing the mutants and wild-type hRFT-2. Confocal images of live Caco-2 cells transiently transfected with hRFT-2 mutants (fused with green fluorescent protein) showed the P28T, E36K, E71K, and R132W mutants were retained within the endoplasmic reticulum, while the W17R and L350M mutants were expressed at the cell membrane; cell surface expression of the W17R mutant was further confirmed by direct determination of cell surface transporter density. These results show for the first time that some of the BVVLS associated mutations in hRFT-2 affect the transporter functionality and that this effect is mediated via alterations in membrane targeting and/or activity of the transporter.
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Affiliation(s)
- Svetlana M Nabokina
- Departments of Medicine, Physiology/Biophysics, University of California, Irvine, CA 92697, USA
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20
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Ghosal A, Said HM. Cys(294) is essential for the function of the human sodium-dependent multivitamin transporter. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2011; 1818:97-102. [PMID: 22015582 DOI: 10.1016/j.bbamem.2011.10.003] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 08/04/2011] [Revised: 09/27/2011] [Accepted: 10/03/2011] [Indexed: 01/04/2023]
Abstract
The sodium-dependent multivitamin transporter (SMVT) plays an important role in biotin uptake in the intestine and other cell types. While significant knowledge has been gained with regard to regulation and cell biology of the SMVT system, there is little known about its structure-function relationships. Here we examined the role of each of the ten conserved (among species) cysteine residues in the function of the human SMVT (hSMVT) using site-directed mutagenesis. Our results showed a significant impairment in biotin uptake only in cells transfected with hSMVT mutated at Cys(294), but not at the other conserved cysteine residues; the impairment in biotin uptake caused by mutating Cys(294) was not related to the polar status of substituting amino acid. The inhibition in hSMVT function upon mutating Cys(294) was mediated via a significant reduction in the V(max), but not the apparent K(m), of the biotin uptake process, suggesting a decrease in the number (and/or activity) of hSMVT but not affinity. Biotinylation assay confirmed this suggestion by showing a marked reduction in the level of expression of the mutated protein at the cell membrane, without affecting total cellular level of induced hSMVT. These results show an important role for Cys(294) in the function and cell biology of hSMVT.
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Affiliation(s)
- Abhisek Ghosal
- Department of Medicine, University of California, Irvine, CA 92697, USA
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21
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Abstract
Our knowledge of the mechanisms and regulation of intestinal absorption of water-soluble vitamins under normal physiological conditions, and of the factors/conditions that affect and interfere with theses processes has been significantly expanded in recent years as a result of the availability of a host of valuable molecular/cellular tools. Although structurally and functionally unrelated, the water-soluble vitamins share the feature of being essential for normal cellular functions, growth and development, and that their deficiency leads to a variety of clinical abnormalities that range from anaemia to growth retardation and neurological disorders. Humans cannot synthesize water-soluble vitamins (with the exception of some endogenous synthesis of niacin) and must obtain these micronutrients from exogenous sources. Thus body homoeostasis of these micronutrients depends on their normal absorption in the intestine. Interference with absorption, which occurs in a variety of conditions (e.g. congenital defects in the digestive or absorptive system, intestinal disease/resection, drug interaction and chronic alcohol use), leads to the development of deficiency (and sub-optimal status) and results in clinical abnormalities. It is well established now that intestinal absorption of the water-soluble vitamins ascorbate, biotin, folate, niacin, pantothenic acid, pyridoxine, riboflavin and thiamin is via specific carrier-mediated processes. These processes are regulated by a variety of factors and conditions, and the regulation involves transcriptional and/or post-transcriptional mechanisms. Also well recognized now is the fact that the large intestine possesses specific and efficient uptake systems to absorb a number of water-soluble vitamins that are synthesized by the normal microflora. This source may contribute to total body vitamin nutrition, and especially towards the cellular nutrition and health of the local colonocytes. The present review aims to outline our current understanding of the mechanisms involved in intestinal absorption of water-soluble vitamins, their regulation, the cell biology of the carriers involved and the factors that negatively affect these absorptive events.
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Affiliation(s)
- Hamid M Said
- School of Medicine, University of California-Irvine, Irvine, CA 92697, USA.
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22
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Ghosal A, Subramanian VS, Said HM. Role of the putative N-glycosylation and PKC-phosphorylation sites of the human sodium-dependent multivitamin transporter (hSMVT) in function and regulation. BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES 2011; 1808:2073-80. [PMID: 21570947 DOI: 10.1016/j.bbamem.2011.04.014] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/07/2011] [Revised: 04/16/2011] [Accepted: 04/22/2011] [Indexed: 10/18/2022]
Abstract
The sodium-dependent multivitamin transporter (SMVT) is a major biotin transporter in a variety of tissues including the small intestine. The human SMVT (hSMVT) polypeptide is predicted to have four N-glycosylation sites and two putative PKC phosphorylation sites but their role in the function and regulation of the protein is not known and was examined in this investigation. Our results showed that the hSMVT protein is glycosylated and that this glycosylation is important for its function. Studies utilizing site-directed mutagenesis revealed that the N-glycosylation sites at positions Asn(138) and Asn(489) are important for the function of hSMVT and that mutating these sites significantly reduces the V(max) of the biotin uptake process. Mutating the putative PKC phosphorylation site Thr(286) of hSMVT led to a significant decrease in the PMA-induced inhibition in biotin uptake. The latter effect was not mediated via changes in the level of expression of the hSMVT protein and mRNA or in its level of expression at the cell membrane. These findings demonstrate that the hSMVT protein is glycosylated, and that glycosylation is important for its function. Furthermore, the study shows a role for the putative PKC-phosphorylation site Thr(286) of hSMVT in the PKC-mediated regulation of biotin uptake.
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Affiliation(s)
- Abhisek Ghosal
- Department of Medicine and Physiology/Biophysics, University of California, Irvine, CA 92697, USA
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Nabokina SM, Subramanian VS, Said HM. Association of PDZ-containing protein PDZD11 with the human sodium-dependent multivitamin transporter. Am J Physiol Gastrointest Liver Physiol 2011; 300:G561-7. [PMID: 21183659 PMCID: PMC3074986 DOI: 10.1152/ajpgi.00530.2010] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Intestinal absorption of biotin is mediated via the sodium-dependent multivitamin transporter (SMVT). Studies from our laboratory and others have characterized different aspects of the human SMVT (hSMVT), but nothing is currently known about protein(s) that may interact with hSMVT and affect its physiology/biology. In this study, a PDZ-containing protein PDZD11 was identified as an interacting partner with hSMVT using yeast two-hybrid screen of a human intestinal cDNA library. The interaction between hSMVT and PDZD11 was confirmed by in vitro GST-pull-down assay and in vivo in a mammalian cell environment by a two-hybrid luciferase and coimmunoprecipitation assays. Furthermore, confocal imaging of live human intestinal epithelial HuTu-80 cells expressing hSMVT-GFP and DsRed-PDZD11 demonstrated colocalization of these two proteins. We also examined the functional consequence of the interaction between hSMVT and PDZD11 in HuTu-80 cells and observed significant induction in [(3)H]biotin uptake upon coexpression of hSMVT and PDZD11. In contrast, knocking down of PDZD11 with gene-specific small interfering RNA led to a significant decrease in biotin uptake; biotinylation assay showed this to be associated with a marked decrease in level of expression of hSMVT at the cell membrane. By truncation approach, we also demonstrated that the PDZ binding domain that is located in the COOH-terminal tail of hSMVT polypeptide is involved in the interaction with PDZD11. These results demonstrate for the first time that PDZD11 is an interacting partner with hSMVT in intestinal epithelial cells and that this interaction affects hSMVT function and cell biology.
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Affiliation(s)
- Svetlana M. Nabokina
- Departments of Medicine, Physiology/Biophysics, University of California, Irvine, and the Department of Veterans Affairs Medical Center, Long Beach, California
| | - Veedamali S. Subramanian
- Departments of Medicine, Physiology/Biophysics, University of California, Irvine, and the Department of Veterans Affairs Medical Center, Long Beach, California
| | - Hamid M. Said
- Departments of Medicine, Physiology/Biophysics, University of California, Irvine, and the Department of Veterans Affairs Medical Center, Long Beach, California
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Subramanya SB, Subramanian VS, Kumar JS, Hoiness R, Said HM. Inhibition of intestinal biotin absorption by chronic alcohol feeding: cellular and molecular mechanisms. Am J Physiol Gastrointest Liver Physiol 2011; 300:G494-501. [PMID: 21148397 PMCID: PMC3064116 DOI: 10.1152/ajpgi.00465.2010] [Citation(s) in RCA: 23] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
The water-soluble vitamin biotin is essential for normal cellular functions and its deficiency leads to a variety of clinical abnormalities. Mammals obtain biotin from exogenous sources via intestinal absorption, a process mediated by the sodium-dependent multivitamin transporter (SMVT). Chronic alcohol use in humans is associated with a significant reduction in plasma biotin levels, and animal studies have shown inhibition in intestinal biotin absorption by chronic alcohol feeding. Little, however, is known about the cellular and molecular mechanisms involved in the inhibition in intestinal biotin transport by chronic alcohol use. These mechanisms were investigated in this study by using rats and transgenic mice carrying the human full-length SLC5A6 5'-regulatory region chronically fed alcohol liquid diets; human intestinal epithelial Caco-2 cells chronically exposed to alcohol were also used as models. The results showed chronic alcohol feeding of rats to lead to a significant inhibition in carrier-mediated biotin transport events across jejunal brush border and basolateral membrane domains. This inhibition was associated with a significant reduction in level of expression of the SMVT protein, mRNA, and heterogenous nuclear RNA. Chronic alcohol feeding also inhibited carrier-mediated biotin uptake in rat colon. Studies with transgenic mice confirmed the above findings and further showed chronic alcohol feeding significantly inhibited the activity of SLC5A6 5'-regulatory region. Finally, chronic exposure of Caco-2 cells to alcohol led to a significant decrease in the activity of both promoters P1 and P2 of the human SLC5A6 gene. These studies identify for the first time the cellular and molecular parameters of the intestinal biotin absorptive processes that are affected by chronic alcohol feeding.
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Affiliation(s)
- Sandeep B. Subramanya
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine; and Department of Veterans Affairs Medical Center, Long Beach, California
| | - Veedamali S. Subramanian
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine; and Department of Veterans Affairs Medical Center, Long Beach, California
| | - Jeyan S. Kumar
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine; and Department of Veterans Affairs Medical Center, Long Beach, California
| | - Robert Hoiness
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine; and Department of Veterans Affairs Medical Center, Long Beach, California
| | - Hamid M. Said
- Departments of Medicine and Physiology/Biophysics, University of California, Irvine; and Department of Veterans Affairs Medical Center, Long Beach, California
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25
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Ghosal A, Said HM. Structure-function activity of the human sodium-dependent multivitamin transporter: role of His¹¹⁵ and His²⁵⁴. Am J Physiol Cell Physiol 2010; 300:C97-104. [PMID: 20962270 DOI: 10.1152/ajpcell.00398.2010] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
Intestinal absorption of biotin occurs via a Na(+)-dependent carrier-mediated process that involves the sodium-dependent multivitamin transporter (SMVT; product of the Slc5a6 gene). The SMVT system is exclusively expressed at the apical membrane domain of the polarized intestinal epithelial cells. Whereas previous studies from our laboratory and others have characterized different physiological and biological aspects of SMVT, little is currently known about its structure-function activity relationship. Using site-directed mutagenesis approach, we examined the role of the positively charged histidine (His) residues of the human SMVT (hSMVT) in transporting the negatively charged biotin. Of the seven conserved (across species) His residues in the hSMVT polypeptide, only His¹¹⁵ and His²⁵⁴ were found to be important for the function of hSMVT as their mutation led to a significant reduction in carrier-mediated biotin uptake. This inhibition was mediated via a significant reduction in the maximal velocity (V(max)), but not the apparent Michaelis constant (K(m)), of the biotin uptake process and was not related to the charge of the His residue. The inhibition was also not due to changes in transcriptional or translational efficiency of the mutated hSMVT compared with wild-type carrier. However, surface biotinylation assay showed a significant reduction in the level of expression of the mutated hSMVT at the cell surface, a finding that was further confirmed by confocal imaging. Our results show important role for His¹¹⁵ and His²⁵⁴ residues in hSMVT function, which is most probably mediated via an effect on level of hSMVT expression at the cell membrane.
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Affiliation(s)
- Abhisek Ghosal
- Department of Medicine, University of California, Irvine, USA
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26
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Fujimura M, Yamamoto S, Murata T, Yasujima T, Inoue K, Ohta KY, Yuasa H. Functional characteristics of the human ortholog of riboflavin transporter 2 and riboflavin-responsive expression of its rat ortholog in the small intestine indicate its involvement in riboflavin absorption. J Nutr 2010; 140:1722-7. [PMID: 20724488 DOI: 10.3945/jn.110.128330] [Citation(s) in RCA: 52] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Riboflavin transporter (RFT) 2 has recently been identified as a transporter that may be, mainly based on the functional characteristics of its rat ortholog (rRFT2), involved in the intestinal absorption of riboflavin. The present study was conducted to further examine such a possible role of RFT2, focusing on the functional characteristics of its human ortholog (hRFT2) and the response of rRFT2 expression in the small intestine to deprivation of dietary riboflavin. When transiently expressed in human embryonic kidney 293 cells, hRFT2 could transport riboflavin efficiently in a pH-sensitive manner, favoring acidic pH and without requiring Na(+). Riboflavin transport by hRFT2 was saturable with a Michaelis constant of 0.77 μmol/L at pH 6.0, and inhibited by some riboflavin derivatives, such as lumiflavin. It was also inhibited, to a lesser extent, by some cationic compounds, such as ethidium. Thus, hRFT2 was suggested to, together with a finding that its mRNA is highly expressed in the small intestine, have characteristics as an intestinal RFT. Furthermore, feeding rats a riboflavin-deficient diet caused an upregulation of the expression of rRFT2 mRNA in the small intestine, presumably as an adaptive response to enhance riboflavin absorption, which would involve rRFT2, and its apically localized characteristic was suggested by the observation of rRFT2 tagged with green fluorescent protein stably expressed in polarized Madin-Darby canine kidney II cells. All these results combined indicate that RFT2 is a transporter involved in the epithelial uptake of riboflavin in the small intestine for its nutritional utilization.
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Affiliation(s)
- Misaki Fujimura
- Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Nagoya City University, Nagoya 467-8603, Japan
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Boudry G, David ES, Douard V, Monteiro IM, Le Huërou-Luron I, Ferraris RP. Role of intestinal transporters in neonatal nutrition: carbohydrates, proteins, lipids, minerals, and vitamins. J Pediatr Gastroenterol Nutr 2010; 51:380-401. [PMID: 20808244 DOI: 10.1097/mpg.0b013e3181eb5ad6] [Citation(s) in RCA: 47] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
To support rapid growth and a high metabolic rate, infants require enormous amounts of nutrients. The small intestine must have the complete array of transporters that absorb the nutrients released from digested food. Failure of intestinal transporters to function properly often presents symptoms as "failure to thrive" because nutrients are not absorbed and as diarrhea because unabsorbed nutrients upset luminal osmolality or become substrates of intestinal bacteria. We enumerate the nutrients that constitute human milk and various infant milk formulas, explain their importance in neonatal nutrition, then describe for each nutrient the transporter(s) that absorbs it from the intestinal lumen into the enterocyte cytosol and from the cytosol to the portal blood. More than 100 membrane and cytosolic transporters are now thought to facilitate absorption of minerals and vitamins as well as products of digestion of the macronutrients carbohydrates, proteins, and lipids. We highlight research areas that should yield information needed to better understand the important role of these transporters during normal development.
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Affiliation(s)
- Gaëlle Boudry
- Institut National de Recherche Agronomique, UMR1079 Système d'Elevage, Nutrition, Animale et Humaine, St-Gilles, France
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Rendic S, Guengerich FP. Update information on drug metabolism systems--2009, part II: summary of information on the effects of diseases and environmental factors on human cytochrome P450 (CYP) enzymes and transporters. Curr Drug Metab 2010; 11:4-84. [PMID: 20302566 PMCID: PMC4167379 DOI: 10.2174/138920010791110917] [Citation(s) in RCA: 63] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2009] [Accepted: 02/22/2010] [Indexed: 12/14/2022]
Abstract
The present paper is an update of the data on the effects of diseases and environmental factors on the expression and/or activity of human cytochrome P450 (CYP) enzymes and transporters. The data are presented in tabular form (Tables 1 and 2) and are a continuation of previously published summaries on the effects of drugs and other chemicals on CYP enzymes (Rendic, S.; Di Carlo, F. Drug Metab. Rev., 1997, 29(1-2), 413-580., Rendic, S. Drug Metab. Rev., 2002, 34(1-2), 83-448.). The collected information presented here is as stated by the cited author(s), and in cases when several references are cited the latest published information is included. Inconsistent results and conclusions obtained by different authors are highlighted, followed by discussion of the major findings. The searchable database is available as an Excel file, for information about file availability contact the corresponding author.
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Affiliation(s)
- S Rendic
- University of Zagreb, Zagreb, Croatia.
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Subramanian VS, Marchant JS, Boulware MJ, Ma TY, Said HM. Membrane targeting and intracellular trafficking of the human sodium-dependent multivitamin transporter in polarized epithelial cells. Am J Physiol Cell Physiol 2009; 296:C663-71. [PMID: 19211916 PMCID: PMC2670647 DOI: 10.1152/ajpcell.00396.2008] [Citation(s) in RCA: 31] [Impact Index Per Article: 1.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2008] [Accepted: 02/06/2009] [Indexed: 11/22/2022]
Abstract
The human sodium-dependent multivitamin transporter (hSMVT) mediates sodium-dependent uptake of biotin in renal and intestinal epithelia. To date, however, there is nothing known about the structure-function relationship or targeting sequences in the hSMVT polypeptide that control its polarized expression within epithelia. Here, we focused on the role of the COOH-terminal tail of hSMVT in the targeting and functionality of this transporter. A full-length hSMVT-green fluorescent protein (GFP) fusion protein was functional and expressed at the apical membrane in renal and intestinal cell lines. Microtubule disrupting agents disrupted the mobility of trafficking vesicles and impaired cell surface delivery of hSMVT, which was also prevented in cells treated with dynamitin (p50), brefeldin, or monensin. Progressive truncation of the COOH-terminal tail impaired the functionality and targeting of the transporter. First, biotin transport decreased by approximately 20-30% on deletion of up to 15 COOH-terminal amino acids of hSMVT, a decrease mimicked solely by deletion of the terminal PDZ motif (TSL). Second, deletions into the COOH-terminal tail (between residues 584-612, containing a region of predicted high surface accessibility) resulted in a further drop in hSMVT transport (to approximately 40% of wild-type). Third, apical targeting was lost on deletion of a helical-prone region between amino acids 570-584. We conclude that the COOH tail of hSMVT contains several determinants important for polarized targeting and biotin transport.
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30
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Liuzzi JP, Guo L, Chang SM, Cousins RJ. Krüppel-like factor 4 regulates adaptive expression of the zinc transporter Zip4 in mouse small intestine. Am J Physiol Gastrointest Liver Physiol 2009; 296:G517-23. [PMID: 19147802 PMCID: PMC2660179 DOI: 10.1152/ajpgi.90568.2008] [Citation(s) in RCA: 49] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 01/31/2023]
Abstract
Epithelial cells of the small intestine are the site of zinc absorption. Intestinal uptake of zinc is inversely proportional to the dietary supply of this essential micronutrient. The mechanism responsible for this adaptive differential in apical zinc transport is not known. The zinc transporter Zip4 (Slc39a4) is essential for adequate enteric zinc uptake. In mice, Zip4 expression is upregulated at low zinc intakes with a concomitant ZIP4 localization to the apical enterocyte plasma membrane. With the present experiments, we show that the zinc finger transcription factor Krüppel-like factor 4 (KLF4), produced in high abundance in the intestine, is expressed at elevated levels in mice fed a low-zinc diet. In the murine intestinal epithelial cell (IEC) line MODE-K, zinc depletion of culture medium with cell-permeant and cell-impermeant chelators increased Zip4 and Klf4 mRNA and Zip4 heterogeneous nuclear RNA expression. Zinc depletion led to increased KLF4 in nuclear extracts. Knockdown of KLF4 using small interfering RNA transfection drastically limited ZIP4 induction upon zinc depletion and reduced 65Zn uptake by depleted IECs. EMSAs with nuclear extracts of IECs showed KLF4 binding to cis elements of the mouse Zip4 promoter, with increased binding under zinc-limited conditions. Reporter constructs with the Zip4 promoter and mutation studies further demonstrated that Zip4 is regulated through a KLF4 response element. These data from experiments with mice and murine IECs demonstrate that KLF4 is induced during zinc restriction and is a transcription factor involved in adaptive regulation of the zinc transporter ZIP4.
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Affiliation(s)
- Juan P. Liuzzi
- Center for Nutritional Sciences and Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida
| | - Liang Guo
- Center for Nutritional Sciences and Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida
| | - Shou-Mei Chang
- Center for Nutritional Sciences and Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida
| | - Robert J. Cousins
- Center for Nutritional Sciences and Food Science and Human Nutrition Department, University of Florida, Gainesville, Florida
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Abstract
Biotin is a water-soluble vitamin and serves as a coenzyme for five carboxylases in humans. Biotin is also covalently attached to distinct lysine residues in histones, affecting chromatin structure and mediating gene regulation. This review describes mammalian biotin metabolism, biotin analysis, markers of biotin status, and biological functions of biotin. Proteins such as holocarboxylase synthetase, biotinidase, and the biotin transporters SMVT and MCT1 play crucial roles in biotin homeostasis, and these roles are reviewed here. Possible effects of inadequate biotin intake, drug interactions, and inborn errors of metabolism are discussed, including putative effects on birth defects.
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Affiliation(s)
- Janos Zempleni
- Department of Nutrition and Health Sciences, University of Nebraska-Lincoln, 316 Ruth Leverton Hall, Lincoln, NE 68583-0806, USA.
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Abstract
Humans cannot synthesize biotin and thus must obtain this vitamin from exogenous sources. The intestine is exposed to 2 sources of biotin: a dietary source and a bacterial source, which is normal microflora of the large intestine. Dietary protein-bound biotin is converted to free biotin prior to absorption. Absorption of free biotin in the small and large intestine involves a saturable and Na(+)-dependent carrier-mediated process that is shared with pantothenic acid and lipoate. For this reason, the involved transport system is referred to as the sodium-dependent multivitamin transporter (SMVT); in humans, it is designated as hSMVT. The hSMVT system has been cloned, demonstrated to be exclusively expressed at the apical membrane of enterocytes, and shown, by means of gene-specific short interfering RNA, to be the main biotin uptake system that operates in human intestinal epithelial cells. The 5'-regulatory region of the hSMVT gene has also been cloned and characterized both in vitro and in vivo. Further, the human intestinal biotin uptake process was adaptively up-regulated in biotin deficiency via a transcriptionally mediated mechanism(s) that involves Kruppel-like factor 4 sites. Studies on cell biology of hSMVT have shown a region in the cytoplasmic C-terminal domain of the polypeptide to be essential for its targeting to the apical membrane domain of epithelial cells. Intracellular trafficking of the hSMVT protein appears to involve distinct trafficking vesicles that require an intact microtubules network and the motor protein dynein for their mobility.
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Affiliation(s)
- Hamid M Said
- University of California School of Medicine, Irvine, California 92697, USA.
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Said ZM, Subramanian VS, Vaziri ND, Said HM. Pyridoxine uptake by colonocytes: a specific and regulated carrier-mediated process. Am J Physiol Cell Physiol 2008; 294:C1192-7. [PMID: 18353902 DOI: 10.1152/ajpcell.00015.2008] [Citation(s) in RCA: 36] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The water-soluble vitamin B6 (pyridoxine) is important for normal cellular functions, growth, and development. The vitamin is obtained from two exogenous sources: a dietary source, which is absorbed in the small intestine, and a bacterial source, where the vitamin is synthesized in significant quantities by the normal microflora of the large intestine. Evidence exists to suggest the bioavailability of the latter source of the vitamin, but nothing is known about the mechanism involved and its regulation. In this study, we addressed these issues using young adult mouse colonic epithelial (YAMC) cells and human colonic apical membrane vesicles (AMV) as models and using [3H]pyridoxine as the uptake substrate. The results showed the initial rate of [3H]pyridoxine uptake by YAMC cells to be 1) energy- and temperature- (but not Na-) dependent and to occur without metabolic alteration in the transported substrate; 2) saturable as a function of concentration with an apparent Km and Vmax of 2.1 +/- 0.5 muM and 53.4 +/- 4.3 pmol.mg protein(-1).3 min(-1), respectively; 3) cis-inhibited by unlabeled pyridoxine and its structural analogs, but not by the unrelated compounds theophylline, penicillamine, and isoniazid; 4) trans-stimulated by unlabeled pyridoxine; 5) amiloride sensitive; and 6) regulated by extracellular and intracellular factors. Uptake of pyridoxine by native human colonic AMV was also found to involve a carrier-mediated process. These studies demonstrate, for the first time, the functional existence of a specific and regulatable carrier-mediated process for pyridoxine uptake by mammalian colonocytes.
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Affiliation(s)
- Zainab M Said
- Department of Medicine and Physiology, University of California, Irvine, California, USA
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Abstract
Although the role of biotin in metabolic reactions has long been recognized, its influence on transcription has only recently been discovered. A key protein in biotin-mediated transcription regulation is the biotin protein ligase, the enzyme responsible for catalyzing covalent linkage of the vitamin to biotin-dependent carboxylases. In the biotin regulatory system of Escherichia coli, the best characterized of the biotin-sensing systems, the biotin protein ligase functions both as the biotinylating enzyme and as a transcription repressor. Detailed mechanistic studies of this system are reviewed. In addition, recent studies have revealed other biotin-sensing systems in organisms ranging from bacteria to humans. These systems and the central role of the biotin protein ligase in each are also reviewed.
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Affiliation(s)
- Dorothy Beckett
- Department of Chemistry and Biochemistry, College of Chemical and Life Sciences, University of Maryland, College Park, MD 20742, USA.
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Reidling JC, Said HM. Regulation of the human biotin transporter hSMVT promoter by KLF-4 and AP-2: confirmation of promoter activity in vivo. Am J Physiol Cell Physiol 2006; 292:C1305-12. [PMID: 17135299 DOI: 10.1152/ajpcell.00360.2006] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
The mechanism of biotin uptake in human intestine has been well characterized and involves the human sodium-dependent multivitamin transporter (hSMVT), yet little is known about the molecular/transcriptional regulation of the system. Previous investigations cloned the 5' regulatory region of the hSMVT gene and identified the minimal promoter. To expand these investigations, we compared activity of the hSMVT promoter in three human intestinal epithelial cell lines (NCM460, Caco-2, and HuTu-80) and contrasted a renal epithelial cell line (HEK-293). We analyzed the role of putative cis-elements in regulating promoter activity and confirmed activity of the cloned hSMVT promoter in vivo. In vitro studies demonstrated that all cell lines utilized the same minimal promoter region, and mutation of specific cis-regulatory elements [Kruppel-like factor 4 (KLF-4) and activator protein-2 (AP-2)] led to a decrease in promoter activity in all intestinal cell types but not in renal cells. Using electrophoretic mobility shift assays, we identified two specific DNA/protein complexes. Using oligonucleotide competition and antibody supershift analysis, we determined that KLF-4 and AP-2 were involved in forming the complexes. In HEK-293 cells, overexpressing KLF-4 increased the endogenous hSMVT message levels threefold and activated a cotransfected hSMVT promoter-reporter construct. In vivo studies using hSMVT promoter-luciferase transgenic mice established physiological relevance and showed the pattern of hSMVT promoter expression to be similar to endogenous mouse SMVT mRNA expression. The results demonstrate, for the first time, the importance of KLF-4 and AP-2 in regulating the activity of the hSMVT promoter in the intestine and provide direct in vivo confirmation of hSMVT promoter activity.
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