American Journal of Physiology-gastrointestinal and Liver Physiology, May 1, 2021
The water-soluble vitamin B1 is essential for normal human health and physiology. In its main bio... more The water-soluble vitamin B1 is essential for normal human health and physiology. In its main biologically active form, i.e., thiamin pyrophosphate (TPP), the vitamin plays many critical roles in cell metabolism; thus, its deficiency leads to a variety of adverse effects. Humans/mammals obtain vitamin B1 from two exogenous sources: diet and gut microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP, and colonocytes can efficiently absorb this TPP via a high-affinity and specific carrier-mediated mechanism that involves the recently cloned colonic TPP transporter (cTPPT; product of SLC44A4 gene). There is nothing currently known about colonic uptake of TPP during early stages of life and whether the process undergoes developmental regulation. We addressed this issue using the mouse as animal model. Our results showed that colonic uptake of TPP undergoes developmental upregulation as the animal moves from the suckling period to weanling and adulthood. This upregulation in uptake was found to be associated with a parallel induction in level of expression of the cTPPT protein, mRNA, and heterogeneous nuclear RNA, suggesting possible involvement of transcriptional mechanism(s). We also found a parallel upregulation in the level of expression of the two nuclear factors that drive activity of the SLC44A4 promoter (i.e., CREB-1 and Elf-3) with maturation. These results demonstrate, for the first time, to our knowledge, that colonic TPP uptake process and cTPPT expression are developmentally upregulated and that this upregulation is likely driven via transcriptional mechanism(s).NEW & NOTEWORTHY The colonic carrier-mediated uptake process of the microbiota-generated and phosphorylated form of vitamin B1, i.e., thiamin pyrophosphate, undergoes ontogenic changes that parallel the development of the gut microbiota (and their ability to generate vitamins) during early stages of life.
Biotin (vitamin B7) is essential for normal human health due to its involvement, as a cofactor, i... more Biotin (vitamin B7) is essential for normal human health due to its involvement, as a cofactor, in a variety of critical cellular metabolic reactions. Previous studies have shown that biotin deficiency enhances inflammation, and that certain chronic inflammatory diseases (like inflammatory bowel diseases) are associated with biotin deficiency. However, the mechanisms that mediate the association between biotin status and inflammation are not well understood. Here, we examined the effect of biotin deficiency on human CD4+ T cell responses to determine their role in biotin deficiency associated inflammation. Our investigations revealed that anti‐CD3/CD28 stimulated human CD4+ T cells cultured in biotin deficient media secreted significantly enhanced levels of pro‐inflammatory cytokines, IFN‐γ, TNF‐α, and IL‐17. Expression of the transcription factors, T‐bet and RORγt, was increased along with a decrease in Foxp3 expression in biotin deficient CD4+ T cells. The percentage of T regulatory cells was also decreased under biotin deficient condition. Further investigations indicated that the enhanced inflammatory response was not a consequence of increased T‐cell differentiation towards Th1 and Th17 or increased proliferation. Instead deficiency of biotin enhanced the production of IFN‐γ from already differentiated cells. Moreover, the results indicated that the increased inflammatory response was due to enhanced activation of the mTOR signaling pathway in biotin deficient CD4+ T cells. Similar increase in T‐bet, RORγt and decrease in Foxp3 transcription factors was also observed in inguinal lymph nodes of mice fed with biotin deficient diet relative to pair‐fed controls. Further, the level of IFN‐γ, IL‐17 cytokines and the expression of mTOR were found to be increased significantly in CD4+T cells isolated from inguinal lymph node of diet induced biotin deficient mice. These findings revealed that biotin deficiency leads to the upregulation of IFN‐γ secreting Th1 and IL‐17 secreting Th‐17 cells via activation of mTOR signaling pathway which may contribute to inflammation associated with biotin deficiencySupport or Funding InformationThis study supported by grants from the DVA and the NIH (DK58057, DK56061 and AA 018071)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of... more This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1. Three complementary models were used: in vitro (human NCM460 cells), ex vivo (human colonoids), and in vivo (mice). The results showed LPS to significantly suppress TPP uptake and the expression of its transporter, and that these effects are mediated via the membrane TLR4 receptor, and involve the NF-κB/p38/JNK signaling pathways.
Thiamin (vitamin B1; also known as the “energy vitamin”) plays a critical role in normal energy m... more Thiamin (vitamin B1; also known as the “energy vitamin”) plays a critical role in normal energy metabolism and ATP production. Like all other human/mammalian cells, pancreatic acinar cells (PACs) cannot synthesize thiamin, and thus, must obtain the vitamin from circulation. Internalized free thiamin is then converted to TPP in the cytoplasm and transported into mitochondria for utilization via a carrier-mediated process that is mediated by the MTPPT (product of the SLC25A19 gene). Previous studies from our laboratory have characterized different aspects of the mitochondrial TPP uptake process in PACs; we have also delineated aspects of the MTPPT transcriptional regulation. To date, however, there has been nothing known about post-transcriptional regulation of MTPPT expression and function in PACs. We began to address these issues and, in this study, we examined the potential role of miRNAs in this regulation. First, we subjected the 458 bp long hMTPPT-3' untranslated region (UTR...
LPS is a major component of the outer membrane of Gram-negative bacteria. The gut is exposed to h... more LPS is a major component of the outer membrane of Gram-negative bacteria. The gut is exposed to high levels of this endotoxin in conditions such as IBD, necrotizing enterocolitis, and sepsis. LPS affects different aspects of cell physiology including transport at the cell membrane. Effects on the latter, however, are differential; and both induction and inhibition in substrate uptake have been observed. Nothing is currently known about the effect of LPS on colonic uptake of the microbiota-generated forms of vitamin B1, i.e., TPP and free thiamine. We investigated this issue in the current study using human-derived colonic epithelial NCM460 cells, human native differentiated and polarized colonoid monolayers, and mouse intact colonic tissue preparation together with appropriate cellular and molecular approaches. The results showed that exposure of colonic epithelial cells to LPS leads to a significant inhibition in carrier-mediated [3H]-TPP uptake. This inhibition was associated with...
The aim of this study was to examine the effect of TNFα (i.e., a predominant proinflammatory cyto... more The aim of this study was to examine the effect of TNFα (i.e., a predominant proinflammatory cytokine produced during chronic gut inflammation) on colonic uptake of thiamin pyrophosphate (TPP) and free thiamin, forms of vitamin B1 that are produced by the gut microbiota and are absorbed via distinct carrier-mediated systems. We utilized human-derived colonic epithelial CCD841 and NCM460 cells, human differentiated colonoid monolayers, and mouse intact colonic tissue preparations together with an array of cellular/molecular approaches in our investigation. The results showed that exposure of colonic epithelial cells to TNFα leads to a significant inhibition in TPP and free thiamin uptake. This inhibition was associated with: 1) a significant suppression in the level of expression of the colonic TPP transporter (cTPPT; encoded by SLC44A4), as well as thiamin transporters-1 & 2 (THTR-1 & -2; encoded by SLC19A2 & SLC19A3, respectively); 2) marked inhibition in activity of the SLC44A4, S...
American Journal of Physiology-Cell Physiology, 2019
The apically localized riboflavin (RF) transporter-3 (RFVT-3) is involved in intestinal absorptio... more 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 coimmunopre...
American Journal of Physiology-gastrointestinal and Liver Physiology, May 1, 2021
The water-soluble vitamin B1 is essential for normal human health and physiology. In its main bio... more The water-soluble vitamin B1 is essential for normal human health and physiology. In its main biologically active form, i.e., thiamin pyrophosphate (TPP), the vitamin plays many critical roles in cell metabolism; thus, its deficiency leads to a variety of adverse effects. Humans/mammals obtain vitamin B1 from two exogenous sources: diet and gut microbiota. Considerable amount of the microbiota-generated vitamin B1 exists in the form of TPP, and colonocytes can efficiently absorb this TPP via a high-affinity and specific carrier-mediated mechanism that involves the recently cloned colonic TPP transporter (cTPPT; product of SLC44A4 gene). There is nothing currently known about colonic uptake of TPP during early stages of life and whether the process undergoes developmental regulation. We addressed this issue using the mouse as animal model. Our results showed that colonic uptake of TPP undergoes developmental upregulation as the animal moves from the suckling period to weanling and adulthood. This upregulation in uptake was found to be associated with a parallel induction in level of expression of the cTPPT protein, mRNA, and heterogeneous nuclear RNA, suggesting possible involvement of transcriptional mechanism(s). We also found a parallel upregulation in the level of expression of the two nuclear factors that drive activity of the SLC44A4 promoter (i.e., CREB-1 and Elf-3) with maturation. These results demonstrate, for the first time, to our knowledge, that colonic TPP uptake process and cTPPT expression are developmentally upregulated and that this upregulation is likely driven via transcriptional mechanism(s).NEW & NOTEWORTHY The colonic carrier-mediated uptake process of the microbiota-generated and phosphorylated form of vitamin B1, i.e., thiamin pyrophosphate, undergoes ontogenic changes that parallel the development of the gut microbiota (and their ability to generate vitamins) during early stages of life.
Biotin (vitamin B7) is essential for normal human health due to its involvement, as a cofactor, i... more Biotin (vitamin B7) is essential for normal human health due to its involvement, as a cofactor, in a variety of critical cellular metabolic reactions. Previous studies have shown that biotin deficiency enhances inflammation, and that certain chronic inflammatory diseases (like inflammatory bowel diseases) are associated with biotin deficiency. However, the mechanisms that mediate the association between biotin status and inflammation are not well understood. Here, we examined the effect of biotin deficiency on human CD4+ T cell responses to determine their role in biotin deficiency associated inflammation. Our investigations revealed that anti‐CD3/CD28 stimulated human CD4+ T cells cultured in biotin deficient media secreted significantly enhanced levels of pro‐inflammatory cytokines, IFN‐γ, TNF‐α, and IL‐17. Expression of the transcription factors, T‐bet and RORγt, was increased along with a decrease in Foxp3 expression in biotin deficient CD4+ T cells. The percentage of T regulatory cells was also decreased under biotin deficient condition. Further investigations indicated that the enhanced inflammatory response was not a consequence of increased T‐cell differentiation towards Th1 and Th17 or increased proliferation. Instead deficiency of biotin enhanced the production of IFN‐γ from already differentiated cells. Moreover, the results indicated that the increased inflammatory response was due to enhanced activation of the mTOR signaling pathway in biotin deficient CD4+ T cells. Similar increase in T‐bet, RORγt and decrease in Foxp3 transcription factors was also observed in inguinal lymph nodes of mice fed with biotin deficient diet relative to pair‐fed controls. Further, the level of IFN‐γ, IL‐17 cytokines and the expression of mTOR were found to be increased significantly in CD4+T cells isolated from inguinal lymph node of diet induced biotin deficient mice. These findings revealed that biotin deficiency leads to the upregulation of IFN‐γ secreting Th1 and IL‐17 secreting Th‐17 cells via activation of mTOR signaling pathway which may contribute to inflammation associated with biotin deficiencySupport or Funding InformationThis study supported by grants from the DVA and the NIH (DK58057, DK56061 and AA 018071)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of... more This study examined the effect of the bacterial lipopolysaccharide (LPS) on the colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1. Three complementary models were used: in vitro (human NCM460 cells), ex vivo (human colonoids), and in vivo (mice). The results showed LPS to significantly suppress TPP uptake and the expression of its transporter, and that these effects are mediated via the membrane TLR4 receptor, and involve the NF-κB/p38/JNK signaling pathways.
Thiamin (vitamin B1; also known as the “energy vitamin”) plays a critical role in normal energy m... more Thiamin (vitamin B1; also known as the “energy vitamin”) plays a critical role in normal energy metabolism and ATP production. Like all other human/mammalian cells, pancreatic acinar cells (PACs) cannot synthesize thiamin, and thus, must obtain the vitamin from circulation. Internalized free thiamin is then converted to TPP in the cytoplasm and transported into mitochondria for utilization via a carrier-mediated process that is mediated by the MTPPT (product of the SLC25A19 gene). Previous studies from our laboratory have characterized different aspects of the mitochondrial TPP uptake process in PACs; we have also delineated aspects of the MTPPT transcriptional regulation. To date, however, there has been nothing known about post-transcriptional regulation of MTPPT expression and function in PACs. We began to address these issues and, in this study, we examined the potential role of miRNAs in this regulation. First, we subjected the 458 bp long hMTPPT-3' untranslated region (UTR...
LPS is a major component of the outer membrane of Gram-negative bacteria. The gut is exposed to h... more LPS is a major component of the outer membrane of Gram-negative bacteria. The gut is exposed to high levels of this endotoxin in conditions such as IBD, necrotizing enterocolitis, and sepsis. LPS affects different aspects of cell physiology including transport at the cell membrane. Effects on the latter, however, are differential; and both induction and inhibition in substrate uptake have been observed. Nothing is currently known about the effect of LPS on colonic uptake of the microbiota-generated forms of vitamin B1, i.e., TPP and free thiamine. We investigated this issue in the current study using human-derived colonic epithelial NCM460 cells, human native differentiated and polarized colonoid monolayers, and mouse intact colonic tissue preparation together with appropriate cellular and molecular approaches. The results showed that exposure of colonic epithelial cells to LPS leads to a significant inhibition in carrier-mediated [3H]-TPP uptake. This inhibition was associated with...
The aim of this study was to examine the effect of TNFα (i.e., a predominant proinflammatory cyto... more The aim of this study was to examine the effect of TNFα (i.e., a predominant proinflammatory cytokine produced during chronic gut inflammation) on colonic uptake of thiamin pyrophosphate (TPP) and free thiamin, forms of vitamin B1 that are produced by the gut microbiota and are absorbed via distinct carrier-mediated systems. We utilized human-derived colonic epithelial CCD841 and NCM460 cells, human differentiated colonoid monolayers, and mouse intact colonic tissue preparations together with an array of cellular/molecular approaches in our investigation. The results showed that exposure of colonic epithelial cells to TNFα leads to a significant inhibition in TPP and free thiamin uptake. This inhibition was associated with: 1) a significant suppression in the level of expression of the colonic TPP transporter (cTPPT; encoded by SLC44A4), as well as thiamin transporters-1 & 2 (THTR-1 & -2; encoded by SLC19A2 & SLC19A3, respectively); 2) marked inhibition in activity of the SLC44A4, S...
American Journal of Physiology-Cell Physiology, 2019
The apically localized riboflavin (RF) transporter-3 (RFVT-3) is involved in intestinal absorptio... more 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 coimmunopre...
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