RESUMEN
Humans and mammals obtain vitamin B1 from dietary and gut microbiota sources. A considerable amount of the microbiota-generated vitamin exists in the form of thiamine pyrophosphate (TPP), and colonocytes are capable of absorbing TPP via a specific carrier-mediated process that involves the colonic TPP transporter (cTPPT encoded by SLC44A4). Little is known about the relative contribution of the SLC44A4 transporter toward total colonic carrier-mediated TPP uptake and its role in colon physiology. To address these issues, we generated an Slc44a4 knockout (KO) mouse model (by Cre-Lox recombination) and found a near-complete inhibition in colonic carrier-mediated [3H]TPP uptake in the Slc44a4 KO compared with wild-type (WT) littermates. We also observed a significant reduction in KO mice's body weight and a shortening of their colon compared with WT. Using RNAseq and Ingenuity pathway analysis (IPA) approaches, we found that knocking out the colonic Slc44a4 led to changes in the level of expression of many genes, including upregulation in those associated with intestinal inflammation and colitis. Finally, we found that the Slc44a4 KO mice were more susceptible to the effect of the colitogenic dextran sodium sulfate (DSS) compared with WT animals, a finding that lends support to the recent prediction by multiple genome-wide association studies (GWAS) that SLC44A4 is a possible colitis susceptibility gene. In summary, the results of these investigations show that Slc44a4 is the predominant or only transporter involved in the colonic uptake of TPP, that the transporter is important for colon physiology, and that its deletion increases susceptibility to inflammation.NEW & NOTEWORTHY This study shows that Slc44a4 is the predominant or only transport system involved in the uptake of the gut microbiota-generated thiamine pyrophosphate (TPP) in the colon and that its deletion affects colon physiology and increases its susceptibility to inflammation.
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Colon , Microbioma Gastrointestinal , Ratones Noqueados , Tiamina Pirofosfato , Animales , Humanos , Masculino , Ratones , Transporte Biológico , Colitis/metabolismo , Colitis/microbiología , Colitis/genética , Colitis/inducido químicamente , Colon/metabolismo , Colon/microbiología , Microbioma Gastrointestinal/fisiología , Absorción Intestinal , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos C57BL , Tiamina Pirofosfato/metabolismoRESUMEN
The sodium-dependent multivitamin transporter encoded by SLC5A6 is responsible for uptake of biotin, pantothenic acid, and α-lipoic acid. Thirteen individuals from eight families are reported with pathogenic biallelic SLC5A6 variants. Phenotype ranges from multisystem metabolic disorder to childhood-onset peripheral motor neuropathy. We report three additional affected individuals with biallelic SLC5A6 variants. In Family A, a male proband (AII:1) presenting in early childhood with gross motor regression, motor axonal neuropathy, recurrent cytopenia and infections, and failure to thrive was diagnosed at 12 years of age via genome sequencing (GS) with a paternal NM_021095.4:c.393+2T>C variant and a maternal c.1285A>G p.(Ser429Gly) variant. An uncle with recurrent cytopenia and peripheral neuropathy was subsequently found to have the same genotype. We also report an unrelated female with peripheral neuropathy homozygous for the c.1285A>G p.(Ser429Gly) recurrent variant identified in seven reported cases, including this study. RT-PCR studies on blood mRNA from AII:1 showed c.393+2T>C caused mis-splicing with all canonically spliced transcripts in AII:1 containing the c.1285A>G variant. SLC5A6 mRNA expression in AII:1 fibroblasts was ~50% of control levels, indicative of nonsense-mediated decay of mis-spliced transcripts. Biotin uptake studies on AII:1 fibroblasts, expressing the p.(Ser429Gly) variant, showed an ~90% reduction in uptake compared to controls. Targeted treatment of AII:1 with biotin, pantothenic acid, and lipoic acid resulted in clinical improvement. Health Economic analyses showed implementation of GS as an early investigation could have saved $ AUD 105,988 and shortened diagnostic odyssey and initiation of treatment by up to 7 years.
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Simportadores , Humanos , Femenino , Masculino , Niño , Simportadores/genética , Linaje , Biotina/uso terapéutico , Transportadores de Anión Orgánico Sodio-Dependiente/genética , Enfermedades del Sistema Nervioso Periférico/genética , Enfermedades del Sistema Nervioso Periférico/diagnóstico , Secuenciación Completa del Genoma , Mutación , Ácido Tióctico/uso terapéutico , Enfermedades de los Ganglios BasalesRESUMEN
Thiamin (vitamin B1) plays a vital role in cellular energy metabolism/ATP production. Pancreatic acinar cells (PACs) obtain thiamin from circulation and convert it to thiamin pyrophosphate (TPP) in the cytoplasm. TPP is then taken up by the mitochondria via a carrier-mediated process that involves the mitochondrial TPP transporter (MTPPT; encoded by the gene SLC25A19). We have previously characterized different aspects of the mitochondrial carrier-mediated TPP uptake process, but nothing is known about its possible regulation at the posttranscriptional level. We address this issue in the current investigations focusing on the role of miRNAs in this regulation. First, we subjected the human (and rat) 3'-untranslated region (3'-UTR) of the SLC25A19 to three in-silico programs, and all have identified putative binding sites for miR-122-5p. Transfecting pmirGLO-hSLC25A19 3'-UTR into rat PAC AR42J resulted in a significant reduction in luciferase activity compared with cells transfected with pmirGLO-empty vector. Mutating as well as truncating the putative miR-122-5p binding sites in the hSLC25A19 3'-UTR led to abrogation of inhibition in luciferase activity in PAC AR42J. Furthermore, transfecting/transducing PAC AR42J and human primary PACs with mimic of miR-122-5p led to a significant inhibition in the level of expression of the MTPPT mRNA and protein as well as in mitochondrial carrier-mediated TPP uptake. Conversely, transfecting PAC AR42J with an inhibitor of miR-122-5p increased MTPPT expression and function. These findings show, for the first time, that expression and function of the MTPPT in PACs are subject to posttranscriptional regulation by miR-122-5p.NEW & NOTEWORTHY This study shows that the expression and function of mitochondrial TPP transporter (MTPPT) are subject to posttranscriptional regulation by miRNA-122-5p in pancreatic acinar cells.
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Células Acinares , MicroARNs , Humanos , Ratas , Animales , Células Acinares/metabolismo , Difosfatos/metabolismo , Tiamina/metabolismo , Tiamina Pirofosfato/metabolismo , Mitocondrias/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Luciferasas/metabolismo , Proteínas de Transporte de Membrana Mitocondrial/genética , Proteínas de Transporte de Membrana Mitocondrial/metabolismoRESUMEN
This study investigated the effect of the bacterial endotoxin lipopolysaccharide (LPS) on colonic uptake of thiamin pyrophosphate (TPP), the biologically active form of vitamin B1 that is generated by gut microbiota. We used three complementary models in our study: in vitro (human-derived colonic epithelial NCM460), ex vivo (human differentiated colonoid monolayers), and in vivo (mouse colonic tissue). The results showed that exposure of NCM460 cells to LPS leads to a significant inhibition of carrier-mediated TPP uptake as well as in decreased expression of the colonic TPP transporter (cTPPT) protein, mRNA, and heterologous nuclear RNA (hnRNA) compared with untreated controls. Similarly, exposure of human differentiated colonoid monolayers and mice to LPS caused significant inhibition in colonic carrier-mediated TPP uptake and in cTPPT protein, mRNA, and hnRNA expression. The effect of LPS on colonic TPP uptake and cTTPT expression was also found to be associated with a significant reduction in activity of the SLC44A4 promoter as well as in decreased expression of the nuclear factor Elf-3 (E74-like ETS transcription factor 3), which is needed for promoter activity. Finally, we found that knocking down the Toll-like receptor 4 (TLR4) and blocking the nuclear factor kappa B (NF-κB), JNK, and p38 signaling pathways with the use of pharmacological inhibitors lead to significant abrogation in the degree of LPS-mediated inhibition in TPP uptake and cTPPT expression. These results demonstrated that exposure of colonic epithelia to LPS inhibits colonic TPP uptake via transcriptional mechanism(s) and that the effect is mediated via TLR4 receptor and NF-κB/p38/JNK signaling pathways.NEW & NOTEWORTHY 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.
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FN-kappa B , Tiamina Pirofosfato , Humanos , Ratones , Animales , Tiamina Pirofosfato/metabolismo , FN-kappa B/metabolismo , Receptor Toll-Like 4/genética , Receptor Toll-Like 4/metabolismo , Lipopolisacáridos/farmacología , Difosfatos , Sistema de Señalización de MAP Quinasas , ARN Nuclear Heterogéneo/metabolismo , Línea Celular , Tiamina/metabolismo , ARN Mensajero/metabolismoRESUMEN
Biotin is an essential vitamin and critical cofactor in several metabolic pathways, and its deficiency has been linked to several disorders including inflammatory bowel disease (IBD). We previously reported that biotin deficiency (BD) in mice, whether modeled through intestine-specific deletion of biotin transporter (SMVT-icKO) or through a biotin-deficient diet, resulted in intestinal inflammation consistent with an IBD-like phenotype. To assess whether the gut microbiome is associated with these BD-induced changes, we collected stool and intestinal samples from both of these mouse models and utilized them for 16S rRNA gene sequencing. We find that both diet-mediated and deletion-mediated BD result in the expansion of opportunistic microbes including Klebsiella, Enterobacter, and Helicobacter, at the expense of mucus-resident microbes including Akkermansia. Additionally, microbiome dysbiosis resulting from diet-mediated BD precedes the onset of the IBD-like phenotypic changes. Lastly, through the use of predictive metagenomics, we report that the resulting BD-linked microbiome perturbations exhibit increased biotin biosynthesis in addition to several other perturbed metabolic pathways. Altogether, these results demonstrate that biotin deficiency results in a specific microbiome composition, which may favor microbes capable of biotin synthesis and which may contribute to intestinal inflammation.
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Biotina , Enfermedades Inflamatorias del Intestino , Animales , Ratones , Disbiosis , ARN Ribosómico 16S/genética , Enfermedades Inflamatorias del Intestino/metabolismo , Fenotipo , InflamaciónRESUMEN
Over time, the structure and function of the broadly dispersed colonization factor (CF) CS6 of enterotoxigenic Escherichia coli (ETEC) have become more significant. CS6 is composed of tightly-associated subunits, CssA and CssB which due to presence of natural point mutation gave rise to CS6 subtypes. In contrast to the other ETEC CFs, CS6 is an afimbrial, spherical-shaped oligomers of (CssA-CssB)n complex where 'n' is concentration dependent. In this study, we have compared AIBI-CS6 and AIIBII-CS6 structurally and functionally. The Mw of CssAI was 18.5 kDa but Mw of CssAII was 15.1 kDa. Both CssBI and CssBII had Mw of 15.9 kDa. The substitution of Gly39 with Ala39 in CssAI leads to reduction in Mw from 18.5 to 15.1 kDa. Due to higher Mw of CssAI, the size of AIBI concentration-dependent oligomers should be higher. However, the Mw of AIIBII oligomers were higher and AIIBII also showed higher oligomeric forms compared to AIBI both in native PAGE and electron microscopy. The oligomers of both subtypes could withstand greater temperatures and denaturant concentrations. In terms of cellular response, the levels of inflammatory cytokines were significantly higher in case of AIBI-CS6 expressing ETEC as compared to AIIBII-CS6 expressing ETEC both in vitro and in vivo. When inflammatory cytokines were evaluated after infecting suckling mice with these ETEC strains, the results were consistent. In conclusion, even though there was subtle structural difference between AIBI-CS6 and AIIBII-CS6 due to natural point mutations but ETEC strains expressing these subtypes displayed great variability in pathogenicity.
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Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Animales , Ratones , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/química , Antígenos Bacterianos/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , EnterotoxinasRESUMEN
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, SLC19A2, and SLC19A3 promoters; and 3) significant suppression in level of expression of nuclear factors that are needed for activity of these promoters (i.e., CREB-1, Elf-3, NF-1A, SP-1). Furthermore, the inhibitory effects were found to be mediated via JNK and ERK1/2 signaling pathways. We also examined the level of expression of cTPPT and THTR-1 & -2 in colonic tissues of patients with active ulcerative colitis and found the levels to be significantly lower than in healthy controls. These findings demonstrate that exposure of colonocytes to TNFα suppresses TPP and free thiamin uptake at the transcriptional level via JNK- and Erk1/2-mediated pathways.
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Tiamina Pirofosfato , Factor de Necrosis Tumoral alfa , Humanos , Ratones , Animales , Tiamina Pirofosfato/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Células Acinares/metabolismo , Tiamina/metabolismo , Tiamina/farmacología , Proteínas de Transporte de Membrana/genética , Proteínas de Transporte de Membrana/metabolismoRESUMEN
Enterotoxigenic E. coli (ETEC) produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and commonly cause diarrhea in resource-poor regions. ETEC have been linked repeatedly to sequelae in children including enteropathy, malnutrition, and growth impairment. Although cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their contributions to sequelae remain unclear. LT increases cellular cAMP to activate protein kinase A (PKA) that phosphorylates ion channels driving intestinal export of salt and water resulting in diarrhea. As PKA also modulates transcription of many genes, we interrogated transcriptional profiles of LT-treated intestinal epithelia. Here we show that LT significantly alters intestinal epithelial gene expression directing biogenesis of the brush border, the major site for nutrient absorption, suppresses transcription factors HNF4 and SMAD4 critical to enterocyte differentiation, and profoundly disrupts microvillus architecture and essential nutrient transport. In addition, ETEC-challenged neonatal mice exhibit substantial brush border derangement that is prevented by maternal vaccination with LT. Finally, mice repeatedly challenged with toxigenic ETEC exhibit impaired growth recapitulating the multiplicative impact of recurring ETEC infections in children. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to prevent major sequelae of these common infections including malnutrition that impact millions of children.
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Escherichia coli Enterotoxigénica , Infecciones por Escherichia coli , Proteínas de Escherichia coli , Desnutrición , Ratones , Animales , Enterotoxinas/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli Enterotoxigénica/genética , Escherichia coli Enterotoxigénica/metabolismo , Infecciones por Escherichia coli/prevención & control , DiarreaRESUMEN
Hypoxia exerts profound effects on cell physiology, but its effect on colonic uptake of the microbiota-generated forms of vitamin B1 (i.e., thiamin pyrophosphate [TPP] and free thiamine) has not been described. Here, we used human colonic epithelial NCM460 cells and human differentiated colonoid monolayers as in vitro and ex vivo models, respectively, and were subjected to either chamber (1% O2, 5% CO2, and 94% N2) or chemically (desferrioxamine; 250 µM)-induced hypoxia followed by determination of different physiological-molecular parameters. We showed that hypoxia causes significant inhibition in TPP and free thiamin uptake by colonic NCM460 cells and colonoid monolayers; it also caused a significant reduction in the expression of TPP (SLC44A4) and free thiamin (SLC19A2 and SLC19A3) transporters and in activity of their gene promoters. Furthermore, hypoxia caused a significant induction in levels of hypoxia-inducible transcription factor (HIF)-1α but not HIF-2α. Knocking down HIF-1α using gene-specific siRNAs in NCM460 cells maintained under hypoxic conditions, on the other hand, led to a significant reversal in the inhibitory effect of hypoxia on TPP and free thiamin uptake as well as on the expression of their transporters. Finally, a marked reduction in level of expression of the nuclear factors cAMP responsive element-binding protein 1 and gut-enriched Krüppel-like factor 4 (required for activity of SLC44A4 and SLC19A2 promoters, respectively) was observed under hypoxic conditions. In summary, hypoxia causes severe inhibition in colonic TPP and free thiamin uptake that is mediated at least in part via HIF-1α-mediated transcriptional mechanisms affecting their respective transporters.
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Subunidad alfa del Factor 1 Inducible por Hipoxia , Microbiota , Tiamina , Transporte Biológico , Hipoxia de la Célula/fisiología , Humanos , Hipoxia/metabolismo , Hipoxia/microbiología , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Tiamina/metabolismo , Tiamina Pirofosfato/metabolismoRESUMEN
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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Biotina/metabolismo , Metilación de ADN , Epigénesis Genética , Etanol/farmacología , Mucosa Intestinal/efectos de los fármacos , Acetaldehído/farmacología , Animales , Células CACO-2 , Células Cultivadas , Etanol/metabolismo , Humanos , Mucosa Intestinal/metabolismo , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Factores de Transcripción de Tipo Kruppel/metabolismo , Ratones , Ratones Endogámicos C57BL , Ácidos Oléicos/farmacología , Ácidos Palmíticos/farmacología , Simportadores/genética , Simportadores/metabolismoRESUMEN
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.
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Colon/metabolismo , Microbioma Gastrointestinal/fisiología , Proteínas de Transporte de Membrana/metabolismo , Tiamina Pirofosfato/metabolismo , Animales , Dieta , Femenino , Masculino , RatonesRESUMEN
BACKGROUND & AIMS: Biotin is a water-soluble vitamin that is indispensable for human health. Biotin deficiency can cause failure-to-thrive, immunodeficiency, alopecia, dermatitis, and conjunctivitis. We previously reported that biotin deficiency also can lead to severe colitis in mice, which is completely reversed with supplementation. Our aim in this study was to determine if high-dose biotin supplementation can provide a therapeutic benefit in a preclinical model for inflammatory bowel disease (IBD) and to identify the molecular mechanism by which this occurs. METHODS: Mice were challenged with dextran sodium sulfate to induce colitis and were treated with 1 mmol/L biotin to induce or maintain remission. Clinical response was monitored by the Disease Activity Index and fecal calprotectin levels. The colon tissue was investigated for histology, length, as well as expression of inflammatory cytokines (interleukin 6, tumor necrosis factor-α, interleukin 1ß), intestinal permeability, tight junctions (zonula occludens-1 and claudin-2), and the transcription factor nuclear factor-κB (NF-κB). RESULTS: Biotin therapy led to delayed onset and severity of colitis as well as accelerated healing. There was improvement in the Disease Activity Index, fecal calprotectin levels, colon length, and histology. In addition, biotin-treated mice had reduced expression of inflammatory cytokines, reduced intestinal permeability, and reduced activation of NF-κB. CONCLUSIONS: Oral supplementation with biotin provides benefit for maintenance and induction of remission in the dextran sodium sulfate preclinical model for IBD. Biotin does this by reducing the activation of NF-κB, which prevents the production of inflammatory cytokines and helps maintain the integrity of the intestinal barrier. Clinically, the NF-κB pathway is important in the development of IBD and this finding suggests that biotin may have therapeutic potential for patients with IBD.
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Biotina , Colitis , Animales , Biotina/farmacología , Biotina/uso terapéutico , Colitis/inducido químicamente , Colitis/tratamiento farmacológico , Sulfato de Dextran , Suplementos Dietéticos , Humanos , Factores de Transcripción de Tipo Kruppel , Ratones , Ratones Endogámicos C57BL , FN-kappa B/metabolismo , Regeneración , Transducción de Señal , Células Madre/metabolismoRESUMEN
Colonocytes possess a specific carrier-mediated uptake process for the microbiota-generated thiamin (vitamin B1) pyrophosphate (TPP) that involves the TPP transporter (TPPT; product of the SLC44A4 gene). Little is known about the effect of exogenous factors (including enteric pathogens) on the colonic TPP uptake process. Our aim in this study was to investigate the effect of Enterohemorrhagic Escherichia coli (EHEC) infection on colonic uptake of TPP. We used human-derived colonic epithelial NCM460 cells and mice in our investigation. The results showed that infecting NCM460 cells with live EHEC (but not with heat-killed EHEC, EHEC culture supernatant, or with non-pathogenic E. Coli) to lead to a significant inhibition in carrier-mediated TPP uptake, as well as in level of expression of the TPPT protein and mRNA. Similarly, infecting mice with EHEC led to a significant inhibition in colonic TPP uptake and in level of expression of TPPT protein and mRNA. The inhibitory effect of EHEC on TPP uptake by NCM460 was found to be associated with reduction in the rate of transcription of the SLC44A4 gene as indicated by the significant reduction in the activity of the SLC44A4 promoter transfected into EHEC infected cells. The latter was also associated with a marked reduction in the level of expression of the transcription factors CREB-1 and ELF3, which are known to drive the activity of the SLC44A4 promoter. Finally, blocking the ERK1/2 and NF-kB signaling pathways in NCM460 cells significantly reversed the level of EHEC inhibition in TPP uptake and TPPT expression. Collectively, these findings show, for the first time, that EHEC infection significantly inhibit colonic uptake of TPP, and that this effect appears to be exerted at the level of SLC44A4 transcription and involves the ERK1/2 and NF-kB signaling pathways.
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Colon/metabolismo , Escherichia coli Enterohemorrágica/aislamiento & purificación , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/metabolismo , Proteínas de Transporte de Membrana/genética , Regiones Promotoras Genéticas , Tiamina Pirofosfato/metabolismo , Animales , Transporte Biológico , Células Cultivadas , Colon/microbiología , Células Epiteliales/microbiología , Infecciones por Escherichia coli/microbiología , Infecciones por Escherichia coli/patología , Regulación de la Expresión Génica , Humanos , Masculino , Proteínas de Transporte de Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/genética , Proteína Quinasa 3 Activada por Mitógenos/metabolismoRESUMEN
Thiamine metabolism dysfunction syndrome-4 (THMD4) includes episodic encephalopathy, often associated with a febrile illness, causing transient neurologic dysfunction and a slowly progressive axonal polyneuropathy. Until now only two mutations (G125S and S194P) have been reported in the SLC25A19 gene as causative for this disease and a third mutation (G177A) as related to the Amish lethal microcephaly. In this work, we describe the clinical and molecular features of a patient carrying a novel mutation (c.576G>C; Q192H) on SLC25A19 gene. Functional studies on this mutation were performed explaining the pathogenetic role of c.576G>C in affecting the translational efficiency and/or stability of hMTPPT protein instead of the mRNA expression. These findings support the pathogenetic role of Q192H (c.576G>C) mutation on SLC25A19 gene. Moreover, despite in other patients the thiamine supplementation leaded to a substantial improvement of peripheral neuropathy, our patient did not show a clinical improvement.
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Predisposición Genética a la Enfermedad , Microcefalia/genética , Proteínas de Transporte de Membrana Mitocondrial/genética , Deficiencia de Tiamina/genética , Adolescente , Encefalopatías/genética , Encefalopatías/fisiopatología , Humanos , Masculino , Microcefalia/fisiopatología , Proteínas de Transporte de Membrana Mitocondrial/química , Mutación , Conformación Proteica , ARN Mensajero/genética , Tiamina/genética , Tiamina/metabolismo , Deficiencia de Tiamina/fisiopatologíaRESUMEN
The sodium-dependent multivitamin transporter (SMVT; SLC5A6) is involved in intestinal absorption of vitamin B7 (biotin). We have previously shown that mice with an embryonic intestinal-specific SMVT knockout (KO) develop biotin deficiency and severe spontaneous intestinal inflammation in addition to growth retardation, developmental delays, and death within the first 6-7 wk of life. The profound morbidity and mortality associated with the SMVT-KO has limited our ability to further characterize the intestinal inflammation and other sequelae of this deletion in adult mice with a mature gut microbiota. To overcome this limitation, we generated an intestine-specific, tamoxifen-inducible, conditional SMVT-KO (SMVT-icKO). Our results showed that adult SMVT-icKO mice have reduced body weight, biotin deficiency, shorter colonic length, and bloody diarrhea compared with age- and sex-matched control littermates. All SMVT-icKO mice also developed spontaneous intestinal inflammation associated with induction of calprotectin (S100a8/S100a9), proinflammatory cytokines (IL-1ß, TNF-α, IFN-γ, and IL-6), and an increase in intestinal permeability. Additionally, the intestines of SMVT-icKO showed activation of the NF-κB pathway and the nucleotide-binding domain and leucine-rich repeat pyrin 3 domain (NLRP3) inflammasome. Notably, administration of broad-spectrum antibiotics reduced lethality and led to normalization of intestinal inflammation, proinflammatory cytokines, altered mucosal integrity, and reduced expression of the NLRP3 inflammasome. Overall, these findings support our conclusion that the biotin transport pathway plays an important role in the maintenance of intestinal homeostasis, and that NF-κB and the NLRP3 inflammasome, as well as gut microbiota, drive the development of intestinal inflammation when SMVT is absent.NEW & NOTEWORTHY This study demonstrates that deletion of the intestinal biotin uptake system in adult mice leads to the development of spontaneous gut inflammation and that luminal microbiota plays a role in its development.
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Enteritis/genética , Antagonistas de Estrógenos/toxicidad , Microbioma Gastrointestinal/efectos de los fármacos , Intestinos/efectos de los fármacos , FN-kappa B/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/genética , Simportadores/metabolismo , Tamoxifeno/toxicidad , Envejecimiento , Animales , Biotina/metabolismo , Peso Corporal/efectos de los fármacos , Colon/patología , Citocinas/metabolismo , Diarrea/inducido químicamente , Diarrea/microbiología , Diarrea/patología , Enteritis/inducido químicamente , Enteritis/microbiología , Intestinos/microbiología , Ratones , Ratones Endogámicos C57BL , Proteína con Dominio Pirina 3 de la Familia NLR/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Simportadores/efectos de los fármacos , Simportadores/genéticaRESUMEN
The apically localized riboflavin (RF) transporter-3 (RFVT-3) is involved in intestinal absorption of vitamin B2. Previous studies have characterized different physiological/biological aspects of the RFVT-3, but there is a lack of knowledge regarding possible existence of interacting partner(s) and consequence of interaction(s) on its function/cell biology. To address the latter, we performed yeast two-hybrid (Y2H) screening of a human colonic cDNA library and have identified transmembrane protein 237 (TMEM237) as a putative interactor with the human (h)RFVT-3; the interaction was further confirmed via "1-by-1" Y2H assay that involved appropriate positive and negative controls. TMEM237 was found to be highly expressed in human native intestine and in human intestinal epithelial cell lines; further, confocal images showed colocalization of the protein with hRFVT-3. The interaction between TMEM237 with hRFVT-3 in human intestinal epithelial HuTu-80 cells was established by coimmunoprecipitation. Expressing TMEM237 in HuTu-80 cells led to a significant induction in RF uptake, while its knockdown (with the use of gene-specific siRNA) led to a significant reduction in uptake. Transfecting TMEM237 into HuTu-80 cells also led to a marked enhancement in hRFVT-3 protein stability (reflected by an increase in the protein half-life). Interestingly, the level of expression of TMEM237 was found to be markedly reduced following treatment with TNF-α (a proinflammatory cytokine that inhibits intestinal RF uptake), while its expression was significantly upregulated following treatment with butyrate (an inducer of intestinal RF uptake). These findings identify TMEM237 as an interactor with the intestinal hRFVT-3 and show that the interaction has physiological/biological significance.
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Mucosa Intestinal/citología , Mucosa Intestinal/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Butiratos/farmacología , Células CACO-2 , Humanos , Mucosa Intestinal/efectos de los fármacos , Proteínas de la Membrana/agonistas , Proteínas de la Membrana/antagonistas & inhibidores , Unión Proteica/efectos de los fármacos , Unión Proteica/fisiología , Receptores Acoplados a Proteínas G , Factor de Necrosis Tumoral alfa/farmacologíaRESUMEN
Intestinal absorption of ascorbic acid (AA) occurs via a Na+-dependent carrier-mediated process facilitated through the human sodium-dependent vitamin C transporters-1 &-2 (hSVCT1 and hSVCT2). Many studies have shown that hSVCT1 (product of the SLC23A1 gene) is expressed on the apical membrane of polarized enterocytes where it mediates AA absorption. hSVCT1 expression levels are therefore an important determinant of physiological vitamin C homeostasis. However, little is known about posttranscriptional mechanisms that regulate hSVCT1 expression in intestinal epithelia. In this study, we investigated regulation of hSVCT1 by microRNA (miRNA). A pmirGLO-SLC23A1-3'-UTR construct transfected into human intestinal cell lines (Caco-2 and NCM460 cells) showed markedly reduced luciferase activity. Bioinformatic analysis of the SLC23A1-3'-UTR predicted five miRNA binding sites (miR-103a, miR-107, miR-328, miR-384, and miR-499-5p) in the 3'-UTR. Expression of mature miR-103a was markedly higher compared to the other four putative miRNA regulators in both intestinal cell lines and mouse jejunal mucosa. Addition of a miR-103a mimic, but not a miR-103a mutant construct, markedly reduced the luminescence of the pmirGLO-SLC23A1-3'-UTR reporter. Reciprocally, addition of a miR-103a inhibitor significantly increased luciferase reporter activity. Addition of the miR-103a mimic led to a significant inhibition in AA uptake, associated with decreased hSVCT1 mRNA and protein expression in Caco-2 cells. In contrast, the miR-103a inhibitor increased AA uptake, associated with increased levels of hSVCT1 mRNA and protein. These findings provide the first evidence for posttranscriptional regulation of hSVCT1 by miRNA in intestinal epithelial cells.
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MicroARNs/genética , Transportadores de Sodio Acoplados a la Vitamina C/genética , Regiones no Traducidas 3' , Animales , Ácido Ascórbico/farmacocinética , Células CACO-2 , Línea Celular , Colon/citología , Regulación de la Expresión Génica , Humanos , Mucosa Intestinal/fisiología , Luciferasas/genética , Luciferasas/metabolismo , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Mutación , Transportadores de Sodio Acoplados a la Vitamina C/metabolismoRESUMEN
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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Ácido Butírico/farmacología , Colon/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Proteínas de Transporte de Membrana/metabolismo , Riboflavina/metabolismo , Animales , Transporte Biológico , Células CACO-2 , Colon/metabolismo , Metilación de ADN/efectos de los fármacos , Epigénesis Genética/efectos de los fármacos , Humanos , Mucosa Intestinal/metabolismo , Masculino , Proteínas de Transporte de Membrana/genética , Ratones Endogámicos C57BL , Organoides , Regulación hacia ArribaRESUMEN
Vitamin C is an antioxidant and acts as a cofactor for many enzymatic reactions. Humans obtain vitamin C from dietary sources via intestinal absorption, a process that involves the sodium-dependent vitamin C transporters-1 and -2 (SVCT1 and SVCT2). Enterotoxigenic Escherichia coli (ETEC) infection impacts intestinal absorption/secretory functions, but nothing is known about its effect on ascorbic acid (AA) uptake. Here we demonstrate that infection of Caco-2 cells with ETEC led to a significant inhibition in intestinal AA uptake. This inhibition was associated with a marked reduction in hSVCT1 and hSVCT2 protein, mRNA, and heterogeneous nuclear RNA (hnRNA) expression levels as well as significant inhibition in the activity of both the SLC23A1 and SLC23A2 promoters. Similarly, exposure of mice to ETEC led to a significant inhibition in intestinal AA uptake and reduction in mSVCT1 and mSVCT2 protein, mRNA, and hnRNA expression levels. Inhibition was caused by the action of heat labile enterotoxin (LT), since infecting Caco-2 cells with LT-deficient ETEC (ΔLT) failed to impact AA uptake. Because LT activates adenylate cyclase, we also examined the effect of dibutyryl-cAMP in AA uptake by Caco-2 cells and observed a significant inhibition. Furthermore, treating the cells with celastrol, a specific NF-κB inhibitor, significantly blocked the inhibition of AA uptake caused by ETEC infection. Together, these data demonstrate that ETEC infection impairs intestinal AA uptake through a cAMP-dependent NF-κB-mediated pathway that regulates both SLC23A1 and SLC23A2 transcription. NEW & NOTEWORTHY Our findings demonstrate that heat-labile enterotoxin produced by enterotoxigenic Escherichia coli inhibits AA uptake in intestinal epithelial cells and mouse intestine. This effect is mediated through transcriptional repression of SLC23A1 (SVCT1) and SLC23A2 (SVCT2) via a cAMP-dependent NF-κB signaling pathway.
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Ácido Ascórbico/farmacología , Escherichia coli Enterotoxigénica/química , Animales , Transporte Biológico/efectos de los fármacos , Células CACO-2 , Enterotoxinas/metabolismo , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Infecciones por Escherichia coli/metabolismo , Humanos , Mucosa Intestinal/efectos de los fármacos , Mucosa Intestinal/metabolismo , Intestinos/efectos de los fármacos , FN-kappa B/metabolismo , Transportadores de Sodio Acoplados a la Vitamina C/efectos de los fármacos , Transportadores de Sodio Acoplados a la Vitamina C/metabolismo , Vitaminas/metabolismoRESUMEN
Intestinal absorption of the water-soluble vitamins biotin and pantothenic acid is carrier mediated and involves the sodium-dependent multivitamin transporter (SMVT; product of the SLC5A6 gene). We recently observed that intestinal-specific (conditional) knockout of the mouse Slc5a6 gene (SMVT-cKO) is associated with growth retardation, the development of spontaneous and severe inflammation, abnormal histology in the large intestine, altered gut permeability, and early death. Our aim in this study was to examine the possibility that biotin and pantothenic acid oversupplementation (BPS) of the SMVT-cKO mice could reverse the above-described abnormalities. BPS was provided in the drinking water to mice before conception, to dams during pregnancy and lactation, and to the SMVT-cKO mice throughout their life. Our findings showed that such a regimen prevents early death, as well as normalizes the growth rate, intestinal integrity, pathology, and inflammation in SMVT-cKO mice. These findings provide clear evidence for a role for biotin and/or pantothenic acid in the maintenance of normal intestinal integrity and health.