Hypoxia inhibits colonic uptake of the microbiota-generated forms of vitamin B1 via HIF-1α-mediated transcriptional regulation of their transporters

Sabui, Subrata; Ramamoorthy, Kalidas; Romero, Javier; Simões, Rita D.; Fleckenstein, James M.; Said, Hamid M.

doi:10.1016/j.jbc.2022.101562

Cited by 9 publications

(7 citation statements)

References 51 publications

(103 reference statements)

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“…Percent expression was normalized by GAPDH and analyzed using the comparative threshold cycle (Ct) method. Amplification of SLC19A3 transcripts from small intestinal enteroids was performed as recently described 83 with relative gene expression normalized to β actin. HNF4a, HNFg, and SMAD4 gene expression was determined by TaqMan (Thermo Fisher) using validated primer and probe sets.…”

Section: Methodsmentioning

confidence: 99%

“…Total protein was extracted from LT toxin-treated (100 ng/ml) human differentiated enteroid monolayers (235D line) and untreated control using radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing protease inhibitor cocktail. An equal amount (∼25 μg) of the proteins were loaded on a NuPAGE 4-12% Bris–Tris gradient gels (Invitrogen) as previously described 83 , then blotted onto polyvinylidene difluoride (PVDF) membranes and probed with anti-SLC19A3 (1:1000; Cat# 13407-1-AP; Proteintech), or anti-SP1 (1:1000; Cat# ab124804; Abcam) antibodies and together with anti-beta actin (1:3000; Cat# sc-47778) primary antibodies. The specificity of the SLC19A3 antibodies was validated in our laboratory previously using different approaches that include overexpression of tagged protein or gene silencing 85 .…”

Section: Methodsmentioning

confidence: 99%

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Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia

et al. 2022

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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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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia

et al. 2022

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“…Percent expression was normalized by GAPDH and analyzed using the comparative threshold cycle (Ct) method. Amplification of SLC19A3 transcripts from small intestinal enteroids was performed as recently described 82 with relative gene expression normalized to β actin. LT_brush border 9/27/22 5:07:19 PM HNF4a, HNFg, and SMAD4 gene expression was determined by TaqMan (ThermoFisher) using validated primer and probe sets.…”

Section: Quantitative Real Time Pcrmentioning

confidence: 99%

“…Total protein was extracted from LT toxin-treated (100ng/ml) human differentiated enteroid monolayers (235D line) and untreated control using radioimmunoprecipitation assay (RIPA) buffer (Sigma) containing protease inhibitor cocktail. An equal amount (∼25 μg) of the proteins were loaded on a NuPAGE 4-12% Bris-Tris gradient gels (Invitrogen) as previously described 82 , then blotted onto polyvinylidene difluoride (PVDF) membranes and probed with anti-SLC19A3 (1:1000; Cat# 13407-1-AP; Proteintech), or anti-SP1 (1:1000; Cat# ab124804; Abcam) 9/27/22 5:07:19 PM antibodies and together with anti-beta actin (1:3000; Cat# sc-47778) primary antibodies. Specificity of the SLC19A3 antibodies was validated in our laboratory previously using different approaches that include overexpression of tagged protein or gene silencing 84 .…”

Section: Immunoblotting Slc19a3 Sp1mentioning

confidence: 99%

“…Enteroid monolayers were then washed with ice-cold Krebs-LT_brush border 9/27/22 5:07:19 PM Ringer buffer followed by lysis with NaOH and neutralization with 10 N HCl. The radioactive content was counted using a liquid scintillation counter as described previously 82 . Uptake of thiamin by its respective and distinct carrier-mediated mechanism was determined by subtracting uptake of [ 3 H]-thiamin in the presence of a high pharmacological concentration (1 mM) of unlabeled thiamin from uptake in their absence; all uptake data points were calculated relative to total protein content (in milligrams) of the different preparations and presented as percentage relative to simultaneously performed controls.…”

Section: Thiamin Uptakementioning

confidence: 99%

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EnterotoxigenicEscherichia coliheat-labile toxin drives enteropathic changes in small intestinal epithelia

Sheikh

Tumala

Vickers

et al. 2022

Preprint

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Enterotoxigenic E. coli (ETEC), produce heat-labile (LT) and/or heat-stable (ST) enterotoxins, and are a common cause of diarrhea in children of resource-poor regions. ETEC have also been linked repeatedly to poorly understood sequelae including enteropathy, malnutrition, and growth impairment. While the cellular actions of ETEC enterotoxins leading to diarrhea are well-established, their potential contribution to subsequent pathology is unclear. LT stimulates cellular cAMP production to activate protein kinase A (PKA) which phosphorylates cellular ion channels that drive export of salt and water into the intestinal lumen resulting in diarrhea. However, as PKA exhibits broad kinase activity and its activated catalytic subunits modulate transcription of many genes, we interrogated the transcriptional profiles of LT-treated small intestinal epithelia. These studies demonstrated toxin-induced changes in hundreds of genes including those required for biogenesis and function of the brush border, the major site absorption of nutrients, and suppression of a key transcription factor, HNF4, critical to differentiation of intestinal epithelia. Accordingly, LT treatment of intestinal epithelial cells significantly disrupted the absorptive microvillus architecture and altered transport of essential nutrients. In addition, challenge of neonatal mice with LT-producing ETEC recapitulated the architectural derangement of the brush border while maternal vaccination with LT prevented brush border disruption in ETEC-challenged neonatal mice. These findings highlight impacts of ETEC enterotoxins beyond acute diarrheal illness and may inform approaches to mitigate and prevent major sequelae including malnutrition that impact millions of young children.

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High-resolution plasma metabolomics and thiamine status in critically Ill adult patients

Gundogan,

Nellis,

Ozer

et al. 2024

Metabolomics

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Introduction Thiamine (Vitamin B1) is an essential micronutrient and is classically considered a co-factor in energy metabolism. The association between thiamine status and whole-body metabolism in critical illness has not been studied. Objectives To determine association between whole blood thiamine pyrophosphate (TPP) concentrations and plasma metabolites and connected metabolic pathways using high resolution metabolomics (HRM) in critically ill patients. Methods Cross-sectional study performed at Erciyes University Hospital, Kayseri, Turkey and Emory University, Atlanta, GA, USA. Participants were critically ill adults with an expected length of intensive care unit stay longer than 48 h and receiving chronic furosemide therapy. A total of 76 participants were included. Mean age was 69 years (range 33–92 years); 65% were female. Blood for TPP and metabolomics was obtained on the day of ICU admission. Whole blood TPP was measured by HPLC and plasma HRM was performed using liquid chromatography/mass spectrometry. Data was analyzed using regression analysis of TPP levels against all plasma metabolomic features in metabolome-wide association studies (MWAS). MWAS using the highest and lowest TPP concentration tertiles was performed as a secondary analysis. Results Specific metabolic pathways associated with whole blood TPP levels in regression and tertile analysis included pentose phosphate, fructose and mannose, branched chain amino acid, arginine and proline, linoleate, and butanoate pathways. Conclusions Plasma HRM revealed that thiamine status, determined by whole blood TPP concentrations, was significantly associated with metabolites and metabolic pathways related to metabolism of energy, carbohydrates, amino acids, lipids, and the gut microbiome in adult critically ill patients.

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Hypoxia inhibits colonic uptake of the microbiota-generated forms of vitamin B1 via HIF-1α-mediated transcriptional regulation of their transporters

Cited by 9 publications

References 51 publications

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia

Enterotoxigenic Escherichia coli heat-labile toxin drives enteropathic changes in small intestinal epithelia

EnterotoxigenicEscherichia coliheat-labile toxin drives enteropathic changes in small intestinal epithelia

High-resolution plasma metabolomics and thiamine status in critically Ill adult patients

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