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

Heskett, Christopher W; Teafatiller, Trevor; Hennessey, Carly; Gareau, Mélanie G.; Marchant, Jonathan S.; Said, Hamid M.; Subramanian, Veedamali S.

doi:10.1007/s10620-020-06389-x

Cited by 12 publications

(11 citation statements)

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“…Thus, the respective proteins appear dispensable for the inhibitory effect. These observations indicate that the effect of EPEC on AA uptake in intestinal epithelia requires a functional T3SS and more than one of the factors it secretes [9].…”

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confidence: 82%

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I Can C Clearly Now: How EPEC Inhibits Gut Vitamin C Transport by Dysregulating SVCT

2020

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confidence: 82%

“…In the study of Heskett and colleagues published in this issue of Digestive Diseases and Sciences, the authors utilized both in vivo and in vitro models in order to address this issue. The authors show for the first time the effect of EPEC infection on the expression of SVCTs [9].…”

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confidence: 89%

I Can C Clearly Now: How EPEC Inhibits Gut Vitamin C Transport by Dysregulating SVCT

2020

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“…After protein transfer, the membrane was blocked for 10 min at room temperature in LI-COR Odyssey Blocking Buffer and then probed with previously characterized primary SVCT2 antibodies (1 : 500 dilution) [ 40 ], anti-IKK αβ antibodies (1 : 1000 dilution; Abcam), anti-NF- κβ p65 antibodies (1 : 1000 dilution; Abcam), anti-laminin antibodies (1 : 300 dilution; Santa Cruz Biotechnology), and anti- β -actin mouse monoclonal antibody (1 : 3000 dilution; ThermoFisher Scientific) used. The respective secondary antibodies (anti-rabbit IRDye-800 and anti-mouse IRDye-680, LI-COR Biosciences) were used in 1 : 30,000 dilutions [ 33 , 34 , 40 ]. Odyssey Infrared imaging system (LI-COR Biosciences) software was used to quantify the densitometry of specific band signal intensities normalized against β -actin.…”

Section: Methodsmentioning

confidence: 99%

Effect of Lipopolysaccharide and TNFα on Neuronal Ascorbic Acid Uptake

Subramanian

Teafatiller

Agrawal

et al. 2021

Mediators of Inflammation

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Vitamin C (ascorbic acid: AA) uptake in neurons occurs via the sodium-dependent vitamin C transporter-2 (SVCT2), which is highly expressed in the central nervous system (CNS). During chronic neuroinflammation or infection, CNS levels of lipopolysaccharide (LPS) and LPS-induced tumor necrosis factor-α (TNFα) are increased. Elevated levels of LPS and TNFα have been associated with neurodegenerative diseases together with reduced levels of AA. However, little is known about the impacts of LPS and TNFα on neuronal AA uptake. The objective of this study was to examine the effect of LPS and TNFα on SVCT2 expression and function using in vitro and in vivo approaches. Treatment of SH-SY5Y cells with either LPS or TNFα inhibited AA uptake. This reduced uptake was associated with a significant decrease in SVCT2 protein and mRNA levels. In vivo exposure to LPS or TNFα also decreased SVCT2 protein and mRNA levels in mouse brains. Both LPS and TNFα decreased SLC23A2 promoter activity. Further, the inhibitory effect of LPS on a minimal SLC23A2 promoter was attenuated when either the binding site for the transcription factor Sp1 was mutated or cells were treated with the NF-κB inhibitor, celastrol. We conclude that inflammatory signals suppress AA uptake by impairing SLC23A2 transcription through opposing regulation of Sp1 and NF-κB factors.

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“…The separated proteins were then transferred to an Immobilon polyvinylidene difluoride (PVDF) membrane (Millipore, Burlington, MA) by electroblotting [18,19]. The membrane was blocked using blocking buffer (Cat# 924-40010; LI-COR Biosciences) then probed with well characterized primary antibodies (raised by Thermo Fisher Scientific, Rockford, IL, USA) against SVCT1 (amino acids: 576-594 RGFSKKTQNQPPVLEDTPD), or SVCT2 (amino acids: 627-645 GYTWKGLRKSDNSRSSDED) (1:500 dilution for both [19]) and/or β-actin (1:2000 dilution) antibodies (Santa Cruz Biotechnology). LI-COR anti-rabbit IRDye 800 (Cat# NC9401842) and/or anti-mouse IRDye 680 (Cat# NC0046410) (1:30000 dilution for both) were used as secondary antibodies to probe the blot at room temperature (RT) for 45 min.…”

Section: Western Blot Analysismentioning

confidence: 99%

“…Representative coronal sections (3-4 sections/brain, 4 brains/group) through the caudal diencephalon were selected for staining and were washed three times at 5 min each in 0.3% Tween-20 + 1X TBS. Tissues were blocked for 30 min at RT in 1X TBS buffer (containing 4% BSA + 0.1% Tween-20) then probed with well characterized custom made rabbit anti-SVCT2 primary antibody (1:1000 dilution; Thermo Fisher Scientific [19]) in 1X TBS buffer (containing 0.1% Tween-20 + 0.2% BSA) for 1 h at RT, then overnight at 4 • C. Sections were then washed three more times, and then stained with secondary antibody Donkey Anti-Rabbit Alexa Flour 568 (Cat# ab175470; Abcam, MA) in 1X TBS buffer (0.1% Tween-20 + 4% BSA) for 1 h at RT. We tested tissue by omission of the primary antibody from the protocol to confirm the absence of any non-specific binding of secondary antibody to the mouse tissue.…”

Section: Immunohistochemistrymentioning

confidence: 99%

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

et al. 2021

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

Cited by 12 publications

References 50 publications

I Can C Clearly Now: How EPEC Inhibits Gut Vitamin C Transport by Dysregulating SVCT

I Can C Clearly Now: How EPEC Inhibits Gut Vitamin C Transport by Dysregulating SVCT

Effect of Lipopolysaccharide and TNFα on Neuronal Ascorbic Acid Uptake

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

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