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).

Background

Enteropathogenic Escherichia coli (EPEC) infection causes prolonged, watery diarrhea leading to morbidity and mortality. Although EPEC infection impacts nutrient transporter function and expression in intestinal epithelial cells, the effects of EPEC infection on intestinal absorption of ascorbic acid (AA) have not yet been investigated.

Aims

To investigate the effect of EPEC infection on intestinal AA uptake process and expression of both AA transporters.

Methods

We used two experimental models: human-derived intestinal epithelial Caco-2 cells and mice. ¹⁴C-AA uptake assay, Western blot, RT-qPCR, and promoter assay were performed.

Results

EPEC (WT) as well as ΔespF and ΔespG/G2 mutant-infected Caco-2 cells showed markedly inhibited AA uptake, while other mutants (ΔescN, ΔespA, ΔespB, and ΔespD) did not affect AA uptake. Infection also reduced protein and mRNA expression levels for both hSVCT1 and hSVCT2. EPEC-infected mice showed marked inhibitory effect on AA uptake and decreased protein and mRNA expression levels for both mSVCT1 and mSVCT2 in jejunum and colon. MicroRNA regulators of SVCT1 and SVCT2 (miR103a, miR141, and miR200a) were upregulated significantly upon EPEC infection in both Caco-2 and mouse jejunum and colon. In addition, expression of the accessory protein glyoxalate reductase/hydroxypyruvate reductase (GRHPR), which regulates SVCT1 function, was markedly decreased by EPEC infection in both models.

Conclusions

These findings suggest that EPEC infection causes inhibition in AA uptake through a multifactorial dysregulation of SVCT1 and SVCT2 expression in intestinal epithelial cells.

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.