No abstract

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 (ChIP) 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).

Pyridoxine (vitamin B6), an essential micronutrient for normal cell physiology, plays an important role in the function of the exocrine pancreas. Pancreatic acinar cells (PACs) obtain vitamin B6 from circulation, but little is known about the mechanism involved in the uptake process; limited information also exists on the effect of pyridoxine availability on the gene expression profile in these cells. We addressed both these issues in the current investigation using mouse-derived pancreatic acinar 266-6 cells (PAC 266-6) and human primary PACs (hPACs; obtained from organ donors), together with appropriate physiological and molecular (RNA-Seq) approaches. The results showed [3H]pyridoxine uptake to be 1) pH and temperature (but not Na+) dependent, 2) saturable as a function of concentration, 3) cis-inhibited by unlabeled pyridoxine and its close structural analogs, 4) trans-stimulated by unlabeled pyridoxine, 5) regulated by an intracellular Ca2+/calmodulin-mediated pathway, 6) adaptively-regulated by extracellular substrate (pyridoxine) availability, and 7) negatively impacted by exposure to cigarette smoke extract. Vitamin B6 availability was found (by means of RNA-Seq) to significantly (FDR < 0.05) modulate the expression profile of many genes in PAC 266-6 cells (including those that are relevant to pancreatic health and development). These studies demonstrate, for the first time, the involvement of a regulatable and specific carrier-mediated mechanism for pyridoxine uptake by PACs; the results also show that pyridoxine availability exerts profound effects on the gene expression profile in mammalian PACs.