Background: Ascorbic acid (AA) is required for normal human health and development. Human intestine expresses two sodium-dependent vitamin C transporters (hSVCT-1 and -2) that mediate cellular AA transport, with hSVCT1 targeting to the apical membrane of polarized epithelia. Studies have shown a role for the Rab8a in the apical membrane targeting of transporters in intestinal cells. Aims: The purpose of this study was to determine whether Rab8a impacts the function and/or targeting of hSVCT1, and intestinal AA uptake. Methods: We used human intestinal cells and cells from a Rab8a knockout mouse. 14C-AA uptake was performed to determine functionality. PCR and western blotting were performed to determine RNA and protein expression, respectively. Confocal imaging was performed to determine co-localization. Results: We show that hSVCT1 co-localized with Rab8a in intestinal cells. Knockdown of Rab8a lead to a significant inhibition in AA uptake and cell surface biotinylation studies revealed a lower cell surface expression of hSVCT1 in Rab8a siRNA-treated cells. Similarly, in the small intestine of a Rab8a knockout mouse, AA uptake was significantly inhibited. This effect again resulted from a decreased expression level of mSVCT1 protein, even though mRNA expression of SVCT1 was similar in intestinal cells from Rab8a knockout and wild-type litter-mates. The latter data are suggestive of enhanced lysosomal degradation of hSVCT1 protein in Rab8a-deficient cells; indeed, confocal imaging of Rab8a siRNA-treated intestinal cells revealed a strong overlap between hSVCT1-YFP and LAMP1-RFP. Conclusions: These findings show a role for Rab8a in the physiological function of hSVCT1 in intestinal epithelia.
Bibliographical noteFunding Information:
Acknowledgments This study was supported by grants from the National Institute of Health (DK84094 to V.S.S., and DK 56061 to H.M.S., GM088790 to J.S.M.) and the Department of Veterans Affairs.
- Ascorbic acid
- Vitamin C