Characterization of the Ascorbic Acid Uptake in Mouse Lens Epithelial Cells

Abstract

Abstract: : Purpose:Aging human lens crystallins progressively form UV–active fluorophores and crosslinks from glycation with reducing sugars or ascorbic acid. Using ¹⁹F–ascorbic acid (¹⁹F–ASA), we previously showed that mouse lens epithelial 17EM15 cells have impaired ascorbic acid uptake, which may be linked to impairment of the high affinity ascorbic acid transporter SVCT2. However, because ¹⁹F–ASA differs in its structure from native ASA, confirmation of these initial findings was crucial prior to embarking in the development of transgenic mouse models of pigmentation, based on ascorbic acid. Thus, we evaluated not only ¹⁹F–ascorbic acid but also native ascorbic acid uptake, which is impaired in mouse lens cells compared to human and determined the presence of SVCT2 by qPCR. Methods: Cells were grown to confluence and incubated with either F–ASA or F–DHA, or ¹⁴C–ASA or the oxidized native compound for 12 hours. Cells were washed, lysed in cold 4% metaphosphoric acid and supernatants were analyzed by 750 MHz NMR spectroscopy. qPCR was performed using the ABI PRISM® 7000 system and primers specifically designed for the human and mouse SVCT2. Results: F–ASA and F–DHA were both taken up in a sodium–dependent manner by human and mouse cells, implicating thus SVCT2. Moreover, F–ASA and native ascorbic acid uptake was specifically impaired in the mouse cells. The oxidized form of ¹⁴C–ASA, ¹⁴C–DHA, was taken up by both cells, albeit in greatly diminished concentration by mouse cells. Mouse ¹⁴C–DHA uptake was further suppressed by high glucose concentrations implicating, thereby, GLUT1 and GLUT3 transporters. Quantitative PCR showed the SVCT2 mRNA was present in human and, surprisingly, also in the mouse cells. Slightly more message was found in the human cells. The presence of the full–length gene has been confirmed by PCR. Mouse and human specific antibodies are being raised to investigate surface expression of SVCT2 protein. Discussion: Our data confirm the existence of a defect in ascorbic acid uptake in 17EM15 cells. In silico research revealed human and mouse share >80% SVCT2 gene homology apart from a 60 N–terminal amino acid deletion in the mouse, and several mutations that may result in defects of glycosylation or ATP binding in mouse. Plasmids containing a construct for the full–length human gene are currently being used for transient and stable overexpression in CHO and mouse cells. A transgenic animal model of lens pigmentation and crosslinking by ascorbylation will be generated by overexpressing the human gene in the mouse. CR : None Support: NEI/NIH EY 07099 and P30EY11373

Keywords: cataract • aging • metabolism