Mechanisms of Human Hepatic Vitamin C Uptake: Studies of the hSCVT Systems

Abstract

Ascorbic acid (AA), the reduced active form of vitamin C, is transported by two sodium‐ascorbate co‐transporters (SVCT1 & 2). Humans do not synthesize AA and the essential micronutrient is both an antioxidant and cofactor for several enzymes. The human liver participates in regulating and maintaining body AA homeostasis. Using a human hepatic cell line (HepG2) we determined, the characteristics of AA uptake, the expression levels of the hSVCT systems and characterized the hSVCT promoters. The initial rate of ascorbic acid uptake by HepG2 cells was: 1) Na+ gradient, pH, and energy dependent, 2) saturable with a Km of 10μM 3) not inhibited by membrane transport inhibitors 4) not stimulated by glucocorticoids 5) PKC, PKA, PTK, Ca‐Camodulin dependant, but not NO dependant. Expression of hSVCT1 and hSVCT2 protein and mRNA in both HepG2 cells and human liver was determined; hSVCT2 is expressed at higher levels. Using cloned promoter fragments, transcriptional activity was determined in HepG2 cells and the minimal regions required for basal activity mapped. In addition, using promoter mutagenesis we determined that Hepatic Nuclear Factor sites in the hSVCT1 promoter and Sp1/KLF sites in the hSVCT2 promoter were essential for activities. Results from these studies are the first determination of the AA uptake process, relative expression of the hSVCT systems and characterization of the hSVCT promoters in human liver cells.