Mechanisms and regulation of vitamin C uptake: studies of the hSVCT systems in human liver epithelial cells

Abstract

Humans use two sodium-ascorbate cotransporters (hSVCT1 and hSVCT2) for transporting the dietary essential micronutrient ascorbic acid, the reduced and active form of vitamin C. Although the human liver plays a pivotal role in regulating and maintaining vitamin C homeostasis, vitamin C transport physiology and regulation of the hSVCT systems in this organ have not been well defined. Thus, this research used a human hepatic cell line (HepG2), confirming certain results with primary human hepatocytes and determined the initial rate of ascorbic acid uptake to be Na(+) gradient, pH dependent, and saturable as a function of concentration over low and high micromolar ranges. Additionally, hSVCT2 protein and mRNA are expressed at higher levels in HepG2 cells and native human liver, and the cloned hSVCT2 promoter has more activity in HepG2 cells. Results using short interfering RNA suggest that in HepG2 cells, decreasing hSVCT2 message levels reduces the overall ascorbic acid uptake process more than decreasing hSVCT1 message levels. Activation of PKC intracellular regulatory pathways caused a downregulation in ascorbic acid uptake not mediated by a single predicted PKC-specific amino acid phosphorylation site in hSVCT1 or hSVCT2. However, PKC activation causes internalization of hSVCT1 but not hSVCT2. Examination of other intracellular regulatory pathways on ascorbic acid uptake determined that regulation also potentially occurs by PKA, PTK, and Ca(2+)/calmodulin, but not by nitric oxide-dependent pathways. These studies are the first to determine the overall ascorbic acid uptake process and relative expression, regulation, and contribution of the hSVCT systems in human liver epithelial cells.

Molecular determinants dictating cell surface expression of the human sodium-dependent vitamin C transporter-2 in human liver cells

“…The Quik change site-directed mutagenesis kit (Stratagene, La Jolla, CA) was used to introduce insertions or deletions of nucleotides into the open reading frame of SLC23A2. Overlapping primers containing the mutated nucleotides to the specified mutation sites (Supplemental Table S2), and full-length hSVCT2-YFP fused plasmid were used as a template for PCR-based site-directed mutagenesis as described (23,29). The nucleotide sequence of each construct was verified by sequencing (Laragen, Los Angeles, CA).…”

“…[ 14 C]ascorbic acid uptake assays were performed on confluent, stably transfected HepG2 cell lines following established procedures (23,30,32). Protein contents were estimated on parallel wells by using a protein assay kit (Bio-Rad, Hercules, CA).…”

“…Both hSVCT1 and hSVCT2 are expressed in human liver, with hSVCT2 expression being higher than hSVCT1 (22,23,38). This abundant expression of hSVCT2 in human liver implies a significant physiological role for this particular transporter in liver vitamin C homeostasis.…”

Vitamin C promotes pluripotency of human induced pluripotent stem cells via the histone demethylase JARID1A

“…Cell clumps were transferred to a new dish on MEF feeder cells. For vitamin C treatment, vitamin C (Sigma) was added to the medium at a final concentration of 50 μg/ml (Reidling et al, 2008).…”

The Role of Free Radicals and Antioxidant in Abortion

“…Thus, the role of folic acid supplementation to prevent recurrent pregnancy loss is inconclusive and requires further analysis. (18)(19)(20)(21) Free radicals and OS have important roles in modulating many physiological functions in reproduction, in this case is recurrent abortion and others pregnancy abnormalities. The evaluation of in vivo OS is difficult.…”