Resveratrol potentiates intracellular ascorbic acid enrichment through dehydroascorbic acid transport and/or its intracellular reduction in HaCaT cells.

L-Ascorbic acid (AsA), a reduced vitamin C (VC), is an important antioxidant, and the internal accumulation and maintenance of AsA are thought to play a significant role in various physiological activities in humans. We focused on resveratrol (RSV), a natural polyphenolic compound, as a candidate for an AsA transport modulator and investigated whether RSV can affect the intracellular VC accumulation after either AsA or dehydroascorbic acid (DHA) addition in HaCaT keratinocytes. Our results demonstrate that RSV treatment could significantly enhance intracellular VC levels after either AsA or DHA supplementation, and intracellular VC accumulated mainly as AsA. Our results also indicate that most of the intracellular transported DHA was reduced to AsA and accumulated after uptake into cells. In addition, RSV could induce several AsA or DHA transport-related and intracellular DHA reduction-related genes including SVCT2, GLUT3, TXNRD2, and TXNRD3, necessary for AsA transport, DHA transport, and DHA reduction/regeneration, respectively. On the other hand, the both protein expression levels and the localizations of sodium-dependent vitamin C transporters 2 (SVCT2) and glucose transporter 3(GLUT3) were scarcely affected by RSV treatment. Furthermore, RSV-induced enrichment of intracellular AsA levels was completely suppressed by a GLUT inhibitor cytochalasin B. These results suggest that RSV can potentiate intracellular AsA accumulation via activation of the DHA transport and subsequent intracellular reduction from DHA to AsA. Thus, RSV might be useful for maintaining substantial AsA accumulation in the skin keratinocytes.

The influence of cellular senescence on intracellular vitamin C transport, accumulation, and function.

In humans, vitamin C (VC) accumulates at higher concentrations in cells than in plasma, and this intracellular accumulation appears critical to several important physiological functions. However, although VC accumulation decreases in the elderly, the influence of cellular senescence on the transport, accumulation, and function of VC is poorly understood. In this study, we investigated the effects of supplementation with both ascorbic acid (AsA) and dehydroascorbic acid (DehAsA) on the uptake and accumulation of VC, AsA, and DehAsA into cells and the effect of AsA on the levels of intracellular reactive oxygen species (ROS) in human fibroblast TIG-1 cells. We also assessed how that supplementation affected senescence-associated changes in intracellular VC transport and accumulation. AsA supplementation significantly increased intracellular levels of AsA, DehAsA, and total VC (i.e., reduced AsA plus oxidized DehAsA) in senescent cells compared with young cells. DehAsA supplementation also significantly increased intracellular AsA and total VC levels in senescent cells, but not DehAsA, and the increases were less than after adding AsA. Among the molecules related to VC accumulation, the mRNA and protein expressions of sodium-dependent VC transporter 2 (SLC23A2) were increased in senescent cells. Furthermore, intracellular peroxide and superoxide anion levels were higher in senescent cells, with AsA supplementation markedly attenuating spontaneous intracellular peroxide accumulation. Overall, our results therefore suggest that VC transport and accumulation improved in senescent human fibroblast TIG-1 cells due to the adaptive upregulation of sodium-dependent VC transporter 2 in response to increased ROS levels. We conclude that adequate supplementation with AsA can effectively mitigate senescence-associated intracellular ROS.