**3. Regulation of VitC Transport by Substrate Concentrations**

VitC transport can potentially be regulated by the amount of the various transporters or by their state of activity, thus relying on the rate of *de novo* synthesis (and degradation) and on activation of non-functional transporters, putatively also involving cellular translocation. Translational regulation of SVCT2 has been demonstrated *in vitro* in human platelets [124], and both SVCT1 and SVCT2 contain potential sites for glycosylation and phosphorylation as putative targets for post-translational regulation (Table 1). *N*-linked glycosylation sites are located in the second and third extracellular loop, and a single protein kinase A-dependent and several protein kinase C-dependent phosphorylation sites have been described in the amino acid sequence [52]. In humans, enzymatic activity of protein kinase C elicits different responses of SVCT1 and SVCT2. For SVCT1, the cytoplasm-to-membrane translocation is reduced, whereas the phosphorylation elicits a conformational alteration of the SVCT2 [97]. An association between the availability of vitC and an effect on transport mechanisms has been suggested, displaying the characteristics of a substrate-dependent regulation [124–130].


**Table 1.** Overview of *in vitro* studies regarding the regulation of the transport of vitamin C (vitC) during deficiency.

Findings *in vitro* have shown a significant decrease in ASC uptake and concurrent mRNA expression-reduction following high-dose ASC culture, suggesting a putative feed-back mechanism and the association of transcriptional repressor elements in the regulation of ASC uptake [125,126] (Table 1). This has been further supported by studies in human hepatic cells, in which ASC depletion significantly increased SVCT1 expression [127]. The involvement of a transcriptional regulatory mechanism was confirmed by the finding of an altered promoter activity associated to the Hepatic Nuclear Factor 1 (HNF-1) binding sites on the SVCT1 promoter region [127]. The involvement of translational regulation has been shown for the SVCT2 transporter in human platelets [124]. A reduction of the ASC concentration in the surrounding medium to 30% increased the *V*max-value by almost 240%, whereas the *K*m-value remained unchanged, indicating a responsive increased transport rate [124]. This was later confirmed by a marked increase in SVCT2 protein expression compared to platelets prior to deprivation of ASC [124]. In renal proximal tubule cells, increasing concentrations of ASC led to a signal translocation from the apical membrane to the cytoplasm before the signal was diminished [128]. The effect was a 50% reduction in transport by cells pretreated with amounts of ASC corresponding to plasma saturation (50–100 μM), suggesting that apical levels play a pivotal role in the regulation of ASC uptake *in vitro*.

A substrate-dependent differential regulation has also been supported by *in vivo* studies (Table 2). Almost four decades ago, Rose and Nahrwold showed that guinea pigs receiving diets containing five or 25 times the vitC of a standard diet reduced the rate of ASC uptake by 32%–52% compared to control animals [129]. Daily intramuscular injections of 300 mg ASC reduced ASC influx into the intestinal mucosa by 16%, suggesting that high circulating ASC levels may inhibit transport across the intestinal epithelium, but not ruling out that ASC concentrations in the intestinal lumen or cytoplasm of epithelial cells contribute to the regulation [129]. In guinea pigs, a diet containing high amounts of vitC (5000 mg/kg feed) reduced ASC uptake across isolated ileac mucosa by 25%–50% in both adult male, lactating female and juvenile animals compared to controls (200 mg vitC/kg feed) [130]. The observed decrease in uptake did not result in alterations of *K*m-values, indicating that the reduced absorption rate was due to either reduced abundance or increased degradation of the transporting molecules [130]. However, guinea pig counterparts receiving low levels of vitC (3.5 mg/week) did not show an increased rate of ASC uptake in response to deficiency [130]. The authors suggested that transporters have a nearly complete extraction efficiency, even under standard conditions (control animals) [130]. Likewise, an absence of up-regulation of SVCT-transporters following long-term vitC deficiency (100 mg vitC/kg feed) in guinea pigs has been shown [31].

In humans, plasma concentrations following oral dosing of vitC are tightly regulated with peak plasma levels of around 200 μmol/L and a steady-state of 70–85 μmol/L, even when excessive amounts (3 g) vitC are ingested [25,133]. A steep decline in ASC bioavailability following increased oral doses suggests that intestinal transport is a key factor in maintaining whole body vitC homeostasis. This regulation may, however, be bypassed by intravenous dosage, achieving plasma concentrations well beyond the threshold of oral administration levels [26,133]. Concentrations in plasma have been shown to pose a direct effect on the tissue accumulation of ASC. Oral supplementation of mice unable to synthesize vitC due to a mutation in the L-gulono-γ-lactone gene (*gulo<sup>−</sup>/<sup>−</sup>* ) has shown that increased plasma levels are required to achieve optimal concentration in several tissues (liver, heart and kidney) compared to levels necessary to obtain saturation in the brain [134]. This confirms the brain as being particularly efficient in retaining ASC, but also points towards a direct association between differences in plasma concentration and tissue-specific uptake [134]. In a recent intervention study in humans, the bioavailability of vitC in skeletal muscle

relative to dietary intake was shown to correspond to plasma concentrations, whereas the same close correlation could not be found in leukocytes [135]. This could indicate an increased sensitivity in muscle towards alterations in plasma concentration or/and a differential uptake in leukocyte cells, possibly linked to differences in SVCT2 expression between the two cell types [135].


**Table 2.** Overview of *in vivo* studies regarding the regulation of vitC transport in response to vitC levels.

An increase in SVCT-expression as a response to a reduction in ASC, followed by a decrease in SVCT mRNA when ASC levels increased, has been reported in developing teleost fish, which naturally are unable to synthesize vitC [136]. Although this could be part of a normal developmental sequence of events, it may also be a substrate-dependent response on SVCT-expression by which ASC uptake is regulated [136]. Studies of senescence marker protein-30 (SMP30)/gluconolactonase (GNL) knockout mice has revealed that vitC depletion increases SVCT1 mRNA levels in liver and small intestine and SVCT2 mRNA in liver [30]. VitC sufficient wild-type (WT) mice had 43% less SVCT2 mRNA compared to ASC-depleted WT mice, suggesting a deficiency associated mRNA upregulation. No changes were found in kidney or cerebellum of either WT or *smp30/gnl<sup>−</sup>/<sup>−</sup>* groups [30]. The study did not find any significant changes in the expression of GLUT-transporters (1, 3 and 4, respectively) [30]. In another study, Meredith *et al*. reported a significant increase in liver

SVCT2 mRNA during vitC deficiency in the *gulo<sup>−</sup>/<sup>−</sup>* mice, whereas no effect was found in either cortex or cerebellum of the brain [29]. These findings may indicate the existence of an alternative transport mechanism involved in the regulation of vitC during deficiency [29]. In a study of fetal *vs*. maternal vitC status in guinea pigs, it was recently found that an inadequate maternal vitC intake during pregnancy resulted in reduced plasma vitC in newborn deficient pups, compared with their mothers [137]. The authors interpreted their findings as an indication that preferential transport of vitC from the mother to the fetus is overridden during a prolonged maternal vitC deficiency, thereby maintaining a basal maternal vitC concentration at the expense of the offspring [137]. Thus, several studies suggest that the exact mechanisms controlling the substrate-mediated regulation of vitC homeostasis in the body remains to be fully disclosed, but it appears likely that there are different mechanisms involved and that regulatory control may vary within specific tissues. Other regulatory mechanisms have also been suggested to contribute to vitC regulation, *i.e.*, feed-back sensing and hormonal regulation [26].
