Sodium-dependent Vitamin C Transporter 2 Research Articles

SVCT-2 determines the sensitivity to ascorbate-induced cell death in cholangiocarcinoma cell lines and patient derived xenografts

Cholangiocarcinoma (CC) is a devastating malignancy with late diagnosis and poor response to conventional chemotherapy. Recent studies have revealed anti-cancer effect of vitamin C (l-ascorbic acid, ascorbate) in several types of cancer. However, the effect of l-ascorbic acid (AA) in CC remains elusive. Herein, we demonstrated that AA induced cytotoxicity in CC cells by generating intracellular reactive oxygen species (ROS), and subsequently DNA damage, ATP depletion, mTOR pathway inhibition. Moreover, AA worked synergistically with chemotherapeutic agent cisplatin to impair CC cells growth both in vitro and in vivo. Intriguingly, sodium-dependent vitamin C transporter 2 (SVCT-2) expression was inversely correlated with IC50 values of AA. Knockdown of SVCT-2 dramatically alleviated DNA damage, ATP depletion, and inhibition of mTOR pathway induced by AA. Furthermore, SVCT-2 knockdown endowed CC cells with the resistance to AA treatment. Finally, the inhibitory effects of AA were further confirmed in patient-derived CC xenograft models. Thus, our results unravel therapeutic potential of AA alone or in combination with cisplatin for CC. SVCT2 expression level may serve as a positive outcome predictor for AA treatment in CC.

An Overview of the Characteristics and Function of Vitamin C in Various Tissues: Relying on its Antioxidant Function

: Vitamin C (L-ascorbic acid or ascorbate) is a biomolecule that participates in many biochemical processes. It is an essential nutrient for humans, however, in some species such as rodents and guinea pigs is synthesized. It has a variety of functions in the body that we might venture to say make it a very important antioxidant nature and pro-oxidant. L-ascorbic acidic a reduced form of vitamin C and dehydroascorbic acid (DHA) is the oxidized form of ascorbate, both L-ascorbic acid and dihydroascorbic acid retain the vitamin C activity. Dehydro-ascorbate is reconverted to ascorbate in the cytosol by cytochrome b reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Ascorbate is transported into the cell via the sodium-dependent vitamin C transporters (SVCTs), which causes accumulation of ascorbate within cells against a concentration gradient. Dehydroascorbic acid, the oxidized form of ascorbate, is transported via glucose transporters family (GLUTs). The highest concentrations of ascorbate in the body are found in brain and adrenal gland. Vitamin C also acts as a co-factor in several enzyme reactions. This vitamin is an essential biochemical factor in the reproductive process. The pharmacophore of vitamin C is the ascorbate, ascorbate is an antioxidant.Ascorbate is a neuromodulator of glutamatergic and dopaminergic system and related behaviors. It also improves components of the immune system. Given the wide role of ascorbate, further investigation is necessary to evaluate the exact mechanism(s) underlying these effects. In this review we will consider a short overview of the characteristics and function of vitamin C (relying on antioxidant function) in various tissues.

Vitamin C enhances the analgesic effect of gabapentin on rats with neuropathic pain

AimsGabapentin (Gap) relieves neuropathic pain, but it has several adverse effects as well. We aimed to investigate whether vitamin C (VitC) supplementation would reduce the effective dose of Gap for analgesia in rats with chronic constriction injury (CCI). Main methodsRats were randomly assigned to Sham, CCI, VitC, Gap, and VitC+Gap treatment groups. CCI, involving the left sciatic nerve, was induced in all animals except the Sham group. VitC (500mg/kg (body weight)), Gap (10, 30, or 100mg/kg), or VitC (500mg/kg)+Gap (10, 30, or 100mg/kg) were injected intraperitoneally twice daily for a week from 7days after sham or CCI surgery. Mechanical paw withdrawal threshold (PWT), thermal paw withdrawal latency (PWL) and malondialdehyde (MDA) content in serum or spinal cord tissues were all measured. The expression of sodium dependent vitamin C transporter 2 (SVCT2) and glucose transporter 3 (GLUT3) in dorsal root ganglion (DRG) were detected by quantitative real-time PCR, Western blot and immunohistochemistry. Key findingsNo more than 30mg/kg Gap could restore the decrease of PWT or PWL induced by CCI so long as combined with 500mg/kg VitC. For mechanism study, we found that VitC supplementation would remarkedly ameliorate oxidative stress in peripheral blood, and possibly cause a positive feedback in VitC uptake of neurons in DRG by promoting SVCT2 expression. SignificanceVitamin C can enhance gabapentin's analgesic effect. And the underlying mechanism may be concerned with antioxidative responses which were more obvious in peripheral blood than in the neurons.

Uptake of ascorbic acid by pancreatic acinar cells is negatively impacted by chronic alcohol exposure.

Vitamin C (ascorbic acid, AA) is indispensable for normal metabolism of all mammalian cells including pancreatic acinar cells (PACs). PACs obtain AA from their surroundings via transport across the cell membrane. Chronic alcohol exposure negatively affects body AA homeostasis; it also inhibits uptake of other micronutrients into PACs, but its effect on AA uptake is not clear. We examined this issue using both in vitro (266-6 cells) and in vivo (mice) models of chronic alcohol exposure. First, we determined the relative expression of the AA transporters 1 and 2 [i.e., sodium-dependent vitamin C transporter-1 (SVCT-1) and SVCT-2] in mouse and human PACs and found SVCT-2 to be the predominant transporter. Chronic exposure of 266-6 cells to alcohol significantly inhibited AA uptake and caused a marked reduction in SVCT-2 expression at the protein, mRNA, and heterogeneous nuclear RNA (hnRNA) levels. Similarly, chronic alcohol feeding of mice significantly inhibited AA uptake and caused a marked reduction in level of expression of the SVCT-2 protein, mRNA, and hnRNA. These findings suggest possible involvement of transcriptional mechanism(s) in mediating chronic alcohol effect on AA uptake by PACs. We also observed significant epigenetic changes (histone modifications) in the Slc23a2 gene (reduction in H3K4me3 level and an increase in H3K27me3 level) in the alcohol-exposed 266-6 cells. These findings show that chronic alcohol exposure inhibits PAC AA uptake and that the effect is mediated, in part, at the level of transcription of the Slc23a2 gene and may involve epigenetic mechanism(s).

L-Ascorbic acid can abrogate SVCT-2-dependent cetuximab resistance mediated by mutant KRAS in human colon cancer cells

Colon cancer patients with mutant KRAS are resistant to cetuximab, an antibody directed against the epidermal growth factor receptor, which is an effective clinical therapy for patients with wild-type KRAS. Numerous combinatorial therapies have been tested to overcome the resistance to cetuximab. However, no combinations have been found that can be used as effective therapeutic strategies. In this study, we demonstrate that L-ascorbic acid partners with cetuximab to induce killing effects, which are influenced by sodium-dependent vitamin C transporter 2 (SVCT-2) in human colon cancer cells with a mutant KRAS. L-Ascorbic acid treatment of human colon cancer cells that express a mutant KRAS differentially and synergistically induced cell death with cetuximab in a SVCT-2-dependent manner. The ectopic expression of SVCT-2 induced sensitivity to L-ascorbic acid treatment in human colon cancer cells that do not express SVCT-2, whereas the knockdown of endogenous SVCT-2 induced resistance to L-ascorbic acid treatment in SVCT-2-positive cells. Moreover, tumor regression via the administration of L-ascorbic acid and cetuximab in mice bearing tumor cell xenografts corresponded to SVCT-2 protein levels. Interestingly, cell death induced by the combination of L-ascorbic acid and cetuximab resulted in both apoptotic and necrotic cell death. These cell death mechanisms were related to a disruption of the ERK pathway and were represented by the impaired activation of RAFs and the activation of the ASK-1–p38 pathway. Taken together, these results suggest that resistance to cetuximab in human colon cancer patients with a mutant KRAS can be bypassed by L-ascorbic acid in an SVCT-2-dependent manner. Furthermore, SVCT-2 in mutant KRAS colon cancer may act as a potent marker for potentiating L-ascorbic acid co-treatment with cetuximab.

Ascorbic acid supplementation improves skeletal muscle oxidative stress and insulin sensitivity in people with type 2 diabetes: Findings of a randomized controlled study

Aim/hypothesisSkeletal muscle insulin resistance and oxidative stress are characteristic metabolic disturbances in people with type 2 diabetes. Studies in insulin resistant rodents show an improvement in skeletal muscle insulin sensitivity and oxidative stress following antioxidant supplementation. We therefore investigated the potential ameliorative effects of antioxidant ascorbic acid (AA) supplementation on skeletal muscle insulin sensitivity and oxidative stress in people with type 2 diabetes. MethodsParticipants with stable glucose control commenced a randomized cross-over study involving four months of AA (2×500mg/day) or placebo supplementation. Insulin sensitivity was assessed using a hyperinsulinaemic, euglycaemic clamp coupled with infusion of 6,6-D2 glucose. Muscle biopsies were measured for AA concentration and oxidative stress markers that included basal measures (2′,7′-dichlorofluorescin [DCFH] oxidation, ratio of reduced-to-oxidized glutathione [GSH/GSSG] and F2-Isoprostanes) and insulin-stimulated measures (DCFH oxidation). Antioxidant concentrations, citrate synthase activity and protein abundances of sodium-dependent vitamin C transporter 2 (SVCT2), total Akt and phosphorylated Akt (ser473) were also measured in muscle samples. ResultsAA supplementation significantly increased insulin-mediated glucose disposal (delta rate of glucose disappearance; ∆Rd) (p=0.009), peripheral insulin-sensitivity index (p=0.046), skeletal muscle AA concentration (p=0.017) and muscle SVCT2 protein expression (p=0.008); but significantly decreased skeletal muscle DCFH oxidation during hyperinsulinaemia (p=0.007) when compared with placebo. Total superoxide dismutase activity was also lower following AA supplementation when compared with placebo (p=0.006). Basal oxidative stress markers, citrate synthase activity, endogenous glucose production, HbA1C and muscle Akt expression were not significantly altered by AA supplementation. Conclusions/interpretationIn summary, oral AA supplementation ameliorates skeletal muscle oxidative stress during hyperinsulinaemia and improves insulin-mediated glucose disposal in people with type 2 diabetes. Findings implicate AA supplementation as a potentially inexpensive, convenient, and effective adjunct therapy in the treatment of insulin resistance in people with type 2 diabetes.

Vitamin C is taken up by human T cells via sodium-dependent vitamin C transporter 2 (SVCT2) and exerts inhibitory effects on the activation of these cells in vitro.

Vitamin C is an essential micronutrient that affects immune responses. T cells are one of the main players in acquired immunity and have been reported to be influenced by in vivo vitamin C supplementation. Yet, the way by which T cells uptake vitamin C and what direct effects vitamin C exerts on the cells are not known. To elucidate, we isolated human peripheral blood T cells and analyzed the expression of sodium-dependent vitamin C transporters (SVCT). T cells were activated in vitro in the absence or presence of vitamin C, before or after activation. As results, human T cells expressed SVCT2, but not SVCT1, and the expression level increased following activation. Vitamin C added in the culture media generally did not affect T-cell behaviors following activation, such as proliferation, apoptosis, expression of CD25 and CD69, and interleukin 2 secretion, regardless whether it was added before or after activation. However, exceptionally, high concentration vitamin C, when it was added before activation, but not after activation, did exert toxic effects on cell activation with respect to the above-mentioned parameters. In conclusion, we showed the expression of SVCT2 in human T cells for the first time. Vitamin C exerted toxic effects, at least in vitro, when the concentration was high and when it was given before activation. These toxic effects are not thought to be via anti-oxidant effects of vitamin C.

Sodium-dependent vitamin C transporter-2 mediates vitamin C transport at the cortical nerve terminal.

It has been shown that vitamin C (VC) is transported at synaptic boutons, but how this occurs has not been elucidated. This study investigates the role of the sodium-dependent vitamin C transporter-2 (SVCT2) in transporting VC at the cortical nerve terminal. Immunostaining of cultured mouse superior cervical ganglion cells showed the SVCT2 to be expressed in presynaptic boutons, colocalizing with the vesicular monoamine transporter-2 and the norepinephrine transporter. Immunoblotting of enriched cortical synaptosomes demonstrated that the SVCT2 was enriched in presynaptic fractions, confirming a predominantly presynaptic location. In crude synaptosomes, known inhibitors of SVCT2 inhibited uptake of VC. Furthermore, the kinetic features of VC uptake were consistent with SVCT2-mediated function. VC was also found to efflux from synaptosomes by a mechanism not involving the SVCT2. Indeed, VC efflux was substantially offset by reuptake of VC on the SVCT2. The presence and function of the SVCT2 at the presynaptic nerve terminal suggest that it is the transporter responsible for recovery of VC released into the synaptic cleft.

The crucial role of vitamin C and its transporter (SVCT2) in bone marrow stromal cell autophagy and apoptosis

Vitamin C is an antioxidant that plays a vital role in various biological processes including bone formation. Previously, we reported that vitamin C is transported into bone marrow stromal cells (BMSCs) through the sodium dependent Vitamin C Transporter 2 (SVCT2) and this transporter plays an important role in osteogenic differentiation. Furthermore, this transporter is regulated by oxidative stress. To date, however, the exact role of vitamin C and its transporter (SVCT2) in ROS regulated autophagy and apoptosis in BMSCs is poorly understood. In the present study, we observed that oxidative stress decreased survival of BMSCs in a dose-dependent manner and induced growth arrest in the G1 phase of the cell cycle. These effects were accompanied by the induction of autophagy, confirmed by P62 and LC3B protein level and punctate GFP-LC3B distribution. The supplementation of vitamin C significantly rescued the BMSCs from oxidative stress by regulating autophagy. Knockdown of the SVCT2 transporter in BMSCs synergistically decreased cell survival even under low oxidative stress conditions. Also, supplementing vitamin C failed to rescue cells from stress. Our results reveal that the SVCT2 transporter plays a vital role in the mechanism of BMSC survival under stress conditions. Altogether, this study has given new insight into the role of the SVCT2 transporter in oxidative stress related autophagy and apoptosis in BMSCs.

Epigenetic reprogramming of melanoma cells by vitamin C treatment.

BackgroundThe loss of 5-hydroxymethylcytosine (5hmC) has been identified as a novel epigenetic hallmark for melanoma. One of the known mechanisms underlying the loss of 5hmC is the decrease in expression of ten-eleven translocation family dioxygenase (TET) genes, which encode enzymes that catalyze the generation of 5hmC. Overexpressing TET2 was shown to partially reestablish a normal 5hmC profile in melanoma and decrease invasiveness in rodents. However, the feasibility to overexpress TETs in patients remains unclear. We and others have recently demonstrated that TETs require vitamin C as a cofactor to generate 5hmC. This finding prompted us to test whether vitamin C, as an alternative to overexpressing TETs, could rebuild 5hmC content in melanoma.ResultsConsistent with previous reports, we found that the expression of TETs was decreased in various melanoma cell lines. In contrast, the expressions of sodium-dependent vitamin C transporters (SVCTs) were down-regulated in cell lines derived from melanoma metastases. Treatment of vitamin C at the physiological level (0.1 mM) promoted the content of 5hmC in melanoma cell lines derived from different stages toward the level of healthy melanocytes, which was comparable to the effect of overexpressing TET2. Vitamin C treatment decreased the malignancy of metastatic A2058 cells by inhibiting migration and anchorage-independent growth, while not exerting any effect on the rate of proliferation. Further, vitamin C treatment caused alterations in genome-wide transcriptions shown by RNA-seq, predominantly in ArhGAP30 and genes involved in extracellular matrix remodeling, which could underlie the decreased malignant phenotypes.ConclusionsOur data support the idea that vitamin C treatment increases 5hmC content in melanoma cells, while causing a decrease in tumor-cell invasiveness and clonogenic growth in soft agar. Thus, vitamin C could be a potential epigenetic treatment for melanoma.

Ascorbic acid transport in brain microvascular pericytes

Intracellular vitamin C, or ascorbic acid, has been shown to prevent the apoptosis of cultured vascular pericytes under simulated diabetic conditions. We sought to determine the mechanism by which ascorbate is transported into pericytes prior to exerting this protective effect. Measuring intracellular ascorbate, we found that pericytes display a linear uptake over 30 min and an apparent transport Km of 21 μM, both of which are consistent with activity of the Sodium-dependent Vitamin C Transporter 2 (SVCT2). Uptake of both radiolabeled and unlabeled ascorbate was prevented by inhibiting SVCT2 activity, but not by inhibiting the activity of GLUT-type glucose transporters, which import dehydroascorbate to also generate intracellular ascorbate. Likewise, uptake of dehydroascorbate was prevented with the inhibition of GLUTs, but not by inhibiting the SVCT2, indicating substrate specificity of both transporters. Finally, presence of the SVCT2 in pericytes was confirmed by western blot analysis, and immunocytochemistry was used to localize it to the plasma membrane and intracellular sites. Together, these data clarify previous inconsistencies in the literature, implicate SVCT2 as the pericyte ascorbate transporter, and show that pericytes are capable of concentrating intracellular ascorbate against a gradient in an energy- and sodium-dependent fashion.

MicroRNAs-141 and 200a regulate the SVCT2 transporter in bone marrow stromal cells

Vitamin C is a micro-nutrient which plays an important role in bone marrow stromal cell (BMSCs) differentiation to osteogenesis. This vitamin is transported into the BMSCs through the sodium dependent vitamin C transporter 2 (SVCT2). We previously reported that knockdown of the SVCT2 transporter decreases osteogenic differentiation. However, our understanding of the post-transcriptional regulatory mechanism of the SVCT2 transporter remains poor. MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally regulate the messenger RNAs of protein-coding genes. In this study, we aimed to investigate the impact of miR-141 and miR-200a on SVCT2 expression. We found that mouse BMSCs expressed miR-141 and miR-200a and repressed SVCT2 expression at the functional level by targeting the 3′-untranslated region of mRNA. We also found that miR-141 and miR-200a decreased osteogenic differentiation. Furthermore, miRNA inhibitors increased SVCT2 and osteogenic gene expression in BMSCs. Taken together, these results indicate that both miRNAs are novel regulators of the SVCT2 transporter and play an important role in the osteogenic differentiation of BMSCs.

High-dose vitamin C supplementation increases skeletal muscle vitamin C concentration and SVCT2 transporter expression but does not alter redox status in healthy males

Antioxidant vitamin C (VC) supplementation is of potential clinical benefit to individuals with skeletal muscle oxidative stress. However, there is a paucity of data reporting on the bioavailability of high-dose oral VC in human skeletal muscle. We aimed to establish the time course of accumulation of VC in skeletal muscle and plasma during high-dose VC supplementation in healthy individuals. Concurrently we investigated the effects of VC supplementation on expression levels of the key skeletal muscle VC transporter sodium-dependent vitamin C transporter 2 (SVCT2) and intramuscular redox and mitochondrial measures. Eight healthy males completed a randomized placebo-controlled, crossover trial involving supplementation with ascorbic acid (2×500mg/day) over 42 days. Participants underwent muscle and blood sampling on days 0, 1, 7, and 42 during each treatment. VC supplementation significantly increased skeletal muscle VC concentration after 7 days, which was maintained at 42 days (VC 3.0±0.2 (mean±SEM) to 3.9±0.4mg/100g wet weight (ww) versus placebo 3.1±0.3 to 2.9±0.2mg/100g ww, p=0.001). Plasma VC increased after 1 day, which was maintained at 42 days (VC 61.0±6.1 to 111.5±10.4µmol/L versus placebo 60.7±5.3 to 59.2±4.8µmol/L, p<0.001). VC supplementation significantly increased skeletal muscle SVCT2 protein expression (main treatment effect p=0.006) but did not alter skeletal muscle redox measures or citrate synthase activity. A main finding of our study was that 7 days of high-dose VC supplementation was required to significantly increase skeletal muscle vitamin C concentration in healthy males. Our findings implicate regular high-dose vitamin C supplementation as a means to safely increase skeletal muscle vitamin C concentration without impairing intramuscular ascorbic acid transport, antioxidant concentrations, or citrate synthase activity.

2D and 3D crystallization of a bacterial homologue of human vitamin C membrane transport proteins.

Most organisms are able to synthesize vitamin C whereas humans are not. In order to contribute to the elucidation of the molecular working mechanism of vitamin C transport through biological membranes, we cloned, overexpressed, purified, functionally characterized, and 2D- and 3D-crystallized a bacterial protein (UraDp) with 29% of amino acid sequence identity to the human sodium-dependent vitamin C transporter 1 (SVCT1). Ligand-binding experiments by scintillation proximity assay revealed that uracil is a substrate preferably bound to UraDp. For structural analysis, we report on the production of tubular 2D crystals and present a first projection structure of UraDp from negatively stained tubes. On the other hand the successful growth of UraDp 3D crystals and their crystallographic analysis is described. These 3D crystals, which diffract X-rays to 4.2Å resolution, pave the way towards the high-resolution crystal structure of a bacterial homologue with high amino acid sequence identity to human SVCT1.

In vivo vitamin C deficiency in guinea pigs increases ascorbate transporters in liver but not kidney and brain

Moderate vitamin C (vitC) deficiency (plasma concentrations less than 23 μmol/L) affects as much as 10% of adults in the Western World and has been associated with an increased mortality in disease complexes such as cardiovascular disease and the metabolic syndrome. The distribution of vitC within the body is subjected to complex and nonlinear pharmacokinetics and largely depends on the sodium-dependent vitC-specific transporters, sodium-dependent vitamin C transporter 1 (SVCT1) and sodium-dependent vitamin C transporter 2 (SVCT2). Although currently not established, it is likely to expect that a state of deficiency may affect the expression of these transporters to preserve vitC concentrations in specific target tissues. We hypothesized that diet-induced states of vitC deficiency lead to alterations in the messenger RNA (mRNA) and/or protein expression of vitC transporters, thereby regulating vitC tissue distribution. Using guinea pigs as a validated model, this study investigated the effects of a diet-induced vitC deficiency (100 mg vitC/kg feed) or depletion (0 mg vitC/kg feed) on the expression of transporters SVCT1 and SVCT2 in selected tissues and the transport from plasma to cerebrospinal fluid (CSF). In deficient animals, SVCT1 was increased in the liver, whereas a decreased SVCT1 expression but increased SVCT2 mRNA in livers of depleted animals suggests a shift in transporter expression as response to the diet. In CSF, a constant plasma:CSF ratio shows unaltered vitC transport irrespective of dietary regime. The study adds novel information to the complex regulation maintaining vitC homeostasis in vivo during states of deficiency.

Preparation, transportation mechanisms and brain-targeting evaluation in vivo of a chemical delivery system exploiting the blood–cerebrospinal fluid barrier

In recent years, specific transportation mechanisms on the blood–brain barrier (BBB) are extensively employed for brain-targeted drug delivery via colloidal nanocarriers. However, in this study, we purposed to exploit the sodium-dependent vitamin C transporter 2 (SVCT2)-mediated transportation on the blood–cerebrospinal fluid barrier to enhance central nervous system penetration of the highly hydrophilic ibuprofen (IBU) by synthesizing a SVCT2-targeted chemical delivery system (CDS), ibuprofen-C6-O-ascorbic acid (IAA). The physicochemical parameters of IAA were determined, and the transporter-mediated transportation mechanism of IAA was explored on a BBB monolayer mode. The overall brain targeting effect of IAA was assayed on mice by measuring the biodistribution of IBU after i.v. administration and calculating the pharmacokinetic parameters and targeting indexes. Results showed that lipophilicity and solubility of IAA was conspicuously improved compared with IBU. At the physiological pH, IAA was stable while in brain homogenates it was easily degraded. Transport studies on the BBB monolayer mode revealed that IAA displayed higher transepithelial permeability than IBU via SVCT2. The biodistribution study in vivo demonstrated that the overall targeting efficiency of IAA was 1.77-fold greater than that of the IBU. In conclusion, the synthetic IAA might be a promising brain-targeted CDS for smuggling small-molecule hydrophilic pharmaceuticals into the brain.

Orally administrated ascorbic acid suppresses neuronal damage and modifies expression of SVCT2 and GLUT1 in the brain of diabetic rats with cerebral ischemia-reperfusion.

Diabetes mellitus is known to exacerbate cerebral ischemic injury. In the present study, we investigated antiapoptotic and anti-inflammatory effects of oral supplementation of ascorbic acid (AA) on cerebral injury caused by middle cerebral artery occlusion and reperfusion (MCAO/Re) in rats with streptozotocin-induced diabetes. We also evaluated the effects of AA on expression of sodium-dependent vitamin C transporter 2 (SVCT2) and glucose transporter 1 (GLUT1) after MCAO/Re in the brain. The diabetic state markedly aggravated MCAO/Re-induced cerebral damage, as assessed by infarct volume and edema. Pretreatment with AA (100 mg/kg, p.o.) for two weeks significantly suppressed the exacerbation of damage in the brain of diabetic rats. AA also suppressed the production of superoxide radical, activation of caspase-3, and expression of proinflammatory cytokines (tumor necrosis factor-α and interleukin-1β) in the ischemic penumbra. Immunohistochemical staining revealed that expression of SVCT2 was upregulated primarily in neurons and capillary endothelial cells after MCAO/Re in the nondiabetic cortex, accompanied by an increase in total AA (AA + dehydroascorbic acid) in the tissue, and that these responses were suppressed in the diabetic rats. AA supplementation to the diabetic rats restored these responses to the levels of the nondiabetic rats. Furthermore, AA markedly upregulated the basal expression of GLUT1 in endothelial cells of nondiabetic and diabetic cortex, which did not affect total AA levels in the cortex. These results suggest that daily intake of AA attenuates the exacerbation of cerebral ischemic injury in a diabetic state, which may be attributed to anti-apoptotic and anti-inflammatory effects via the improvement of augmented oxidative stress in the brain. AA supplementation may protect endothelial function against the exacerbated ischemic oxidative injury in the diabetic state and improve AA transport through SVCT2 in the cortex.

The Molecular Mechanism Underlying the Proliferating and Preconditioning Effect of Vitamin C on Adipose-Derived Stem Cells

Although adipose-derived stem cells (ASCs) show promise for cell therapy, there is a tremendous need for developing ASC activators. In the present study, we investigated whether or not vitamin C increases the survival, proliferation, and hair-regenerative potential of ASCs. In addition, we tried to find the molecular mechanisms underlying the vitamin C-mediated stimulation of ASCs. Sodium-dependent vitamin C transporter 2 (SVCT2) is expressed in ASCs, and mediates uptake of vitamin C into ASCs. Vitamin C increased the survival and proliferation of ASCs in a dose-dependent manner. Vitamin C increased ERK1/2 phosphorylation, and inhibition of the mitogen-activated protein kinase (MAPK) pathway attenuated the proliferation of ASCs. Microarray and quantitative polymerase chain reaction showed that vitamin C primarily upregulated expression of proliferation-related genes, including Fos, E2F2, Ier2, Mybl1, Cdc45, JunB, FosB, and Cdca5, whereas Fos knock-down using siRNA significantly decreased vitamin C-mediated ASC proliferation. In addition, vitamin C-treated ASCs accelerated the telogen-to-anagen transition in C3H/HeN mice, and conditioned medium from vitamin C-treated ASCs increased the hair length and the Ki67-positive matrix keratinocytes in hair organ culture. Vitamin C increased the mRNA expression of HGF, IGFBP6, VEGF, bFGF, and KGF, which may mediate hair growth promotion. In summary, vitamin C is transported via SVCT2, and increased ASC proliferation is mediated by the MAPK pathway. In addition, vitamin C preconditioning enhanced the hair growth promoting effect of ASCs. Because vitamin C is safe and effective, it could be used to increase the yield and regenerative potential of ASCs.

Gene expression profiles of sodium-dependent vitamin C transporters in mice after alcohol consumption

Alcoholic liver disease (ALD) is a serious liver problem in western countries. Our previous study has demonstrated that vitamin C plays a protective role in ALD. The vitamin C homeostasis is tightly regulated by sodium-dependent vitamin C transporters (SVCTs) 1 and 2. But the role of two SVCTs in ALD is less understood. In this study, we examined the expression patterns of two SVCTs in mice after alcohol consumption. Our results suggested that alcohol consumption obviously increased the expression of two SVCTs in liver and SVCT1 in kidney and intestine, which is important for vitamin C absorption. Vitamin C supplement increased the sera vitamin C content and ameliorated the symptom of ALD. Intestinal absorption and renal re-absorption mediated by SVCT1 are key factors to increase the sera vitamin C content after alcohol consumption. We proposed that both reactive oxygen species and low vitamin C concentration regulate the expression of SVCTs, and the protective role of vitamin C is mediated by suppressing the stability of hypoxia-inducible factor-1α. Thus, our study is significant for the understanding of vitamin C homeostasis in ALD and for better use of other antioxidants in ALD therapy.

Regulation of embryonic neurotransmitter and tyrosine hydroxylase protein levels by ascorbic acid

Scope: Ascorbic acid (ascorbate) is required to recycle tetrahydrobiopterin, which is necessary for neurotransmitter synthesis by the rate-limiting enzymes tyrosine and tryptophan hydroxylases. We sought to determine whether ascorbate might regulate embryonic brain cortex monoamine synthesis utilizing transgenic mouse models with varying intracellular ascorbate levels. Methods and results: In embryos lacking the sodium-dependent vitamin C transporter 2 (SVCT2), very low levels of brain ascorbate decreased cortex levels of norepinephrine and dopamine by approximately 33%, but had no effect on cortex serotonin or its metabolite, 5-hydroxyindole acetic acid. This decrease in ascorbate also led to a decrease in protein levels of tyrosine hydroxylase, but not of tryptophan hydroxylase. Increased cortex ascorbate in embryos carrying extra copies of the SVCT2 resulted in increased levels of dopamine and its metabolite, 3,4-dihydroxyphenylacetic acid (DOPAC), as well as serotonin and 5-hydroxyindole acetic acid. Conclusion: The dependence of embryonic brain cortex neurotransmitter synthesis and tyrosine hydroxylase expression on intracellular ascorbate emphasizes the importance of receiving adequate ascorbate during development.