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(The FASEB Journal. 2005;19:1657-1667.)
© 2005 FASEB

Vitamin C enters mitochondria via facilitative glucose transporter 1 (Glut1) and confers mitochondrial protection against oxidative injury

Sagun KC*,{dagger}, Juan M. Cárcamo{ddagger},1 and David W. Golde*,{ddagger},§

* Department of Pharmacology, Weill Medical College, Cornell University, New York, New York, USA; and
{dagger} Program in Molecular Pharmacology and Chemistry,
{ddagger} Department of Clinical Laboratories, and
§ Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, New York, USA

1 Correspondence: Memorial Sloan-Kettering Cancer Center, Box 451, 1275 York Ave., New York, NY 10021, USA. E-mail: jcarcamo{at}enzobio.com


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Reactive oxygen species (ROS)-induced mitochondrial abnormalities may have important consequences in the pathogenesis of degenerative diseases and cancer. Vitamin C is an important antioxidant known to quench ROS, but its mitochondrial transport and functions are poorly understood. We found that the oxidized form of vitamin C, dehydroascorbic acid (DHA), enters mitochondria via facilitative glucose transporter 1 (Glut1) and accumulates mitochondrially as ascorbic acid (mtAA). The stereo-selective mitochondrial uptake of D-glucose, with its ability to inhibit mitochondrial DHA uptake, indicated the presence of mitochondrial Glut. Computational analysis of N-termini of human Glut isoforms indicated that Glut1 had the highest probability of mitochondrial localization, which was experimentally verified via mitochondrial expression of Glut1-EGFP. In vitro mitochondrial import of Glut1, immunoblot analysis of mitochondrial proteins, and cellular immunolocalization studies indicated that Glut1 localizes to mitochondria. Loading mitochondria with AA quenched mitochondrial ROS and inhibited oxidative mitochondrial DNA damage. mtAA inhibited oxidative stress resulting from rotenone-induced disruption of the mitochondrial respiratory chain and prevented mitochondrial membrane depolarization in response to a protonophore, CCCP. Our results show that analogous to the cellular uptake, vitamin C enters mitochondria in its oxidized form via Glut1 and protects mitochondria from oxidative injury. Since mitochondria contribute significantly to intracellular ROS, protection of the mitochondrial genome and membrane may have pharmacological implications against a variety of ROS-mediated disorders.—KC, S., Cárcamo, J. M., Golde, D. W. Vitamin C enters mitochondria via facilitative glucose transporter 1 (Glut1) and confers mitochondrial protection against oxidative injury.


Key Words: ROS • oxidative stress • DNA damage • antioxidants • cellular redox


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MITOCHONDRIA are centrally involved in the oxidative synthesis of ATP and play a critical role in regulating apoptosis, redox homeostasis, and maintaining cellular transmembrane potential (1) . The human mitochondrion contains 2 to 10 molecules of double-stranded, closed circular DNA (mtDNA). The 16.6 kb self-replicating mitochondrial genome encodes 13 proteins, 2 rRNAs, and 22 tRNAs. Proteins encoded by mtDNA are associated exclusively with oxidative phosphorylation and the electron transport chain (ETC), and the remaining mitochondrial proteins are encoded by nuclear DNA (2) . Nuclear-encoded proteins enter mitochondria through specialized import pathways (3) using an N-terminal targeting sequence that comprises four or five positively charged amino acid residues aligned isofacially in an amphipathic {alpha}-helix (4 , 5) . Eukaryotic mitochondria have two morphologically distinct membranes. The outer membrane contains nonselective proton pumps, or porins, whereas the inner membrane contains proteins involved in the selective transport of solutes, nucleotides, water, and ions into the mitochondrial matrix. The precise number of transport proteins localized in the mitochondrial inner membrane remains unknown.

Reactive oxygen species (ROS) are generated continuously by intracellular oxidative events and mitochondria contribute significantly to the production of intracellular ROS (6) . ROS can modify the biological activity of enzymes, modulate intracellular signaling events, and damage biological macromolecules (7) . The mtDNA has been shown to be extremely susceptible to the mutagenic effects of ROS (8) . To protect itself from the damaging effects of ROS, mitochondria possess elaborate defense mechanisms including ROS-eliminating enzymes (9 10 11 12) , thiols (13) , and water and lipid-soluble antioxidants (14) .

Vitamin C is a potent antioxidant known to protect tissues from oxidative injury (15 , 16) . Loading cells with vitamin C reduces oxidative cell death (17 , 18) , inhibits FAS-induced apoptosis (19) , and confers genomic protection (20) through the quenching of intracellular ROS. Vitamin C is a cofactor to enzymes involved in the synthesis of collagen (21) and carnitine (22) , and is postulated to be involved in the mitochondrial reduction of {alpha}-tocopherol (23) and ferricytochrome c (24) . In specialized cells, vitamin C is directly transported as ascorbic acid (AA) via sodium-dependent vitamin C transporters (SVCT) (25 , 26) . However, most cells transport vitamin C in its oxidized form, dehydroascorbic acid (DHA), via facilitative glucose transporters (Glut), including Glut1 (27) . Once inside cells, DHA is reduced and accumulated as ascorbic acid (AA) (28) . Glut1 is ubiquitously expressed in cells and up-regulated by malignant transformation (29) . Recently, Glut1 was discovered to serve as a receptor for human T cell leukemia virus 1 (HTLV-1) (30) .

In vivo studies show that mammalian mitochondria contain AA at concentrations that can be increased with dietary vitamin C supplementation (31 32 33) . Mitochondria and mitoplasts isolated from rat liver transport vitamin C (34) and purified plant mitochondria were shown to uptake DHA and glucose (35) . However, the precise mechanism of vitamin C uptake by mammalian mitochondria is not known. Here we report that vitamin C enters mitochondria via Glut1 in its oxidized form, DHA, which is reduced and accumulated as mitochondrial AA (mtAA). Our data indicate that by quenching ROS, mtAA protects the mitochondrial genome and membrane from oxidative damages.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Human kidney (293T) and murine fibroblast (NIH/3T3) cells were grown in Dulbecco’s modified Eagle media containing high glucose (DME-HG) and minimum Eagle media (MEM), respectively, supplemented with 10% fetal bovine serum (FBS), and 1% each of penicillin/streptomycin, L-glutamine, and sodium pyruvate. Human myeloid leukemia (HL-60) and hepatocarcinoma (HepG2) cells were grown in Iscove’s modified Dulbecco’s media (IMDM) and MEM, respectively, supplemented with 10% FBS, and 1% each of penicillin/streptomycin and L-glutamine.

Isolation of mitochondria and purification mitochondrial DNA (mtDNA)
Cells were harvested, pelleted, and mechanically homogenized in 10 pellet volumes of homogenization buffer (HB) (0.25 M sucrose, 10 mM HEPES, 0.3 mM EDTA, pH 7.5). The lysate was centrifuged twice at 700 g to pellet the cell debris and generate a cytoplasmic fraction. Mitochondria were pelleted by centrifuging the cytoplasmic fraction at 12,000 g for 15 min. Pellets were pooled and resuspended in two volumes of sucrose TE buffer (20% sucrose, 50 mM Tris-HCl, 5 mM EDTA, pH 7.5). Aliquots of 5.5 mL of this mitochondria-enriched fraction were layered between sucrose cushion A (1.0 M sucrose, 10 mM Tris-HCl, 5 mM EDTA, pH 7.5) and sucrose cushion B (1.5 M sucrose, 10 mM Tris-HCl, 6 mM EDTA, pH 7.5) and centrifuged for 30 min at 25,000 rpm using a 55.2 Ti ultracentrifuge rotor (Beckman Coulter, Fullerton, CA, USA). The mitochondrial layer above cushion B (5 mL) was extracted, combined with five volumes of HB, and centrifuged for 20 min at 15,000 rpm to pellet mitochondria. Pooled mitochondrial pellets were resuspended in appropriate buffers for further studies, or dissolved in guanidine HCl buffer (0.8 M guanidine-HCl, 1% Triton X-100, 1 mM EDTA, 200 µg/mL proteinase K) for isolation of mtDNA. The mtDNA was isolated and purified using a plasmid isolation kit (Qiagen Inc., Valencia, CA, USA).

Measurement of AA accumulation
AA levels were measured electrochemically using HPLC (Beckman-Coulter) linked to Coul-Array (ESA Inc., Chelmsford, MA, USA). Cells were incubated for 30 min at room temperature in incubation buffer (IB) (15 mM HEPES, 135 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, pH 7.4) supplemented with DHA (Sigma-Aldrich, St. Louis, MO, USA) to achieve a defined concentration of intracellular AA. Control uptakes of AA (Sigma-Aldrich) were conducted in the presence of 1 mM DTT. Cells were washed in cold PBS, harvested and counted using a hemocytometer, lysed in ice-cold 60% HPLC-grade methanol (J.T. Baker, Philipsburg, NJ, USA), repelleted, and the supernatant was filtered through a cellulose-acetate filter (Sigma-Aldrich). To determine compartmentalization of intracellularly accumulated AA in mitochondria, mitochondria from control and AA-loaded cells were purified, lysed, and the mitochondrially accumulated AA (mtAA) quantified with HPLC-ECD.

To measure in vitro uptake of vitamin C in mitochondria, mitochondria were purified as described above and incubated for 30 min in IB containing specified concentrations of DHA. After DHA treatment, mitochondria were washed, lysed, filtered, and the levels of mtAA were measured electrochemically using HPLC-ECD (19) . Intracellular and mitochondrial concentrations of AA were calculated using published values for cell volume (20) , and mitochondrial volume (34) , respectively. For competition assays, purified mitochondria were preincubated for 10 min with D- or L-glucose (0.1, 1.0 mM), followed by a 30 min uptake of 0.5 mM DHA (28) . After DHA uptake, mitochondria were washed, lysed, and mtAA content was quantified by electrochemical detection, as described previously.

Mitochondrial uptake of 14C-labeled D- or L-glucose
Purified mitochondria were incubated for 30 min at room temperature in IB containing specified concentrations of 14C-labeled D- or L-glucose (50 µCi/mol) (Perkin-Elmer, Wellesley, MA, USA). Mitochondria were pelleted, washed with PBS, and lysed with lysis buffer (50 mM Tris-HCl, 0.2% SDS, pH 7.4). Mitochondrial incorporation of 14C-labeled glucose was determined using scintillation spectroscopy (Beckman-Coulter).

Mitochondrial targeting of Glut1
A DNA fragment coding the first 19 amino acids of Glut1 was ligated into an EGFP vector (Invitrogen, Carlsbad, CA, USA) to generate chimeric pGlut1-EGFP using the following primers: Glut1-forward: 5'-CCG GAA TTC AGC GCT GCC ATG GAG CCC AGC AGC AAG AAG CTG ACG GGT CGC CTC ATG CTG GCT GTG GGA TCC ATC-3' and Glut1-reverse: 5'-GAT GGA TCC CAC AGC CAG CAT GAG GCG ACC CGT CAG CTT CTT GCT GCT GGG CTC CAT GGC AGC GCT GAA TTC CGG-3'. pEGFP and pCMV/Myc/Mito/EGFP (CoxVIII-EGFP) were used as negative and positive controls, respectively. Cells (293T) were transiently transfected with the appropriate plasmid vectors using Lipofectamine 2000 (Invitrogen). After 48 h, cells were harvested and incubated for 1 h with 100 nM Mitotracker Red (Molecular Probes, Eugene, OR, USA), washed twice with PBS, and mounted onto microscope slides. Cellular localization of proteins was detected by fluorescence microscopy using Retiga 1300 digital camera (Qimaging Inc., Burnaby, B.C., Canada). Images were analyzed with Adobe Photoshop 7.0 (Adobe Inc., San Jose, CA, USA) and assembled in Canvas 9.0 (Deneba Inc., Miami, FL, USA).

Immunoblotting
Proteins in fractionated cellular extracts were resolved by SDS-PAGE (Bio-Rad, Hercules, CA, USA) and transferred to nitrocellulose membranes. Glut1 polyclonal antibody (Charles River Labs, Wilmington, MA, USA) was used to detect Glut1. The mitochondrial fraction was tested with anti-Mcl1 antibody (Upstate Biotech, Waltham, MA, USA). As controls, antibodies against Lamp1 and Grp78 proteins (BD Biosciences, San Jose, CA, USA) were used to detect lysosome and endoplasmic reticulum proteins, respectively. HRP-conjugated donkey secondary antibodies were used and proteins were visualized by enhanced chemiluminescence detection system (Amersham Biosciences, Piscataway, NJ, USA).

Immunolocalization of Glut1 in cells
Cells (NIH/3T3) cultured in 8-well chambers (Nunc Nalgene, Naperville, IL, USA) were fixed with 4% para-formaldehyde for 10 min, washed, and treated with 50% acetone for 30 s. Cells were blocked at room temperature in PBS supplemented with 5% BSA and 0.3% Triton-X, followed by an overnight incubation with Glut1 antibody (1:200). Cells were washed twice with PBS and incubated for 6 h at 4°C with 1:2000 dilution of FITC-conjugated goat-anti-rabbit secondary antibody (Sigma-Aldrich). Alternatively, rhodamine-conjugated goat-anti-rabbit secondary antibody (Molecular Probes) was used in cells transfected with CoxVIII-EGFP. Intracellular colocalization of mitochondria, as detected by the mitochondrially targeted CoxVIII-EGFP or Mitotracker Red, with Glut1 was visualized with a Leica TCS SP2 confocal microscope (Leica Microsystems, Wetzlar, Germany).

In vitro mitochondrial import of Glut1
Glut1 was transcribed and translated in vitro using the TNT rabbit reticulocyte lysate system supplemented with canine microsomal membranes (Promega, Madison, WI, USA). In vitro synthesized CoxVIII-EGFP and EGFP were used as positive and negative controls, respectively. Mitochondrial pellets purified from 293T cells were resuspended in import buffer (250 mM sorbitol, 10 mM ATP, 2 mM MgSO4, 50 mM HEPES-KOH, pH 7.6, supplemented with 0.2% BSA) and incubated with the in vitro synthesized proteins in the TNT reaction mixture (3:1 ratio v/v) for 30 min at room temperature. Mitochondrial pellets were washed twice with import buffer to remove nonspecifically bound proteins and lysed with 0.2% SDS buffer (50 mM Tris, 0.2% SDS, pH 7.4). A negative control import assay was performed in the absence of mitochondria. Equal amounts of mitochondrial lysates were resolved using SDS-PAGE and probed with antibodies against Glut1. For analysis of mitochondrial import of control proteins (CoxVIII-EGFP or EGFP), the mitochondrial lysate retrieved after the translocation reaction was spotted on a nitrocellulose membrane and the imported GFP-tagged proteins were visualized by fluorescence microscopy. The relative mitochondrial localization of control proteins is indicated by their fluorescence intensities, as quantified by NIH Image.

Mitochondrial production of ROS
Mitochondrial ROS was quantified by measuring superoxide (–•O2), hydrogen peroxide (H2O2), and hydroxyl radical (•OH) generated by purified mitochondria using these fluoremetric probes (Molecular Probes): dihydroethidium for –•O2 formation, Amplex Red for H2O2 generation, and dihydrorhodamine 123 for •OH formation (36) . All measurements were made with Fluoroskan Ascent FL fluorometer (ThermoElectron Corp., Waltham, MA, USA). To test the effect of AA on mitochondrial ROS, equal amounts of purified mitochondria were preloaded with a defined concentration of mtAA, washed, and incubated for 30 min in the presence or absence of 10 µM 2,3-dimethoxy-1-naphthoquinone (DMNQ) as an oxidative stressor (37) (Alexis Biochemicals, San Diego, CA, USA). Alternatively, to analyze the role of vitamin C against oxidative stress resulting from the disruption of the mitochondrial respiratory chain (ETC), cells or mitochondrial suspensions were loaded with or without vitamin C and treated for 1 h with 1 µM of the complex I inhibitor, rotenone (38) . Cells or purified mitochondria were pelleted, washed, layered equally onto 96-well plates (Corning Inc., Corning, NY, USA), and incubated for 30 min at 37°C with the appropriate fluoremetric probes. Background fluorescence of the cellular/mitochondrial suspension and the reduced probe were subtracted from the measured fluorescence. Measurements are expressed as a percentage of controls.

Mitochondrial DNA damage
Purified mitochondria were preloaded with a defined concentration of mtAA, washed, and treated for 60 min with either H2O2 (Fisher Scientific) or a combination of 0.1 mM Cu2+ and H2O2. Mitochondrial DNA was treated with nuclease P1 and alkaline phosphatase (Sigma-Aldrich) using previously described methods (20) . Equal amounts of digested samples were eluted through a modified C-18 column (Phenomenex) using HPLC mobile phase buffer (0.1 M lithium acetate, 10% methanol, pH 5.0) at a flow rate of 1.0 mL/min. The 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG) content in mtDNA was measured using peak areas derived from standard 8-oxo-dG samples (ESA) and expressed as µg 8-oxo-dG/mg of total mtDNA. Alternatively, the levels of apurinic/apyrimidic (AP) sites in isolated mtDNA were quantified colorimetrically using an AP-site quantification kit (Dojindo Inc., Gaithersburg, MD, USA). For analysis of H2O2-induced mtDNA fragmentation, purified mitochondria were incubated with 1.0 mM H2O2 for 1 h and 1 µg of the isolated mtDNA was resolved using a 1% agarose gel. The resolved mtDNA was stained with SYBR Green I (Molecular Probes).

Loss of mitochondrial membrane potential ({Delta}{Psi}m)
Human myeloid HL-60 cells were preloaded with or without AA, washed, and incubated in the dark for 30 min in media containing 100 nM 3,3'-dihexyloxacarbocyanine iodide (DiOC(6) (3); Molecular Probes). A 10 min pretreatment with carbonyl cyanide m-chlorophenylhydrazone (CCCP) in 0–50 µM concentrations (Sigma Aldrich) was used to disrupt mitochondrial membrane potential. A threshold level of {Delta}{Psi}m, derived from the mitochondrial retention of DIOC(6) (3) in control population of live cells, was used to analyze the loss of {Delta}{Psi}m in response to CCCP, as determined by flow cytometry (BD Immunocytometry).

Statistical tests and analysis
All statistical tests were performed with Graphpad Prism 4.0 (Graphpad Inc., San Diego, CA, USA). All values are denoted as means ± SD unless otherwise noted.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
DHA is transported into mitochondria and accumulates as AA
Cells (293T) accumulate ascorbic acid (AA) intracellularly through the uptake of DHA, the oxidized form of vitamin C (Fig. 1 A). We observed a dose-dependent increase in intracellular AA in response to extracellularly administered DHA. However, incubation with extracellular AA did not elevate intracellular AA levels after 30 min of incubation. Mitochondria isolated from cells loaded with AA via DHA treatment also showed a concentration-dependent increase in mitochondrially accumulated AA (mtAA) with respect to the extracellularly administered DHA (Fig. 1B ). These results indicate that cellular uptake of DHA results in cytosolic and mitochondrial accumulation of AA in 293T cells.



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  		Figure 1. Mitochondrial transport of vitamin C involves a facilitative glucose transporter. A) Intracellular and B) mitochondrial accumulation of ascorbic acid (AA) in 293T cells incubated with extracellular DHA (•) or AA ({circ}). AA levels were measured electrochemically with HPLC-ECD. C) Mitochondrial ascorbic acid levels in purified mitochondria incubated for 30 min with DHA. Asterisks indicate statistical significance (Dunnett’s multiple comparison test, P<0.01). D) Stereo-selective inhibition of 0.5 mM DHA uptake by D-glucose ({blacksquare}) or L-glucose ({square}) in purified mitochondria. Asterisk indicates statistical significance (Dunnett’s multiple comparison test, P<0.01). E) Stereo-selective uptake of labeled D-glucose ({blacksquare}) or L-glucose ({square}) in purified mitochondria. *Statistical significance (Student’s t test, n=3, P<0.05).

It was previously reported that purified rat mitochondria take up DHA but not AA (33 , 34) . Therefore, to determine the mechanism of DHA transport in mitochondria, we first performed in vitro DHA uptake studies with purified mitochondria. Incubating purified mitochondria with 0.1 or 0.5 mM DHA for 30 min led to 5- and 10-fold increases in mtAA levels compared with control, respectively (Fig. 1C ) (Dunnett’s multiple comparison test, P<0.01). A mitochondrial concentration of 2.0±0.5 mM AA was achieved with 0.5 mM DHA (Fig. 1C ) whereas treatment with 0.5 mM AA did not appreciably increase levels of mtAA (data not shown). Our results show that DHA is the form of vitamin C transported in purified human mitochondria, in agreement with previous reports on DHA uptake in rat (33) and plant (35) mitochondria.

Mitochondrial uptake of DHA involves a facilitative glucose transporter
Since vitamin C is universally transported in cells via plasma membrane facilitative glucose transporters (Glut), we reasoned that mitochondrial uptake of DHA also occurs through a facilitative glucose transporter. Since Gluts are stereo-selective in their transport properties, we investigated the competitive effect of D-glucose on the mitochondrial uptake of DHA. Preincubation of purified mitochondria with 1.0 mM D-glucose before treatment with 0.5 mM DHA led to a significant reduction in mtAA accumulation (Dunnett’s multiple comparison test, P<0.01) (Fig. 1D ). However, preincubation with the same concentration of the nontransportable L-glucose had no effect, suggesting that the transport of DHA into mitochondria involves a facilitative glucose transporter. Furthermore, we found that purified mitochondria preferably uptake D-glucose, but not L-glucose (Student’s t test, P<0.05) (Fig. 1E ). These studies with purified mitochondria indicate the involvement of a functional Glut in the mitochondrial uptake of vitamin C.

Glut1 localizes to the mitochondrial membrane
Localization of cellular proteins in mitochondria is partially dependent on the N-terminal sequence of the targeted proteins. We therefore analyzed the probability of mitochondrial localization of glucose transporters on the basis of their N-terminal sequences using prediction programs available through the Expert Protein Analysis System (EXPASY) server. The first 60 amino acids of human glucose transporter isoforms were analyzed for mitochondrial localization using TargetP (39) , Mitoprot (40) , and Predotar (41) . The output scores obtained from individual programs indicate probability of mitochondrial localization. We used N-terminal sequence (60 amino acids) of human cytochrome oxidase subunit VIII (CoxVIII) and myeloid cell leukemia 1 (Mcl1) as positive controls. Computational analysis of N-terminal sequences of human Glut isoforms revealed that Glut1 has the highest probability of mitochondrial localization (Fig. 2 A). The mitochondrial localization probability for Glut1 was consistently higher in all prediction assays than any other isoform of glucose transporters analyzed. For example, based on TargetP analysis, the mitochondrial localization probability of myeloid cell leukemia 1 (Mcl1) protein (0.161), a Bcl2 homologue known to localize to the mitochondrial membrane (42) , was similar to the output score of Glut1 (0.169). We have highlighted several positively charged amino-acid residues in the N terminus of Glut1 that may be involved in mitochondrial targeting. Based on these analyses, our theoretical prediction is that Glut1 localizes to mitochondria in human cells.



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  		Figure 2. Glucose transporter 1 (Glut1) localizes to mitochondria in human cells. A) Prediction of mitochondrial localization of human facilitative glucose transporter isoforms using TargetP, Mitoprot, and Predotar. Multiple sequence alignment of the N-termini of glucose transporters with the positively charged amino acids (boldface) and the consensus amino acids (highlighted). The N-termini of two mitochondrial proteins, Mcl1 and cytochrome oxidase subunit VIII (CoxVIII), are included as reference. The probability of mitochondrial localization correlates with the output scores of individual programs. B) Intracellular localization of transiently expressed EGFP, Glut1-EGFP, and CoxVIII-EGFP in 293T cells, as measured by GFP fluorescence (upper panel). Cytosolic localization of mitochondria is shown with Mitotracker Red (lower panel). C) Cellular (Total), mitochondrial (Mito), plasma membrane (PM), and endoplasmic reticulum (ER) proteins were analyzed by immunoblotting using specific antibodies against Glut1, Mcl1, Lamp1 (lysosome-specific protein), and Grp78 (ER-specific protein). D) Immunolocalization of Glut1 in NIH/3T3 cells was analyzed by confocal microscopy using Mitotracker Red to visualize mitochondria. Control cells were incubated with FITC-conjugated secondary antibody alone. E) Immunolocalization studies performed on CoxVIII-EGFP transfected NIH/3T3 cells using CoxVIII-EGFP as a mitochondrial marker. Control cells were incubated with rhodamine-conjugated secondary antibody alone. F) Mitochondria purified from 293T cells were incubated with in vitro synthesized Glut1, CoxVIII-EGFP, and EGFP and mitochondrial import of Glut1 was assessed by Glut1 immunoblotting. A null import assay performed in absence of mitochondria is indicated as control. Fluorescence intensities of GFP-tagged control proteins (CoxVIII-EGFP and EGFP), as quantified by NIH Image, were used to assess mitochondrial import.

To experimentally test for the role of N-terminal Glut1 in mitochondrial targeting, we created a chimeric vector (pGlut1-EGFP) encoding the first 19 amino acids of Glut1 fused to enhanced green fluorescent protein (EGFP). Cells (293T) transiently transfected with pGlut1-EGFP revealed differential pattern of expression compared with the cells transfected with pEGFP. EGFP was ubiquitously expressed in both the cytoplasm and the nuclei of transfected 293T cells, whereas Glut1-EGFP was expressed in distinct cytosolic areas. Cells stained with Mitotracker Red revealed a strong colocalization of Glut1-EGFP with mitochondria whereas EGFP did not colocalize with mitochondria (Fig. 2B ). The expression pattern of Glut1-EGFP was similar to CoxVIII-EGFP, a mitochondrially targeted chimeric protein. These results indicate that the N terminus of Glut1 functions as a mitochondrial targeting sequence.

To physically confirm that Glut1 localizes to mitochondria, we analyzed for the presence of Glut1 in purified mitochondrial extracts via immunoblotting. Glut1 was detected as a 54 kDa protein in mitochondrial (Mito), plasma membrane (PM), and endoplasmic reticulum (ER) fractions (Fig. 2C ). The mitochondrial extract was positive for Mcl1 but negative for Grp78 (ER protein) and Lamp1 (lysosomal protein), indicating the purity of the mitochondrial fractions. These results indicate physical presence of Glut1 in the mitochondrial fractions. Immunodetection of Glut1 in NIH/3T3 cells with confocal microscopy revealed strong overlapping of Glut1 with mitochondria, as detected by Mitotracker Red (Fig. 2D ). Immunodetection of Glut1 overlapped with mitochondrial expression of CoxVIII-EGFP (Fig. 2E ). Taken together, these results indicate that Glut1 localizes to mitochondria in mammalian cells.

To examine the translocation of Glut1 into mammalian mitochondria, we performed a mitochondrial import assay of in vitro translated Glut1, CoxVIII-EGFP or EGFP. In vitro synthesized proteins were incubated with purified mitochondria for 1 h and the mitochondrial import of full-length Glut1 was assessed with immunodetection. The presence of a denser band in Glut1-supplemented mitochondrial fraction indicates that Glut1 was imported into mitochondria (Fig. 2F ). Endogenous mitochondrial Glut1 in equal amounts was observed in CoxVIII-EGFP and EGFP-supplemented mitochondrial fractions but absent in the control reaction, which was not supplemented with purified mitochondria. Analysis of mitochondrial import of CoxVIII-EGFP or EGFP by fluorescence detection indicated that CoxVIII-EGFP translocated to mitochondria whereas EGFP did not. These results indicate that Glut1 is capable of mitochondrial import in human cells.

Mitochondrial AA quenches ROS generated by purified mitochondria
Oxidative events in mitochondria produce ROS that can damage the mitochondrial membrane and mtDNA. To study the effect of mtAA on mitochondrial ROS, purified mitochondria were loaded with or without AA, and the levels of ROS were detected upon treatment with the oxidative stressor, DMNQ. We analyzed levels of superoxide (–•O2), hydroxyl radical (•OH), and hydrogen peroxide (H2O2) in control and oxidatively stressed purified mitochondria. Levels of –•O2 were significantly increased by treating mitochondria with the superoxide generator DMNQ, which was inhibited by preloading mitochondria with 2.0 mM AA (Fig. 3 A). A significant reduction in •OH levels was achieved by preloading mitochondria with AA (Fig. 3B ). Mitochondrial AA also significantly reduced levels of H2O2 in oxidatively stressed purified mitochondria (Fig. 3C ). Overall, we found that levels of mitochondrial ROS were attenuated when purified mitochondria were preloaded with AA (Student’s t test, n=8, P<0.05). These data indicate that mtAA inhibits mitochondrial ROS production during both normal and oxidative conditions.



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  		Figure 3. Mitochondrial AA (mtAA) quenches ROS in control, oxidatively stressed, and ETC-disrupted purified mitochondria. Fluoremetric measurement of A) superoxide (–•O2), B) hydroxyl radical (•OH), and C) hydrogen peroxide (H2O2) in purified mitochondria preloaded with or without 2.0 mM AA and treated with buffer or 10 µM DMNQ. D) Measurement of H2O2 liberated by control and AA-loaded mitochondria treated with or without 1 µM rotenone. *Statistical significance (Student’s t test, n=8, P<0.05).

Intracellular oxidative stress and energetic decline resulting from defective or disrupted mitochondrial ETC is considered to be the cause of aging and age-related degenerative diseases (8) . Hence, we tested the effect of vitamin C against oxidative stress resulting from rotenone-mediated disruption of the mitochondrial ETC (38) . We found that rotenone-induced generation of H2O2 in purified mitochondria, as measured by Amplex red assay, was significantly quenched by loading mitochondria with 2.0 mM AA (Student’s t test, n=8, P<0.05) (Fig. 3D ).

mtAA protects mitochondrial genome and membrane from oxidative damage
ROS-induced oxidation of mitochondrial DNA (mtDNA) results in a wide variety of adductive and structural DNA damages (8) . We reasoned that by quenching mitochondrial ROS, mtAA could protect the mtDNA against oxidative damages. The protective effect of AA was assessed by treating control or AA-loaded mitochondria with an oxidative stressor and measuring levels of 8-oxo-dG in the mtDNA. We found that compared with controls, purified mitochondria treated with 0.1 mM H2O2 had significantly higher levels of mitochondrial 8-oxo-dG, a biomarker of oxidative DNA damage (Fig. 4 A). However, preloading mitochondria with 2.0 mM AA significantly reduced 8-oxo-dG levels in oxidatively stressed samples (Fig. 4A ) (Student’s t test, n=3, P<0.05). With the knowledge that transition metals can facilitate the production of ROS and hence exacerbate the damaging effects of ROS on DNA (20) , we tested the effect of mtAA against Cu2+/H2O2-induced oxidative mtDNA damage. We found that in the presence of 0.1 mM Cu2+, 8-oxo-dG levels in the mtDNA increased with increasing concentrations of H2O2, while preloading mitochondria with vitamin C reduced 8-oxo-dG levels at all concentrations of the oxidative stressor (Fig. 4B ). As an additional biomarker of oxidative DNA damage, we measured levels of apurinic/apyrimidic (AP) sites in mtDNA isolated from control and oxidatively stressed mitochondria. Treatment of purified mitochondria with 0.1 mM Cu2+ and 0.5 mM H2O2 increased mitochondrial AP sites twofold, which was significantly reduced in mitochondria preloaded with 2.0 mM AA (Student’s t test, n=5, P<0.05) (Fig. 4C ). Our results show that mtAA protects mtDNA against ROS-induced elevation of 8-oxo-dG and AP sites.



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  		Figure 4. Mitochondrial AA (mtAA) protects mitochondrial DNA (mtDNA) and membrane from oxidative damage. A) Mitochondria purified from 293T cells were loaded with or without AA, treated with 0.1 mM H2O2, and 8-oxo-dG levels in mtDNA were measured with HPLC-ECD. *Statistical significance (Student’s t test, n=3, P<0.05). B) Levels of 8-oxo-dG in the mtDNA of mitochondria preloaded with (•) or without ({circ}) AA and incubated with 0.1 mM Cu2+ and specified concentrations of H2O2. C) Levels of apurinic/apyrimidic (AP) sites in mtDNA of control and AA preloaded mitochondria after treatment with 0.1 mM each of Cu2+ and H2O2. AP sites were quantified by a colorimetric assay. *Statistical significance (Student’s t test, n=5, P<0.05). D) Mitochondrial DNA fragmentation in control and AA preloaded purified mitochondria treated with 1.0 mM H2O2 was analyzed by gel electrophoresis. E) Fluoremetric measurement of ROS in control and AA preloaded HL-60 cells treated with or without 1 µM rotenone. Statistical significance was assessed with a Student’s t test (n=8, P<0.05). F) CCCP-induced disruption of mitochondrial membrane potential ({Delta}{psi}m) in control ({square}) or AA preloaded ({blacksquare}) HL-60 cells, as measured by FACS analysis. The percentage of cells with disrupted {Delta}{psi}m was measured by mitochondrial retention of the fluorochrome DIOC(6) (3). Statistical significance was assessed with a Student’s t test (n=3, P<0.05).

Oxidative stress in mitochondria can also lead to DNA double-strand breaks, resulting in shearing of mtDNA. Therefore, we examined whether mtAA could protect against ROS-mediated fragmentation of mtDNA using gel electrophoresis (43) . Purified mitochondria loaded with or without AA were treated with 1.0 mM H2O2. We found that H2O2 treatment increased shearing of mtDNA, which was significantly reduced by preloading mitochondria with 2.0 mM mtAA (Fig. 4D ).

To analyze the protective effect of vitamin C against intracellular oxidative stress stemming from the disruption of the mitochondrial ETC, we performed fluoremetric measurements of ROS in control and vitamin C preloaded HL-60 cells treated with or without 1 µM rotenone. We found that cells preloaded with 23 mM intracellular AA, achieved after a 30 min incubation with 0.5 mM DHA (44) , were significantly protected from rotenone-induced intracellular oxidative stress (Student’s t test, n=8, P<0.05) (Fig. 4E ). Our results point to the protective role of vitamin C in preventing oxidative disorders stemming from a defective or disrupted mitochondrial ETC.

Vitamin C inhibits protonophore-induced loss of membrane potential ({Delta}{Psi}m)
Since apoptotic and necrotic pathways can frequently be triggered in response to mitochondrial membrane damage, the stability of the mitochondrial membrane is essential for the survival of the cell (45) . To study the effect of AA on mitochondrial membrane stability, we analyzed the loss of {Delta}{psi}m in HL-60 cells in response to a protonophore, CCCP. Treatment with CCCP resulted in a dose-dependent increase in the percentage of cells with disrupted {Delta}{psi}m, which was significantly inhibited by loading cells with 23 mM AA (Student’s t test, n=3, P<0.05) (Fig. 4F ). Taken together, these results suggest a protective role of vitamin C in preventing mitochondrial membrane depolarization in response to uncouplers such as CCCP.

Generality of vitamin C-mediated genomic protection
To study the generality of vitamin C-mediated protection of mtDNA against oxidative damage, we measured 8-oxo-dG levels in mitochondria isolated from HepG2 and HL-60 cell-lines. Consistent with our observations with mitochondria isolated from 293T cells, mitochondria purified from HepG2 and HL-60 cells transport DHA, which is accumulated mitochondrially as AA (Fig. 5 A, B). Similarly, loading mitochondria with AA via DHA treatment conferred protection against H2O2-induced 8-oxo-dG formation (Fig. 5C, D ). HepG2 mitochondria preloaded with 0.8 mM AA showed a 60% reduction in 8-oxo-dG levels when treated with 0.1 mM H2O2. Likewise, HL-60 mitochondria preloaded with 1.5 mM AA showed a 70% reduction in 8-oxo-dG levels (Student’s t test, n=3, P<0.05). These results collectively point to a protective role of mtAA in preventing ROS-induced mtDNA damage in mammalian mitochondria.



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  		Figure 5. Generality of mitochondrial uptake of vitamin C and its role in genomic protection. Mitochondria purified from A) HepG2 and B) HL-60 cells were incubated with specified concentrations of DHA for 30 min and the accumulation of AA was measured by HPLC-ECD. Purified mitochondria loaded with or without AA were then oxidatively stressed with H2O2/Cu2+ and the levels of 8-oxo-dG in mtDNA isolated from C) HepG2 and D) HL-60 mitochondria were measured via electrochemical detection. Asterisks indicate statistical significance (Student’s t test, n=3, P<0.05).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Glut1 localizes to the mitochondrial membrane in mammalian cells
Although it is well documented that mammalian mitochondria contain vitamin C, the precise mechanism of its transport into mitochondria is poorly understood. We have previously shown that vitamin C is universally transported into cells in its oxidized form, DHA, through facilitative glucose transporters (27) . Using theoretical, functional, and physical studies, we have discovered that a parallel mechanism exists for the mitochondrial transport of vitamin C. We show that facilitative glucose transporter 1 (Glut1) localizes to mitochondria in mammalian cells and participates in the mitochondrial transport of vitamin C. Our theoretical analysis and experimental studies of chimeric Glut1-EGFP localization indicate a role of N-terminal Glut1 in mitochondrial targeting. Analogous studies indicate that the N-terminal sequence of the plastidic glucose transporter (pGlcT) may be involved in the translocation of the protein to the chloroplasts inner membrane (46) . It was also recently shown that plant mitochondria transport glucose and DHA in a facilitative manner (35) , which we postulate to be mediated by a functional mitochondrial glucose transporter. Our studies collectively point to the localization of Glut1 to mitochondria of mammalian cells.

Glut1 confers mitochondrial uptake of vitamin C
Our results on the uptake of vitamin C in mitochondria isolated from human cells agree with previous findings using purified mammalian (32 , 33) and plant (35) mitochondria. We postulate that the localization of Glut1 to mitochondria confers uncompeted transport of DHA into mitochondria since glucose in the cytosolic milieu exists almost entirely in the nontransportable form, glucose-6-phosphate (47) . Due to the apparent lack of a glycolytic pathway in mitochondria, we speculate that Glut1 on the mitochondrial membrane likely exists for the transport of DHA. Once inside mitochondria, using potential reductive mechanisms (48 49 50) , DHA is reduced and mitochondrially accumulated as AA (mtAA). During oxidative stress, mtAA is oxidized to DHA (14) , which we postulate to be recycled in either the mitochondrial compartment or the cytosol, and thus confer differential compartmentalization of vitamin C during normal and oxidative conditions (17) . Our proposed mechanism of uptake, reduction, and recycling of vitamin C by the mitochondrion appears to be a recapitulation of the metabolism of vitamin C in the cytosolic compartment (Fig. 6 ).



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  		Figure 6. Schematic illustration of vitamin C uptake and recycling in the cell. Vitamin C in its oxidized form, DHA, is transported into mitochondria via facilitative glucose transporter 1 and reduced to mitochondrial AA. Mitochondrial AA quenches ROS, protects the mitochondrial genome, and inhibits mitochondrial membrane depolarization. The mechanisms involved in the uptake, trapping, and recycling of vitamin C in mitochondria appear to recapitulate the metabolism of AA in the cytosolic compartment.

Vitamin C protects mitochondrial genome from oxidative injury
ROS generated metabolically and during oxidative stress can react with nucleic acids to generate a variety of cytotoxic and mutagenic DNA adducts (7) . For example, hydroxyl radical (•OH), which is generated continuously by the mitochondrial respiratory chain, is a major contributor of oxidative mtDNA damage (51) . Oxidation of guanosine yields potentially mutagenic 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxo-dG), which results in GC->TA transversions in 50% of the replicating DNA (52 , 53) . Mitochondrial ROS can also increase levels of apurinic/apyrimidic (AP) sites (54) , followed by mtDNA double-strand breaks (55) . Although mitochondria possess DNA repair pathways for combating oxidative damage (56 57 58) , lack of protective histones and proximity to ROS make the mtDNA particularly susceptible to oxidative injury (8) . Our studies of vitamin C-mediated quenching of mitochondrial ROS during normal and oxidative conditions correlate with the protective effect of vitamin C in inhibiting oxidative insults on the mtDNA. Furthermore, since levels of mitochondrial AA can be augmented with dietary vitamin C supplementation, our data suggest the pharmacological relevance of vitamin C in the protection of the mitochondrial genome against oxidative injury.

Vitamin C prevents mitochondrial membrane depolarization
Integrity of the mitochondrial membrane plays a key role in diverse cellular processes such as growth, differentiation, apoptosis, and necrosis (45 , 59) . Loss of mitochondrial membrane potential ({Delta}{Psi}m) and cytochrome c release are important events during apoptosis, both of which are inhibited by vitamin C (60) . Protonophores such as CCCP disrupt {Delta}{Psi}m (61) , increase mitochondrial respiration (62) , and induce membrane permeability transition (MPT) (63 , 64) . Our studies indicate that vitamin C inhibits CCCP-induced disruption of {Delta}{Psi}m, which we postulate to be mediated through the quenching of ROS. Since CCCP-induced increase in mitochondrial respiration is inhibited by respiratory substrates (62) , vitamin C may also be eliciting a similar effect. Alternatively, protection of mitochondrial membrane by vitamin C may involve inactivation of MPT, which is brought about by the redox switching of the vicinal thiols of the PT pore complex (65) .

Mitochondrial AA and its projected role in cellular redox homeostasis
Redox homeostasis is an intricate balance between oxidative and reductive events in the cell and vitamin C plays an important role in the modulation of the cellular redox state (66) . Since most pro-oxidants are liberated by mitochondria during oxidative metabolism (6) , quenching of mitochondrial ROS might favorably shift the cellular redox state. Oxidative imbalance is an important contributor to aging and degenerative diseases (67) , and our studies suggest a role of mtAA against a variety of ROS-mediated mitochondrial disorders.


   ACKNOWLEDGMENTS
 
We thank G. Stratis for his technical assistance and the Sloan-Kettering Molecular Cytology Core Facility for confocal imaging. This work was supported by grants from the National Institutes of Health (CA 30388), the New York State Department of Health, and the Lebensfeld Foundation. Our mentor David W. Golde, who was the motivating inspiration behind this work, passed away on August 9, 2004.

Received for publication May 19, 2005. Accepted for publication May 27, 2005.


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 MATERIALS AND METHODS
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 DISCUSSION
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