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© 2004 The American Society for Nutritional Sciences J. Nutr. 134:2216-2221, September 2004

Nutrient-Gene Interactions

Gender and Sodium-Ascorbate Transporter Isoforms Determine Ascorbate Concentrations in Mice1

Shiu-Ming Kuo*,{dagger}, Marlene E. MacLean*, Kathleen McCormick* and John X. Wilson**,2

* Department of Exercise and Nutrition Sciences and {dagger} Department of Biochemistry, University at Buffalo, Buffalo, NY; and ** Department of Physiology and Pharmacology, Faculty of Medicine and Dentistry, University of Western Ontario, London, ON, Canada

2To whom correspondence should be addressed. E-mail: John.Wilson{at}fmd.uwo.ca.


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 
We evaluated the hypothesis that sodium-dependent vitamin C (ascorbate) transporters SVCT1 and SVCT2 (encoded by genes Slc23a1 and Slc23a2) regulate ascorbate concentrations in tissues of adult mice. Slc23a2+/– and Slc23a2+/+ mice were fed an ascorbate-free diet for 10–12 wk, and then segregated according to gender and genome, and were placed in groups of 3–4 in metabolic cages for 24-h urine collection. RT-PCR analysis showed that liver and kidney expressed mainly SVCT1, and brain, skeletal muscle, and spleen expressed predominantly SVCT2. Slc23a2+/– mice had low SVCT2 but normal SVCT1 messenger RNA (mRNA) levels, which did not differ between genders. Ascorbate concentrations were lower in Slc23a2+/– than Slc23a2+/+ mice in tissues where SVCT2 was the main isoform. Compared with males, females had lower ascorbate excretion and ascorbate:creatinine ratio in urine and had higher ascorbate concentrations in plasma and SVCT1-predominant tissues. SVCT2 contributed to a gender effect in spleen because males had higher spleen ascorbate concentration than females in wild-type but not in Slc23a2+/– mice. Hepatic gulonolactone oxidase mRNA and activity levels did not differ with genotype or gender, indicating no differences in ascorbate synthesis. We concluded that SVCT2 is a major determinant of ascorbate accumulation in tissues lacking SVCT1. The SVCT isoforms appear to function independently of one another because SVCT1 expression and ascorbate concentrations in SVCT1-predominant organs were not affected by SVCT2 deficiency. Additionally, lower ascorbate excretion in females may elevate the vitamin’s concentrations in plasma and tissues expressing SVCT1 that, unlike SVCT2, is not saturated by plasma ascorbate concentrations.

KEY WORDS: • vitamin C • ascorbate • transport • sexual dimorphism

Ascorbate (vitamin C) is an antioxidant and enzyme cofactor. Its physiological importance as an antioxidant is indicated by the inverse correlation between plasma ascorbate concentration and lipid peroxidation in healthy people (1). High intracellular concentrations of ascorbate are required for its cofactor role in enzymatic processes, e.g., collagen synthesis (2). Cells may accumulate ascorbate through the sodium-dependent vitamin C transporters SVCT1 and SVCT2, which are encoded by the genes Slc23a1 and Slc23a2, respectively (3,4). In situ hybridization and Northern blot analysis of human, rat, and mouse tissues have found messenger RNA (mRNA) for one or both SVCT isoforms in most organs (5–9). However, SVCT1 and SVCT2 may not be the sole regulators of ascorbate concentration. Northern blot analyses have not found either SVCT isoform in skeletal muscle (6,9,10), although this tissue contains 40% of the body’s ascorbate (11). There is also evidence that volume-regulated anion channels can mediate diffusion of ascorbate into and out of cells and that these channels translocate ascorbate at faster rates than do SVCT isoforms when cells are swollen (4,12). Additionally, many cell types take up dehydroascorbic acid through facilitative glucose transporters and reduce both it and the ascorbyl radical to ascorbate intracellularly (13,14). Finally, the hepatocytes of most animal species (but not humans) can synthesize ascorbate de novo from glucose, through a pathway in which gulonolactone oxidase is the rate-limiting enzyme (4).

The importance of SVCT1 and SVCT2 to ascorbate homeostasis in vivo is uncertain. It has been reported that SVCT2-deficient (Slc23a2+/–) mice have low ascorbate levels in several tissues prior to birth, as well as in the brain at 9–11 mo of age, but the gender of these mice was not specified (15). Gender is a potential confounder, because male gender has been identified as a risk factor for hypovitaminosis C (plasma ascorbate < 30 µmol/L) in hospitalized patients (16). Therefore, we tested the hypothesis that SVCT1 and SVCT2 regulate ascorbate concentrations in adult mice. Our approach was to measure SVCT1 and SVCT2 expression by RT-PCR, plasma, and tissue ascorbate concentrations, and renal ascorbate excretion in wild-type and in SVCT2-deficient mice of both genders. Heterozygous SVCT2 knockout (Slc23a2+/–) mice are a valuable model because they survive to adulthood, unlike the homozygous Slc23a2–/– mice, which die shortly after birth.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 LITERATURE CITED
 
    Animals. The protocols were approved by the Institutional Animal Care and Use Committee of the University at Buffalo. Slc23a2+/– mice for breeding were generous gifts from Dr. Robert Nussbaum (National Institutes of Health/National Human Genome Research Institute/Genetic Disease Research Branch) (15). They were mated with wild-type 129S6 mice from Taconic and progeny were maintained in the specific pathogen-free facility of the University at Buffalo. All mice were maintained under 12-h light/dark cycle (the light period began at 1100 h and ended at 2300 h) with free access to water and to a diet (PicoLab Mouse Chow 20) that did not contain detectable ascorbate (<1 nmol/L). Thus, the mice were fed an ascorbate-free diet to negate intestinal absorption until they were adults. They were used for experiments at 10–12 wk of age. Genotyping was performed with PCR of DNA extracted from ear pieces (MasterPure DNA Purification Kit, Epicenter). The primers used for detection of mRNA are shown in Table 1.


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  		TABLE 1 PCR primers used in the study

 
Urine was collected for a 24-h period according to a published method (17). Three or four mice of the same gender and genotype were housed together in a metabolic cage with a wire-mesh bottom, and their urine was pooled. The mice were killed by halothane overdose and cervical dislocation at the end of the light period. Trunk blood was collected into heparinized vials and centrifuged immediately to separate plasma. Bladder urine was collected by syringe and was combined with the voided urine for analysis. Liver, kidney, spleen, brain, and hindlimb skeletal muscle were also collected, so that the study included organs that had been identified previously as expressing predominantly SVCT1 or SVCT2 (15). Urine, plasma, and tissue samples were immediately frozen in liquid nitrogen and then were transferred to a –80°C freezer for storage until analysis. All subsequent analyses were performed by personnel without knowledge of the genotype or the gender of the samples.

    Biochemical analyses. Urinary creatinine was measured by the modified Jaffe method adapted for a 96-well plate reader measuring absorption at 490 nm (18). We confirmed that the concentrations of ascorbate and urate present in the samples did not interfere with this assay of creatinine.

Gulonolactone oxidase activity was measured in fresh liver (19). Minced liver was incubated with 5 mmol/L L-gulonolactone or a vehicle in phosphate-buffered saline for 30 min at 37°C, and the reaction was terminated by rapid freezing. Subsequently, ascorbate was assayed by HPLC with electrochemical detection.

Ascorbate and urate were measured by HPLC-based electrochemical assay with a Waters M460 amperometric detector, according to the procedures we described previously (20). Tissues were homogenized in metaphosphoric acid solution (8.5 g/L) that contained 3,4-dihydroxybenzylamine as an internal standard and, subsequently, their ascorbate concentrations were corrected for 3,4-dihydroxybenzylamine recovery. Total glutathione in the spleen was measured by using an enzyme-based spectrophotometric method adapted for a 96-well plate reader (21).

    RT-PCR. RNA was extracted by a modified guanidinium thiocyanate-phenol-chloroform method (22). Tissues were pulverized under liquid nitrogen prior to the extraction. The RT reaction was carried out with an Omniscript RT kit from Qiagen by using 2 ug total RNA per 20 uL reaction mixture. cDNA was then quantified by real-time PCR by using iQ SYBR Green Supermix and iCycler PCR equipment (Bio-Rad). A single melt curve was observed for each primer set in all real-time PCR reactions. Duplicate PCR reactions were performed for each sample, and the mean threshold cycles (as determined by the linear portion of the fluorescence absorbance curve) were used for the final calculation. The expression of SVCT1 and SVCT2 in kidney was also determined by using different primer sets to amplify longer RT-PCR products and then by visualizing the products in agarose gel (20 g/L) by ethidium bromide staining. The results were consistent with the real-time quantitative RT-PCR. The cDNA levels of SVCT1 and SVCT2 in brain, spleen, and skeletal muscle (pooled from forelimbs and hind limbs) were determined by using primer sets to amplify longer RT-PCR products that were then detected by ethidium bromide staining. RT-PCR was done twice on each sample, with identical results.

    Statistical analysis. All numerical results are expressed as means ± SE. Differences between means were evaluated by two-way ANOVA (gender x genotype) and the Student-Newman-Keuls multiple comparison test. Student’s pooled t-test was used to compare the expression of SVCT1 and SCVT2 in each organ. Differences with P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 LITERATURE CITED
 
RT-PCR analyses detected abundant SVCT2 but relatively little SVCT1 mRNA in brain and spleen of wild-type (Slc23a2+/+) mice (Figs. 1and 2) and, within each tissue, the difference between expression levels of the two isoforms was significant (P < 0.001). SVCT2 expression in skeletal muscle was low but clearly present (Fig. 3A). As in brain and spleen, SVCT2 was the predominant isoform in skeletal muscle. This finding was supported by RT-PCR analysis performed after skeletal muscle mRNA was enriched (Fig. 3B). In contrast, liver and kidney expressed SVCT1 at much higher levels than they did SVCT2 (compare Tables 2, and 3). Quantitative RT-PCR showed that SVCT2 mRNA levels in liver and kidney were <5% of SVCT1 levels (Tables 2, and 3), and the difference between the two isoforms was significant (P < 0.001). Heterozygous SVCT2 knockout (Slc23a2+/–) mice had lower SVCT2 but normal SVCT1 mRNA levels relative to wild-type controls (Fig. 1 and Tables 2, and 3).



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  		FIGURE 1 RT-PCR analysis of SVCT1 and SVCT2 in brain (panel A) and spleen (panel B) of male (M) and female (F) Slc23a2+/+ (+/+) and Slc23a2+/– (+/–) mice. Each lane represents one mouse. The kidney (K) is a predominantly SVCT1-expressing organ included for comparison with brain and spleen.

 


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  		FIGURE 2 Quantification of SVCT1 and SVCT2 mRNA expression in brain gel (panel A) and spleen gel (panel B) band intensities for the gels of Figure 1. SVCT1 and SVCT2 expression are normalized to GAPDH expression of the same mouse. Values are means ± SE or range of the normalized expression levels for mice of the same gender and genotype (n = 3 males and 3 females for Slc23a2+/– mice, and n = 2 males and 2 females for Slc23a2+/+ mice). In each group, expression levels were greater (P < 0.001) for SVCT2 than for SVCT1 in both brain and spleen.

 


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  		FIGURE 3 RT-PCR determined expression of SVCT1 and SVCT2 in liver and skeletal muscle from a male wild-type mouse. Panel A: RT-PCR using total RNA from liver and muscle. The same amount of cDNA was used for the PCR of SVCT1 and SVCT2 of liver and muscle samples. Panel B: RT-PCR using skeletal muscle mRNA enriched by oligo-dT column. PCR was carried out with decreasing amounts of cDNA in the ratio of 2:1:0.5:0.2.

 

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  		TABLE 2 SVCT1 expression level in liver and kidney of male and female Slc23a2+/+ and Slc23a2+/– mice determined by real-time quantitative RT-PCR of Slc23a1 mRNA1, 2

 

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  		TABLE 3 SVCT2 expression level in liver and kidney of male and female Slc23a2+/+ and Slc23a2+/– mice determined by real-time quantitative RT-PCR of Slc23a2 mRNA1, 2

 
Ascorbate concentrations in brain and skeletal muscle were significantly higher for wild type than for heterozygote (Slc23a2+/–) but were not dependent on gender (Table 4). A significant interaction between gender and genotype was found in spleen. A simple main effect of genotype on spleen ascorbate concentration occurred in both genders. Additionally, males had a significantly higher spleen ascorbate concentration than did females in wild-type mice but not in heterozygotes (Table 4). In contrast, spleen glutathione concentration was not affected by either genotype or gender. Concentrations were 2.80 ± 0.13 nmol/mg in male wild-type mice, 3.10 ± 0.20 nmol/mg in male heterozygotes, 3.16 ± 0.35 nmol/mg in female wild-type mice, and 2.80 ± 0.15 nmol/mg in female heterozygotes (n = 6–9). Female mice had higher ascorbate concentrations in plasma, kidney, and liver than did males, and there was no effect of genotype on these variables (Table 5).


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  		TABLE 4 Ascorbate concentrations in predominantly SVCT2-expressing tissues of male and female Slc23a2+/+ and Slc23a2+/– mice1

 

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  		TABLE 5 Ascorbate concentrations in predominantly SVCT1-expressing tissues and in plasma of male and female Slc23a2+/+ and Slc23a2+/–1

 
For urine, each individual measurement (n = 1) was from a group of 3–4 mice housed together in a metabolic cage. The volume of urine produced over 24 h (i.e., the combination of voided and bladder urine) per mouse was larger in females, whether expressed as mL/mouse (Table 6) or mL/g body weight (data not shown). Body weight was lower in female than male mice but was not affected by genotype (Table 6). Females excreted more creatinine than males (Table 7). Females also had much lower urinary ascorbate excretion and ascorbate:creatine ratio than males, and these variables were not affected by genotype (Table 7). Gender and genotype did not affect the urinary excretion of urate or the urate:creatinine ratio (Table 7).


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  		TABLE 6 Body weight and 24-h urinary fluid excretion of male and female Slc23a2+/+ and Slc23a2+/– mice1, 2

 

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  		TABLE 7 Urinary excretion of ascorbate, urate, and creatinine during 24-h collection period of male and female Slc23a2+/+ and Slc23a2+/– mice1

 
Mouse liver can synthesize ascorbate de novo from glucose, and gulonolactone oxidase catalyzes the rate-limiting step. We observed that hepatic gulonolactone oxidase mRNA and enzyme activity levels did not differ with genotype or gender (data not shown).


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
LITERATURE CITED
 
To test our hypothesis that SVCT1 and SVCT2 regulate ascorbate concentrations in adult mice, we first examined the tissue expression of these transporters. Except for skeletal muscle, the distribution revealed by our RT-PCR analyses confirmed what was found previously by Northern blot analysis (5–10). While Northern blot analysis did not detect either isoform in skeletal muscle (6,9,10), the greater sensitivity of RT-PCR revealed a low level of SVCT2 expression in this tissue. Subsequent assay of ascorbate concentration indicated that SVCT2 mediates accumulation of this vitamin in skeletal muscle, because male and female SVCT2 heterozygote (Slc23a2+/–) mice had less ascorbate than did wild-type (Slc23a2+/+) mice. This interpretation is consistent with studies of the L6 skeletal muscle cell line that showed ascorbate stimulates muscle differentiation (23). Because 40% of the ascorbate in the body is contained in skeletal muscle (11), SVCT2 expression in this tissue is also an important contributor to the body’s total stores of this vitamin.

The SVCT isoforms appear to function independently of each other, because we observed that the SVCT1 expression in all examined organs and the ascorbate concentrations in SVCT1-predominant organs (kidney, liver) were not affected by SVCT2 deficiency. In contrast, ascorbate concentrations were significantly lower in SVCT2 heterozygote than in wild-type mice in organs that mainly or exclusively expressed SVCT2 (brain, skeletal muscle, spleen). These results indicate that SVCT2 is a major pathway of ascorbate accumulation in adult tissues that lack SVCT1. This interpretation is consistent with a previous report that ascorbate concentrations in adult SVCT2 heterozygote mice were decreased in brain but normal in liver (15).

In spleen, female gender was associated with lower ascorbate concentration in wild-type mice but not in SVCT2-heterozygote mice. Glutathione is a low-molecular weight antioxidant, like ascorbate, but the glutathione concentration in spleen was not affected by either gender or SVCT2 genotype. Taken together, our data indicate that SVCT2 contributes to a specific effect of gender on ascorbate concentration in spleen. This is likely of physiological importance because spleen uptake of ascorbate regulates plasma ascorbate concentration (24,25), and increases in spleen ascorbate concentration enhance the immune response to infection (26,27).

Urinary fluid volume and creatinine excretion were greater for female for than male mice, reflecting a higher glomerular filtration rate in females. These findings are consistent with rat studies showing females had higher rates of water consumption and urine production than did males when provided unrestricted access to drinking water (28). Furthermore, we found that female mice had decreased urinary ascorbate excretion but similar urate excretion compared with males. The effect of gender on urinary excretion of ascorbate was significant whether the data were expressed as nmol per mouse per 24 h or ascorbate:creatinine ratio and thus was likely due to sexual dimorphism in renal tubular transport.

The higher plasma ascorbate concentration in females may be attributed to decreased urinary excretion of ascorbate, as well as decreased uptake of ascorbate by cells in the spleen (and perhaps by other tissues that we did not assay). Hepatic gulonolactone oxidase mRNA and enzyme activity levels did not differ with genotype or gender, suggesting that de novo ascorbate synthesis was not greater in females than in males. On the contrary, the smaller livers of females may have produced less ascorbate than did the livers of males. It may be because the Slc23a2 genotype did not affect urinary ascorbate excretion that this genotype also did not affect plasma ascorbate concentration in the 10- to 12-wk-old mice of our study. Sotiriou et al. (15) reported that the Slc23a2+/– genotype was associated with low plasma ascorbate concentration; however, because they did not state the gender of their mice, their finding of abnormal plasma ascorbate may have been caused by gender differences between the compositions of the wild-type and the SVCT2-heterozygote mice groups.

SVCT2 has such a high affinity for ascorbate that it is virtually saturated at the concentrations of the vitamin that normally occur in rodent plasma (3,4). Because SVCT1 has a lower affinity and is not saturated at plasma ascorbate concentrations, the higher plasma ascorbate concentration we observed in female mice may have increased the intracellular ascorbate concentration through this transporter. Therefore, in our study, the higher plasma ascorbate concentration in female mice may have caused the gender difference in tissue ascorbate levels in organs that expressed SVCT1 predominantly.

In conclusion, this study showed that SVCT2 is a major determinant of ascorbate accumulation in adult mouse tissues that lack SVCT1. Additionally, the lower ascorbate excretion rate in female mice may elevate the vitamin’s concentrations in plasma and in tissues expressing SVCT1 that, unlike SVCT2, are not saturated by the ascorbate concentrations found in plasma.


   ACKNOWLEDGMENTS
 
We thank James Boyer, Ewa Jaworski, and Chee-Ho Tan for technical support. Real-time PCR was performed in the Confocal Microscopy and 3-D Imaging Laboratory of the University at Buffalo.


   FOOTNOTES
 
1 Supported by NIH DK54728 and Natural Sciences and Engineering Research Council of Canada Operating Grant 2200. Back

Manuscript received 11 March 2004. Initial review completed 4 June 2004. Revision accepted 23 June 2004.


   LITERATURE CITED
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 LITERATURE CITED
 

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