Corpe CP et al. (2013), Intestinal dehydroascorbic acid (DHA) transport...

XB-ART-46663

J Biol Chem 2013 Mar 29;28813:9092-101. doi: 10.1074/jbc.M112.436790.

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Intestinal dehydroascorbic acid (DHA) transport mediated by the facilitative sugar transporters, GLUT2 and GLUT8.

Corpe CP , Eck P , Wang J , Al-Hasani H , Levine M .

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Intestinal vitamin C (Asc) absorption was believed to be mediated by the Na(+)-dependent ascorbic acid transporter SVCT1. However, Asc transport across the intestines of SVCT1 knock-out mice is normal indicating that alternative ascorbic acid transport mechanisms exist. To investigate these mechanisms, rodents were gavaged with Asc or its oxidized form dehydroascorbic acid (DHA), and plasma Asc concentrations were measured. Asc concentrations doubled following DHA but not Asc gavage. We hypothesized that the transporters responsible were facilitated glucose transporters (GLUTs). Using Xenopus oocyte expression, we investigated whether facilitative glucose transporters GLUT2 and GLUT5-12 transported DHA. Only GLUT2 and GLUT8, known to be expressed in intestines, transported DHA with apparent transport affinities (Km) of 2.33 and 3.23 mm and maximal transport rates (Vmax) of 25.9 and 10.1 pmol/min/oocyte, respectively. Maximal rates for DHA transport mediated by GLUT2 and GLUT8 in oocytes were lower than maximal rates for 2-deoxy-d-glucose (Vmax of 224 and 32 pmol/min/oocyte for GLUT2 and GLUT8, respectively) and fructose (Vmax of 406 and 116 pmol/min/oocyte for GLUT2 and GLUT8, respectively). These findings may be explained by differences in the exofacial binding of substrates, as shown by inhibition studies with ethylidine glucose. DHA transport activity in GLUT2- and GLUT8-expressing oocytes was inhibited by glucose, fructose, and by the flavonoids phloretin and quercetin. These studies indicate intestinal DHA transport may be mediated by the facilitative sugar transporters GLUT2 and GLUT8. Furthermore, dietary sugars and flavonoids in fruits and vegetables may modulate Asc bioavailability via inhibition of small intestinal GLUT2 and GLUT8.

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Species referenced: Xenopus
Genes referenced: slc23a1 slc2a1 slc2a2 slc2a5 slc2a8 sts

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	FIGURE 2. DHA and 2-DG uptake in oocytes injected to express sugar transporters. Xenopus oocytes previously injected with GLUT1, -2, and -5ï¿½12 were incubated for 10 min at room temperature with 300 Î¼M [14C]DHA (A) or 1 mM 2-[3H]DG (B). Oocytes were then washed, and intracellular radioactivity was quantified. Control was sham water-injected oocytes. G, facilitative sugar transporter; m, mouse; r, rat; w, wild type GLUT (di-leucine motif); m, mutant GLUT (di-alanine motif); HA, human influenza hemagglutinin. Results are mean ï¿½ S.D. of 10ï¿½20 oocytes.
	FIGURE 3. DHA, 2-deoxy-D-glucose, and D-fructose transport kinetics in GLUT2-injected oocytes. Oocytes previously injected with GLUT2 mRNA were incubated for 10 min with 0ï¿½8 mM [14C]DHA (A), 0ï¿½100 mM 2-[3H]DG (B), and 0ï¿½300 mM D-[14C]fructose (C). Oocytes were then washed and intracellular radioactivity quantified. A best fit curve was fitted to the collected data using the nonlinear regression function Y = Vmaxï¿½X/Kt + X (SigmaPlot). Data represent mean ï¿½ S.D. of 10ï¿½20 oocytes.
	FIGURE 4. DHA, 2-deoxy-D-glucose, and D-fructose transport kinetics in GLUT8-injected oocytes. Oocytes previously injected with GLUT8 mRNA were incubated for 10 min with 0ï¿½8 mM [14C]DHA (A), 0ï¿½100 mM 2-[3H]DG (B), and 0ï¿½300 mM D-[14C]fructose (C). Oocytes were then washed, and intracellular radioactivity was quantified. A best fit curve was fitted to the collected data using the nonlinear regression function Y = Vmaxï¿½X/Kt + X (SigmaPlot). Data represent mean ï¿½ S.D. of 10ï¿½20 oocytes.
	FIGURE 5. Inhibition of GLUT2- and GLUT8-mediated DHA transport by exo- and endofacial sugar transporter inhibitors. Oocytes injected with GLUT2 (â—) and GLUT8 (â–´) were incubated with 300 Î¼M [14C]DHA for 10 min in the presence of 0ï¿½100 mM of the exofacial sugar transporter inhibitor, ethyldiene glucose (A) and 0ï¿½50 Î¼M of the endofacial sugar transporter inhibitor, cytochalasin B (B). Oocytes were washed, and internalized radioactivity was quantified. A best fit curve for the collected data were obtained using the nonlinear regression analysis equation, y = min + (max âˆ’ min)/1 + (x/IC50) âˆ’ Hill slope four-parameter logistic curve (SigmaPlot). Data represent mean ï¿½ S.D. of 10ï¿½20 oocytes.
	FIGURE 6. Inhibition of GLUT2- and GLUT8-mediated DHA uptake by sugars. Oocytes injected with GLUT2 (â—) and GLUT8 (â–´) were incubated with 300 Î¼M [14C]DHA for 10 min in the presence of 0ï¿½100 mM D-glucose (A) and 0ï¿½100 mM D-fructose (B). Oocytes were washed, and internalized radioactivity was quantified. A best fit curve for the collected data was obtained using nonlinear regression analysis. Data represent mean ï¿½ S.D. of 10ï¿½20 oocytes.
	FIGURE 7. Inhibition of GLUT2- and GLUT8-mediated DHA uptake by flavonoids. Oocytes injected with GLUT2 (â—) and GLUT8 (â–´) were incubated with 300 Î¼M [14C]DHA for 10 min in the presence of 0ï¿½100 Î¼M phloretin (A) and 0ï¿½100 Î¼M quercetin (B). Oocytes were washed, and internalized radioactivity was quantified. A best fit curve for the collected data was obtained using nonlinear regression analysis. Data represent mean ï¿½ S.D. of 10ï¿½20 oocytes.

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