Dehydroascorbic acid transport by GLUT4 in Xenopus oocytes and isolated rat adipocytes

Dehydroascorbic acid (DHA), the first stable oxidation product of vitamin C , was transported by GLUT1 and GLUT3 in Xenopus laevis oocytes with transpo rt rates similar to that of P-deoxyglucose (2-DG), but due to inherent diff iculties with GLUT4 expression in oocytes it was uncertain whether GLUT4 tr ansported DHA (Rumsey, S, C., Kwon, O., Xu, G. W., Burant, C. F., Simpson, I., and Levine, M. (1997) J. Biol. Chem. 272, 18982-18989). We therefore st udied DHA and 2-DG transport in rat adipocytes, which express GLUT4. Withou t insulin, rat adipocytes transported 2-DG 2-3-fold faster than DHA. Preinc ubation with insulin (0.67 mu M) increased transport of each substrate simi larly: 7-10-fold for 2-DG and 6-8-fold for DHA, Because intracellular reduc tion of DHA in adipocytes was complete before and after insulin stimulation , increased transport of DHA was not explained by increased internal reduct ion of DHA to ascorbate, To determine apparent transport kinetics of GLUT4 for DHA, GLUT4 expression in Xenopus oocytes was reexamined. Preincubation of oocytes for >4 h with insulin (1 mu M) augmented GLUT4 transport of 2-DG and DKA by up to 5-fold. Transport of both substrates was inhibited by cyt ochalasin B and displayed saturable kinetics. GLUT4 had a higher apparent t ransport affinity (K-m of 0.98 versus 5.2 mM) and lower maximal transport r ate (V-max of 66 versus 880 pmol/oocyte/10 min) for DHA compared with 2-DG. The lower transport rate for DHA could not be explained by binding differe nces at the outer membrane face, as shown by inhibition with ethylidene glu cose, or by transporter trans-activation and therefore was probably due to substrate-specific differences in transporter/substrate translocation or re lease. These novel data indicate that the insulin-sensitive transporter GLU T4 transports DHA in both rat adipocytes and Xenopus oocytes, Alterations o f this mechanism in diabetes could have clinical implications for ascorbate utilization.