Asparagine uptake in rat hepatocytes: Resolution of a paradox and insightsinto substrate-dependent transporter regulation

Extracellular asparagine has previously been shown to markedly stimulate bo th ornithine decarboxylase and System N-mediated glutamine transport activi ties in hepatocytes by a transport-dependent mechanism. However, as a weak substrate of its inferred transporter System N, the specific route of aspar agine uptake has remained enigmatic. In this study, asparagine transport wa s studied in detail and shown to be Na+-dependent, Li+-tolerant, stereospec ific, and inhibited profoundly by glutamine and histidine. Coupled with com petitive inhibition by glutamine (K-i = 2.63 +/- 1.11 mM), the data indicat ed that asparagine was indeed slowly transported by System N in rat hepatoc ytes, albeit at rates an order of magnitude less than for glutamine. The di fferential substrate transport velocities were shown to be attributable to a low transporter asparagine affinity (K-m = 9.3 - 17.5 mM) compared to glu tamine (K-m similar to 1mM). Consistent with its slow uptake, asparagine ac cumulated to a fivefold lesser degree than glutamine after 60 mins, yet sti mulated System N activity to the same extent as glutamine. The transaminase inhibitor aminooxyacetate and starvation of the donor animal each enhanced asparagine uptake twofold and augmented subsequent transporter activation. Conversely, asparagine-dependent System N stimulation was abrogated by hyp erosmotic media and blunted 30%-40% by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Collectively, the data suggest that Sy stem N-mediated asparagine uptake serves an autostimulatory role, mediated by cellular swelling and in part by a PI3K-dependent signal transduction pa thway.