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

T. M. Pawlik, W. W. Souba, B. P. Bode

Research output: Contribution to journalArticlepeer-review

Abstract

Extracellular asparagine has previously been shown to markedly stimulate both ornithine decarboxylase and System N-mediated glutamine transport activities in hepatocytes by a transport-dependent mechanism. However, as a weak substrate of its inferred transporter System N, the specific route of asparagine uptake has remained enigmatic. In this study, asparagine transport was studied in detail and shown to be Na1-dependent, Li1-tolerant, stereospecific, and inhibited profoundly by glutamine and histidine. Coupled with competitive inhibition by glutamine Ki = 2.63 ± 1.11mM), the data indicated that asparagine was indeed slowly transported by System N in rat hepatocytes, albeit at rates an order of magnitude less than for glutamine. The differential substrate transport velocities were shown to be attributable to a low transporter asparagine affinity Km = 9.32 - 17.5mM) compared to glutamine Km ∼ 1mM). Consistent with its slow uptake, asparagine accumulated to a fivefold lesser degree than glutamine after 60min, yet stimulated 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 hyperosmotic media and blunted 30%-40% by phosphatidylinositol 3-kinase (PI3K) inhibitors wortmannin and LY294002. Collectively, the data suggest that System N-mediated asparagine uptake serves an autostimulatory role, mediated by cellular swelling and in part by a PI3K-dependent signal transduction pathway.

Original languageEnglish (US)
Pages (from-to)335-352
Number of pages18
JournalAmino Acids
Volume20
Issue number4
DOIs
StatePublished - 2001
Externally publishedYes

Keywords

  • Amino acids
  • Asparagine
  • Cell volume
  • Glutamine
  • Hepatocytes
  • Phosphatidylinositol 3-kinase
  • Transport

ASJC Scopus subject areas

  • Clinical Biochemistry
  • Biochemistry
  • Endocrinology

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