Insulin action on activity and cell surface dispositon of human HepG2 glucose transporters expressed in Chinese hamster ovary cells

Scott A. Harrison, Joanne M. Buxton, Amy L. Helgerson, Richard G MacDonald, Francis J. Chlapowski, Anthony Carruthers, Michael P. Czech

Research output: Contribution to journalArticle

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Abstract

Complementary DNA encoding a facilitative glucose transporter was isolated from a human hepatoma cell line (HepG2) cDNA library and subcloned into a metal-inducible mammalian expression vector, pLEN (California Biotechnology) containing human metallothionein gene II promoter sequences. Chinese hamster ovary (CHO) cells transfected with this transporter expression vector, pLENGT, exhibited a 2-17-fold increase in immunoreactive HepG2-type glucose transporter protein, as measured by protein immunoblotting with antipeptide antibodies directed against the HepG2-type glucose transporter C-terminal domain. Expression of the human glucose transporter was verified by protein immunoblotting with a mouse polyclonal antiserum that recognizes the human but not the rodent HepG2-type transporter. 2-Deoxy-D-glucose uptake was increased 2-7-fold in transfected cell lines. Polyclonal antisera directed against purified red blood cell glucose transporter were raised in several rabbits. Antiserum from one rabbit, δ, was found to bind to the surface of intact red cells but not to inside-out red cell ghosts. Using this δ-antiserum in intact cell-binding assays, 1.6-9-fold increases in cell surface expression of the human glucose transporter were measured in CHO-K1 cell lines transfected with the transporter expression vector. Measurements of total cellular glucose transporter immunoreactive protein using anti-HepG2 transporter C-terminal peptide serum, cell surface glucose transporter protein using δ-antiserum and 2-deoxyglucose uptake revealed proportional relationships among these parameters in transfected cell lines expressing different levels of transporter protein. Insulin increased 2-deoxyglucose uptake 40% in control CHO-K1 cells and in CHO-K1 cells expressing modest levels of the human glucose transporter protein. However, stimulation of sugar uptake by insulin was only 10% in cells overexpressing human glucose transporter protein 9-fold, and no effect of insulin on sugar uptake was detected in several cell lines expressing very high levels (12-17-fold over controls) of human HepG2 glucose transporter protein. No insulin stimulation of anti-cell surface glucose transporter antibody binding was detected in any control or transfected CHO-K1 cell lines. These data indicate that a glucose transporter protein that is insensitive to insulin in HepG2 cells is regulated by insulin when expressed at low but not at high levels in insulin-response CHO-K1 cells. Additionally, the results suggest that insulin does not increase 2-deoxyglucose uptake by increasing the number of cell surface HepG2-type glucose transporters in CHO-K1 fibroblasts.

Original languageEnglish (US)
Pages (from-to)5793-5801
Number of pages9
JournalJournal of Biological Chemistry
Volume265
Issue number10
StatePublished - Apr 25 1990

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Facilitative Glucose Transport Proteins
Cricetulus
Ovary
Cells
Insulin
Deoxyglucose
Immune Sera
Proteins
Cell Line
Hep G2 Cells
Immunoblotting
Sugars
human SLC2A1 protein
Rabbits
Antibodies
Metallothionein
Erythrocyte Membrane
Fibroblasts
Biotechnology
Gene Library

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Harrison, S. A., Buxton, J. M., Helgerson, A. L., MacDonald, R. G., Chlapowski, F. J., Carruthers, A., & Czech, M. P. (1990). Insulin action on activity and cell surface dispositon of human HepG2 glucose transporters expressed in Chinese hamster ovary cells. Journal of Biological Chemistry, 265(10), 5793-5801.

Insulin action on activity and cell surface dispositon of human HepG2 glucose transporters expressed in Chinese hamster ovary cells. / Harrison, Scott A.; Buxton, Joanne M.; Helgerson, Amy L.; MacDonald, Richard G; Chlapowski, Francis J.; Carruthers, Anthony; Czech, Michael P.

In: Journal of Biological Chemistry, Vol. 265, No. 10, 25.04.1990, p. 5793-5801.

Research output: Contribution to journalArticle

Harrison, SA, Buxton, JM, Helgerson, AL, MacDonald, RG, Chlapowski, FJ, Carruthers, A & Czech, MP 1990, 'Insulin action on activity and cell surface dispositon of human HepG2 glucose transporters expressed in Chinese hamster ovary cells', Journal of Biological Chemistry, vol. 265, no. 10, pp. 5793-5801.
Harrison, Scott A. ; Buxton, Joanne M. ; Helgerson, Amy L. ; MacDonald, Richard G ; Chlapowski, Francis J. ; Carruthers, Anthony ; Czech, Michael P. / Insulin action on activity and cell surface dispositon of human HepG2 glucose transporters expressed in Chinese hamster ovary cells. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 10. pp. 5793-5801.
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