Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel: Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester

Geoffrey D. Holman, Izabela J. Kozka, Avril E. Clark, Carolyn J. Flower, John Saltis, Alan D. Habberfield, Ian Simpson, Samuel W. Cushman

Research output: Contribution to journalArticle

252 Citations (Scopus)

Abstract

A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases ≈5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a ≈4-fold increase in GLUT4 while GLUT1 increases ≈5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport ≈3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).

Original languageEnglish (US)
Pages (from-to)18172-18179
Number of pages8
JournalJournal of Biological Chemistry
Volume265
Issue number30
StatePublished - Oct 25 1990

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Facilitative Glucose Transport Proteins
Phorbol Esters
Mannose
Labeling
Rats
Protein Isoforms
Insulin
Acetates
Glucose
3-O-Methylglucose
Photoaffinity Labels
Immunoprecipitation
phorbol-12-myristate
Antibodies

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Holman, Geoffrey D. ; Kozka, Izabela J. ; Clark, Avril E. ; Flower, Carolyn J. ; Saltis, John ; Habberfield, Alan D. ; Simpson, Ian ; Cushman, Samuel W. / Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel : Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. In: Journal of Biological Chemistry. 1990 ; Vol. 265, No. 30. pp. 18172-18179.
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title = "Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel: Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester",
abstract = "A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases ≈5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a ≈4-fold increase in GLUT4 while GLUT1 increases ≈5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport ≈3-fold to only 13{\%} of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).",
author = "Holman, {Geoffrey D.} and Kozka, {Izabela J.} and Clark, {Avril E.} and Flower, {Carolyn J.} and John Saltis and Habberfield, {Alan D.} and Ian Simpson and Cushman, {Samuel W.}",
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Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel : Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. / Holman, Geoffrey D.; Kozka, Izabela J.; Clark, Avril E.; Flower, Carolyn J.; Saltis, John; Habberfield, Alan D.; Simpson, Ian; Cushman, Samuel W.

In: Journal of Biological Chemistry, Vol. 265, No. 30, 25.10.1990, p. 18172-18179.

Research output: Contribution to journalArticle

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T1 - Cell surface labeling of glucose transporter isoform GLUT4 by bis-mannose photolabel

T2 - Correlation with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester

AU - Holman, Geoffrey D.

AU - Kozka, Izabela J.

AU - Clark, Avril E.

AU - Flower, Carolyn J.

AU - Saltis, John

AU - Habberfield, Alan D.

AU - Simpson, Ian

AU - Cushman, Samuel W.

PY - 1990/10/25

Y1 - 1990/10/25

N2 - A new impermeant photoaffinity label has been used for identifying cell surface glucose transporters in isolated rat adipose cells. This compound is 2-N-4(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2- propylamine. We have used this reagent in combination with immunoprecipitation by specific antibodies against the GLUT4 and GLUT1 glucose transporter isoforms to estimate the relative abundance of these two transporters on the surface of the intact adipose cell following stimulation by insulin and phorbol 12-myristate 13-acetate (PMA). In the basal state, GLUT4 and GLUT1 are both present at the cell surface but GLUT4 is more abundant than GLUT1. In response to insulin, GLUT4 increases 15-20-fold and GLUT1 increases ≈5-fold while 3-O-methyl-D-glucose transport is stimulated 20-30-fold. By contrast, PMA only induces a ≈4-fold increase in GLUT4 while GLUT1 increases ≈5-fold to the same level as seen with insulin. In addition, PMA stimulates 3-O-methyl-D-glucose transport ≈3-fold to only 13% of the insulin-stimulated state. Thus GLUT4 is the major glucose transporter isoform under all conditions, and it is selectively and markedly enriched in response to insulin but not PMA which increases GLUT1 and GLUT4 equally. Furthermore, stimulation of glucose transport activity correlates closely with the appearance of GLUT4 on the cell surface in response to both insulin and PMA but does not correlate with the sum of GLUT1 and GLUT4 appearance. These results suggest that GLUT4 may be inherently more active than GLUT1 due to a higher TK (turnover/Km).

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