Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells

Evidence that exocytosis is a critical site of hormone action

Shinobu Satoh, Haruo Nishimura, Avril E. Clark, Izabela J. Kozka, Susan J. Vannucci, Ian Simpson, Michael J. Quon, Samuel W. Cushman, Geoffrey D. Holman

Research output: Contribution to journalArticle

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Abstract

The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low-density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with the [3H]bismannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos-4-yloxy)-2- propylamine. The half-time for internalization of tracer-tagged GLUT4 when insulin is removed by collagenase treatment is similar to that observed for the decrease in immunodetectable GLUT4 in the plasma membranes and the decrease in glucose transport activity in the intact cells. In contrast, internalization of tracer-tagged GLUT4 also occurs when cells are maintained in the continuous presence of insulin even though the plasma membrane level of immunodetectable GLUT4 and glucose transport activity in the intact cells are unaltered. These data show, for the first time, that insulin has little, if any, effect on the rate constant for GLUT4 endocytosis, but instead, primarily increases the rate constant for exocytosis. Tracer-tagged GLUT4 that is returned to the low-density microsomes can be restimulated with fresh insulin to recycle to the plasma membranes and to a steady-state distribution level that is the same as that observed in cells that are maintained in the continuous presence of insulin. These data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Following insulin stimulation of adipose cells initially in the basal state, the increase in immunodetectable GLUT4 in the plasma membranes precedes the increase in accessibility of GLUT4 to exofacial 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2- propylamine photolabeling, and this in turn precedes the increase in cellular glucose transport activity. Such time course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathway. The kinetic properties of GLUT4 translocation and its recycling have been interpreted in terms of a subcellular trafficking model that identifies exocytosis, possibly involving-hypothetical "docking" and "fusion" steps, as the critical site of hormone action.

Original languageEnglish (US)
Pages (from-to)17820-17829
Number of pages10
JournalJournal of Biological Chemistry
Volume268
Issue number24
StatePublished - Aug 25 1993

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Exocytosis
Cell membranes
Rats
Hormones
Insulin
Kinetics
Cell Membrane
Glucose
Recycling
Microsomes
Rate constants
Collagenases
Endocytosis
Fusion reactions

All Science Journal Classification (ASJC) codes

  • Biochemistry

Cite this

Satoh, Shinobu ; Nishimura, Haruo ; Clark, Avril E. ; Kozka, Izabela J. ; Vannucci, Susan J. ; Simpson, Ian ; Quon, Michael J. ; Cushman, Samuel W. ; Holman, Geoffrey D. / Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells : Evidence that exocytosis is a critical site of hormone action. In: Journal of Biological Chemistry. 1993 ; Vol. 268, No. 24. pp. 17820-17829.
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abstract = "The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low-density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with the [3H]bismannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos-4-yloxy)-2- propylamine. The half-time for internalization of tracer-tagged GLUT4 when insulin is removed by collagenase treatment is similar to that observed for the decrease in immunodetectable GLUT4 in the plasma membranes and the decrease in glucose transport activity in the intact cells. In contrast, internalization of tracer-tagged GLUT4 also occurs when cells are maintained in the continuous presence of insulin even though the plasma membrane level of immunodetectable GLUT4 and glucose transport activity in the intact cells are unaltered. These data show, for the first time, that insulin has little, if any, effect on the rate constant for GLUT4 endocytosis, but instead, primarily increases the rate constant for exocytosis. Tracer-tagged GLUT4 that is returned to the low-density microsomes can be restimulated with fresh insulin to recycle to the plasma membranes and to a steady-state distribution level that is the same as that observed in cells that are maintained in the continuous presence of insulin. These data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Following insulin stimulation of adipose cells initially in the basal state, the increase in immunodetectable GLUT4 in the plasma membranes precedes the increase in accessibility of GLUT4 to exofacial 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2- propylamine photolabeling, and this in turn precedes the increase in cellular glucose transport activity. Such time course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathway. The kinetic properties of GLUT4 translocation and its recycling have been interpreted in terms of a subcellular trafficking model that identifies exocytosis, possibly involving-hypothetical {"}docking{"} and {"}fusion{"} steps, as the critical site of hormone action.",
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Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells : Evidence that exocytosis is a critical site of hormone action. / Satoh, Shinobu; Nishimura, Haruo; Clark, Avril E.; Kozka, Izabela J.; Vannucci, Susan J.; Simpson, Ian; Quon, Michael J.; Cushman, Samuel W.; Holman, Geoffrey D.

In: Journal of Biological Chemistry, Vol. 268, No. 24, 25.08.1993, p. 17820-17829.

Research output: Contribution to journalArticle

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T1 - Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells

T2 - Evidence that exocytosis is a critical site of hormone action

AU - Satoh, Shinobu

AU - Nishimura, Haruo

AU - Clark, Avril E.

AU - Kozka, Izabela J.

AU - Vannucci, Susan J.

AU - Simpson, Ian

AU - Quon, Michael J.

AU - Cushman, Samuel W.

AU - Holman, Geoffrey D.

PY - 1993/8/25

Y1 - 1993/8/25

N2 - The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low-density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with the [3H]bismannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos-4-yloxy)-2- propylamine. The half-time for internalization of tracer-tagged GLUT4 when insulin is removed by collagenase treatment is similar to that observed for the decrease in immunodetectable GLUT4 in the plasma membranes and the decrease in glucose transport activity in the intact cells. In contrast, internalization of tracer-tagged GLUT4 also occurs when cells are maintained in the continuous presence of insulin even though the plasma membrane level of immunodetectable GLUT4 and glucose transport activity in the intact cells are unaltered. These data show, for the first time, that insulin has little, if any, effect on the rate constant for GLUT4 endocytosis, but instead, primarily increases the rate constant for exocytosis. Tracer-tagged GLUT4 that is returned to the low-density microsomes can be restimulated with fresh insulin to recycle to the plasma membranes and to a steady-state distribution level that is the same as that observed in cells that are maintained in the continuous presence of insulin. These data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Following insulin stimulation of adipose cells initially in the basal state, the increase in immunodetectable GLUT4 in the plasma membranes precedes the increase in accessibility of GLUT4 to exofacial 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2- propylamine photolabeling, and this in turn precedes the increase in cellular glucose transport activity. Such time course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathway. The kinetic properties of GLUT4 translocation and its recycling have been interpreted in terms of a subcellular trafficking model that identifies exocytosis, possibly involving-hypothetical "docking" and "fusion" steps, as the critical site of hormone action.

AB - The subcellular trafficking of tracer-tagged GLUT4 between the plasma membranes and low-density microsomes of rat adipose cells has been studied. Cell-surface GLUT4 have been initially tracer-tagged in the insulin-stimulated state with the [3H]bismannose photolabel 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos-4-yloxy)-2- propylamine. The half-time for internalization of tracer-tagged GLUT4 when insulin is removed by collagenase treatment is similar to that observed for the decrease in immunodetectable GLUT4 in the plasma membranes and the decrease in glucose transport activity in the intact cells. In contrast, internalization of tracer-tagged GLUT4 also occurs when cells are maintained in the continuous presence of insulin even though the plasma membrane level of immunodetectable GLUT4 and glucose transport activity in the intact cells are unaltered. These data show, for the first time, that insulin has little, if any, effect on the rate constant for GLUT4 endocytosis, but instead, primarily increases the rate constant for exocytosis. Tracer-tagged GLUT4 that is returned to the low-density microsomes can be restimulated with fresh insulin to recycle to the plasma membranes and to a steady-state distribution level that is the same as that observed in cells that are maintained in the continuous presence of insulin. These data suggest that the cells' entire complement of GLUT4 is involved in the recycling process. Following insulin stimulation of adipose cells initially in the basal state, the increase in immunodetectable GLUT4 in the plasma membranes precedes the increase in accessibility of GLUT4 to exofacial 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4-yloxy)-2- propylamine photolabeling, and this in turn precedes the increase in cellular glucose transport activity. Such time course data suggest that there may be plasma membrane intermediate states in the GLUT4 trafficking pathway. The kinetic properties of GLUT4 translocation and its recycling have been interpreted in terms of a subcellular trafficking model that identifies exocytosis, possibly involving-hypothetical "docking" and "fusion" steps, as the critical site of hormone action.

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