Abstract
We have recently described a monoclonal antibody (1F8) that recognizes a form of glucose transporter unique to fat and muscle (James, D. E., Brown, R., Navarro, J., and Pilch, P. F. (1988) Nature 333, 183-185), tissues that respond acutely to insulin by markedly increasing their glucose uptake. Here, we report that rat adipocytes possess two immunologically distinct glucose-transporters: one recognized by 1F8, and one reactive with antibodies raised against the human erythrocyte glucose transporter. Immunoadsorption experiments indicate that these glucose transporters reside in different vesicle populations and that both transporter isoforms translocate from intracellular sites to the plasma membrane in response to insulin. The insulin-regulatable transporter resides in a unique vesicle that comprises 3% or less of the low density microsomes of fat cells and has a limited protein composition that does not include the bulk of another translocatable protein, the insulin-like growth factor II receptor. Immunoprecipitation with 1F8 of microsomal glucose transporters photoaffinity labeled with [3H]cytochalasin B brings down 90% of the label. Similarly, immunoprecipitation with 1F8 of glucose transporters from insulin-stimulated plasma membranes photolabeled with 3-[125I]iodo-4-azidophenethylamido-7-O-succinyldeacetyl-f ors kolin, another transporter-selective reagent, results in 75% of the labeled transporter localized in the immunoprecipitate. Thus, insulin action involves the combined effect of translocation from at least two vesicle pools each containing different glucose transporters. The 1F8-reactive transporter comprises the majority of the total transporter pool that is responsible for the insulin-induced increase in glucose transporter number.
Highlights
Insulin-regulated Glucose Uptake in Rat AdipocytesIs Mediated by Two Transporter Isoforms Present inat Least Two Vesicle Populations*
Reactivewith theanti-red cell antibodyisnotin vesicles the glucose transporter recognized by the anti-erythrocyte containingthefat/muscletypetransporter (Fig. l B, right antibody gives a strong signal in the basal PMwhich is not panel).thetwo fat cell glucose transporter typesreside dramatically increased by insulin, a result seen by others in two intracellular compartments
The purified erythrocyte glucose transporter stainsvery poorly with protein reagents, and we arenotsure if the stained proteinin Fig. 5 is actuallytransporter.Inany case, in contrast to the 1F8-adsorbed vesicles, the vesicle population that was not adsorbed contains numerous proteins, and few, if any, of these show obvious insulin regulation
Summary
Reactivewith theanti-red cell antibodyisnotin vesicles the glucose transporter recognized by the anti-erythrocyte containingthefat/muscletypetransporter (Fig. l B , right antibody gives a strong signal in the basal PMwhich is not panel).thetwo fat cell glucose transporter typesreside dramatically increased by insulin, a result seen by others in two (or more) intracellular compartments. Since the translocation of transporters from the LDM to the PM accountsfor essentially all the transporter redistribution due to insulin(above and Ref. 1)and most (90%o)f the LDM transporters areimmunoprecipitable with 1F8, the data in Fig. 3 support the notion that theinsulin-regulatable transporter is the quantitatively predominanfot rm responsible for the observed increase intotal cell surface transporters. Protein from the gels was transferred to nitrocellulose and immunoblotted with 1F8 (top panel) or with the anti-red cell antibody ( A b )(nCT,bottom panel).This experiment has been performed >10 times with qualitatively similar results and 5
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