The aim of the proposed research is to develop and test impermeant hexose carrier affinity labels for use in studying hexose carrier turnover and the mechanism of insulin action in rat eipdidymal adipocytes. The methods involve coupling the disaccharide maltose (which binds the substrate site of the hexose carrier, but does not undergo transport) to one or more cross-linking agents, which can in turn covalently attach maltose to the hexose carrier. This two-step process allows highly specific radio-labeling of cell surface hexose carriers. The proposed methodology has significant advantages over available techniques for quantitating cell surface hexose carrier numbers in intact cells, particularly as these are increased in response to insulin stimulation. Furthermore, the impermeant affinity labels can be used to follow hexose carrier internalization, degradation, and possible recycling back to the plasma membrane. In this regard it is planned to test the effects of insulin on adipocyte hexose carrier turnover, in light of the recent evidence suggesting that insulin enhances hexose transport in insulin-responsive cells by increasing the translocation of intracellular hexose carriers to the plasma membrane, where they can function to allow glucose entry. Specifically, the rates of hexose carrier internalization and insertion into the plasma membrane of intact adipocytes will be assessed using the covalent carrier affinity labels with the aim of determining whether hexose carriers might undergo a recycling process. The effects of insulin on hexose carrier turnover will then be evaluated to determine whether insulin-stimulated hexose transport can be explained by an acceleration of the reinsertion phase of a carrier recycling mechanism. Experiments will also be performed to establish whether such recycling might occur by an endocytic-exocytic process involving other membrane protein as well as hexose carriers. These investigations should help to elucidate the molecular mechanism of insulin-stimulated hexose transport, and perhaps prove reasonable to adopt for use in studies of pathologic conditions such as diabetes mellitus and obesity, in which abnormalities of hexose transport have been documented.
