Gino Vallega

Immunopurification and Characterization of Rat Adipocyte Caveolae Suggest Their Dissociation from Insulin Signaling

Adipocytes play an important role in the insulin-dependent regulation of organismal fuel metabolism and express caveolae at levels as high or higher than any other cell type. Recently, a link between insulin signaling and caveolae has been suggested; nevertheless, adipocyte caveolae have been the subject of relatively few studies, and their contents have been minimally characterized. With the aid of a new monoclonal antibody, we developed a rapid procedure for the immunoisolation of caveolae derived from the plasma membrane of adipocytes, and we characterized their protein content. We find that immunopurified adipocyte caveolae have a relatively limited protein composition, and they lack the raft protein, flotillin, and insulin receptors. Immunogold labeling and electron microscopy of the adipocyte plasma membrane confirmed the lack of insulin receptors in caveolae. In addition to caveolins, the structural components of caveolae, their major protein constituents, are the semicarbazide-sensitive amine oxidase and the scavenger lipoprotein receptor CD36. The results are consistent with a role for caveolae in lipid flux in and of adipocytes.

Insulin-dependent protein trafficking in skeletal muscle cells

We have established a simple procedure for the separation of intracellular pool(s) of glucose transporter isoform GLUT-4-containing vesicles from the surface sarcolemma and T tubule membranes of rat skeletal myocytes. This procedure enabled us to immunopurify intracellular GLUT-4-containing vesicles and to demonstrate that 20-30% of the receptors for insulin-like growth factor II/mannose 6-phosphate and transferrin are colocalized with GLUT-4 in the same vesicles. Using our new fractionation procedure as well as cell surface biotinylation, we have shown that these receptors are translocated from their intracellular compartment(s) to the cell surface along with GLUT-4 after insulin stimulation in vivo. Denervation causes a considerable downregulation of GLUT-4 protein in skeletal muscle but does not affect the level of expression of other known component proteins of the corresponding vesicles. Moreover, the sedimentation coefficient of these vesicles remains unchanged by denervation. We suggest that the normal level of GLUT-4 expression is not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment.

Dissociation of Insulin Receptor Expression and Signaling from Caveolin-1 Expression

The presence of cell surface caveolin/caveolae has been postulated to influence the localization, expression levels, and kinase activity of numerous receptors, including the insulin receptor. However, there are conflicting data concerning the effects of caveolin on insulin receptor expression and function. To help clarify this issue, we created a gain of function situation by expressing caveolin-1 at various levels in HEK-293 cells where the endogenous level of caveolin-1 is very low. We generated four permanent lines of this cell expressing amounts of caveolin-1 ranging from 10 to 40 times that of parental cells. The amount of caveolin-1 in the human embryonic kidney cells expressing the highest caveolin levels is comparable with that of adipocytes, cells that naturally express one of the highest levels of caveolin-1. We measured insulin receptor amount and insulin-dependent receptor autophosphorylation as well as insulin receptor substrate 1 (IRS1) tyrosine phosphorylation as an index of insulin signaling. We found that the insulin receptor level was essentially the same in the parental and all four derived cell lines. Likewise, we determined that insulin-dependent insulin receptor and IRS1 tyrosine phosphorylation was not significantly different in the four cell lines representing parental, low, medium, and high levels of caveolin-1 expression. We conclude that insulin receptor expression and ligand-dependent signaling is independent of caveolin-1 expression.