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.

We have created by transfection a series of HEK 293 cell lines that express varying amounts of caveolin-1 to test the possible effect of this protein on the transport and metabolism of long chain fatty acids (FA) in cells with this gain of function. We used an extracellular fluorescent probe (ADIFAB) to monitor binding of exogenous FA to the plasma membrane and an intracellular pH probe to monitor FA equilibration across the plasma membrane. Real-time fluorescence measurements showed rapid binding of oleic acid to the extracellular side of the plasma membrane and a rapid translocation across the lipid bilayer by the flip-flop mechanism (<5 s). Two cell lines expressing levels of caveolin-1 roughly comparable to that of adipocytes, which have a very high level of endogenous expression of caveolin-1, showed a relatively slow change in intracellular pH (t(1/2)< 100 s) in addition to the fast changes in fluorescence. We interpret this additional second phase to represent translocation of additional FA from the outer to inner leaflet of the plasma membrane. The slower kinetics could represent either slower flip-flop of FA across highly organized, rigid regions of the plasma membrane or binding of FA to caveolin-1 in the intracellular leaflet of the plasma membrane. The kinetics of palmitate and elaidate (a trans FA) transmembrane movement were identical to that for oleate. These results were observed in the absence of the putative FA transport protein, CD36, and in the absence of any changes in expression of fatty acid transport proteins (FATP) 2 and 4, and are in direct correlation with increased cellular free cholesterol content. FA metabolism was slow in all cell lines and was not enhanced by caveolin-1 expression. We conclude that transport of FA across the plasma membrane is modulated by caveolin-1 and cholesterol and is not dependent on the putative FA transport proteins CD36 and FATP.

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 insulindependent 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.

One of the four glutathione-S-transferases (GST) that is overproduced in the insecticide-resistant Cornell-R strain of the housefly (Musca domestica) produces an activity that degrades the insecticide dimethyl parathion and conjugates glutathione to lindane. In earlier work, it was shown that the resistant Cornell-R carries an amplification, probably a duplication, of one or more of its GST loci and that this amplification is directly related to resistance. Using polymerase chain reaction (PCR) amplification with genomic DNA, multiple copies of the gene encoding the parathion-degrading activity (called MdGst-3) were subcloned from both the ancestral, insecticide-susceptible strain BPM and from the insecticide-resistant Cornell-R. In BPM, three different MdGst-3 genes were identified while in Cornell-R, 12 different MdGst-3 sequences were found that, though closely related to ancestral genes, had diverged by a few nucleotides. This diversity in MdGst-3 genomic sequences in Cornell-R is reflected in the expressed sequences, as sampled through a cDNA bank. Population heterozygosity cannot account for these multiple GST genes. We suggest that selection for resistance to insecticides has resulted in not only amplification of the MdGst-3 genes but also in the divergence of sequence between the amplified copies.

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