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

Abstract

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. 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. plasma membrane(s) bovine serum albumin glucose transporter isoform 4 heavy microsome(s) light microsome(s) N-hydroxysuccinimide phosphate-buffered saline secretory compartment-associated membrane protein semicarbazide-sensitive amine oxidase vesicle-associated membrane protein Caveolae are 50–100-nm invaginations of the plasma membrane (PM)1 which are formed by the expression of one or more isoforms of caveolin, the protein that produces their distinct structure (1Galbiati F. Razani B. Lisanti M.P. Cell. 2001; 106: 403-411Abstract Full Text Full Text PDF PubMed Scopus (517) Google Scholar). The membrane lipid composition of caveolae is enriched in sphingolipids and cholesterol, and caveolae represent a subtype of membrane lipid rafts, that is, subdomains of the PM with specific lipid and protein compositions (2Brown D.A. London E. J. Biol. Chem. 2000; 275: 17221-17224Abstract Full Text Full Text PDF PubMed Scopus (2065) Google Scholar). The physiological roles of caveolae remain uncertain (3Parton R.G. Nat. Rev. Mol. Cell. Biol. 2003; 4: 162-167Crossref PubMed Scopus (140) Google Scholar), but they have been suggested to participate in a large number of important cellular functions. These include the formation of transcytotic/endocytic vesicles in endothelial cells (4Henley J.R. Krueger E.W. Oswald B.J. McNiven M.A. J. Cell Biol. 1998; 141: 85-99Crossref PubMed Scopus (628) Google Scholar, 5Oh P. McIntosh D.P. Schnitzer J.E. J. Cell Biol. 1998; 141: 101-114Crossref PubMed Scopus (557) Google Scholar), the organization/localization of numerous transmembrane signaling complexes in many cell types (6Anderson R.G. Annu. Rev. Biochem. 1998; 67: 199-225Crossref PubMed Scopus (1727) Google Scholar, 7Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. 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Zhang X.L. Marks C.B. Macaluso F. Russell R.G. Li M. Pestell R.G. Di Vizio D. Hou Jr., H. Kneitz B. Lagaud G. Christ G.J. Edelmann W. Lisanti M.P. J. Biol. Chem. 2001; 276: 38121-38138Abstract Full Text Full Text PDF PubMed Scopus (258) Google Scholar, 14Drab M. Verkade P. Elger M. Kasper M. Lohn M. Lauterbach B. Menne J. Lindschau C. Mende F. Luft F.C. Schedl A. Haller H. Kurzchalia T.V. Science. 2001; 293: 2449-2452Crossref PubMed Scopus (1318) Google Scholar). On the other hand, these animals do have vascular abnormalities, particularly in the lung, and consequently, they have a reduced ability to exercise. Interestingly, with age they show abnormalities in lipid metabolism as a result of apparent adipocyte pathology (15Razani B. Combs T.P. Wang X.B. Frank P.G. Park D.S. Russell R.G. Li M. Tang B. Jelicks L.A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 2002; 277: 8635-8647Abstract Full Text Full Text PDF PubMed Scopus (472) Google Scholar). 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In that membrane with as and have to caveolae, with large by by of the and we are in the of In and of adipocyte caveolae their from insulin signaling in these we find other signaling the major protein components of caveolae. we a receptor in lipid and of physiological with other the of adipocyte caveolae as cellular for of lipid Indeed, of from adipocytes has to an and a of these to the We all with from of for with the electron microscopy and from the at for with