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W2023286320 abstract "Lipid rafts/caveolae are found to be essential for insulin-like growth factor (IGF)-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction. In 3T3-L1 cells, IGF-1 receptor is located in lipid rafts/caveolae of the plasma membrane and can directly interact with caveolin-1, the major protein component in caveolae. Disruption of lipid rafts/caveolae by depleting cellular cholesterol with cholesterol-binding reagent, β-methylcyclodextrin or filipin, blocks the IGF-1 receptor signaling in 3T3-L1 preadipocyte. Both hormonal induced adipocyte differentiation and mitotic clonal expansion are inhibited by lipid rafts/caveolae disruption. However, a nonspecific lipid binding reagent, xylazine, does not affect adipocyte differentiation or mitotic clonal expansion. Further studies indicate that lipid rafts/caveolae are required only for IGF-1 receptor downstream signaling and not the activation of receptor itself by ligand. Thus, our results suggest that localization in lipid rafts/caveolae and association with caveolin enable IGF-1 receptor to have a close contact with downstream signal molecules recruited into lipid rafts/caveolae and transmit the signal through these signal molecule complexes. Lipid rafts/caveolae are found to be essential for insulin-like growth factor (IGF)-1 receptor signaling during 3T3-L1 preadipocyte differentiation induction. In 3T3-L1 cells, IGF-1 receptor is located in lipid rafts/caveolae of the plasma membrane and can directly interact with caveolin-1, the major protein component in caveolae. Disruption of lipid rafts/caveolae by depleting cellular cholesterol with cholesterol-binding reagent, β-methylcyclodextrin or filipin, blocks the IGF-1 receptor signaling in 3T3-L1 preadipocyte. Both hormonal induced adipocyte differentiation and mitotic clonal expansion are inhibited by lipid rafts/caveolae disruption. However, a nonspecific lipid binding reagent, xylazine, does not affect adipocyte differentiation or mitotic clonal expansion. Further studies indicate that lipid rafts/caveolae are required only for IGF-1 receptor downstream signaling and not the activation of receptor itself by ligand. Thus, our results suggest that localization in lipid rafts/caveolae and association with caveolin enable IGF-1 receptor to have a close contact with downstream signal molecules recruited into lipid rafts/caveolae and transmit the signal through these signal molecule complexes. insulin-like growth factor-1 extracellular signal-regulated kinase fluorescein isothiocyanate 4-morpholineethanesulfonic acid Lipid rafts are plasma membrane microdomains, principally composed of cholesterol and sphingolipids, which form liquid-ordered domains of decreased membrane fluidity (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 5Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 6van Meer G. Science. 2002; 296: 855-857Google Scholar). With integration of caveolins into lipid rafts, these microdomains will form caveolae, which are flask-shaped vesicular invaginations in the plasma membrane (2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar). Caveolae are a specific form of lipid rafts and are now considered to be broader than just vesicular membrane invaginations (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar). The long saturated lipid tails of sphingolipids impart the lipid rafts a high degree of order further stabilized by interacting cholesterol. This property leads a light buoyant density on sucrose density gradient centrifugation.Cholesterol is an essential component in lipid rafts/caveolae. In caveolae, cholesterol binds directly to caveolins and facilitates the integration of caveolins into membrane (8Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Google Scholar, 9Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Google Scholar). Depletion of cellular cholesterol with cholesterol-binding reagents, such as methylcyclodextrin or filipin, will remove cholesterol from lipid rafts/caveolae, dissemble the striated caveolin coats, and eventually lead to the disruption of both lipid rafts and caveolae (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar,10Chang W.J. Rothberg K.G. Kamen B.A. Anderson R.G.W. J. Cell Biol. 1992; 118: 63-69Google Scholar, 11Fielding C.J. Fielding P.E. Biochim. Biophys. Acta. 2000; 1529: 210-222Google Scholar, 12Parpal S. Karlsson M. Thorn H. Stralfors P. J. Biol. Chem. 2001; 276: 9670-9678Google Scholar, 13Hailstones D. Sleer L.S. Parton R.G. Stanley K.K. J. Lipid Res. 1998; 39: 369-379Google Scholar, 14Furuchi T. Anderson R.G.W. J. Biol. Chem. 1998; 273: 21099-21104Google Scholar, 15Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Google Scholar).During recent years, more and more reports confirmed that many signaling molecules are found to be enriched in lipid rafts/caveolae, which serve as platforms and play an important role in regulating signal cascade (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar, 16Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Google Scholar, 17Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Google Scholar). Signal molecules, such as heterotrimeric G-proteins (18Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Google Scholar), protein kinase C (19Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.L. Hermanoski- Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Google Scholar, 20Smart E.J. Ying Y. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Google Scholar), Shc (21Liu P. Ying Y.S. Ko Y.G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Google Scholar), SOS (22Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Google Scholar), Raf1 (22Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Google Scholar, 23Mineo C. Anderson R.G.W. White M.A. J. Biol. Chem. 1997; 272: 10345-10348Google Scholar), and Src family tyrosine kinases (19Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.L. Hermanoski- Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Google Scholar, 24Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Google Scholar, 25Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Google Scholar, 26Robbins S.M. Quintrell N.A. Bishop M.J. Mol. Cell. Biol. 1995; 15: 3507-3515Google Scholar), are recruited into caveolae by caveolins, which, through the scaffolding domain, interact with the caveolin-binding motifs in these signal molecules (17Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Google Scholar). These clusters of signal molecules can form “preassembled signaling complexes” on the plasma membrane. In addition, many growth factor receptors (epidermal growth factor receptor, platelet-derived growth factor receptor, insulin receptor, etc.) (12Parpal S. Karlsson M. Thorn H. Stralfors P. J. Biol. Chem. 2001; 276: 9670-9678Google Scholar, 20Smart E.J. Ying Y. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Google Scholar, 21Liu P. Ying Y.S. Ko Y.G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Google Scholar, 27Gustavsson J. Parpal S. Karlsson M. Ramsing C. Thorn H. Borg M. Lindroth M. Peterson K.H. Magnusson K. Stralfors P. FASEB J. 1999; 13: 1961-1971Google Scholar, 29Liu P. Ying Y.S. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13666-13670Google Scholar) are found to be located in lipid rafts/caveolae. Thus, the enrichment of receptors and signal molecules in lipid rafts/caveolae enables them to be in close contact with each other and makes lipid rafts/caveolae the gateways for signals entering into the cells.Lipid rafts/caveolae are indicated to be important for insulin receptor signaling (30Czech M.P. Nature. 2000; 407: 147-148Google Scholar, 31Bickel P.E. Am. J. Physiol. 2002; 282: E1-E10Google Scholar). Insulin receptors are found to be located in caveolae of adipocyte plasma membrane (27Gustavsson J. Parpal S. Karlsson M. Ramsing C. Thorn H. Borg M. Lindroth M. Peterson K.H. Magnusson K. Stralfors P. FASEB J. 1999; 13: 1961-1971Google Scholar), and many signal molecules involved in insulin receptor signal cascade are also found to be enriched in caveolin-enriched plasma membrane domain (32Smith R.M. Harada S. Smith J.A. Zhang S. Jarett L. Cell. Signalling. 1998; 10: 355-362Google Scholar). In 3T3-L1 adipocytes, caveolin-1 is phosphorylated by insulin receptor (33Kimura A. Mora S. Shigematsu S. Pessin J.E. Saltiel A.R. J. Biol. Chem. 2002; 277: 30153-30158Google Scholar, 34Mastick C.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 20706-20714Google Scholar) and is an activator of insulin receptor signaling (35Yamamoto M. Toya Y. Schwencke C. Lisanti M.P. Meyers Jr., M.G. Ishikawa Y. J. Biol. Chem. 1998; 273: 26962-26968Google Scholar). In addition, caveolin-enriched lipid raft microdomains and lipid rafts are required for insulin signaling and Glut4 translocation (36Karlsson M. Thorn H. Parpal S. Stralfors P. Gustavsson J. FASEB J. 2002; 16: 249-251Google Scholar, 37Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Google Scholar, 38Chiang S. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Google Scholar, 39Watson R.T. Shigematsu S. Chiang S. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Google Scholar). These results provide compelling evidence that lipid rafts/caveolae are essential for insulin receptor signaling.IGF-11 receptor tyrosine kinase signaling (along with glucocorticoid and cAMP signaling) is required for 3T3-L1 preadipocyte differentiation induction (40Smith P.J. Wise L.S. Berkowitz R. Wan C. Rubin C.S. J. Biol. Chem. 1988; 263: 9402-9408Google Scholar, 41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar, 42Mackall J.C. Student A.K. Polakis S.E. Lane M.D. J. Biol. Chem. 1976; 251: 6462-6464Google Scholar, 43Rosen O.M. Smith C.J. Hirsch A. Lai E. Rubin C.S. Rec. Prog. Horm. Res. 1979; 35: 477-499Google Scholar, 44Student A.K. Hsu R.Y. Lane M.D. J. Biol. Chem. 1980; 255: 4745-4750Google Scholar, 45Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Google Scholar). High level insulin or IGF-1 at physiological concentration activates the IGF-1 receptor on the plasma membrane, leading to the initiation of the differentiation program (40Smith P.J. Wise L.S. Berkowitz R. Wan C. Rubin C.S. J. Biol. Chem. 1988; 263: 9402-9408Google Scholar, 41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar). Two events occur after the activation of IGF-1 receptor in 3T3-L1 preadipocytes: mitotic clonal expansion and adipocyte differentiation. Previously, we have identified that these two events are both activated by the IGF-1 receptor signaling and can be separately blocked without affecting the other (41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar, 46Qiu Z. Wei Y. Chen N. Jiang M. Wu J. Liao K. J. Biol. Chem. 2001; 276: 11988-11995Google Scholar, 47Liao K. Lane M.D. J. Biol. Chem. 1995; 270: 12123-12132Google Scholar). The mitotic clonal expansion is activated by IGF-1 receptor through a signal pathway involving the activation of ERK1 and -2, whereas adipocyte differentiation is initiated through the reversible tyrosine phosphorylation of the adapter protein c-Crk by IGF-1 receptor tyrosine kinase and tyrosine phosphatase. These results suggest that IGF-1 receptor activates two separate signal pathways in 3T3-L1 preadipocytes simultaneously.Although some of the cellular functions for IGF-1 receptor and insulin receptor are different, structural and signaling similarities between these two receptors have long been recognized. Recently, it has been reported that IGF-1 may also induce caveolin-1 tyrosine phosphorylation and its translocation in the lipid rafts (48Maggi D. Biedi C. Segat D. Barbero D. Panetta D. Cordera R. Biochem. Biophys. Res. Commun. 2002; 295: 1085-1089Google Scholar). Since in 3T3-L1 cells IGF-1 receptor activates more than one signal pathway at the same time, it is likely that IGF-1 receptor on plasma membrane interacts or cross-talks with several signaling pathways. With their preassembled signaling complexes on the intracellular side, lipid rafts/caveolae provide the structural foundation for simultaneous activation or cross-talking of multiple signal pathways by IGF-1 receptor.In the present study, we reported that IGF-1 receptor was located in lipid rafts/caveolae in 3T3-L1 preadipocyte and adipocyte. The integrity of lipid rafts/caveolae was essential for IGF-1 receptor signal transduction during 3T3-L1 preadipocyte differentiation induction. Disruption of lipid rafts/caveolae by cholesterol depletion effectively blocked the downstream signaling of IGF-1 receptor but not IGF-1 receptor activation itself.DISCUSSIONIn many ways, caveolae are lipid rafts enriched with structural protein caveolins, which are the defining protein components in caveolae. Although caveolins have three types, general types caveolin-1 and -2 and muscle-specific caveolin-3 (7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar), cells completely lack caveolae in caveolin-1 knockout mice (53Drab 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: 2449-2452Google Scholar). This result from caveolin-1-deficient mice has demonstrated the importance of caveolin-1 in the formation of caveolae and provided support for using caveolin-1 as an indicator for caveolae. By using immunofluorescence staining (Fig. 1), sucrose density gradient centrifugation (Figs. 2 and 3), and co-immunoprecipitation (Fig. 4), we have identified that IGF-1 receptor was located in lipid rafts/caveolae in 3T3-L1 cells. To provide further support, we have also shown that IGF-1 receptor and insulin receptor, which has been indicated to be located in caveolae of adipocyte plasma membrane (26Robbins S.M. Quintrell N.A. Bishop M.J. Mol. Cell. Biol. 1995; 15: 3507-3515Google Scholar, 51Vainio S. Heino S. Mansson J.E. Fredman P. Kuismanen E. Vaarala O. Ikonen E. EMBO Rep. 2002; 3: 95-100Google Scholar), were associated with the same membrane structures in 3T3-L1 adipocytes (Fig. 3 B). These results provided strong evidence that IGF-1 was located in lipid rafts/caveolae in 3T3-L1 preadipocytes and adipocytes.It was consistently observed that, besides in caveolae, IGF-1 receptor also appeared to be in the membrane structure slightly lighter than caveolae. The peak of IGF-1 receptor and insulin receptor was in fractions 6 and 7, whereas the peak of caveolin was in fractions 7 and 8 (Figs. 2 and 3 B). However, in high level insulin-stimulated adipocytes, IGF-1 receptor, insulin receptor, and caveolin were better correlated in density gradient separation (Fig.3 B). These results were observed in several independently repeated experiments (results not shown). Thus, it is likely that IGF-1 receptor was also located in the caveolin-free lipid rafts around the caveolae and might further converge into caveolae upon ligand stimulation. Currently, we are investigating this translocation induced by ligand.Although lipid rafts and caveolae are important in signal transduction (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 5Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 6van Meer G. Science. 2002; 296: 855-857Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar), their roles in IGF-1 receptor cellular signaling are not fully understood. The identification of IGF-1 receptor in lipid rafts/caveolae provided us an opportunity to study the role of lipid rafts/caveolae in IGF-1 signaling. In 3T3-L1 preadipocytes, IGF-1 receptor signal is essential for inducing two cellular responses: adipocyte differentiation and mitotic clonal expansion. However, these two cellular responses can be separately blocked by inhibitors without affecting the other (41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar, 46Qiu Z. Wei Y. Chen N. Jiang M. Wu J. Liao K. J. Biol. Chem. 2001; 276: 11988-11995Google Scholar). PD98059, an inhibitor of MEK-1, blocked mitotic clonal expansion but not adipocyte differentiation, whereas vanadate, a protein-tyrosine phosphatase inhibitor, only blocked adipocyte differentiation. Since lipid rafts/caveolae are on the plasma membrane, which is on the top of the IGF-1 receptor signal cascade, disruption at the level of lipid rafts/caveolae will more likely block all of the cellular responses induced by IGF-1 receptor signaling. Our results clearly supported this hypothesis. Disruption of lipid rafts/caveolae by cholesterol-binding reagents led to the blockade of both cellular responses simultaneously (Figs. 5 and 6). Taken together, these results composed a hierarchy of IGF-1 receptor signaling system in 3T3-L1 cells. The signal generated by IGF-1 receptor requires the assistance of lipid rafts/caveolae on plasma membrane to transmit into the cell and activates different signal pathways, which lead to adipocyte differentiation and mitotic clonal expansion, respectively.It has been reported that β-methylcyclodextrin treatment does not inhibit insulin receptor-induced ERK1 and -2 activation in primary rat adipocytes (12Parpal S. Karlsson M. Thorn H. Stralfors P. J. Biol. Chem. 2001; 276: 9670-9678Google Scholar). We observed similar results in 3T3-L1 adipocytes (results not shown). However, in 3T3-L1 preadipocytes, β-methylcyclodextrin treatment dramatically inhibited IGF-1 receptor-induced ERK1 and -2 activation (Fig. 7). It should be noted that 3T3-L1 adipocytes contain more cholesterol than preadipocytes, and under the same β-methylcyclodextrin treatment, less caveolin-1 was displaced from the low density centrifugation fractions in 3T3-L1 in adipocytes (results not shown). Thus, it is likely that lipid rafts/caveolae in adipocytes were not disrupted by β-methylcyclodextrin treatment as completely as in preadipocytes. Although IGF-1 receptor may employ a different signal pathway from insulin receptor to activate ERK1 and -2, we believe that the discrepancy between 3T3-L1 adipocyte and preadipocyte in the activation of ERK is more likely due to the incomplete disruption of lipid rafts/caveolae in adipocyte.Based on our present studies, only the downstream signal transduction of the receptor required lipid rafts/caveolae (Fig. 7). A possible function of lipid rafts/caveolae in IGF-1 receptor signaling is to recruit intracellular signal molecules for the receptor. Interestingly, studies of the phosphorylation of c-Crk, an endogenous IGF-1 receptor tyrosine kinase substrate, suggest that physical contact between IGF-1 receptor and c-Crk is essential for the phosphorylation of the substrate by the receptor kinase (54Koval A.-P. Blakesley V.-A. Roberts Jr., C.-T. Zick Y. LeRoith D. Biochem. J. 1998; 330: 923-932Google Scholar, 55Anafi M. Rosen M.-K. Gish G.-D. Kay L.-E. Pawson T. J. Biol. Chem. 1996; 271: 21365-21374Google Scholar). IGF-1 receptor tyrosine kinase can only phosphorylate c-Crk that has bound to the receptor through its Src homology 2 domain. c-Crk protein with the Src homology 2 domain deleted is not phosphorylated by IGF-1 receptor tyrosine kinase. Recruitment of signal molecules by lipid rafts/caveolae not only brings the downstream signal molecules into the receptor but also allows the receptor to be in close contact with signal molecules of many signal pathways for which lipid rafts/caveolae act like a signaling hub.Recent studies from caveolin-1 knockout mice indicate that the caveolin-1-deficient mice show some abnormalities in adiposity (56Razani 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: 8635-8647Google Scholar). The younger caveolin-1-deficient mice have a relatively intact adipocyte tissue except in a few places like in mammary gland and hypodermal fat layers and show similar body weight to their wild type littermates. However, the older caveolin-1-deficient mice have much smaller body sizes than their normal cohorts. This reduced body weight in older caveolin-1-deficient mice is due to reduced adiposity. The Cav-1 knockout mice also appear to be resistant to diet-induced obesity. These results indicate a relatively normal fetal development of adipose tissue in caveolin-1-deficient mice but a problem in adulthood adipose tissue metabolism. In our present studies, lipid rafts/caveolae appeared to be required for adipocyte differentiation induction. Therefore, the caveolaeless knockout mice are probably having problem in adulthood adipocyte differentiation.Adulthood adipocyte differentiation has more and more been considered as one of the leading causes in obesity, especially in hyperplastic obesity (28Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Google Scholar, 45Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Google Scholar). New adipocytes can differentiated from the residual preadipocytes in adipose tissue throughout the life span. The phenotype of caveolin-1-deficient mice suggests that the lipid rafts/caveolae-dependent adipocyte differentiation mechanism in 3T3-L1 cells might more closely resemble adipocyte differentiation in adult animal rather than in embryonic development. Further studies are needed to verify this hypothesis. Taken together with the strong evidence from Cav-1 knockout mice, our present study has established the role of lipid rafts/caveolae in the adipocyte differentiation process. Lipid rafts are plasma membrane microdomains, principally composed of cholesterol and sphingolipids, which form liquid-ordered domains of decreased membrane fluidity (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 5Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 6van Meer G. Science. 2002; 296: 855-857Google Scholar). With integration of caveolins into lipid rafts, these microdomains will form caveolae, which are flask-shaped vesicular invaginations in the plasma membrane (2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar). Caveolae are a specific form of lipid rafts and are now considered to be broader than just vesicular membrane invaginations (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar). The long saturated lipid tails of sphingolipids impart the lipid rafts a high degree of order further stabilized by interacting cholesterol. This property leads a light buoyant density on sucrose density gradient centrifugation. Cholesterol is an essential component in lipid rafts/caveolae. In caveolae, cholesterol binds directly to caveolins and facilitates the integration of caveolins into membrane (8Murata M. Peranen J. Schreiner R. Weiland F. Kurzchalia T. Simons K. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10339-10343Google Scholar, 9Li S. Song K.S. Lisanti M.P. J. Biol. Chem. 1996; 271: 568-573Google Scholar). Depletion of cellular cholesterol with cholesterol-binding reagents, such as methylcyclodextrin or filipin, will remove cholesterol from lipid rafts/caveolae, dissemble the striated caveolin coats, and eventually lead to the disruption of both lipid rafts and caveolae (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar,10Chang W.J. Rothberg K.G. Kamen B.A. Anderson R.G.W. J. Cell Biol. 1992; 118: 63-69Google Scholar, 11Fielding C.J. Fielding P.E. Biochim. Biophys. Acta. 2000; 1529: 210-222Google Scholar, 12Parpal S. Karlsson M. Thorn H. Stralfors P. J. Biol. Chem. 2001; 276: 9670-9678Google Scholar, 13Hailstones D. Sleer L.S. Parton R.G. Stanley K.K. J. Lipid Res. 1998; 39: 369-379Google Scholar, 14Furuchi T. Anderson R.G.W. J. Biol. Chem. 1998; 273: 21099-21104Google Scholar, 15Rothberg K.G. Heuser J.E. Donzell W.C. Ying Y.S. Glenney J.R. Anderson R.G.W. Cell. 1992; 68: 673-682Google Scholar). During recent years, more and more reports confirmed that many signaling molecules are found to be enriched in lipid rafts/caveolae, which serve as platforms and play an important role in regulating signal cascade (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar, 16Okamoto T. Schlegel A. Scherer P.E. Lisanti M.P. J. Biol. Chem. 1998; 273: 5419-5422Google Scholar, 17Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Google Scholar). Signal molecules, such as heterotrimeric G-proteins (18Li S. Okamoto T. Chun M. Sargiacomo M. Casanova J.E. Hansen S.H. Nishimoto I. Lisanti M.P. J. Biol. Chem. 1995; 270: 15693-15701Google Scholar), protein kinase C (19Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.L. Hermanoski- Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Google Scholar, 20Smart E.J. Ying Y. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Google Scholar), Shc (21Liu P. Ying Y.S. Ko Y.G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Google Scholar), SOS (22Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Google Scholar), Raf1 (22Mineo C. James G.L. Smart E.J. Anderson R.G.W. J. Biol. Chem. 1996; 271: 11930-11935Google Scholar, 23Mineo C. Anderson R.G.W. White M.A. J. Biol. Chem. 1997; 272: 10345-10348Google Scholar), and Src family tyrosine kinases (19Lisanti M.P. Scherer P.E. Vidugiriene J. Tang Z.L. Hermanoski- Vosatka A. Tu Y.H. Cook R.F. Sargiacomo M. J. Cell Biol. 1994; 126: 111-126Google Scholar, 24Sargiacomo M. Sudol M. Tang Z.-L. Lisanti M.P. J. Cell Biol. 1993; 122: 789-807Google Scholar, 25Shenoy-Scaria A.M. Dietzen D.J. Kwong J. Link D.C. Lublin D.M. J. Cell Biol. 1994; 126: 353-363Google Scholar, 26Robbins S.M. Quintrell N.A. Bishop M.J. Mol. Cell. Biol. 1995; 15: 3507-3515Google Scholar), are recruited into caveolae by caveolins, which, through the scaffolding domain, interact with the caveolin-binding motifs in these signal molecules (17Couet J. Li S. Okamoto T. Ikezu T. Lisanti M.P. J. Biol. Chem. 1997; 272: 6525-6533Google Scholar). These clusters of signal molecules can form “preassembled signaling complexes” on the plasma membrane. In addition, many growth factor receptors (epidermal growth factor receptor, platelet-derived growth factor receptor, insulin receptor, etc.) (12Parpal S. Karlsson M. Thorn H. Stralfors P. J. Biol. Chem. 2001; 276: 9670-9678Google Scholar, 20Smart E.J. Ying Y. Mineo C. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 10104-10108Google Scholar, 21Liu P. Ying Y.S. Ko Y.G. Anderson R.G.W. J. Biol. Chem. 1996; 271: 10299-10303Google Scholar, 27Gustavsson J. Parpal S. Karlsson M. Ramsing C. Thorn H. Borg M. Lindroth M. Peterson K.H. Magnusson K. Stralfors P. FASEB J. 1999; 13: 1961-1971Google Scholar, 29Liu P. Ying Y.S. Anderson R.G.W. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 13666-13670Google Scholar) are found to be located in lipid rafts/caveolae. Thus, the enrichment of receptors and signal molecules in lipid rafts/caveolae enables them to be in close contact with each other and makes lipid rafts/caveolae the gateways for signals entering into the cells. Lipid rafts/caveolae are indicated to be important for insulin receptor signaling (30Czech M.P. Nature. 2000; 407: 147-148Google Scholar, 31Bickel P.E. Am. J. Physiol. 2002; 282: E1-E10Google Scholar). Insulin receptors are found to be located in caveolae of adipocyte plasma membrane (27Gustavsson J. Parpal S. Karlsson M. Ramsing C. Thorn H. Borg M. Lindroth M. Peterson K.H. Magnusson K. Stralfors P. FASEB J. 1999; 13: 1961-1971Google Scholar), and many signal molecules involved in insulin receptor signal cascade are also found to be enriched in caveolin-enriched plasma membrane domain (32Smith R.M. Harada S. Smith J.A. Zhang S. Jarett L. Cell. Signalling. 1998; 10: 355-362Google Scholar). In 3T3-L1 adipocytes, caveolin-1 is phosphorylated by insulin receptor (33Kimura A. Mora S. Shigematsu S. Pessin J.E. Saltiel A.R. J. Biol. Chem. 2002; 277: 30153-30158Google Scholar, 34Mastick C.C. Saltiel A.R. J. Biol. Chem. 1997; 272: 20706-20714Google Scholar) and is an activator of insulin receptor signaling (35Yamamoto M. Toya Y. Schwencke C. Lisanti M.P. Meyers Jr., M.G. Ishikawa Y. J. Biol. Chem. 1998; 273: 26962-26968Google Scholar). In addition, caveolin-enriched lipid raft microdomains and lipid rafts are required for insulin signaling and Glut4 translocation (36Karlsson M. Thorn H. Parpal S. Stralfors P. Gustavsson J. FASEB J. 2002; 16: 249-251Google Scholar, 37Baumann C.A. Ribon V. Kanzaki M. Thurmond D.C. Mora S. Shigematsu S. Bickel P.E. Pessin J.E. Saltiel A.R. Nature. 2000; 407: 202-207Google Scholar, 38Chiang S. Baumann C.A. Kanzaki M. Thurmond D.C. Watson R.T. Neudauer C.L. Macara I.G. Pessin J.E. Saltiel A.R. Nature. 2001; 410: 944-948Google Scholar, 39Watson R.T. Shigematsu S. Chiang S. Mora S. Kanzaki M. Macara I.G. Saltiel A.R. Pessin J.E. J. Cell Biol. 2001; 154: 829-840Google Scholar). These results provide compelling evidence that lipid rafts/caveolae are essential for insulin receptor signaling. IGF-11 receptor tyrosine kinase signaling (along with glucocorticoid and cAMP signaling) is required for 3T3-L1 preadipocyte differentiation induction (40Smith P.J. Wise L.S. Berkowitz R. Wan C. Rubin C.S. J. Biol. Chem. 1988; 263: 9402-9408Google Scholar, 41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar, 42Mackall J.C. Student A.K. Polakis S.E. Lane M.D. J. Biol. Chem. 1976; 251: 6462-6464Google Scholar, 43Rosen O.M. Smith C.J. Hirsch A. Lai E. Rubin C.S. Rec. Prog. Horm. Res. 1979; 35: 477-499Google Scholar, 44Student A.K. Hsu R.Y. Lane M.D. J. Biol. Chem. 1980; 255: 4745-4750Google Scholar, 45Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Google Scholar). High level insulin or IGF-1 at physiological concentration activates the IGF-1 receptor on the plasma membrane, leading to the initiation of the differentiation program (40Smith P.J. Wise L.S. Berkowitz R. Wan C. Rubin C.S. J. Biol. Chem. 1988; 263: 9402-9408Google Scholar, 41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar). Two events occur after the activation of IGF-1 receptor in 3T3-L1 preadipocytes: mitotic clonal expansion and adipocyte differentiation. Previously, we have identified that these two events are both activated by the IGF-1 receptor signaling and can be separately blocked without affecting the other (41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar, 46Qiu Z. Wei Y. Chen N. Jiang M. Wu J. Liao K. J. Biol. Chem. 2001; 276: 11988-11995Google Scholar, 47Liao K. Lane M.D. J. Biol. Chem. 1995; 270: 12123-12132Google Scholar). The mitotic clonal expansion is activated by IGF-1 receptor through a signal pathway involving the activation of ERK1 and -2, whereas adipocyte differentiation is initiated through the reversible tyrosine phosphorylation of the adapter protein c-Crk by IGF-1 receptor tyrosine kinase and tyrosine phosphatase. These results suggest that IGF-1 receptor activates two separate signal pathways in 3T3-L1 preadipocytes simultaneously. Although some of the cellular functions for IGF-1 receptor and insulin receptor are different, structural and signaling similarities between these two receptors have long been recognized. Recently, it has been reported that IGF-1 may also induce caveolin-1 tyrosine phosphorylation and its translocation in the lipid rafts (48Maggi D. Biedi C. Segat D. Barbero D. Panetta D. Cordera R. Biochem. Biophys. Res. Commun. 2002; 295: 1085-1089Google Scholar). Since in 3T3-L1 cells IGF-1 receptor activates more than one signal pathway at the same time, it is likely that IGF-1 receptor on plasma membrane interacts or cross-talks with several signaling pathways. With their preassembled signaling complexes on the intracellular side, lipid rafts/caveolae provide the structural foundation for simultaneous activation or cross-talking of multiple signal pathways by IGF-1 receptor. In the present study, we reported that IGF-1 receptor was located in lipid rafts/caveolae in 3T3-L1 preadipocyte and adipocyte. The integrity of lipid rafts/caveolae was essential for IGF-1 receptor signal transduction during 3T3-L1 preadipocyte differentiation induction. Disruption of lipid rafts/caveolae by cholesterol depletion effectively blocked the downstream signaling of IGF-1 receptor but not IGF-1 receptor activation itself. DISCUSSIONIn many ways, caveolae are lipid rafts enriched with structural protein caveolins, which are the defining protein components in caveolae. Although caveolins have three types, general types caveolin-1 and -2 and muscle-specific caveolin-3 (7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar), cells completely lack caveolae in caveolin-1 knockout mice (53Drab 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: 2449-2452Google Scholar). This result from caveolin-1-deficient mice has demonstrated the importance of caveolin-1 in the formation of caveolae and provided support for using caveolin-1 as an indicator for caveolae. By using immunofluorescence staining (Fig. 1), sucrose density gradient centrifugation (Figs. 2 and 3), and co-immunoprecipitation (Fig. 4), we have identified that IGF-1 receptor was located in lipid rafts/caveolae in 3T3-L1 cells. To provide further support, we have also shown that IGF-1 receptor and insulin receptor, which has been indicated to be located in caveolae of adipocyte plasma membrane (26Robbins S.M. Quintrell N.A. Bishop M.J. Mol. Cell. Biol. 1995; 15: 3507-3515Google Scholar, 51Vainio S. Heino S. Mansson J.E. Fredman P. Kuismanen E. Vaarala O. Ikonen E. EMBO Rep. 2002; 3: 95-100Google Scholar), were associated with the same membrane structures in 3T3-L1 adipocytes (Fig. 3 B). These results provided strong evidence that IGF-1 was located in lipid rafts/caveolae in 3T3-L1 preadipocytes and adipocytes.It was consistently observed that, besides in caveolae, IGF-1 receptor also appeared to be in the membrane structure slightly lighter than caveolae. The peak of IGF-1 receptor and insulin receptor was in fractions 6 and 7, whereas the peak of caveolin was in fractions 7 and 8 (Figs. 2 and 3 B). However, in high level insulin-stimulated adipocytes, IGF-1 receptor, insulin receptor, and caveolin were better correlated in density gradient separation (Fig.3 B). These results were observed in several independently repeated experiments (results not shown). Thus, it is likely that IGF-1 receptor was also located in the caveolin-free lipid rafts around the caveolae and might further converge into caveolae upon ligand stimulation. Currently, we are investigating this translocation induced by ligand.Although lipid rafts and caveolae are important in signal transduction (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 5Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 6van Meer G. Science. 2002; 296: 855-857Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar), their roles in IGF-1 receptor cellular signaling are not fully understood. The identification of IGF-1 receptor in lipid rafts/caveolae provided us an opportunity to study the role of lipid rafts/caveolae in IGF-1 signaling. In 3T3-L1 preadipocytes, IGF-1 receptor signal is essential for inducing two cellular responses: adipocyte differentiation and mitotic clonal expansion. However, these two cellular responses can be separately blocked by inhibitors without affecting the other (41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar, 46Qiu Z. Wei Y. Chen N. Jiang M. Wu J. Liao K. J. Biol. Chem. 2001; 276: 11988-11995Google Scholar). PD98059, an inhibitor of MEK-1, blocked mitotic clonal expansion but not adipocyte differentiation, whereas vanadate, a protein-tyrosine phosphatase inhibitor, only blocked adipocyte differentiation. Since lipid rafts/caveolae are on the plasma membrane, which is on the top of the IGF-1 receptor signal cascade, disruption at the level of lipid rafts/caveolae will more likely block all of the cellular responses induced by IGF-1 receptor signaling. Our results clearly supported this hypothesis. Disruption of lipid rafts/caveolae by cholesterol-binding reagents led to the blockade of both cellular responses simultaneously (Figs. 5 and 6). Taken together, these results composed a hierarchy of IGF-1 receptor signaling system in 3T3-L1 cells. The signal generated by IGF-1 receptor requires the assistance of lipid rafts/caveolae on plasma membrane to transmit into the cell and activates different signal pathways, which lead to adipocyte differentiation and mitotic clonal expansion, respectively.It has been reported that β-methylcyclodextrin treatment does not inhibit insulin receptor-induced ERK1 and -2 activation in primary rat adipocytes (12Parpal S. Karlsson M. Thorn H. Stralfors P. J. Biol. Chem. 2001; 276: 9670-9678Google Scholar). We observed similar results in 3T3-L1 adipocytes (results not shown). However, in 3T3-L1 preadipocytes, β-methylcyclodextrin treatment dramatically inhibited IGF-1 receptor-induced ERK1 and -2 activation (Fig. 7). It should be noted that 3T3-L1 adipocytes contain more cholesterol than preadipocytes, and under the same β-methylcyclodextrin treatment, less caveolin-1 was displaced from the low density centrifugation fractions in 3T3-L1 in adipocytes (results not shown). Thus, it is likely that lipid rafts/caveolae in adipocytes were not disrupted by β-methylcyclodextrin treatment as completely as in preadipocytes. Although IGF-1 receptor may employ a different signal pathway from insulin receptor to activate ERK1 and -2, we believe that the discrepancy between 3T3-L1 adipocyte and preadipocyte in the activation of ERK is more likely due to the incomplete disruption of lipid rafts/caveolae in adipocyte.Based on our present studies, only the downstream signal transduction of the receptor required lipid rafts/caveolae (Fig. 7). A possible function of lipid rafts/caveolae in IGF-1 receptor signaling is to recruit intracellular signal molecules for the receptor. Interestingly, studies of the phosphorylation of c-Crk, an endogenous IGF-1 receptor tyrosine kinase substrate, suggest that physical contact between IGF-1 receptor and c-Crk is essential for the phosphorylation of the substrate by the receptor kinase (54Koval A.-P. Blakesley V.-A. Roberts Jr., C.-T. Zick Y. LeRoith D. Biochem. J. 1998; 330: 923-932Google Scholar, 55Anafi M. Rosen M.-K. Gish G.-D. Kay L.-E. Pawson T. J. Biol. Chem. 1996; 271: 21365-21374Google Scholar). IGF-1 receptor tyrosine kinase can only phosphorylate c-Crk that has bound to the receptor through its Src homology 2 domain. c-Crk protein with the Src homology 2 domain deleted is not phosphorylated by IGF-1 receptor tyrosine kinase. Recruitment of signal molecules by lipid rafts/caveolae not only brings the downstream signal molecules into the receptor but also allows the receptor to be in close contact with signal molecules of many signal pathways for which lipid rafts/caveolae act like a signaling hub.Recent studies from caveolin-1 knockout mice indicate that the caveolin-1-deficient mice show some abnormalities in adiposity (56Razani 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: 8635-8647Google Scholar). The younger caveolin-1-deficient mice have a relatively intact adipocyte tissue except in a few places like in mammary gland and hypodermal fat layers and show similar body weight to their wild type littermates. However, the older caveolin-1-deficient mice have much smaller body sizes than their normal cohorts. This reduced body weight in older caveolin-1-deficient mice is due to reduced adiposity. The Cav-1 knockout mice also appear to be resistant to diet-induced obesity. These results indicate a relatively normal fetal development of adipose tissue in caveolin-1-deficient mice but a problem in adulthood adipose tissue metabolism. In our present studies, lipid rafts/caveolae appeared to be required for adipocyte differentiation induction. Therefore, the caveolaeless knockout mice are probably having problem in adulthood adipocyte differentiation.Adulthood adipocyte differentiation has more and more been considered as one of the leading causes in obesity, especially in hyperplastic obesity (28Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Google Scholar, 45Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Google Scholar). New adipocytes can differentiated from the residual preadipocytes in adipose tissue throughout the life span. The phenotype of caveolin-1-deficient mice suggests that the lipid rafts/caveolae-dependent adipocyte differentiation mechanism in 3T3-L1 cells might more closely resemble adipocyte differentiation in adult animal rather than in embryonic development. Further studies are needed to verify this hypothesis. Taken together with the strong evidence from Cav-1 knockout mice, our present study has established the role of lipid rafts/caveolae in the adipocyte differentiation process. In many ways, caveolae are lipid rafts enriched with structural protein caveolins, which are the defining protein components in caveolae. Although caveolins have three types, general types caveolin-1 and -2 and muscle-specific caveolin-3 (7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar), cells completely lack caveolae in caveolin-1 knockout mice (53Drab 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: 2449-2452Google Scholar). This result from caveolin-1-deficient mice has demonstrated the importance of caveolin-1 in the formation of caveolae and provided support for using caveolin-1 as an indicator for caveolae. By using immunofluorescence staining (Fig. 1), sucrose density gradient centrifugation (Figs. 2 and 3), and co-immunoprecipitation (Fig. 4), we have identified that IGF-1 receptor was located in lipid rafts/caveolae in 3T3-L1 cells. To provide further support, we have also shown that IGF-1 receptor and insulin receptor, which has been indicated to be located in caveolae of adipocyte plasma membrane (26Robbins S.M. Quintrell N.A. Bishop M.J. Mol. Cell. Biol. 1995; 15: 3507-3515Google Scholar, 51Vainio S. Heino S. Mansson J.E. Fredman P. Kuismanen E. Vaarala O. Ikonen E. EMBO Rep. 2002; 3: 95-100Google Scholar), were associated with the same membrane structures in 3T3-L1 adipocytes (Fig. 3 B). These results provided strong evidence that IGF-1 was located in lipid rafts/caveolae in 3T3-L1 preadipocytes and adipocytes. It was consistently observed that, besides in caveolae, IGF-1 receptor also appeared to be in the membrane structure slightly lighter than caveolae. The peak of IGF-1 receptor and insulin receptor was in fractions 6 and 7, whereas the peak of caveolin was in fractions 7 and 8 (Figs. 2 and 3 B). However, in high level insulin-stimulated adipocytes, IGF-1 receptor, insulin receptor, and caveolin were better correlated in density gradient separation (Fig.3 B). These results were observed in several independently repeated experiments (results not shown). Thus, it is likely that IGF-1 receptor was also located in the caveolin-free lipid rafts around the caveolae and might further converge into caveolae upon ligand stimulation. Currently, we are investigating this translocation induced by ligand. Although lipid rafts and caveolae are important in signal transduction (1Brown D.A. London E. Annu. Rev. Cell Dev. Biol. 1998; 14: 111-136Google Scholar, 2Galbiati F. Razani B. Lisanti M. Cell. 2001; 106: 403-411Google Scholar, 3Simons K. Toomre D. Nat. Rev. Mol. Cell. Biol. 2000; 1: 31-39Google Scholar, 4Smart E.J. Graf G.A. McNiven M.A. Sessa W.C. Engelman J.A. Scherer P.E. Okamoto T. Lisanti M.P. Mol. Cell. Biol. 1999; 19: 7289-7304Google Scholar, 5Simons K. Ikonen E. Nature. 1997; 387: 569-572Google Scholar, 6van Meer G. Science. 2002; 296: 855-857Google Scholar, 7Anderson R.G.W. Annu. Rev. Biochem. 1998; 67: 199-225Google Scholar), their roles in IGF-1 receptor cellular signaling are not fully understood. The identification of IGF-1 receptor in lipid rafts/caveolae provided us an opportunity to study the role of lipid rafts/caveolae in IGF-1 signaling. In 3T3-L1 preadipocytes, IGF-1 receptor signal is essential for inducing two cellular responses: adipocyte differentiation and mitotic clonal expansion. However, these two cellular responses can be separately blocked by inhibitors without affecting the other (41Jin S. Zhai B. Qiu Z. Wu J. Lane M.D. Liao K. J. Biol. Chem. 2000; 275: 34344-34352Google Scholar, 46Qiu Z. Wei Y. Chen N. Jiang M. Wu J. Liao K. J. Biol. Chem. 2001; 276: 11988-11995Google Scholar). PD98059, an inhibitor of MEK-1, blocked mitotic clonal expansion but not adipocyte differentiation, whereas vanadate, a protein-tyrosine phosphatase inhibitor, only blocked adipocyte differentiation. Since lipid rafts/caveolae are on the plasma membrane, which is on the top of the IGF-1 receptor signal cascade, disruption at the level of lipid rafts/caveolae will more likely block all of the cellular responses induced by IGF-1 receptor signaling. Our results clearly supported this hypothesis. Disruption of lipid rafts/caveolae by cholesterol-binding reagents led to the blockade of both cellular responses simultaneously (Figs. 5 and 6). Taken together, these results composed a hierarchy of IGF-1 receptor signaling system in 3T3-L1 cells. The signal generated by IGF-1 receptor requires the assistance of lipid rafts/caveolae on plasma membrane to transmit into the cell and activates different signal pathways, which lead to adipocyte differentiation and mitotic clonal expansion, respectively. It has been reported that β-methylcyclodextrin treatment does not inhibit insulin receptor-induced ERK1 and -2 activation in primary rat adipocytes (12Parpal S. Karlsson M. Thorn H. Stralfors P. J. Biol. Chem. 2001; 276: 9670-9678Google Scholar). We observed similar results in 3T3-L1 adipocytes (results not shown). However, in 3T3-L1 preadipocytes, β-methylcyclodextrin treatment dramatically inhibited IGF-1 receptor-induced ERK1 and -2 activation (Fig. 7). It should be noted that 3T3-L1 adipocytes contain more cholesterol than preadipocytes, and under the same β-methylcyclodextrin treatment, less caveolin-1 was displaced from the low density centrifugation fractions in 3T3-L1 in adipocytes (results not shown). Thus, it is likely that lipid rafts/caveolae in adipocytes were not disrupted by β-methylcyclodextrin treatment as completely as in preadipocytes. Although IGF-1 receptor may employ a different signal pathway from insulin receptor to activate ERK1 and -2, we believe that the discrepancy between 3T3-L1 adipocyte and preadipocyte in the activation of ERK is more likely due to the incomplete disruption of lipid rafts/caveolae in adipocyte. Based on our present studies, only the downstream signal transduction of the receptor required lipid rafts/caveolae (Fig. 7). A possible function of lipid rafts/caveolae in IGF-1 receptor signaling is to recruit intracellular signal molecules for the receptor. Interestingly, studies of the phosphorylation of c-Crk, an endogenous IGF-1 receptor tyrosine kinase substrate, suggest that physical contact between IGF-1 receptor and c-Crk is essential for the phosphorylation of the substrate by the receptor kinase (54Koval A.-P. Blakesley V.-A. Roberts Jr., C.-T. Zick Y. LeRoith D. Biochem. J. 1998; 330: 923-932Google Scholar, 55Anafi M. Rosen M.-K. Gish G.-D. Kay L.-E. Pawson T. J. Biol. Chem. 1996; 271: 21365-21374Google Scholar). IGF-1 receptor tyrosine kinase can only phosphorylate c-Crk that has bound to the receptor through its Src homology 2 domain. c-Crk protein with the Src homology 2 domain deleted is not phosphorylated by IGF-1 receptor tyrosine kinase. Recruitment of signal molecules by lipid rafts/caveolae not only brings the downstream signal molecules into the receptor but also allows the receptor to be in close contact with signal molecules of many signal pathways for which lipid rafts/caveolae act like a signaling hub. Recent studies from caveolin-1 knockout mice indicate that the caveolin-1-deficient mice show some abnormalities in adiposity (56Razani 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: 8635-8647Google Scholar). The younger caveolin-1-deficient mice have a relatively intact adipocyte tissue except in a few places like in mammary gland and hypodermal fat layers and show similar body weight to their wild type littermates. However, the older caveolin-1-deficient mice have much smaller body sizes than their normal cohorts. This reduced body weight in older caveolin-1-deficient mice is due to reduced adiposity. The Cav-1 knockout mice also appear to be resistant to diet-induced obesity. These results indicate a relatively normal fetal development of adipose tissue in caveolin-1-deficient mice but a problem in adulthood adipose tissue metabolism. In our present studies, lipid rafts/caveolae appeared to be required for adipocyte differentiation induction. Therefore, the caveolaeless knockout mice are probably having problem in adulthood adipocyte differentiation. Adulthood adipocyte differentiation has more and more been considered as one of the leading causes in obesity, especially in hyperplastic obesity (28Spiegelman B.M. Flier J.S. Cell. 1996; 87: 377-389Google Scholar, 45Gregoire F.M. Smas C.M. Sul H.S. Physiol. Rev. 1998; 78: 783-809Google Scholar). New adipocytes can differentiated from the residual preadipocytes in adipose tissue throughout the life span. The phenotype of caveolin-1-deficient mice suggests that the lipid rafts/caveolae-dependent adipocyte differentiation mechanism in 3T3-L1 cells might more closely resemble adipocyte differentiation in adult animal rather than in embryonic development. Further studies are needed to verify this hypothesis. Taken together with the strong evidence from Cav-1 knockout mice, our present study has established the role of lipid rafts/caveolae in the adipocyte differentiation process." @default.
W2023286320 created "2016-06-24" @default.
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W2023286320 title "Lipid Rafts/Caveolae Are Essential for Insulin-like Growth Factor-1 Receptor Signaling during 3T3-L1 Preadipocyte Differentiation Induction" @default.
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