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. 1994 Dec 1;127(5):1233–1243. doi: 10.1083/jcb.127.5.1233

Induction of caveolin during adipogenesis and association of GLUT4 with caveolin-rich vesicles

PMCID: PMC2120260  PMID: 7962086

Abstract

Caveolae, also termed plasmalemmal vesicles, are small, flask-shaped, non-clathrin-coated invaginations of the plasma membrane. Caveolin is a principal component of the filaments that make up the striated coat of caveolae. Using caveolin as a marker protein for the organelle, we found that adipose tissue is the single most abundant source of caveolae identified thus far. Caveolin mRNA and protein are strongly induced during differentiation of 3T3-L1 fibroblasts to adipocytes; during adipogenesis there is also a dramatic increase in the complexity of the protein composition of caveolin-rich membrane domains. About 10- 15% of the insulin-responsive glucose transporter GLUT4 is found in this caveolin-rich fraction, and immuno-isolated vesicles containing GLUT4 also contain caveolin. However, in non-stimulated adipocytes the majority of caveolin fractionates with the plasma membrane, while most GLUT4 associates with low-density microsomes. Upon addition of insulin to 3T3-L1 adipocytes, there is a significant increase in the amount of GLUT4 associated with caveolin-rich membrane domains, an increase in the amount of caveolin associated with the plasma membrane, and a decrease in the amount of caveolin associated with low-density microsomes. Caveolin does not undergo a change in phosphorylation upon stimulation of 3T3-L1 adipocytes with insulin. However, after treatment with insulin it is associated with a 32-kD phosphorylated protein. Caveolae thus may play an important role in the vesicular transport of GLUT4 to or from the plasma membrane. 3T3-L1 adipocytes offer an attractive system to study the function of caveolae in several cellular trafficking and signaling events.

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Selected References

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  1. Anderson R. G., Kamen B. A., Rothberg K. G., Lacey S. W. Potocytosis: sequestration and transport of small molecules by caveolae. Science. 1992 Jan 24;255(5043):410–411. doi: 10.1126/science.1310359. [DOI] [PubMed] [Google Scholar]
  2. Baldini G., Hohl T., Lin H. Y., Lodish H. F. Cloning of a Rab3 isotype predominantly expressed in adipocytes. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5049–5052. doi: 10.1073/pnas.89.11.5049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baldini G., Hohman R., Charron M. J., Lodish H. F. Insulin and nonhydrolyzable GTP analogs induce translocation of GLUT 4 to the plasma membrane in alpha-toxin-permeabilized rat adipose cells. J Biol Chem. 1991 Mar 5;266(7):4037–4040. [PubMed] [Google Scholar]
  4. Benito M., Porras A., Nebreda A. R., Santos E. Differentiation of 3T3-L1 fibroblasts to adipocytes induced by transfection of ras oncogenes. Science. 1991 Aug 2;253(5019):565–568. doi: 10.1126/science.1857988. [DOI] [PubMed] [Google Scholar]
  5. Brown D. A., Rose J. K. Sorting of GPI-anchored proteins to glycolipid-enriched membrane subdomains during transport to the apical cell surface. Cell. 1992 Feb 7;68(3):533–544. doi: 10.1016/0092-8674(92)90189-j. [DOI] [PubMed] [Google Scholar]
  6. Brown S. J., Gould G. W., Davies A., Baldwin S. A., Lienhard G. E., Gibbs E. M. Characterization of vesicles containing insulin-responsive intracellular glucose transporters isolated from 3T3-L1 adipocytes by an improved procedure. Biochim Biophys Acta. 1988 Oct 7;971(3):339–350. doi: 10.1016/0167-4889(88)90150-4. [DOI] [PubMed] [Google Scholar]
  7. Bruns R. R., Palade G. E. Studies on blood capillaries. I. General organization of blood capillaries in muscle. J Cell Biol. 1968 May;37(2):244–276. doi: 10.1083/jcb.37.2.244. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cain C. C., Trimble W. S., Lienhard G. E. Members of the VAMP family of synaptic vesicle proteins are components of glucose transporter-containing vesicles from rat adipocytes. J Biol Chem. 1992 Jun 15;267(17):11681–11684. [PubMed] [Google Scholar]
  9. Chamberlain J. P. Fluorographic detection of radioactivity in polyacrylamide gels with the water-soluble fluor, sodium salicylate. Anal Biochem. 1979 Sep 15;98(1):132–135. doi: 10.1016/0003-2697(79)90716-4. [DOI] [PubMed] [Google Scholar]
  10. Chang W. J., Ying Y. S., Rothberg K. G., Hooper N. M., Turner A. J., Gambliel H. A., De Gunzburg J., Mumby S. M., Gilman A. G., Anderson R. G. Purification and characterization of smooth muscle cell caveolae. J Cell Biol. 1994 Jul;126(1):127–138. doi: 10.1083/jcb.126.1.127. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Charron M. J., Brosius F. C., 3rd, Alper S. L., Lodish H. F. A glucose transport protein expressed predominately in insulin-responsive tissues. Proc Natl Acad Sci U S A. 1989 Apr;86(8):2535–2539. doi: 10.1073/pnas.86.8.2535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Chen J. W., Murphy T. L., Willingham M. C., Pastan I., August J. T. Identification of two lysosomal membrane glycoproteins. J Cell Biol. 1985 Jul;101(1):85–95. doi: 10.1083/jcb.101.1.85. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Corvera S., Graver D. F., Smith R. M. Insulin increases the cell surface concentration of alpha 2-macroglobulin receptors in 3T3-L1 adipocytes. Altered transit of the receptor among intracellular endocytic compartments. J Biol Chem. 1989 Jun 15;264(17):10133–10138. [PubMed] [Google Scholar]
  14. Czech M. P., Buxton J. M. Insulin action on the internalization of the GLUT4 glucose transporter in isolated rat adipocytes. J Biol Chem. 1993 May 5;268(13):9187–9190. [PubMed] [Google Scholar]
  15. Davis R. J., Corvera S., Czech M. P. Insulin stimulates cellular iron uptake and causes the redistribution of intracellular transferrin receptors to the plasma membrane. J Biol Chem. 1986 Jul 5;261(19):8708–8711. [PubMed] [Google Scholar]
  16. Fan J. Y., Carpentier J. L., van Obberghen E., Grunfeld C., Gorden P., Orci L. Morphological changes of the 3T3-L1 fibroblast plasma membrane upon differentiation to the adipocyte form. J Cell Sci. 1983 May;61:219–230. doi: 10.1242/jcs.61.1.219. [DOI] [PubMed] [Google Scholar]
  17. Frost S. C., Lane M. D. Evidence for the involvement of vicinal sulfhydryl groups in insulin-activated hexose transport by 3T3-L1 adipocytes. J Biol Chem. 1985 Mar 10;260(5):2646–2652. [PubMed] [Google Scholar]
  18. Fujimoto T. Calcium pump of the plasma membrane is localized in caveolae. J Cell Biol. 1993 Mar;120(5):1147–1157. doi: 10.1083/jcb.120.5.1147. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Fujimoto T., Nakade S., Miyawaki A., Mikoshiba K., Ogawa K. Localization of inositol 1,4,5-trisphosphate receptor-like protein in plasmalemmal caveolae. J Cell Biol. 1992 Dec;119(6):1507–1513. doi: 10.1083/jcb.119.6.1507. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Garcia de Herreros A., Birnbaum M. J. The regulation by insulin of glucose transporter gene expression in 3T3 adipocytes. J Biol Chem. 1989 Jun 15;264(17):9885–9890. [PubMed] [Google Scholar]
  21. Glenney J. R., Jr The sequence of human caveolin reveals identity with VIP21, a component of transport vesicles. FEBS Lett. 1992 Dec 7;314(1):45–48. doi: 10.1016/0014-5793(92)81458-x. [DOI] [PubMed] [Google Scholar]
  22. Glenney J. R., Jr Tyrosine phosphorylation of a 22-kDa protein is correlated with transformation by Rous sarcoma virus. J Biol Chem. 1989 Dec 5;264(34):20163–20166. [PubMed] [Google Scholar]
  23. Goldberg R. I., Smith R. M., Jarett L. Insulin and alpha 2-macroglobulin-methylamine undergo endocytosis by different mechanisms in rat adipocytes: I. Comparison of cell surface events. J Cell Physiol. 1987 Nov;133(2):203–212. doi: 10.1002/jcp.1041330202. [DOI] [PubMed] [Google Scholar]
  24. Hare J. F., Holocher A. Solubility in non-ionic detergents distinguishes between slowly and rapidly degraded plasma membrane proteins. J Biol Chem. 1994 Feb 25;269(8):5981–5988. [PubMed] [Google Scholar]
  25. Holman G. D., Lo Leggio L., Cushman S. W. Insulin-stimulated GLUT4 glucose transporter recycling. A problem in membrane protein subcellular trafficking through multiple pools. J Biol Chem. 1994 Jul 1;269(26):17516–17524. [PubMed] [Google Scholar]
  26. Hunt C., Calderwood S. Characterization and sequence of a mouse hsp70 gene and its expression in mouse cell lines. Gene. 1990 Mar 15;87(2):199–204. doi: 10.1016/0378-1119(90)90302-8. [DOI] [PubMed] [Google Scholar]
  27. James D. E., Brown R., Navarro J., Pilch P. F. Insulin-regulatable tissues express a unique insulin-sensitive glucose transport protein. Nature. 1988 May 12;333(6169):183–185. doi: 10.1038/333183a0. [DOI] [PubMed] [Google Scholar]
  28. Jhun B. H., Rampal A. L., Liu H., Lachaal M., Jung C. Y. Effects of insulin on steady state kinetics of GLUT4 subcellular distribution in rat adipocytes. Evidence of constitutive GLUT4 recycling. J Biol Chem. 1992 Sep 5;267(25):17710–17715. [PubMed] [Google Scholar]
  29. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  30. Lisanti M. P., Scherer P. E., Tang Z., Sargiacomo M. Caveolae, caveolin and caveolin-rich membrane domains: a signalling hypothesis. Trends Cell Biol. 1994 Jul;4(7):231–235. doi: 10.1016/0962-8924(94)90114-7. [DOI] [PubMed] [Google Scholar]
  31. Lisanti M. P., Scherer P. E., Vidugiriene J., Tang Z., Hermanowski-Vosatka A., Tu Y. H., Cook R. F., Sargiacomo M. Characterization of caveolin-rich membrane domains isolated from an endothelial-rich source: implications for human disease. J Cell Biol. 1994 Jul;126(1):111–126. doi: 10.1083/jcb.126.1.111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Lodish H. F., Kong N. Cyclosporin A inhibits an initial step in folding of transferrin within the endoplasmic reticulum. J Biol Chem. 1991 Aug 15;266(23):14835–14838. [PubMed] [Google Scholar]
  33. Mackall J. C., Student A. K., Polakis S. E., Lane M. D. Induction of lipogenesis during differentiation in a "preadipocyte" cell line. J Biol Chem. 1976 Oct 25;251(20):6462–6464. [PubMed] [Google Scholar]
  34. Mastick C. C., Aebersold R., Lienhard G. E. Characterization of a major protein in GLUT4 vesicles. Concentration in the vesicles and insulin-stimulated translocation to the plasma membrane. J Biol Chem. 1994 Feb 25;269(8):6089–6092. [PubMed] [Google Scholar]
  35. Mayor S., Rothberg K. G., Maxfield F. R. Sequestration of GPI-anchored proteins in caveolae triggered by cross-linking. Science. 1994 Jun 24;264(5167):1948–1951. doi: 10.1126/science.7516582. [DOI] [PubMed] [Google Scholar]
  36. Merril C. R., Goldman D., Van Keuren M. L. Silver staining methods for polyacrylamide gel electrophoresis. Methods Enzymol. 1983;96:230–239. doi: 10.1016/s0076-6879(83)96021-4. [DOI] [PubMed] [Google Scholar]
  37. Montesano R., Roth J., Robert A., Orci L. Non-coated membrane invaginations are involved in binding and internalization of cholera and tetanus toxins. Nature. 1982 Apr 15;296(5858):651–653. doi: 10.1038/296651a0. [DOI] [PubMed] [Google Scholar]
  38. Payne G. S., Baker D., van Tuinen E., Schekman R. Protein transport to the vacuole and receptor-mediated endocytosis by clathrin heavy chain-deficient yeast. J Cell Biol. 1988 May;106(5):1453–1461. doi: 10.1083/jcb.106.5.1453. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Robinson L. J., Pang S., Harris D. S., Heuser J., James D. E. Translocation of the glucose transporter (GLUT4) to the cell surface in permeabilized 3T3-L1 adipocytes: effects of ATP insulin, and GTP gamma S and localization of GLUT4 to clathrin lattices. J Cell Biol. 1992 Jun;117(6):1181–1196. doi: 10.1083/jcb.117.6.1181. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Rothberg K. G., Heuser J. E., Donzell W. C., Ying Y. S., Glenney J. R., Anderson R. G. Caveolin, a protein component of caveolae membrane coats. Cell. 1992 Feb 21;68(4):673–682. doi: 10.1016/0092-8674(92)90143-z. [DOI] [PubMed] [Google Scholar]
  41. Rothberg K. G., Ying Y. S., Kolhouse J. F., Kamen B. A., Anderson R. G. The glycophospholipid-linked folate receptor internalizes folate without entering the clathrin-coated pit endocytic pathway. J Cell Biol. 1990 Mar;110(3):637–649. doi: 10.1083/jcb.110.3.637. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sargiacomo M., Sudol M., Tang Z., Lisanti M. P. Signal transducing molecules and glycosyl-phosphatidylinositol-linked proteins form a caveolin-rich insoluble complex in MDCK cells. J Cell Biol. 1993 Aug;122(4):789–807. doi: 10.1083/jcb.122.4.789. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Satoh S., Nishimura H., Clark A. E., Kozka I. J., Vannucci S. J., Simpson I. A., Quon M. J., Cushman S. W., Holman G. D. Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J Biol Chem. 1993 Aug 25;268(24):17820–17829. [PubMed] [Google Scholar]
  44. Simionescu N. Cellular aspects of transcapillary exchange. Physiol Rev. 1983 Oct;63(4):1536–1579. doi: 10.1152/physrev.1983.63.4.1536. [DOI] [PubMed] [Google Scholar]
  45. Simpson I. A., Cushman S. W. Hormonal regulation of mammalian glucose transport. Annu Rev Biochem. 1986;55:1059–1089. doi: 10.1146/annurev.bi.55.070186.005211. [DOI] [PubMed] [Google Scholar]
  46. Slot J. W., Geuze H. J., Gigengack S., James D. E., Lienhard G. E. Translocation of the glucose transporter GLUT4 in cardiac myocytes of the rat. Proc Natl Acad Sci U S A. 1991 Sep 1;88(17):7815–7819. doi: 10.1073/pnas.88.17.7815. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Slot J. W., Geuze H. J., Gigengack S., Lienhard G. E., James D. E. Immuno-localization of the insulin regulatable glucose transporter in brown adipose tissue of the rat. J Cell Biol. 1991 Apr;113(1):123–135. doi: 10.1083/jcb.113.1.123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Smith R. M., Charron M. J., Shah N., Lodish H. F., Jarett L. Immunoelectron microscopic demonstration of insulin-stimulated translocation of glucose transporters to the plasma membrane of isolated rat adipocytes and masking of the carboxyl-terminal epitope of intracellular GLUT4. Proc Natl Acad Sci U S A. 1991 Aug 1;88(15):6893–6897. doi: 10.1073/pnas.88.15.6893. [DOI] [PMC free article] [PubMed] [Google Scholar]
  49. Strosberg A. D. Structure/function relationship of proteins belonging to the family of receptors coupled to GTP-binding proteins. Eur J Biochem. 1991 Feb 26;196(1):1–10. doi: 10.1111/j.1432-1033.1991.tb15778.x. [DOI] [PubMed] [Google Scholar]
  50. Tanner L. I., Lienhard G. E. Localization of transferrin receptors and insulin-like growth factor II receptors in vesicles from 3T3-L1 adipocytes that contain intracellular glucose transporters. J Cell Biol. 1989 Apr;108(4):1537–1545. doi: 10.1083/jcb.108.4.1537. [DOI] [PMC free article] [PubMed] [Google Scholar]
  51. Towbin H., Staehelin T., Gordon J. Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci U S A. 1979 Sep;76(9):4350–4354. doi: 10.1073/pnas.76.9.4350. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Urayama O., Shutt H., Sweadner K. J. Identification of three isozyme proteins of the catalytic subunit of the Na,K-ATPase in rat brain. J Biol Chem. 1989 May 15;264(14):8271–8280. [PubMed] [Google Scholar]
  53. Walker P. S., Ramlal T., Donovan J. A., Doering T. P., Sandra A., Klip A., Pessin J. E. Insulin and glucose-dependent regulation of the glucose transport system in the rat L6 skeletal muscle cell line. J Biol Chem. 1989 Apr 15;264(11):6587–6595. [PubMed] [Google Scholar]
  54. Wardzala L. J., Simpson I. A., Rechler M. M., Cushman S. W. Potential mechanism of the stimulatory action of insulin on insulin-like growth factor II binding to the isolated rat adipose cell. Apparent redistribution of receptors cycling between a large intracellular pool and the plasma membrane. J Biol Chem. 1984 Jul 10;259(13):8378–8383. [PubMed] [Google Scholar]
  55. YAMADA E. The fine structure of the gall bladder epithelium of the mouse. J Biophys Biochem Cytol. 1955 Sep 25;1(5):445–458. doi: 10.1083/jcb.1.5.445. [DOI] [PMC free article] [PubMed] [Google Scholar]
  56. Yang J., Holman G. D. Comparison of GLUT4 and GLUT1 subcellular trafficking in basal and insulin-stimulated 3T3-L1 cells. J Biol Chem. 1993 Mar 5;268(7):4600–4603. [PubMed] [Google Scholar]
  57. Ying Y. S., Anderson R. G., Rothberg K. G. Each caveola contains multiple glycosyl-phosphatidylinositol-anchored membrane proteins. Cold Spring Harb Symp Quant Biol. 1992;57:593–604. doi: 10.1101/sqb.1992.057.01.065. [DOI] [PubMed] [Google Scholar]

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