Cholesterol depletion disrupts caveolae and insulin receptor signaling for metabolic control via insulin receptor substrate-1, but not for mitogen-activated protein kinase control.
Insulin exerts its cellular control through receptor binding in caveolae in plasmalemma of target cells (Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., and Strålfors, P. (1999) FASEB. J. 13, 1961-1971). We now report that a progressive cholesterol depletion of 3T3-L1 adipocytes with beta-cyclodextrin gradually destroyed caveolae structures and concomitantly attenuated insulin stimulation of glucose transport, in effect making cells insulin-resistant. Insulin access to or affinity for the insulin receptor on rat adipocytes was not affected as determined by (125)I-insulin binding. By immunoblotting of plasma membranes, total amount of insulin receptor and of caveolin remained unchanged. Receptor autophosphorylation in response to insulin was not affected by cholesterol depletion. Insulin treatment of isolated caveolae preparations increased autophosphorylation of receptor before and following cholesterol depletion. Insulin-increased tyrosine phosphorylation of an immediate downstream signal transducer, insulin receptor substrate-1, and activation of the further downstream protein kinase B were inhibited. In contrast, insulin signaling to mitogenic control as determined by control of the extracellular signal-related kinases 1/2, mitogen-activated protein kinase pathway was not affected. Insulin did not control Shc phosphorylation, and Shc did not control extracellular signal-related kinases 1/2, whereas cholesterol depletion constitutively phosphorylated Shc. In conclusion, caveolae are critical for propagating the insulin receptor signal to downstream targets and have the potential for sorting signal transduction for metabolic and mitogenic effects.
Parpal
S
S
Department of Cell Biology, Faculty of Health Sciences, Linköping University, S-58185 Linköping, Sweden.
Karlsson
M
M
Thorn
H
H
Strålfors
P
P
D016475:3T3 Cells; D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D017667:Adipocytes / Q000378:metabolism; D000818:Animals; D002273:Carcinogens; D021941:Caveolae / Q000378:metabolism*; D002454:Cell Differentiation / Q000187:drug effects; D002455:Cell Division / Q000187:drug effects; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002784:Cholesterol / Q000378:metabolism*; D003505:Cyclodextrins; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D005947:Glucose / Q000378:metabolism / Q000493:pharmacokinetics; D015151:Immunoblotting; D007150:Immunohistochemistry; D007328:Insulin / Q000378:metabolism / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D020935:MAP Kinase Signaling System; D051379:Mice; D008854:Microscopy, Electron; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation; D011485:Protein Binding; D017346:Protein Serine-Threonine Kinases; D011506:Proteins / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D011817:Rabbits; D051381:Rats; D017207:Rats, Sprague-Dawley; D011972:Receptor, Insulin / Q000378:metabolism*; D012694:Serine / Q000378:metabolism; D055765:Shc Signaling Adaptor Proteins; D015398:Signal Transduction / Q000187:drug effects; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D013912:Threonine / Q000378:metabolism; D013997:Time Factors; D014443:Tyrosine / Q000378:metabolism; D047392:beta-Cyclodextrins
D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D002273:Carcinogens; D003505:Cyclodextrins; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C526296:Irs1 protein, rat; D010750:Phosphoproteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D055765:Shc Signaling Adaptor Proteins; C528034:Shc1 protein, mouse; C528035:Shc1 protein, rat; D000071425:Src Homology 2 Domain-Containing, Transforming Protein 1; D047392:beta-Cyclodextrins; D013912:Threonine; D014443:Tyrosine; D012694:Serine; D002784:Cholesterol; D011972:Receptor, Insulin; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases; D005947:Glucose; C031215:betadex