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(The FASEB Journal. 1999;13:1961-1971.)
© 1999 FASEB

Localization of the insulin receptor in caveolae of adipocyte plasma membrane

JOHANNA GUSTAVSSON, SANTIAGO PARPAL, MARGARETA KARLSSON, CECILIA RAMSING, HANS THORN, MARIE BORG, MARGARETHA LINDROTH, KAJSA HOLMGREN PETERSON*, KARL-ERIC MAGNUSSON* and PETER STRåLFORS1

Department of Cell Biology and
* Department of Medical Microbiology, Faculty of Health Sciences, Linköping University, S-58185 Linköping, Sweden

1Correspondence: Department of Cell Biology, Faculty of Health Sciences, S-58185 Linkoping, Sweden. E-mail: peter.stralfors{at}mcb.liu.se


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The insulin receptor is a transmembrane protein of the plasma membrane, where it recognizes extracellular insulin and transmits signals into the cellular signaling network. We report that insulin receptors are localized and signal in caveolae microdomains of adipocyte plasma membrane. Immunogold electron microscopy and immunofluorescence microscopy show that insulin receptors are restricted to caveolae and are colocalized with caveolin over the plasma membrane. Insulin receptor was enriched in a caveolae-enriched fraction of plasma membrane. By extraction with ß-cyclodextrin or destruction with cholesterol oxidase, cholesterol reduction attenuated insulin receptor signaling to protein phosphorylation or glucose transport. Insulin signaling was regained by spontaneous recovery or by exogenous replenishment of cholesterol. ß-Cyclodextrin treatment caused a nearly complete annihilation of caveolae invaginations as examined by electron microscopy. This suggests that the receptor is dependent on the caveolae environment for signaling. Insulin stimulation of cells prior to isolation of caveolae or insulin stimulation of the isolated caveolae fraction increased tyrosine phosphorylation of the insulin receptor in caveolae, demonstrating that insulin receptors in caveolae are functional. Our results indicate that insulin receptors are localized to caveolae in the plasma membrane of adipocytes, are signaling in caveolae, and are dependent on caveolae for signaling.—Gustavsson, J., Parpal, S., Karlsson, M., Ramsing, C., Thorn, H., Borg, M., Lindroth, M., Peterson, K. H., Magnusson, K.-E., Strålfors, P. Localization of the insulin receptor in caveolae of adipocyte plasma membrane.


Key Words: cholesterol • signal transduction • insulin resistance • diabetes


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
WHEN INSULIN BINDS to its specific receptors in the plasma membrane of target cells, control signals are passed to the cellular signaling network for regulation of metabolic machinery and the genome of the cell (1 2 3) . Insulin binding leads to tyrosine-specific autophosphorylation of the receptor, which initiates intracellular signal transduction cascades. Signaling to mitogenic control has been explained by consecutive molecular interactions via insulin receptor substrate-1 (IRS-1), the GTP binding protein Ras, and the MAP-kinase associated phosphorylation (2 , 3) . The acute effects on intermediary metabolism are largely controlled through regulation of reversible phosphorylation of key metabolic enzymes. For instance, glycogen synthase phosphorylation may be controlled by insulin via activation of phosphatidylinositide 3-kinase (PI3-kinase) and PDK1 kinase cascade (4) . It has been established that uptake of glucose is controlled by the number of glucose transporters (insulin-regulated glucose transporter type 4, or GLUT4) in the plasma membrane (5 , 6) , but the molecular mechanisms for insulin receptor activation of GLUT4 translocation or target protein phosphorylation are yet not completely understood (7 , 8) .

Caveolae are often seen as small caves or flask-shaped invaginations of the plasma membrane. They appear to be particularly abundant in the plasma membrane of adipocytes (9) . Caveolae have high concentrations of cholesterol, and caveolar function has been shown to be critically dependent on the level of cholesterol in the plasma membrane (10 , 11) . A number of proteins involved in signal transduction are localized to caveolae, which suggests that caveolae are involved in cellular signaling (reviewed in refs 12 13 14 15 ). Cholesterol- and sphingolipid-rich microdomains or rafts are described in the plasma membrane of many cell types. There appears to be a dynamic relation between rafts and caveolae, and it has been proposed that caveolae formation from rafts at the cell surface is induced by caveolin (14) . Caveolae/rafts have been isolated either in the presence of detergent, as an insoluble fraction of the cell membranes, or in the absence of detergent (reviewed in ref 14 ).

Here we demonstrate that insulin receptors are to a large extent localized to caveolae, are functional in caveolae, and are dependent on caveolae for their signaling.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Materials
Rabbit anti-insulin receptor ß chain polyclonal antibodies (immunization with cytoplasmic domain residues 441–620) were from Transduction laboratories (Lexington, Ky.); monoclonal antibodies (29B4, immunization with human placental insulin receptor) and rabbit anti-insulin receptor {alpha} chain were from Santa Cruz Biotechnology (Santa Cruz, Calif.). Rabbit anti-caveolin polyclonal antibodies, anti-clathrin monoclonal antibodies, mouse anti-phosphotyrosine (PY20) monoclonal antibodies, and mouse anti-PI3-kinase monoclonal antibodies were from Transduction Laboratories; Na,K-ATPase {alpha}2 polyclonal antibodies were from Upstate Biotechnology Inc. (Lake Placid, N.Y.). Rabbit anti-IRS-1 polyclonal antibodies were from Santa Cruz Biotech. Cholesterol oxidase (Rhodococus erythropolis) was from Boehringer Mannheim (Mannheim, Germany). DMEM culture medium and sera were from Life Technologies (Stockholm, Sweden). 2-Deoxy-D-[1-3H]glucose, D-[3-3H]glucose, [{gamma}-32P]ATP, and [32P]phosphate were from Amersham (Little Chalfont, U.K.). Insulin, ß-cyclodextrin, hydroxypropyl-ß-cyclodextrin, and other chemicals were from Sigma-Aldrich Chem. Co. (St. Louis, Mo.), Boehringer Mannheim, or as indicated in the text. Sprague-Dawley rats were from B&K Universal (Stockholm, Sweden).

Isolation and incubation of rat adipocytes
Adipocytes were isolated by collagenase digestion from epididymal fat pads of Sprague Dawley rats (160–200 g) (16) . At a final concentration of 100 µl packed cell volume per milliliter, cells were freshly incubated in Krebs-Ringer solution (0.12 M NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4) containing 20 mM HEPES, pH 7.40, 3.5% (w/v) fatty acid-free bovine serum albumin, 100 nM phenylisopropyladenosine, 0.5 U.ml-1 adenosine deaminase with 2 mM D-glucose, at 37°C in a shaking water bath. Freshly isolated rat adipocytes were used when possible, since they are physiologically highly relevant target cells of insulin. 3T3-L1 adipocytes were used for morphological examination of plasma membrane and experiments requiring long incubation time.

3T3-L1 cell culture and differentiation
3T3-L1 fibroblasts were grown on 16 mm (diam.) glass coverslips in DMEM with 25 mM D-glucose supplemented with 10% newborn calf serum, 50 IU/ml of penicillin, and 50 µg/ml of streptomycin in 10% CO2/humidified atmosphere at 37°C. The medium was changed every 2–3 days. Two days after the fibroblasts reached confluence, differentiation was induced with slight modifications to (17) : cells were incubated for 2 days in DMEM containing 10% fetal bovine serum, 5 µg/ml insulin, 0.25 µM dexamethasone, and 0.1 mM 3-isobutyl-1-metylxanthine. The cells were then incubated for an additional 2 days in the same medium excluding dexamethasone and 3-isobutyl-1-metylxanthine. Cells were maintained for 8–10 days in DMEM with 10% fetal bovine serum in order to attain maximal differentiation. More than 95% of the cells expressed the adipocyte phenotype, as determined from accumulation of triacylglycerol droplets.

Double immunofluorescence microscopy
Differentiated 3T3-L1 adipocytes were kept for 2 h in serum-free DMEM supplemented with 0.5% bovine serum albumin (fatty acid free), then washed and incubated in 120 mM NaCl, 4.7 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4, 1.2 mM KH2PO4 containing 1% bovine serum albumin and 20 mM HEPES, pH 7.4, with additions as indicated. Plasma membranes attached to coverslips were prepared by incubation for 30 s in 0.5 mg/ml poly-L-lysine, 137 mM NaCl, 2.7 mM KCl, 10 mM Na2PO4, 1.8 mM KH2PO4, pH 7.3, followed by 20 s in hypotonic solution A (solution A diluted to 1/3). The coverslips were then placed in solution A (70 mM KCl, 5 mM MgCl2, 3 mM EGTA, 30 mM HEPES, pH 7.5) with 1 mM dithioerythritol and 0.1 mM phenylmethylsulfonyl fluoride and probe-sonicated for 2 s (18) . Membranes were fixed in 2% paraformaldehyde on ice (19) [we have also used 4% paraformaldehyde or 4% paraformaldehyde + 0.5% glutaraldehyde (19) with the same results].

Autofluorescence was quenched by incubating fixed plasma membranes in 1 mg/ml NaBH4. Nonspecific binding was blocked with 1% bovine serum albumin. Membranes were incubated with mouse anti-insulin receptor ß chain monoclonal antibodies (10 µg/ml) and rabbit anti-caveolin polyclonal antibodies (40 µg/ml) for 90 min at 37°C. Primary antibodies were detected with fluorescein-isothiocyanate-conjugated goat anti-mouse antibody and tetramethyl-rhodamine-conjugated goat anti-rabbit antibody for 2 h at room temperature. The coverslips were mounted in glycerol solution with antifading agent (Cityfluor; University of Kent, U.K.) before examination of plasma membranes with a 60x objective (numerical aperture = 1.3) in the confocal laser scanning microscope (CLSM; Phoibos 2000; Sunnyvale, Calif.). The CLSM was equipped with an argon laser, which was used at 30% intensity of total 10.5 mW, with all lines open. The primary beam splitter for excitation and the secondary for emission were DRLP 535 (nm) and B/S 565 (nm), respectively. The barrier filter for the tetramethyl-rhodamine-channel (No. 1) was EFPL 570 (nm) and for the fluorescein-isothiocyanate-channel (No. 2), DF30 540 (nm). The photomultiplier voltages were set to minimize crossover between the channels using single-labeled samples. The spatial resolution was 80–100 nm.

Immunogold transmission electron microscopy
Cells grown on Formvar-coated nickel grids (300 mesh) were treated as described above so as to generate plasma membranes attached to the grids. Membranes were then fixed in 4% paraformaldehyde, 0.5% glutaraldehyde for 30 min at room temperature. Nonspecific binding was blocked with 1% bovine serum albumin and 0.1% gelatin before incubation with primary antibodies, as described above. These were detected with 15 nm gold-conjugated anti-mouse antibody and 6 nm gold-conjugated anti-rabbit antibody for 15 h at 4°C. The membrane preparation was finally fixed in 2% glutaraldehyde for 10 min and 1% OsO4 for 30 min at room temperature. After rinsing, the grids with membranes were frozen, lyophilized, and covered with 2 nm tungsten. Transmission electron microscopy was done with JEOL EX1200 TEM-SCAN (Tokyo, Japan). No labeling was observed in the absence of the primary antibodies.

Isolation of caveolae-enriched membrane fraction
Adipocytes were homogenized in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, 0.5 mM EGTA, 0.25 M sucrose, 25 mM NaF, 1 mM Na2-pyrophosphate, with protease inhibitors, 10 µM leupeptin, 1 µM pepstatin, 1 µM aprotinin, 4 mM iodoacetate, and 50 µM phenylmethylsulfonyl fluoride using a motor-driven Teflon/glass homogenizer at room temperature. Subsequent procedures were carried out at 0–4°C. A plasma membrane-containing pellet, obtained by centrifugation at 16,000 x g for 20 min, was resuspended in 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, and protease inhibitors. Purified plasma membranes were isolated by sucrose density gradient centrifugation (20) . Aliquots of this fraction were pelleted and resuspended in 0.5 M Na2CO3 (pH 11) and protease inhibitors (21) and sonicated with a probe-type sonifier (MSE, Soniprep 150) 3 x 20 s. The homogenate was then adjusted to 45% sucrose in 12 mM Mes, pH 6.5, 75 mM NaCl, 0.25 M Na2CO3, loaded under a 5–35% discontinuous sucrose gradient in the same buffer solution, and centrifuged at 39,000 rpm for 16–20 h in a SW41 rotor (Beckman Instruments, Fullerton, Calif.). The light-scattering band at the 5–35% sucrose interface was collected (caveolae-enriched fraction).

For insulin treatment of isolated caveolae, the caveolae-enriched fraction was suspended in the Krebs-Ringer HEPES-buffered cell incubation medium (above) with 0.01% (w/v) bovine serum albumin, 0.2 mM NaF, 0.2 mM Na3VO4, 1 mM MgCl2, and 0.1 mM ATP. Insulin or vehicle was added as indicated; the suspension was sonicated with the probe sonifier 5 x 3 s (sonication was necessary, presumably for insulin to gain access to the interior of caveolae vesicles) on ice and incubated for 10 min at 37°C.

SDS-PAGE and immunoblotting
After sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) (10% acrylamide), separated proteins were electrophoretically transferred to a polyvinylidene difluoride blotting membrane (Immobilone-P, Millipore, Bedford, Mass.) (22) and incubated with the indicated antibodies. Bound antibodies were detected according to the ECL protocol and reagents from Amersham, using horseradish peroxidase-conjugated anti-IgG as secondary antibody. Blots were evaluated by chemiluminescence imaging (Las 1000, Fuji, Japan) or, in some cases, by densitometric analysis (LKB Ultroscan XL, Sweden) of films. Silver staining after SDS-PAGE was performed according to ref 23 .

Protein phosphorylation
For analysis of protein phosphorylation, adipocytes (30 µl packed cell volume/ml) were prelabeled with [32P]phosphate for 1 h (24) . Total cell protein was prepared for SDS-PAGE, as described (16) . 32P Incorporation in ATP citrate-lyase (25) was evaluated by radioimaging (Bas 1000, Fuji).

Glucose transport
Glucose transport was determined in 3T3-L1 adipocytes as uptake of 2-deoxy-D-[1-3H]glucose (17) . Cells were grown on 13 mm plastic coverslips (Thermanox Coverslips, Nunc Inc., Copenhagen, Denmark) in 24-well culture dishes. 2-Deoxy-D-[1-3H]glucose was added to a final concentration of 50 µM (0.5 µCi/ml) and the cells were incubated for 6 min. Glucose uptake was stopped by rinsing the coverslips in three successive solutions of ice-cold buffer. Nonspecific uptake was determined in the presence of 25 µM of cytochalasin B. Coverslips were transferred to scintillation vials and the cells were dissolved in 1% SDS. Radioactivity was measured after adding 5 ml of scintillant (Ready Gel, Beckman).

Miscellaneous procedures
To determine cholesterol content, membranes were pelleted and lipids were extracted with 2-propanol. The extract was evaporated to dryness, dissolved in hexane, and applied to 100 mg silica column (Isolute SPE columns, Sorbent AB, V. Frölunda, Sweden). The cholesterol-containing fraction was eluted according to ref 26 , with a > 95% yield. Cholesterol was then quantitated spectrofluorometrically by measuring the amount of H2O2 produced by cholesterol oxidase (27) .

PI3-kinase activity was determined as described (28) . Briefly, the substrate phosphatidylinositol was emulsified by probe sonication; caveolae were collected by centrifugation and resuspended in assay solution with [{gamma}-32P]ATP (28) . Reaction products were identified by thin-layer chromatography (29) and evaluated by radioimaging (Bas 1000, Fuji). Protein was determined by Coomassie blue binding (39) using bovine serum albumin as standard.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Localization of insulin receptor in caveolae by electron microscopy and immunogold labeling
Double immunogold transmission electron microscopy was used to examine the localization of insulin receptors in the plasma membrane of 3T3-L1 adipocytes. Cells were grown on grids and plasma membranes attached to the grids were prepared by sonication of the cultured cell layer (18) . Insulin receptors and the caveola marker protein caveolin in the membranes were gold-labeled with antibodies. By electron microscopy, we found the membrane to be characterized by frequent roundish structures of ~50 nm diameter (Fig. 1 , stereo image), appearing in stereo viewing as sacs protruding into the cell. Caveolin (small grains) was detected in these structures, identified as caveolae (Fig. 1) . In Fig. 1b , a caveolae cluster is shown; clusters of caveolae have been described in adipocytes (31) . Insulin receptor labeling (large grains) is less prevalent than caveolin labeling (Fig. 1) , but is only found in the caveolae structures: 90 of 91 identified particles, from 14 micrographs representing 5 preparations, were associated with the caveolae-structures.



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  		Figure 1. Localization of insulin receptor in caveolae of 3T3-L1 adipocyte plasma membrane examined by transmission electron microscopy. Stereo image of the inner surface of plasma membrane with gold-labeled antibodies (see Materials and Methods) against insulin receptor (15 nm, arrows) and caveolin (6 nm). a) An area of plasma membrane with individual caveolae. b) Caveolae cluster.

When we used a different antibody against the insulin receptor ß subunit (rabbit polyclonal vs. mouse monoclonal) or antibodies against the insulin receptor {alpha} chain, similar results were obtained: the insulin receptor was labeled in the caveolae only (not shown). Immunoblotting of isolated caveolae or plasma membrane fractions showed that antibodies against the insulin receptor ß subunit labeled a single major protein migrating as the receptor ß subunit (not shown).

Fan et al. (9) have calculated that the number of caveolae is ~10 per µm2 of cell surface, corresponding to ~105 caveolae per adipocyte, which is of the same order of magnitude as the estimated number of insulin receptors (105) on an adipocyte (32) . Labeling of caveolae with anti-insulin receptor antibodies compared to anti-caveolin antibodies is therefore a comparatively rare event. Caveolae with insulin receptor labeling frequently exhibited multiple labeling (c.f. Fig. 1 ), indicating that the insulin receptor may be confined to a subpopulation of caveolae rather than being evenly dispersed between caveolae.

Colocalization of insulin receptor and caveolin by immunofluorescence microscopy
Since the number of insulin receptors present on a cell is limited and an electron microscopic picture is limiting, we used double immunofluorescence confocal microscopy to examine the global colocalization of the insulin receptor with caveolin in the plasma membrane. Plasma membranes were prepared in essentially the same way as described for the examination by electron microscopy. We used differentially fluorescing antibodies against the insulin receptor (Fig. 2 A) and caveolin (Fig. 2B ). To compare the localization of the much more prevalent caveolin protein with the rare insulin receptor protein, fluorescence labeling and signal amplification were carefully adjusted to ensure comparable intensities in both channels while minimizing crossover. A typical membrane fragment is illustrated; the insulin receptor was colocalized with caveolin in the plasma membrane, as demonstrated by the yellow areas in the superimposed images (Fig. 2C ). The insulin receptor and caveolin were clustered in spots of 0.3–0.5 µM, which appear to be organized in ring-like structures of varying size. The size of the spots corresponds to that of the clusters of caveolae identified by electron microscopy above (Fig. 1b ). The colocalization within the membrane was not affected by incubation with 100 nM insulin for 5 or 20 min (not shown).



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  		Figure 2. Colocalization of insulin receptor and caveolin in 3T3-L1 adipocyte plasma membrane examined by immunofluorescence microscopy. The inside surface of the plasma membrane (illustrated here with cells incubated without insulin) was labeled with fluorescent antibodies (see Materials and Methods) against A) the insulin receptor (green) and B) caveolin (red). C) The superimposed images of panels A and B turn yellow where the green and red antibodies are colocalized. White square, 1 x 1 µM.

The noncaveolar protein clathrin exhibited a distribution in the plasma membrane (Fig. 3 a) clearly distinguished from that of caveolin (Fig. 2 and Fig. 3b ) or of the insulin receptor (Fig. 2) . This indicates that the typical spots/clusters of caveolin and insulin receptor labeling are not a preparation artifact. As a further control, plasma membranes were fixed in different concentrations of paraformaldehyde or paraformaldehyde in combination with glutaraldehyde (see Materials and Methods) before the incubation with antibodies. We found no effect on the pattern of immunofluorescence labeling (not shown), indicating that the insulin receptor did not artifactually aggregate in caveolae as a result of antibody cross-linking, which has been shown to occur with phosphatidylinositol glycan-anchored proteins (19) . As we will show later, the insulin receptor also differs from those proteins in that it is solubilized by treatment of membranes with cold non-ionic detergent.



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  		Figure 3. Localization of clathrin in 3T3-L1 adipocyte plasma membrane examined by immunofluorescence microscopy. Plasma membranes were treated as in Fig. 2 and double-labeled with antibodies against clathrin (A) and caveolin (B) and photographed by fluorescence microscopy.

Insulin receptor in isolated caveolae-enriched fraction
The morphological demonstration of localization of the insulin receptor in caveolae was further affirmed after isolation of a caveolae-enriched fraction of rat adipocyte plasma membranes using a procedure that avoids detergent treatment (Fig. 4 A). The insulin receptor recovered in the caveolae fraction was enriched 36 ± 11-fold (mean ± SE of six preparations) over the intact plasma membrane fraction. We found no significant effect of insulin on the distribution of insulin receptors between caveolae and the rest of the plasma membrane, which is in accordance with the immunofluorescence analysis above; within the plasma membrane, localization of the insulin receptor to caveolae does not appear to be affected by ligand binding. At the concentration of insulin and incubation time used, loss of insulin receptors from the plasma membrane through receptor down-regulation is too small (33) to be detectable by this methodology.



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  		Figure 4. Insulin receptor in caveolae-enriched fraction of plasma membrane. A) Isolated adipocytes were subfractionated into plasma membrane and caveolae-enriched fraction of plasma membrane (without detergent treatment). Aliquots of 7 µg protein were subjected to SDS-PAGE and immunoblotting with antibodies against the ß subunit of the insulin receptor (upper) or phosphotyrosine (lower). Adipocytes were preincubated with (+) or without (-) 10 nM insulin for 5 min. pm, plasma membrane. B) Caveolae-enriched fractions were prepared from purified plasma membranes without detergent treatment (0), as in panel A, or after treatment with Triton X-100 (0.1, 0.3) at the percent (w/v) concentrations indicated (22) . Aliquots corresponding to equal amounts of cells were subjected to SDS-PAGE and immunoblotting with antibodies against the ß subunit of the insulin receptor (upper) or caveolin (lower).

We have verified in different ways the enrichment of caveolae by using the detergent-free method. 1) The caveolae marker protein caveolin was enriched seven- ± twofold (mean ± SE, n=6) (Fig. 5 A), whereas noncaveolar proteins Na, K-ATPase {alpha}2 subunit (Fig. 5A ) and clathrin (Fig. 5A ) were depleted >10-fold in the caveolar fraction compared to the plasma membrane in general. 2) The cholesterol content was 1.1 ± 0.20 µmol/mg protein (mean ± SE, n=3) in the caveolae fraction compared to 0.3 ± 0.05 µmol/mg protein (mean ± SE, n=3) in the plasma membrane in general. 3) 7.5 ± 1.2% (mean ± SE, n=4) of the plasma membrane protein was recovered in the caveolae fraction, with a distinct protein composition (Fig. 5B ). 4) By electron microscopy and immunogold labeling of caveolin, 50-70% (in different preparations) of the caveolar fraction consisted of caveolae membranes (J. Gustavsson, unpublished results).



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  		Figure 5. Characterization of caveolae-enriched fraction. Isolated adipocytes were subfractionated into a plasma membrane (pm) and a caveolae-enriched (cav) fraction of the plasma membrane fraction (without detergent treatment). A) Aliquots were subjected to SDS-PAGE and immunoblotting with antibodies against caveolin, Na, K-ATPase, or clathrin, as indicated. B) Aliquots of 5 µg protein were subjected to SDS-PAGE and silver staining of protein. Indicated are the molecular masses of reference proteins and proteins that are different in the plasma membrane and caveolae fractions (arrowheads).

The number of caveolae in the adipocyte plasma membrane was determined to be around 10 caveolae per µm2 (9) . Assuming an average diameter of 50 nm and a depth of 50 nm for each caveola (Fig. 1) , caveolae are conservatively calculated to constitute ~10% of the plasma membrane of an adipocyte. This indicates that the caveolae-enriched fraction we used is rather pure. This also implies that the concentration of cholesterol is fivefold higher in caveolae than the average of the surrounding plasma membrane.

When we isolated caveolae as the detergent-resistant fraction of plasma membranes, the insulin receptor (but not caveolin) was selectively lost (Fig. 4B ). This detergent-solubility of the insulin receptor distinguishes it from many other proteins localized to caveolae or rafts—including GLUT4 (22) and in particular phosphatidylinositol glycan-anchored proteins (14 , 34) —but it has also been described for other proteins (35) . The detergent solubility also explains earlier failures to identify the receptor in caveolae (36) .

Insulin receptor signaling in caveolae
The insulin receptor was functional in caveolae, since in response to insulin stimulation of the intact cells, tyrosine phosphorylation of the insulin receptor ß subunit (95 kDa) was increased 3.3 ± 0.5-fold (mean ± SE, n=3) in the caveolar fraction. This equals the 3.4 ± 0.8-fold (mean ± SE, n=3) increase found in the plasma membrane in general, i.e., in plasma membranes with caveolae (Fig. 4A ).

Insulin treatment of the isolated caveolae fraction similarly caused an increase in the level of tyrosine phosphorylation of the insulin receptor ß subunit (Fig. 6 ): a 3.3 ± 0.7-fold (mean ± SE, n=3) increase compared to non-insulin-stimulated caveolae fraction.



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  		Figure 6. Insulin stimulation of the isolated caveolae fraction induces receptor autophosphorylation. A caveola-enriched fraction was isolated and incubated with (+) or without (-) 1 µM insulin (see Materials and Methods) for 10 min, subjected to SDS-PAGE, and immunoblotted with antibodies against phosphotyrosine. Indicated are the molecular masses of reference proteins.

IRS-1 was present at very low levels in the total plasma membrane and caveolae fraction, in keeping with the findings of others (37) . However, the amount did not appear to be affected by insulin (not shown). We found that PI3-kinase was present in the caveolae-enriched fraction (2.7 ± 0.7-fold, mean ± SE, n=7, enriched over the plasma membrane fraction), as determined by immunoblotting against the p85 subunit. Insulin stimulation of intact cells appeared to increase the amount of PI3-kinase protein in the caveolae fraction by 1.7 ± 0.4-fold (mean ± SE, n=7), but the activity of PI3-kinase in the caveolae-enriched fraction was unaffected. Also, when the isolated caveolae fraction was treated with insulin, the activity of resident PI3-kinase was not affected (not shown) even though the insulin receptor was autophosphorylated.

Insulin signaling dependence on cholesterol
Insulin signaling appeared to be critically dependent on the integrity of caveolae as shown next. Partial removal of cholesterol from cells has been shown to disrupt the function of caveolae (10 , 11) . When we incubated rat adipocytes with the cholesterol-sequestering ß-cyclodextrin (38) , plasma membrane cholesterol was reduced by 60%: 0.4 µmol cholesterol/mg protein in plasma membranes from control cells and 0.15 µmol/mg protein from treated cells. Such treatment of 3T3-L1 adipocytes caused invaginated caveolae of the plasma membrane to be nearly annihilated, as determined by electron microscopy; in a typical micrograph, the occasional invaginated caveola was still found together with what appears to be the remains of caveolae (Fig. 7 ). Caveolin remained clustered in the plasma membrane (Fig. 7) , presumably associated with noninvaginated caveolar patches (39) .



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  		Figure 7. Effect of cholesterol depletion on caveolae morphology examined by electron microscopy. 3T3-L1 adipocytes were treated with 10 mM ß-cyclodextrin for 1 h, when cells were subjected to immunogold labeling of caveolin and electron microscopy as in Fig. 1 . The micrograph is typical, but some variation was found, with more caveolar structures remaining in some preparations and less in others.

Normally, insulin causes a dose-dependent increase of ATP citrate-lyase phosphorylation in the intact rat adipocytes. This in situ response was attenuated by ß-cyclodextrin pretreatment (Fig. 8 A). Also, destruction of cell surface cholesterol with cholesterol oxidase (40) inhibited subsequent in situ insulin signaling to protein phosphorylation (Fig. 8B ) in a dose-dependent manner (not shown). In a separate experiment, adipocytes were cholesterol-depleted by incubating with hydroxypropyl-ß-cyclodextrin and then replenished with cholesterol by incubating with hydroxypropyl-ß-cyclodextrin saturated with cholesterol (18:1, mol:mol) (38) for 40 min. This partially restored insulin signaling to protein phosphorylation, from 9% of maximal insulin effect after cholesterol depletion to 66% of maximal effect after cholesterol replenishment.



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  		Figure 8. Effect of cholesterol depletion on insulin stimulation of protein phosphorylation. A) [32P]Phosphate-labeled rat adipocytes were preincubated with (filled) or without (open) 10 mM ß-cyclodextrin for 50 min, when insulin was added at the concentration indicated for 15 min and phosphorylation of ATP citrate-lyase was determined (mean ± SE, n=6). B) [32P]Phosphate-labeled adipocytes were preincubated with (filled) or without (open) 1 U/ml cholesterol oxidase for 1 h in the dark; cells were incubated with (columns 2, 4) or without (columns 1, 3) 500 pM insulin for 10 min after which the phosphorylation of ATP citrate-lyase was determined (mean ± SE, n=6). Phosphorylation is expressed in arbitrary densitometric units.

Insulin stimulation of glucose uptake was also attenuated in the fat cells after ß-cyclodextrin pretreatment (not shown) as well as in 3T3-L1 adipocytes in cell culture (Fig. 9 ). The capacity of insulin to stimulate glucose transport was almost completely regained after washing the 3T3-L1 adipocytes and 24 h additional culturing in the absence of ß-cyclodextrin (Fig. 9) , presumably through resynthesis and replenishment of caveolar cholesterol.



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  		Figure 9. Reversible effect of cholesterol depletion on insulin stimulation of glucose uptake. 3T3-L1 adipocytes were kept serum-free for 2 h; during the last hour, they were incubated with (filled) or without (open) 10 mM ß-cyclodextrin, when (columns 1, 2) 100 nM insulin was added for 20 min and the uptake of 2-deoxy-D-[1-3H]glucose was determined or (columns 3, 4) cells were washed and incubated in DMEM culture medium with 10% fetal calf serum for 22 h in order to restore cellular cholesterol and kept serum-free for another 2 h, when 100 nM insulin was added for 20 min and uptake of 2-deoxyglucose was determined (mean ± SE, n=8). Glucose transport was not affected in control cells not stimulated by insulin.

The findings that the two independent techniques for cholesterol depletion—cholesterol extraction or specific cholesterol destruction—inhibit insulin signaling, that cholesterol replenishment restores signaling, and that cholesterol depletion seriously affects caveolar morphology suggest a critical role for cholesterol/caveolae in insulin receptor signaling. Since plasma membrane cholesterol is concentrated in caveolae and caveolae structure/function depends on cholesterol, the functional dependence on cholesterol that the insulin receptor exhibits also indirectly supports a localization of the insulin receptor to caveolae microdomains of the plasma membrane.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The morphological, biochemical, and functional correlates presented here strongly suggest that the insulin receptor is located and is signaling in the caveolae of the plasma membranes of adipocytes. The adipocyte represents a key physiological target cell of the hormone. An ensemble of earlier experiments agree with this model. It has been shown, for instance, that gold-labeled insulin predominantly binds to plasma membrane invaginations of adipocytes (41) . A caveolin binding amino acid sequence motif found in the insulin receptor sequence (42) can explain how the insulin receptors are targeted to caveolae. The insulin receptor was also demonstrated to increase the state of phosphorylation of caveolin in 3T3-L1 adipocytes (36) . Recently Wu et al. (43) showed the presence of insulin receptors in caveolae-like domains from neuronal plasma membranes. It was reported very recently that caveolin of subtypes 1 and 3, but not 2, could bind and activate the insulin receptor (44) . The presence of insulin receptors in caveolae can explain the long-standing question of how cryptic receptors could be unmasked by treatment of intact cells with phospholipase or digitonin (45) . Filipin, which binds to cholesterol, blocked insulin uptake by transcytosis in endothelium, a caveolae-mediated process (11) . Insulin-sensitive adipocytes exhibit large amounts of caveolae, and caveolin and caveolae are present to a greater extent in 3T3-L1 cells when they differentiate to insulin-responsive adipocytes (9 , 46 , 47) or when differentiation of myoblasts into insulin-responsive muscle cells is induced (48) .

Radiolabelled insulin was found to bind to microvilli and to clathrin-coated pits in different cell types (49–51). It may be that insulin binds to clathrin-coated pits for its subsequent degradation rather than signaling per se, in line with endothelial insulin-degradation via coated pits and transendothelial insulin-transport (for subsequent signaling) via caveolae (11) . Evidence has indeed been presented to implicate two distinct pathways in internalization of the receptor (52) .

Numerous hormone and growth factor receptors and signal mediating molecules are believed to reside in caveolae (12 , 13 , 15) , but with limited functional consequences described earlier. The insulin receptor appears to be critically dependent on caveolar cholesterol or caveolae integrity for its ability to signal. This is in contrast to the epidermal growth factor receptor, for example, which after cholesterol depletion of caveolae caused hyperactivation of ERK (extracellular signal-related kinase) (53) . It appears that enzymes (e.g., tyrosine kinases) with caveolin binding sequence motifs can interact with and be inhibited by caveolin (54 , 55) . We have not addressed this specific question with respect to the insulin receptor; however, in the absence of ligand the receptor may be kept in a subdued state through its interaction with caveolin, but insulin receptors with insulin bound are obviously actively signaling in caveolae. Indeed, our results suggest that caveolae are required for insulin receptor signaling, which agrees with recent findings that the insulin receptor can be activated by caveolin (44) .

Platelet-derived growth factor was recently shown to bind to its receptor and to activate mitogen-activated protein kinase in isolated caveolae of fibroblasts (56 , 57) , demonstrating the presence of the entire signaling pathway in a caveolae preparation. Insulin similarly activates its receptor in caveolae when the intact cell or the isolated caveolar fraction is treated with insulin. Our finding that PI3-kinase is enriched in the caveolae-enriched fraction of adipocyte plasma membranes agrees with earlier findings with human fibroblasts (56 , 57) . Insulin has, however, been found to mainly affect PI3-kinase levels and activity in cytosol compartment and intracellular membrane fractions (58 59 60 61 62) , and this is not contradicted by our present findings. Although smaller effects on PI3-kinase activity in the plasma membrane have been reported (58 , 59) , we found no effect of insulin on local caveolar PI3-kinase activity. The low level of association of IRS-1 with the plasma membrane or with our caveolae fraction is also in line with earlier findings (37 , 59 , 60) , but does not preclude an in situ association that does not survive the plasma membrane or caveolae isolation procedure (c.f. ref 60 ). The present findings thus introduce the caveola as a structural framework for insulin receptor signal generation, but leave open the involvement of caveolae in the downstream signal propagation.

We have earlier reported that a specific phosphatidylinositol glycan, a precursor of potential insulin-mediators (63) , may be localized to caveolae (64) . Moreover, we have explained that insulin-stimulated translocation of GLUT4 to the plasma membrane is followed by a relocation of GLUT4 to caveolae where glucose uptake appears to take place (22) . In that study, we isolated caveolae as a detergent-resistant fraction, we have now verified those findings using the detergent-free isolation procedure described here and immunofluorescence colocalization of GLUT4 with caveolin (J. Gustavsson, M. Karlsson, C. Ramsing, M. Borg, S. Parpal, K.-E. Magnusson, and P. Strålfors, unpublished results). Taken together, our results suggest a novel concept for insulin action in that caveolae may provide a necessary scaffold at the plasma membrane for both insulin signal generation and insulin-stimulated glucose transport. Perturbation of caveolar cholesterol, and hence caveolar integrity, in effect make the cells insulin-resistant.

Financial support was obtained from Lions Foundation, Swedish Society for Medical Research, Östergötland County Council's Medical Research Funds, Swedish Diabetes Association, and the Swedish Medical Research Council.


   FOOTNOTES
 
Received for publication March 9, 1999. Accepted for publication June 14, 1999.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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