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INTRODUCTION |
Insulin controls target cells by binding to its cell surface
receptor. The further intracellular transmission of the insulin signal
involves phosphorylation of the receptor as well as other proteins, in
particular the insulin receptor substrate
(IRS),1 on specific tyrosine
residues. After tyrosine phosphorylation IRS is recognized by Src
homology 2 domain-containing proteins for metabolic and glucose
transport control, or activation of the mitogen-activated protein
kinase (MAP kinase) pathway and mitogenic control (1-4). In type 2 diabetes target cells of the hormone are not fully responsive, which is
compensated temporarily by enhanced insulin secretion. The pathogenic
mechanisms for this insulin resistance are not known, but an important
common feature appears to be a reduced activation/tyrosine
phosphorylation of IRS-1 (5).
The insulin receptors are sequestered in the caveolae microdomains of
the plasma membrane in adipocytes, and caveolae appear to be critical
for insulin control (6). By thin-section electron microscopy, caveolae
appear as omega-shaped invaginations of 50-100 nm diameter in the
plasma membrane (7). Caveolae invaginations are found in the plasma
membrane of many cell types, but are particularly abundant in
adipocytes where they increase in number in conjunction with the
differentiation of 3T3-L1 fibroblasts to mature adipocytes (8-10).
Caveolae are involved in receptor-mediated uptake of solutes into the
cytosol (11) and in transcytosis (12). A number of proteins, in
addition to the insulin receptor, involved in signal transduction are
localized to caveolae, which suggests that they may be involved in
cellular signaling and control (reviewed in Refs. 13-16).
Caveolae are rich in cholesterol and sphingolipids. Caveolae may indeed
form from cholesterol- and sphingolipid-rich rafts in the membrane in a
process requiring the caveolae-specific structural protein caveolin.
Caveolin is found in the plasma membrane and intracellularly, but in
the plasma membrane is confined to caveolae; it is therefore used as a
marker for these structures. The function of caveolae is dependent on a
sufficient level of cholesterol in the plasma membrane and caveolae
(12, 17). We have also demonstrated a critical dependence of the
insulin receptor signal transduction on cholesterol; depletion of
cholesterol from the plasma membrane of rat adipocytes reversibly
inhibited insulin stimulation of glucose transport and metabolic
protein phosphorylation control (6). The importance of caveolae for
insulin receptor signaling is further indicated by a consensus binding
site for interaction with caveolin (18), and coprecipitation of the
receptor with caveolin (4) indicates that the interaction may be
physiological. Moreover, the insulin receptor appears to phosphorylate
caveolin (19), whereas caveolin was shown to activate the isolated
receptor, although the physiological relevance of this is not known
(20).
Herein we examine in detail the dependence of the insulin receptor on
caveolae for signal transduction: the effects of cholesterol depletion
on plasma membrane and caveolae morphology, on the insulin receptor and
on the downstream propagation of the insulin signal. In short, the
insulin receptor is unaffected, but interaction with the immediate
downstream mediator molecule IRS-1 and metabolic control is inhibited
by cholesterol depletion and caveolae destruction, while insulin
signaling via the MAP kinase pathway remains intact. We also discuss
implications for the pathogenesis of insulin-resistant states.
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EXPERIMENTAL PROCEDURES |
Materials--
Rabbit anti-insulin receptor
-chain polyclonal
antibodies were from Transduction Laboratories, and mouse monoclonal
antibodies were from Santa Cruz Biotechnology. Rabbit anti-caveolin
polyclonal antibodies, and mouse anti-phosphotyrosine (PY20) monoclonal
antibodies were from Transduction Laboratories, and rabbit anti-IRS-1
polyclonal antibodies were from Santa Cruz Biotechnology. Rabbit
polyclonal antibodies against phospho-p44 and phospho-p42 MAP kinases
(ERK1 and ERK2) were from New England Biolabs, Inc., and
anti-phospho-Akt1/protein kinase B
sheep polyclonal antibodies and
rabbit polyclonal antibodies against Shc were from Upstate
Biotechnology Inc. DMEM culture medium and sera were from Life
Technologies, Inc. 2-Deoxy-D-[1-3H]glucose
and [32P]phosphate were from Amersham Pharmacia Biotech.
Insulin,
-cyclodextrin, and other chemicals were from Sigma-Aldrich
or as indicated in the text. Harlan Sprague-Dawley rats were from B&K
Universal (Stockholm, Sweden).
3T3-L1 Cell Culture and Differentiation--
3T3-L1 fibroblasts
were grown on 13 mm (diam.) glass coverslips in DMEM with 25 mM D-glucose, supplemented with 10% newborn calf serum, 50 UI/ml penicillin, and 50 µg/ml 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
the procedure in Ref. 21; 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-methylxanthine. The cells were then incubated for an
additional 2 days in the same medium excluding dexamethasone and
3-isobutyl-1-methylxanthine. Cells were maintained for 8-10 days in
DMEM with 10% fetal bovine serum to attain maximal differentiation. More than 95% of the cells expressed the adipocyte phenotype, as
determined from accumulation of triacylglycerol droplets.
Immunogold Transmission Electron Microscopy--
3T3-L1 cells
were grown on Formvar-coated grids (300-mesh) and differentiated to
adipocytes. They were kept for 2 h in serum-free DMEM supplemented
with 0.5% bovine serum albumin (fatty acid-free) and 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 7.5, with additions as
indicated. Plasma membranes attached to grids were prepared by
incubation for 30 s in 1 mg/ml poly-L-lysine, 137 mM NaCl, 2.7 mM KCl, 10 mM
Na2PO4, 1.8 mM
KH2PO4, pH 7.5, followed by 20 s in
hypotonic solution A (solution A diluted to 1/3). The grids 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
(22).
Membranes were then fixed in 3% paraformaldehyde, 0.05%
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: mouse anti-insulin receptor
-chain monoclonal antibodies (20 µg/ml) and rabbit anti-caveolin polyclonal antibodies (20 µg/ml) for 90 min at 37 °C. These were detected with gold(15 nm)-conjugated anti-mouse antibody and gold (6 nm)-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), equipped with Gatan Bioscan CCD camera;
images were obtained with GATAN Digital Micrograph software.
Isolation and Incubation of Rat Adipocytes--
Adipocytes were
isolated by collagenase digestion from epididymal fat pads of Harlan
Sprague-Dawley rats (160-200 g) (23). Cells, at a final concentration
100 µl of packed cell volume/ml, 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 units·ml
1
adenosine deaminase with 2 mM D-glucose, at
37 °C on a shaking water bath.
125I-Insulin Binding to Adipocytes--
Rat
adipocytes were incubated in a shaking water bath with
125I-insulin and unlabeled insulin as indicated at
37 °C. Cells were rapidly separated from incubation medium by
centrifugation through dinonylphthalate. 125I in cell cake
and medium was separately determined. Unspecific binding, determined on
cells incubated with an excess of unlabeled insulin (1 µM), was on average 0.2% of total and was subtracted. The amount of cells was determined as packed cell volume at the end of
each incubation.
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 × 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 (24). Aliquots of this
fraction were pelleted and resuspended in 0.5 M
Na2CO3, pH 11, and protease inhibitors (25) and
sonicated with a probe-type sonifier (MSE, Soniprep 150) three times
for 20 s each. The homogenate was then adjusted to 45% sucrose in
12 mM Mes, pH 6.5, 75 mM NaCl, 0.25 M Na2CO3 and 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). The light-scattering band, enriched in caveolae, at the
5-35% sucrose interface was collected. Enrichment of caveolae was
demonstrated by the enrichment of caveolin (7-fold) and cholesterol
(3-fold) compared with the intact plasma membrane fraction; by electron
microscopy and immunogold labeling of caveolin, between 50% and 70%
of the isolated membranes constituted caveolae (6).
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 and the suspension sonicated with the
probe sonifier five times for 3 s each time (the sonication was
necessary, presumably for insulin to gain access to the interior of
caveolae vesicles) on ice, and then incubated for 10 min at 37 °C.
Equal amounts of caveolae protein was subjected to SDS-PAGE and immunoblotting.
Additional Procedures--
Total cell protein was prepared for
SDS-PAGE as described (23); cells were immediately spun through
dinonylphthalate and frozen. The cell cake was thawed by boiling in
SDS-PAGE sample solution (26). IRS-1 tyrosine-specific phosphorylation,
phosphoprotein kinase B, or phospho-ERK1/2 was thus determined by
immunoblotting after SDS-PAGE of total cell protein (23). Equal amounts
of cells were loaded within each experiment.
For immunoprecipitation cells were rapidly rinsed in Krebs-Ringer
solution without serum albumin (with or without insulin). Cells were
lysed in 50 mM Hepes, pH 7.4, 10 mM EDTA, 100 mM NaF, 10 mM sodium pyrophosphate, 2 mM Na3VO4, 0.5 mM
phenylmethylsulfonyl fluoride, 1 µM aprotinin, 1 µM pepstatin, 10 µM leupeptin, 1% Nonidet
P-40 with or without 100 µM genistein (Calbiochem). The solubilized cell lysate was filtered through a 0.22-µm pore size filter to remove fat. Equal amounts of protein were incubated with
antibodies for 14 h at 4 °C, when Sepharose G-Plus (Santa Cruz
Biotechnology) was added for 1 h at 4 °C with constant mixing (turning over). The beads were washed and boiled with SDS-PAGE sample
solution and subjected to SDS-PAGE.
After SDS-PAGE (7-11% acrylamide), separated proteins were
electrophoretically transferred to a polyvinylidene difluoride blotting
membrane (Immobilon-P, Millipore) (27) and then incubated with
indicated antibodies. Bound antibodies were detected according to the
ECL+ protocol and reagents from Amersham Pharmacia Biotech or
Renaissance+ from PerkinElmer Life Sciences, using horseradish peroxidase-conjugated anti-IgG as secondary antibodies. Blots were
quantitated by chemiluminescence imaging (Las 1000, Fuji, Japan) and
Image Gauge software (Fuji).
Total and phosphoamino acid-specific phosphorylation of IRS-1 was
determined after immunoprecipitation of IRS-1 from cells preincubated
with [32P]phosphate for 1 h (28). Phosphoamino acid
identification was by electrophoresis on thin-layer silica plates at pH
1.9 (29) after partial acid hydrolysis in 5.7 M HCl at
110 °C for 2 h, using authentic internal standards (Sigma) that
were identified by ninhydrin staining.
Glucose transport was determined in 3T3-L1 adipocytes as uptake of
2-deoxy-D-[1-3H]glucose (21). 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 (0.3 µCi/ml), and the cells were
incubated for 9 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 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).
For determination of cholesterol content, membranes were pelleted and
lipids were extracted with 2-propanol. Cholesterol was then quantitated
spectrofluorometrically by measuring the amount of
H2O2 produced by cholesterol oxidase (30).
Protein was determined by Coomassie Blue binding (31) using bovine
serum albumin as standard.
 |
RESULTS |
Effect of Cholesterol Depletion on Caveolae Structure in Adipocyte
Plasma Membrane--
Here we in detail examine effects of cholesterol
depletion on caveolar structure in relation to insulin signaling. We
have chosen to study cultured 3T3-L1 adipocytes because their plasma membranes can be prepared intact for electron microscopic examination (6). Incubation of 3T3-L1 adipocytes with increasing concentrations of
-cyclodextrin in a dose-dependent manner reduced the
cholesterol content of the plasma membrane (Fig.
1). Concomitantly the structural integrity of caveolae in the plasma membrane was progressively corrupted, as revealed by transmission electron microscopy of isolated
plasma membranes (Fig. 2). Control
membranes incubated with vehicle typically displayed an abundance of
caveolar structures of 50-100 nm diameter (Fig. 2a),
identified by labeling with antibodies against the caveolar structural
protein caveolin (6). Incubation with 2 mM
-cyclodextrin
had no visible effect on caveolar morphology (data not shown), whereas
5 mM
-cyclodextrin was seen to clearly have an effect on
the caveolae, which became flattened, less distinct, and reduced in
number (Fig. 2b; cf. Refs. 32 and 33). At 10 mM
-cyclodextrin, caveolae invaginations have almost
vanished and may be discerned as minor irregular structures at the
membrane surface (Fig. 2c); after 20 mM
-cyclodextrin treatment, no invaginations were seen (data not
shown).

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Fig. 1.
Effects of
-cyclodextrin on plasma membrane cholesterol.
3T3-L1 adipocytes were incubated with the indicated concentration of
-cyclodextrin for 50 min at 37 °C, when cells were harvested,
homogenized, and plasma membranes isolated after which their content of
cholesterol was determined. Figure shows mean ± S.E.
(n = 3).
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Fig. 2.
Effect of cholesterol depletion on caveolar
structure in the plasma membrane. 3T3-L1 adipocytes were incubated
with 0 (a), 5 mM (b), or 10 mM (c) -cyclodextrin for 50 min, when cells
were prepared for transmission electron microscopy as under
"Experimental Procedures." Arrows indicate insulin
receptor labeling. Scale bars = 100 nm.
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Immunogold labeling of caveolin showed that the caveolin protein
remained clustered, indicating that underlying caveolar structures existed in the membrane after 50% reduction of the amount of plasma membrane cholesterol by treatment of the cells with 10 mM
-cyclodextrin (Fig. 2c), perhaps reflecting flattened
caveolar patches or "rafts" remaining in the membrane after the
extensive but partial cholesterol depletion (34).
Insulin-stimulated Glucose Uptake after Cholesterol
Depletion--
The cholesterol depletion concomitantly also inhibited
progressively the ability of insulin to enhance glucose uptake (Fig. 3a). The extent of insulin
stimulation of glucose transport was correlated to the amount of
cholesterol, and hence to degree of caveolar intactness, in the plasma
membrane of the cells (Fig. 3b).

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Fig. 3.
Effects of cholesterol depletion on insulin
signaling. 3T3-L1 adipocytes were incubated with the indicated
concentration of -cyclodextrin for 50 min at 37 °C, followed by
incubation with indicated concentration of insulin for 20 min, when
cellular uptake of 2-deoxyglucose was determined (a).
Mean ± S.E. (n = 3) is shown. b,
replot of data from panel a and from Fig.
1.
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Effect of Cholesterol Depletion on Insulin Receptors in the Plasma
Membrane--
By immunogold labeling and electron microscopy
examination, it appeared that the insulin receptors remained associated
with the caveolin clusters and hence with the underlying caveolar
remains or rafts after the depletion of cholesterol (Fig. 2). This was affirmed by immunoprecipitation of the insulin receptor, which coprecipitated caveolin to the same extent with or without prior cholesterol depletion (data not shown). We used competitive
125I-insulin binding to isolated rat adipocytes to
determine the effect of cholesterol depletion on the accessibility of
insulin receptors at the cell surface (Fig.
4). Cholesterol depletion (with 10 mM
-cyclodextrin, which reduced plasma membrane
cholesterol concentration by ~60% (Ref. 6)) did not affect the time
taken for steady state binding of insulin (Fig. 4a). We
subsequently incubated the cells with 125I-insulin for 30 min when near-maximal binding was obtained. Specific insulin binding
was not affected by prior cholesterol depletion (Fig. 4b).
We used nonlinear regression to fit a one-site binding equation
(GraphPad Software, Inc.) to the data in Fig. 4b, to determine the effect on number of insulin receptors and on the dissociation constant for insulin binding. Neither the number of
receptors nor the binding affinity was significantly affected by the
cholesterol depletion.

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Fig. 4.
Effects of cholesterol depletion on
125I-insulin binding to intact adipocytes. Freshly
isolated rat adipocytes were incubated without ( ) or with
( ) 10 mM -cyclodextrin for 50 min (which
reduces plasma membrane cholesterol by 60% (Ref. 6)) when 2.5 pM (a) or 58 pM (b)
125I-insulin with unlabeled insulin at the indicated total
concentrations, was added for the indicated length of time
(a), or 30 min (b). b, illustrated are
the results of three separate experiments (each the mean of three
samples). The fraction of 125I-insulin bound has been
corrected for the determined amount of packed cell volume in each
sample.
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After subcellular fractionation of rat adipocytes and isolation of the
plasma membranes, SDS-PAGE and immunoblotting with antibodies against
the insulin receptor indicated that the total amount of receptor
protein in the plasma membrane fraction was not affected by the 10 mM
-cyclodextrin treatment (Fig.
5a). This agrees well with the
electron microscopy and 125I-insulin binding results.
Similarly, the amount of caveolin in the plasma membrane fraction was
not affected by treatment with up to 10 mM
-cyclodextrin
(Fig. 5b), also in agreement with the electron microscopic
findings.

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Fig. 5.
Effects of cholesterol depletion on the
amount of insulin receptor and caveolin in the plasma membrane.
Freshly isolated rat adipocytes were incubated without or with 10 mM -cyclodextrin for 50 min and then with or without 1 nM insulin for 10 min when plasma membranes were isolated,
and the amount of insulin receptor and caveolin was determined by
immunoblotting after SDS-PAGE. a, insulin receptor;
b, caveolin. The experiments were repeated three times with
similar results.
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Effect of Cholesterol Depletion on Insulin Receptor
Function--
The
-cyclodextrin depletion of cholesterol from the
isolated rat adipocytes did not affect the ability of subsequent
insulin treatment to stimulate tyrosine-specific receptor
autophosphorylation (Fig. 6a).
Nor did 10 mM
-cyclodextrin extraction of cholesterol from the isolated caveolae preparation affect the subsequent ability of
insulin to stimulate receptor autophosphorylation in the caveolae fraction (Fig. 6b).

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Fig. 6.
Effects of cholesterol depletion of intact
cells or of isolated caveolae fraction on insulin stimulation of
insulin receptor autophosphorylation. Freshly isolated rat
adipocytes (a) or caveolae-fraction isolated from purified
plasma membranes of rat adipocytes (b) were incubated
without or with 10 mM -cyclodextrin for 50 min and then
with (+) or without ( ) 1 nM insulin (a) or 1 µM insulin (b) for 10 min (see "Experimental
Procedures"). Plasma membranes were isolated (a). SDS-PAGE
and immunoblotted with antibodies against phosphotyrosine. The
experiments were repeated three times with similar results.
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Effect of Cholesterol Depletion on Downstream IRS-1 and Protein
Kinase B--
We concluded that cholesterol depletion does not
adversely affect the number of insulin receptors at the cell surface,
the insulin receptor's interaction with or activation by insulin, or
its ability to autophosphorylate. We therefore next examined the
ability of the receptor to tyrosine phosphorylate an immediate downstream target for insulin's metabolic control, IRS-1. After cholesterol depletion of rat adipocytes, the tyrosine-specific phosphorylation of IRS-1 in response to insulin treatment was severely
reduced (Fig. 7). The depletion of
cholesterol inhibited the 1 nM insulin-stimulated increase
in IRS-1 phosphorylation by 71 ± 6% (mean ± S.E.,
n = 10). Cholesterol or intact caveolae are apparently
required for signal transfer from the insulin receptor to IRS-1. As an
expected consequence of the inhibition of IRS-1 phosphorylation,
cholesterol depletion also completely blocked insulin stimulation to
phosphorylation/activation of the further downstream protein kinase B
(Akt) (Fig. 8).

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Fig. 7.
Effects of cholesterol depletion on insulin
stimulation of IRS-1 phosphorylation. Freshly isolated adipocytes
were incubated without ( ) or with ( ) 10 mM
-cyclodextrin for 50 min and then with insulin at the indicated
concentration for 10 min, after which cells were subjected to SDS-PAGE
and immunoblotted with antibodies against phosphotyrosine. IRS-1 was
positively identified by immunoblotting with antibodies against IRS-1.
a, immunoblot of one representative experiment;
b, quantitation of two separate experiments.
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Fig. 8.
Effects of cholesterol depletion on insulin
stimulation of protein kinase B phosphorylation. Isolated rat
adipocytes were incubated without or with 10 mM
-cyclodextrin for 50 min when the cells were incubated with or
without 1 nM insulin for another 10 min. After SDS-PAGE the
state of protein kinase B phosphorylation was determined by
immunoblotting with antibodies against the phosphorylated form of the
protein. The experiment has been repeated three times with the same
result.
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It has been suggested that serine phosphorylation of IRS-1 acts to
inhibit the insulin receptor (35, 36), and serine/threonine phosphorylation of IRS-1 has been found to inhibit IRS-1 binding to the
insulin receptor and hence IRS-1 tyrosine phosphorylation (37, 38).
Similarly, an unknown serine kinase activity against IRS-1 was detected
in insulin-resistant cells (39), and it has been suggested that
caveolin acts as a general serine/threonine protein kinase inhibitor
(40). Cholesterol depletion had, however, no effect on the total
phosphorylation of IRS-1 or on the distribution of phosphorylation
between serine, threonine and tyrosine (Fig. 9). An enhanced degradation and lowered
levels of IRS-1 in states of insulin resistance have been reported (41,
42). We found no effect of cholesterol depletion on the total cellular
levels of IRS-1; the amount of IRS-1 protein in cholesterol-depleted rat adipocytes determined by immunoblotting after immunoprecipitation of IRS-1 and SDS-PAGE was not significantly different from control cells (107 ± 6%, mean ± S.E., n = 3). To
directly determine the effect of cholesterol depletion on IRS-1 binding
to the insulin receptor, we immunoprecipitated the insulin receptor and
looked for IRS-1. The effect of insulin to increase the amount of IRS-1 and tyrosine-phosphorylated IRS-1 that coimmunoprecipitated with the
insulin receptor was much reduced in cholesterol-depleted cells (Fig.
10), which suggests that IRS-1 binding
to the insulin receptor is inhibited in cholesterol/caveolae-depleted
cells.

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Fig. 9.
Effects of cholesterol depletion on IRS-1
serine/threonine phosphorylation. Isolated rat adipocytes were
incubated with [32P]phosphate and then with 10 mM -cyclodextrin for 50 min, when cells were lysed and
immunoprecipitated with antibodies against IRS-1. After SDS-PAGE and
transfer to PVDF-membrane (a), total
32P-phosphorylation was analyzed by phosphorimaging and
IRS-1 protein by immunoblotting with antibodies against IRS-1.
Indicated are the molecular masses of reference proteins (Rainbow,
Amersham Pharmacia Biotech). b, the
32P-phosphorylated IRS-1 protein band was cut out and the
protein partially hydrolyzed in HCl. Phosphoamino acids were separated
by silica thin-layer electrophoresis and analyzed by phosphorimaging.
Lane 1, control incubation; lane
2, -cyclodextrin-treated; lane 3,
ninhydrin stain of internal standards.
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Fig. 10.
Effect of cholesterol depletion on IRS-1
binding to the insulin receptor. Isolated rat adipocytes were
incubated without or with or without 10 mM -cyclodextrin
for 50 min when the cells were incubated with or without 1 nM insulin for another 10 min. Cells were homogenized in
lysis buffer (see "Experimental Procedures"), without detergent,
containing 100 µM genistein, centrifuged at 4000 × g for 10 min to remove fat, and immunoprecipitated with
polyclonal anti-insulin receptor antibodies as under "Experimental
Procedures." Immunoblotting against IRS-1 (upper
panel) or against phosphotyrosine (PY)
(lower panel). Indicated is the molecular mass of
reference protein (Rainbow, Amersham Pharmacia Biotech).
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Lack of Effect of Cholesterol Depletion on MAP Kinase
Pathway--
IRS-1 is believed to transmit insulin signaling for
metabolic as well as mitogenic control. However, there is a redundancy among insulin receptor substrates and mitogenic control by insulin has
in some cell-types been described via, e.g., the protein
Shc. It has also been shown that cholesterol depletion of fibroblasts activates the MAP kinase pathway and epidermal growth factor causes its
hyperactivation (43). We therefore examined the effect of cholesterol
depletion of rat adipocytes on the extracellular signal-related kinases
(ERK1 and ERK2) of the MAP kinase pathway. Extensive cholesterol depletion (60% reduction) did not by itself have an effect on ERK1/2
phosphorylation (Fig. 11). Moreover,
insulin enhanced the phosphorylation/activation of ERK1/2 to the same
extent whether cholesterol was depleted or not (Fig. 11). It was
verified in the same experiments that insulin-stimulated
tyrosine-specific phosphorylation of IRS-1 was inhibited.

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Fig. 11.
Effects of cholesterol depletion on insulin
stimulation of ERK1 and ERK2 phosphorylation. Isolated rat
adipocytes were incubated without or with 10 mM
-cyclodextrin for 50 min when the cells were incubated with the
indicated concentration of insulin for another 10 min. After SDS-PAGE,
the state of ERK1/2 phosphorylation was determined by immunoblotting
with antibodies against the phosphorylated form of the two proteins.
The experiment was repeated five times with similar results.
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In contrast to ERK1/2, the cholesterol depletion strongly enhanced the
state of Shc 52-kDa protein tyrosine phosphorylation (Fig.
12). Inclusion of the protein tyrosine
kinase inhibitor genistein in the lysis buffer and during
immunoprecipitation did not affect the increase in Shc phosphorylation,
making a postlysis artifact unlikely. Insulin on the other hand had no
effect on Shc phosphorylation, neither before nor after cholesterol
depletion, even at a supraphysiological insulin concentration (Fig.
12), in accordance with recent findings in rat adipocytes (44).

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Fig. 12.
Effects of cholesterol depletion on Shc
tyrosine phosphorylation. Isolated rat adipocytes were incubated
without or with 10 mM -cyclodextrin for 50 min, when the
cells were incubated with the indicated concentration of insulin for
another 10 min. Shc was immunoprecipitated, and after SDS-PAGE the
state of Shc tyrosine phosphorylation was determined by immunoblotting
with antibodies against phosphotyrosine. The identity of Shc 52-kDa
protein was determined by immunoblotting with antibodies against Shc
(no phosphotyrosine corresponding to Shc 46-kDa protein was detected).
a, immunoblot against phosphotyrosine. Indicated is the
molecular mass of reference protein (Rainbow, Amersham Pharmacia
Biotech). b, quantitation of three separate experiments.
Incubation with 1 nM insulin as indicated. Each experiment
was normalized by setting the control without insulin to 100%. Figure
shows mean ± S.E. (n = 3).
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 |
DISCUSSION |
-Cyclodextrin is a widely used tool for control of cellular
levels of cholesterol through its ability to extract cholesterol from
the plasma membrane of intact cells. Without itself incorporating into
the membrane,
-cyclodextrin selectively extracts cholesterol from
the surface of the cells (45, 46). We have shown previously that the
inhibition of insulin signaling by cholesterol extraction using
-cyclodextrin was reproduced by cholesterol oxidation using cholesterol oxidase (6). Moreover, cholesterol-loaded
-cyclodextrin did not inhibit insulin signaling (6), attesting against
cholesterol-unrelated effects on insulin signaling by the
-cyclodextrin treatment. We now demonstrate that a reduction of
adipocyte plasma membrane cholesterol concentration with
-cyclodextrin in a dose-dependent manner affected the
structural integrity of caveolae in the plasma membrane. Eventually,
the caveolar structure was lost by cholesterol depletion as determined
by transmission electron microscopy of plasma membranes. Likewise,
insulin signaling to enhanced glucose uptake was progressively
inhibited in an apparently linear manner by cholesterol depletion.
Hence, there was a correlation between the level of plasma membrane
cholesterol, caveolae structural integrity, and insulin stimulation of
glucose transport. Earlier studies have indicated that experimental
modulation of membrane physical properties affects insulin signaling
(47-49), but caveolae or cholesterol levels were not examined.
Cholesterol depletion and loss of the caveolae structures in the plasma
membrane did not reduce the amount of insulin receptors in the
membrane, or their affinity for insulin, as determined by
125I-insulin binding to intact adipocytes, by
immunogold-electron microscopy, or by immunoblotting for the receptor
in isolated plasma membranes after SDS-PAGE. Nor was insulin receptor
interaction with caveolin affected as shown by coimmunoprecipitation
and by electron microscopy. Insulin-stimulated autophosphorylation of the insulin receptor was not affected by cholesterol depletion either
of intact cells or of isolated caveolae. This is compatible with the
demonstrated insulin-responsiveness of the detergent-solubilized receptor (50). In contrast, cholesterol has been found to be necessary
for ligand binding (51) or receptor activity (52), with GalaninR2 and
the nicotinic acetylcholine receptor, respectively.
Hence, intact caveolae are necessary not for the insulin receptor to
bind insulin or to signal per se, but for the further downstream propagation of the signal. Activation of the immediate downstream protein IRS-1 was clearly abrogated, since after cholesterol depletion its insulin-stimulated tyrosine phosphorylation was inhibited. As a consequence, the further downstream propagation of the
signal to protein kinase B phosphorylation was also blocked. There is a
redundancy of IRS proteins in mediating metabolic control by insulin;
IRS-2, -3, and -4 have also to varying degrees been implicated,
although IRS-4 appears to have a very restricted tissue expression. The
fact that cholesterol depletion inhibits the end effects of metabolic
signal transduction suggests that insulin receptor interaction with all
involved IRS proteins was inhibited.
Insulin-resistant states and reduced insulin-stimulated tyrosine
phosphorylation of IRS-1 have been associated with reduced levels of
IRS-1 (41, 42) or with serine-phosphorylation of IRS-1 (37, 38). We
have ruled out these possibilities for the effect on IRS-1 of caveolae
destruction/cholesterol depletion. A pleckstrin homology and
phosphotyrosine-binding domain of IRS-1 have been shown to direct
binding to the insulin receptor (53, 54). We assessed if IRS-1 binding
to the insulin receptor was affected by cholesterol/caveolae depletion.
The reduced amount of IRS-1 that coprecipitated with anti-insulin
receptor antibodies suggests that cholesterol/caveolae are required for
IRS-1 binding to the receptor. The exact function of
caveolae/cholesterol in insulin receptor IRS-1 interaction in intact
cells will require further investigation.
Interestingly, our findings demonstrate that, although intact
caveolae/caveolar cholesterol was necessary for metabolic control, it
was not required for insulin's mitogenic control via ERK1 and ERK2 and
the MAP kinase pathway. Shc has been suggested to dominate over IRS-1
in mediating insulin's control via the MAP kinase pathway in some cell
types (55-58), but apparently not in, e.g., skeletal muscle
(59). Our findings demonstrate that, although Shc is present, insulin
does not affect Shc in isolated adipocytes and Shc does not control ERK
and the MAP kinase pathway in adipocytes. Lack of insulin effect on Shc
phosphorylation in adipocytes was recently described (44). The
differential effects of caveolae destruction on insulin IRS/metabolic
and ERK/mitogenic control indicate that caveolae may have a role in
insulin signal sorting for metabolic versus mitogenic
control. Hence, in cell types with no or little caveolae insulin
signaling may be mainly via the MAP kinase pathway, at the expense of
IRS-1-mediated metabolic signaling. It remains to identify the
protein(s) which transmits insulin receptor activation of the MAP
kinase pathway and is immune to caveolae destruction (e.g.
the insulin receptor substrates Gab1 (Ref. 60),
p62dok (Ref. 61), c-Cbl (Ref. 62), or Tub (Ref.
63)).
The dependence of the insulin receptor on intact caveolae/cholesterol
for transmission of its metabolic signals stands in contrast to other
tyrosine kinase receptors, which are inhibited by
cholesterol/caveolae/caveolin: Cholesterol depletion (using cyclodextrin) or caveolin-1 depletion (using caveolin-1 antisense expression) therefore constitutively activated the p42/44 MAP kinase
cascade (43, 64), and epidermal growth factor caused hyperactivation of
ERK in cholesterol-depleted cells (43). The adipocyte p42/44 ERK1 and
ERK2 were, however, not phosphorylated/activated by cholesterol
depletion. Cholesterol depletion of adipocytes did not significantly
reduce the amount of caveolin in the plasma membrane (herein), nor the
amount of caveolin associated with the insulin receptor (herein),
whereas depletion or oxidation of cholesterol was found to reduce
plasma membrane-localized caveolin in fibroblasts (43, 65) and in MDCK
cells (66). It can be argued that, in our experiments, the adipocyte
plasma membrane concentration of cholesterol and caveolae were reduced,
but not caveolin, and hence we do not see constitutive activation or
hyperactivation of ERK in cholesterol-depleted adipocytes. However,
this does not explain the reduced ability of active insulin receptors
to tyrosine phosphorylate IRS-1. Moreover, caveolin has been shown to
activate the isolated insulin receptor (20). The strong increase in Shc
phosphorylation after cholesterol depletion may be explained by
activation of a Shc kinase other than the insulin receptor (an
interpretation supported by the lack of insulin effect on Shc
phosphorylation in adipocytes) or by cholesterol depletion making Shc
accessible as a substrate for kinases.
A pronounced threshold effect with caveolae structures vanishing after
more than 30% reduction of the normal cellular cholesterol concentration was described in MDCK cells (66). In MA104 epithelial cells, the number of caveolae was reduced to 10% of normal after lowering total cellular cholesterol by 55% through inhibition of
endogenous cholesterol synthesis (67). In the 3T3-L1 adipocytes, we
found that the stucture of caveolae was not affected after 20%
reduction, was partially destroyed after 40%, and was almost completely destroyed after 50% reduction of the plasma membrane cholesterol. In MDCK cells, caveolin expression was correlated to the
cellular cholesterol concentration (66), but was in rat or 3T3-L1
adipocytes apparently not affected by a 50% reduction of the plasma
membrane concentration of cholesterol. This discrepancy may be due to
the different cell types and/or to the acute effects studied herein and
the long term (overnight to several days) effects of cholesterol
reduction examined in the MDCK cells. Ilangumaran and Hoessli (68)
found that methyl-
-cyclodextrin treatment of lymphocytes caused a
release of apparently raft-associated proteins and lipids, but not
caveolin, from the cells. The adipocytes, however, remained intact
after
-cyclodextrin extraction of up to 50% (3T3-L1 adipocytes
(herein)) or 60% (rat adipocytes (Ref. 6)) of the plasma membrane
cholesterol, when the cells were able to regain insulin control upon
cholesterol replenishment (6). The insulin receptor, moreover, was
quantitatively retained in the plasma membrane after cholesterol
depletion. However, extraction of more than 60% of the plasma membrane
cholesterol caused the adipocytes to easily rupture (data not shown).
Apparently the cholesterol extraction made the adipocyte membranes brittle.
A uniform finding in insulin resistance has been that IRS-1 tyrosine
phosphorylation in response to insulin is decreased in adipocytes and
skeletal muscle in obesity and type 2 diabetes (5). Indeed, our
findings demonstrate that cholesterol depletion induces insulin
resistance with decreased insulin-stimulated IRS-1 tyrosine
phosphorylation. It is also interesting to note that in skeletal muscle
of type 2 diabetic patients insulin stimulation to phosphorylation of
MAP kinase was normal, while insulin-stimulated tyrosine
phosphorylation of IRS-1 was severely impaired (69, 70). Similarly, in
the vasculature of insulin-resistant obese Zucker fa/fa rats,
insulin-stimulated tyrosine phosphorylation of IRS-1/2 were reduced,
while the phosphorylation of ERK-1/2 were equal to that in normal rats
(71). Insulin-resistant state induced by
3-adrenergic
stimulation blocked insulin-stimulated phosphorylation of IRS1/2
without affecting MAP kinase (72). Severely insulin-resistant patients
with pseudoacromegaly had impaired insulin activation of
IRS-1-associated phosphatidylinositol 3-kinase, with intact
activation/phosphorylation of MAP kinase (73). Furthermore,
glucocorticoid-induced insulin resistance blocked protein
synthesis-stimulation by insulin without affecting the MAP kinase
pathway (74). These findings are similar to the insulin resistance
incurred on adipocytes by cholesterol depletion.
In conclusion, our findings point to the caveola as forming a hub,
which is critical for insulin's cellular control, apparently with the
potential to sort for metabolic versus mitogenic control. Our findings, moreover, suggest that caveolae dysfunction can be
involved in the pathogenesis of insulin resistance.