 |
INTRODUCTION |
Lipid rafts are plasma membrane microdomains, principally composed
of cholesterol and sphingolipids, which form liquid-ordered domains of
decreased membrane fluidity (1-6). With integration of caveolins into
lipid rafts, these microdomains will form caveolae, which are
flask-shaped vesicular invaginations in the plasma membrane (2, 4, 7).
Caveolae are a specific form of lipid rafts and are now considered to
be broader than just vesicular membrane invaginations (1, 7). The long
saturated lipid tails of sphingolipids impart the lipid rafts a high
degree of order further stabilized by interacting cholesterol. This
property leads a light buoyant density on sucrose density gradient centrifugation.
Cholesterol is an essential component in lipid rafts/caveolae. In
caveolae, cholesterol binds directly to caveolins and facilitates the
integration of caveolins into membrane (8, 9). Depletion of cellular
cholesterol with cholesterol-binding reagents, such as
methylcyclodextrin or filipin, will remove cholesterol from lipid
rafts/caveolae, dissemble the striated caveolin coats, and eventually
lead to the disruption of both lipid rafts and caveolae (1, 7,
10-15).
During recent years, more and more reports confirmed that many
signaling molecules are found to be enriched in lipid rafts/caveolae, which serve as platforms and play an important role in regulating signal cascade (1-4, 7, 16, 17). Signal molecules, such as
heterotrimeric G-proteins (18), protein kinase C (19, 20), Shc (21),
SOS (22), Raf1 (22, 23), and Src family tyrosine kinases (19, 24-26),
are recruited into caveolae by caveolins, which, through the
scaffolding domain, interact with the caveolin-binding motifs in these
signal molecules (17). These clusters of signal molecules can form
"preassembled signaling complexes" on the plasma membrane. In
addition, many growth factor receptors (epidermal growth factor
receptor, platelet-derived growth factor receptor, insulin receptor,
etc.) (12, 20, 21, 27, 29) are found to be located in lipid
rafts/caveolae. Thus, the enrichment of receptors and signal molecules
in lipid rafts/caveolae enables them to be in close contact with each
other and makes lipid rafts/caveolae the gateways for signals entering
into the cells.
Lipid rafts/caveolae are indicated to be important for insulin receptor
signaling (30, 31). Insulin receptors are found to be located in
caveolae of adipocyte plasma membrane (27), and many signal molecules
involved in insulin receptor signal cascade are also found to be
enriched in caveolin-enriched plasma membrane domain (32). In 3T3-L1
adipocytes, caveolin-1 is phosphorylated by insulin receptor (33, 34)
and is an activator of insulin receptor signaling (35). In addition,
caveolin-enriched lipid raft microdomains and lipid rafts are required
for insulin signaling and Glut4 translocation (36-39). These results
provide compelling evidence that lipid rafts/caveolae are essential for
insulin receptor signaling.
IGF-11 receptor tyrosine
kinase signaling (along with glucocorticoid and cAMP signaling) is
required for 3T3-L1 preadipocyte differentiation induction (40-45).
High level insulin or IGF-1 at physiological concentration activates
the IGF-1 receptor on the plasma membrane, leading to the initiation of
the differentiation program (40, 41). Two events occur after the
activation of IGF-1 receptor in 3T3-L1 preadipocytes: mitotic clonal
expansion and adipocyte differentiation. Previously, we have identified that these two events are both activated by the IGF-1 receptor signaling and can be separately blocked without affecting the other
(41, 46, 47). The mitotic clonal expansion is activated by IGF-1
receptor through a signal pathway involving the activation of ERK1 and
-2, whereas adipocyte differentiation is initiated through the
reversible tyrosine phosphorylation of the adapter protein c-Crk by
IGF-1 receptor tyrosine kinase and tyrosine phosphatase. These results
suggest that IGF-1 receptor activates two separate signal pathways in
3T3-L1 preadipocytes simultaneously.
Although some of the cellular functions for IGF-1 receptor and insulin
receptor are different, structural and signaling similarities between
these two receptors have long been recognized. Recently, it has been
reported that IGF-1 may also induce caveolin-1 tyrosine phosphorylation
and its translocation in the lipid rafts (48). Since in 3T3-L1 cells
IGF-1 receptor activates more than one signal pathway at the same time,
it is likely that IGF-1 receptor on plasma membrane interacts or
cross-talks with several signaling pathways. With their preassembled
signaling complexes on the intracellular side, lipid rafts/caveolae
provide the structural foundation for simultaneous activation or
cross-talking of multiple signal pathways by IGF-1 receptor.
In the present study, we reported that IGF-1 receptor was located in
lipid rafts/caveolae in 3T3-L1 preadipocyte and adipocyte. The
integrity of lipid rafts/caveolae was essential for IGF-1 receptor
signal transduction during 3T3-L1 preadipocyte differentiation induction. Disruption of lipid rafts/caveolae by cholesterol depletion effectively blocked the downstream signaling of IGF-1 receptor but not
IGF-1 receptor activation itself.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Anti-IGF-1 receptor
-subunit antibody was
purchased from Oncogene Research Products. Anti-phosphotyrosine
antibody was from Upstate Biotechnology, Inc. (Lake Placid, NY).
Anti-caveolin-1
isoform, anti-clathrin light chain, anti-insulin
receptor
-subunit, anti-ERK, and anti-p-ERK (against the critical
Tyr residue-phosphorylated peptide) antibodies were from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Anti-c-Crk antibody was from
Transduction Laboratories. Horseradish peroxidase conjugate, FITC
conjugate, or rhodamine conjugate secondary antibodies,
-methylcyclodextrin, filipin, xylazine, dexamethasone,
1-methyl-3-isobutylxanthine, and insulin were from Sigma. Dulbecco's
modified Eagle's medium and protein A-agarose were from Invitrogen.
Cell Culture, Differentiation Induction of 3T3-L1 Preadipocytes,
and Oil Red-O Staining--
3T3-L1 preadipocytes were cultured and
induced to differentiate following the protocol described previously
(41-44). The differentiated 3T3-L1 adipocytes were stained with Oil
Red-O to show triglyceride droplets (41).
Double Immunofluorescence Staining--
For caveolin-1 and IGF-1
receptor double immunofluorescence staining, 3T3-L1 preadipocytes were
cultured on glass coverslips. The coverslips were rinsed with
phosphate-buffered saline and fixed for 10 min in 3.7% formaldehyde
and 0.18% Triton X-100 in phosphate-buffered saline solution. The
fixed cells were incubated in blocking buffer (1% bovine serum albumin
in Tween/Tris-buffered saline) for 30 min at room temperature. The
cells were then incubated with anti-Cav-1 antibody (rabbit) and
anti-IGF-1R
antibody (mouse) in blocking buffer for 1 h at room
temperature. After washing, the coverslips were incubated with
rhodamine conjugate anti-mouse and FITC conjugate anti-rabbit secondary
antibodies. The cells were visualized by confocal microscope
(Bio-Rad).
For caveolin-1 and clathrin double immunofluorescence staining, 3T3-L1
preadipocytes cultured on glass coverslips were induced to
differentiate into adipocytes following the standard differentiation protocol. Day 8 adipocytes on coverslips were rinsed, fixed, and blocked as mentioned before. The cells were then incubated with anti-Cav-1 antibody (rabbit) and anti-clathrin antibody (mouse) in
blocking buffer for 1 h at room temperature. After washing, the
coverslips were incubated with rhodamine conjugate anti-rabbit and FITC
conjugate anti-mouse secondary antibodies. The cells were visualized by
confocal microscope.
Sodium Carbonate Extraction and Sucrose Density Gradient
Fractionation of Lipid Rafts/Caveolae--
The experiments
were carried out following the detergent-free protocol developed by
Song et al. (49). Briefly, two 10-cm monolayers of 3T3-L1
preadipocytes or adipocytes with different treatment as indicated in
the Figs. 2 and 3 were washed with ice-cold phosphate-buffered
saline, scraped into 2 ml of 0.5 M
Na2CO3, pH 11.0, and sonicated. The homogenized
cell sample was mixed with an equal volume of 90% sucrose solution in
Mes-buffered saline (25 mM Mes, pH 6.5, 0.15 M
NaCl), placed at the bottom of an ultracentrifuge tube, and overlaid
with 4 ml of 35% sucrose and 4 ml of 5% sucrose in Mes-buffered
saline containing 0.25 M Na2CO3.
The gradient was centrifuged at 39,000 rpm for 20 h in a SW41
rotor (Beckman). Fractions were collected from the top of the gradient,
and proteins were precipitated with 12.5% trichloroacetic acid,
dissolved in 1× Laemmli SDS sample buffer containing 20 mM
dithiothreitol, and subjected to Western immunoblot.
Immunoprecipitation and Western Immunoblot--
10-cm 3T3-L1
cell monolayers were treated as described in Fig. 4. Cell
extracts were prepared and immunoprecipitated as described before (41).
500 µg of cell extract was immunoprecipitated with 1 µg of
anti-IGF-1R
antibody. The immunoprecipitated samples were subjected
to SDS-PAGE and Western blot analysis, which was conducted as described
previously (46).
Analysis of IGF-1 Receptor Signaling--
Two-day postconfluent
3T3-L1 preadipocytes were fed with Dulbecco's modified Eagle's medium
containing 10% fetal bovine serum for 48 h to minimize the serum
effect. 10 mM
-methylcyclodextrin was added to the cells
for 45 min to deplete the cellular cholesterol and disrupt lipid
rafts/caveolae (12-14). The cells were then treated with 10 µg/ml
insulin (to activate the IGF-1 receptor) or 0.5 mM
3-isobutyl-1-methylxanthine (to inhibit cAMP phosphodiesterase and
increase cellular cAMP concentration). At the indicated time point as
described in Fig. 7, cells were harvested for
immunoprecipitation analysis of IGF-1 receptor and its
autophosphorylation or harvested directly in 1× boiling SDS sample
buffer with 20 mM dithiothreitol for analysis of ERK and
its activation. In control cells, the experiments were conducted in the
same way without the
-methylcyclodextrin treatment.
Effect of
-Methylcyclodextrin, Filipin, and Xylazine on the
3T3-L1 Preadipocyte Differentiation and Mitotic Clonal
Expansion--
Postconfluent 3T3-L1 preadipocytes were pretreated with
cholesterol binding reagents (
-methylcyclodextrin or filipin) or nonspecific lipid binding reagent (xylazine) for 24 h and then were induced to differentiate following the standard differentiation induction protocol. Lipid binding reagents were supplemented in the
differentiation induction medium during the first 4 days
(i.e. 2 days of induction with 1-methyl-3-isobutylxanthine,
dexamethasone, and insulin and an additional 2 days with insulin alone)
and removed after day 4. During differentiation induction, cell numbers
were counted to analyze the mitotic clonal expansion. By day 8, the cells were stained with Oil Red-O.
 |
RESULTS |
Co-localization and Association of IGF-1 Receptor with
Caveolin-1--
Since they are predominantly found in caveolae on
plasma membrane (7), caveolins could be used to indicate caveolae on cell surface. Caveolin-1, the most abundant form of caveolins in
caveolae, has two isoforms,
and
, which are produced by RNA
alternative splicing (50). Using double immunofluorescence staining
with rabbit anti-caveolin-1 (reacting only with
isoform) and mouse
anti-IGF-1 receptor
-subunit antibodies, we could simultaneously reveal the location of caveolin-1 and IGF-1 receptor on the cell surface. As shown in Fig. 1, A
and B, IGF-1 receptor was co-localized with caveolin-1
protein in 3T3-L1 preadipocytes. To validate this double
immunofluorescence staining result, localization of caveolin-1 and
clathrin (the component protein in coated pits, which are different
plasma membrane structures from caveolae) was analyzed. Since clathrin
was highly expressed in 3T3-L1 adipocyte, but not preadipocyte (Fig.
3A), 3T3-L1 adipocytes were used in double
immunofluorescence staining for caveolin-1 and clathrin. Fig. 1,
C and D, showed the confocal images of caveolin-1
and clathrin double immunofluorescence in 3T3-L1 adipocytes. Under the
confocal microscope, ball-shaped 3T3-L1 adipocytes were revealed as a
ring. Caveolin-1 staining and clathrin staining were quite different,
and their two images were not well overlapped.

View larger version (85K):
[in this window]
[in a new window]
|
Fig. 1.
Co-localization of IGF-1 receptor with
caveolin-1. Double immunofluorescence staining was conducted on
3T3-L1 cells as described under "Experimental Procedures."
A and B, IGF-1 receptor was detected by rhodamine
red fluorescence (IGF-1R) and caveolin-1 by FITC green
fluorescence (Caveolin-1). These two images were merged into
one overlapped picture (merged) to show the relative
location of IGF-1 receptor and caveolin-1. A and
B, high and low magnification view, respectively.
C and D, caveolin-1 was detected by rhodamine red
fluorescence (Caveolin-1), and clathrin was detected by FITC
green fluorescence (Clathrin). The overlapped picture of
these two images is shown (merged). C, high
magnification view; D, low magnification view.
|
|
Due to the large difference in the protein amount of IGF-1 receptor and
caveolin-1 (on plasma membrane, caveolin-1 is much more abundant than
IGF-1 receptor), the immunofluorescence signal of IGF-1 receptor had to
be highly amplified by the confocal microscope to match that of
caveolin-1. Thus, there was a possibility that the cross-reactive
signal from rhodamine anti-mouse IgG secondary antibody to the rabbit
anti-caveolin primary antibody might interfere with the IGF-1 receptor
signal. To rule out this possibility, sucrose density gradient
isolation of lipid rafts/caveolae was used to confirm the result of
double immunofluorescence. The results from sodium carbonate extraction
and sucrose density gradient separation of lipid rafts/caveolae
confirmed the immunofluorescence result. As indicated in Fig.
2, almost all of the IGF-1 receptors were
extracted and isolated in the same fractions of lipid rafts/caveolae (identified by the presence of caveolin-1), whereas the majority of
cytosolic protein c-Crk was in the high density fractions. Treatment with
-methylcyclodextrin significantly disrupted lipid rafts/caveolae, since the protein amounts of caveolin-1 and IGF-1 receptor were decreased in the fractions corresponding to lipid rafts/caveolae (Fig. 2). In addition, a significant amount of caveolin-1 protein appeared in the high density fractions (Fig. 2), and
IGF-1 receptor could also be detected in the high density fractions
with a large amount of sample loaded on the SDS-PAGE (results not
shown).

View larger version (49K):
[in this window]
[in a new window]
|
Fig. 2.
Association of IGF-1 receptor with lipid
rafts/caveolae by density gradient centrifugation. Two-day
postconfluent 3T3-L1 preadipocytes were treated with or without 10 mM -methylcyclodextrin for 45 min, and the cells were
subjected to Na2CO3 extraction and
centrifugation as described under "Experimental Procedures."
Samples were then analyzed by Western blot. Control, cells
without any treatment; +MCD, cells treated with
-methylcyclodextrin. Fraction No. 1-18, fractions
collected from the density gradient. Fraction 1 is the top of the gradient. Cav-1, Western blot by
anti-caveolin-1 isoform antibody; IGF-1R , Western
blot by anti-IGF-1 receptor -subunit; c-Crk, Western blot
by anti-c-Crk antibody. The arrows indicate the proteins
detected by the antibodies. All of these Western blots used the samples
from the same density gradient centrifugation.
|
|
It has been reported that insulin receptor is located in lipid
rafts/caveolae on adipocyte plasma membrane (26, 51). We compared the
location of IGF-1 receptor with that of insulin receptor in 3T3-L1
adipocyte, because insulin receptor exists in high amounts only in
3T3-L1 adipocytes, not preadipocytes. In addition, clathrin, a major
protein component of coated pits on cell surface, was also abundant in
adipocytes (Fig. 3A). Using
clathrin protein as a control, we could analyze the relationship of
IGF-1 receptor with clathrin-coated pits, which are also cell surface
membrane structure but have not been associated with membrane
signal transduction. As shown in Fig. 3B, caveolin-1,
insulin receptor, and IGF-1 receptor were all separated by density
gradient centrifugation into the same low density fractions, whereas
clathrin, apparently without any association to a particular membrane
domain, was distributed into all of the fractions. This result clearly
indicated that IGF-1 receptor, like insulin receptor, located in lipid
rafts/caveolae but not in the clathrin-coated pits.

View larger version (45K):
[in this window]
[in a new window]
|
Fig. 3.
Association of IGF-1 receptor and insulin
receptor with lipid rafts/caveolae but not clathrin-coated pits in
3T3-L1 adipocytes. A, protein amount of clathrin in
adipocytes and preadipocytes. 3T3-L1 preadipocytes and adipocytes were
lysed directly in Laemmli SDS sample buffer, and the samples were
subjected to SDS-PAGE and immunoblotted with anti-clathrin light chain
antibody. Preadip. and Adip., protein samples
from 3T3-L1 preadipocytes and adipocytes, respectively.
Clathrin, the clathrin protein light chain. B,
fully differentiated 3T3-L1 adipocytes (day 8 cells) were serum-starved
for 24 h and then treated with or without 1 µg/ml insulin for
2 h. The cells were then subjected to extraction, density gradient
centrifugation, and Western blot as described in the legend to Fig. 2.
Control, cells without any treatment; +insulin,
cells treated with insulin. Fraction No. 1-20, fractions
collected from the density gradient. Fraction 1 is the top of the gradient. Cav-1, Western blot by
anti-caveolin-1 isoform antibody; IR , Western blot by
anti-insulin receptor -subunit; IGF-1R , Western blot
by anti-IGF-1 receptor -subunit; Clathrin, Western blot
by anti-clathrin light chain; c-Crk, Western blot by
anti-c-Crk antibody. The arrows indicate the proteins detected by the
antibodies. All of these Western blots used the samples from the same
density gradient centrifugation.
|
|
In order to ascertain whether IGF-1 receptors directly interact with
caveolin, IGF-1 receptors were extracted from 3T3-L1 cells and
immunoprecipitated by anti-IGF-1 receptor antibody. Fig.
4C shows that
immunoprecipitation of IGF-1 receptor brought down caveolin-1 protein.
Furthermore, this interaction between IGF-1 receptor and caveolin-1 was
independent from the receptor's activation (Fig. 4, A and
C). There was no significant difference between the
association of caveolin-1 to the unactivated quiescent receptor (0 min,
Fig. 4C) or activated receptor with tyrosine autophosphorylation (30 min to 4 h; Fig. 4C). This
immunoprecipitation result suggested the direct physical interaction
between these two proteins. With the density gradient finding that the
majority of IGF-1 receptor was located in caveolae/lipid rafts, it was most likely that interaction of IGF-1 receptor with caveolin was a
general event for these two proteins.

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 4.
Association of IGF-1 receptor with caveolin-1
protein. A, Western blot analysis of IGF-1 receptor
autophosphorylation after stimulation with high level insulin.
+Insulin, 2-day postconfluent 3T3-L1 preadipocytes
stimulated with insulin; 0, control prior to insulin
stimulation; 30', 4h, 8h, and
24h, 30-min, 4-h, 8-h, and 24-h insulin stimulation time,
respectively. IP, immunoprecipitation; IB,
Western blot. -IGF-1R , antibody against IGF-1 receptor
subunit; -P-Tyr, antibody against phosphotyrosine.
B, extraction of IGF-1 receptor and caveolin-1 by Triton
X-100 and ultrasonic homogenization. Cells were lysed with Triton
buffer as described under "Experimental Procedures" and homogenized
with 3× ultrasonic pulse. P, insoluble material;
S, soluble extract. C, caveolin-1
co-immunoprecipitation with IGF-1 receptor. B,
immunoprecipitation with lysis buffer as a blank control; C,
cell lysate without immunoprecipitation as control for caveolin-1.
+Insulin, 0, 30' 2h and 4h, same as in A. IgG, the antibody used for immunoprecipitation recognized by
the secondary antibody in Western blot.
|
|
Blockade of 3T3-L1 Preadipocyte Differentiation Induction by Lipid
Rafts/Caveolae Disruption--
To analyze the function of
lipid rafts/caveolae in IGF-1 receptor signaling, cholesterol binding
reagent,
-methylcyclodextrin or filipin, was used to deplete the
cellular cholesterol, which is essential for the structure integrity of
lipid rafts/caveolae. As a control to cholesterol-binding reagents, a
general lipid binding reagent, xylazine, was also used in the
experiments. Following the reports by other researchers (12-14), 10 mM
-methylcyclodextrin was used to treat 3T3-L1
preadipocytes. After the treatment, lipid rafts/caveolae were
significantly disrupted, as indicated by dislocation of caveolin in
density gradient separation (Fig. 2). As a control, xylazine had no
effect on the integrity of lipid rafts/caveolae (result not shown).
Thus, only cholesterol-binding reagent (
-methylcyclodextrin), but
not general lipid-binding reagent (xylazine), disrupted lipid rafts/caveolae.
IGF-1 receptor signaling plays a key role in inducing 3T3-L1
preadipocytes to differentiate into adipocytes (40, 41, 46). To study
the function of lipid rafts/caveolae in IGF-1 receptor signaling during
3T3-L1 preadipocyte differentiation induction, cells were induced to
differentiate in the presence of cholesterol-binding reagent
(
-methylcyclodextrin or filipin) or control reagent (xylazine). Since these reagents were used at different concentrations, the concentrations used in experiments were determined by their effect on
cell proliferation of dividing 3T3-L1 preadipocytes. In order to
compare the results of these reagents, we used these reagents at the
concentrations that had similar effect on cell proliferation (Fig. 6,
A, C, and E). At high concentrations,
they all severely inhibited cell proliferation of exponentially growing
cells and did not allow cells to survive the differentiation induction
process (results not shown). Therefore, in our experiments, these
reagents were used at concentrations at which cells could proliferate
and survive the differentiation induction process (Fig. 6).
Before the differentiation induction, cells were pretreated with
-methylcyclodextrin or filipin at the indicated concentrations for
24 h to deplete cellular cholesterol. With this treatment (e.g. 4 mM
-methylcyclodextrin for 24 h), significant amounts of lipid rafts/caveolae were disrupted (results
not shown).
-Methylcyclodextrin or filipin was then kept in the
differentiation induction medium to maintain a low cellular cholesterol
level. Since the major triglyceride accumulation in the 3T3-L1
adipocyte differentiation process occurred after day 4, the reagent was
removed from the culture medium after the initial 4-day induction
period to prevent any side effect on the lipid accumulation. The
effect on 3T3-L1 adipocyte differentiation by these lipid-binding
reagents was evaluated by the triglyceride accumulation in the cells
after differentiation induction (Fig.
5A). At 2.5 mM,
-methylcyclodextrin significantly reduced the triglyceride
accumulation in the cells. At 4 mM
-methylcyclodextrin
almost completely inhibited the adipocyte differentiation. For filipin,
a similar effect on adipocyte differentiation was observed (Fig.
5B). At 1.5 µg/ml (~2.3 µM), filipin
completely blocked the induced adipocyte differentiation. At a
concentration of 1 µg/ml (~1.5 µM), filipin still
exhibited some inhibitory effect on adipocyte differentiation (results
not shown). In contrast, xylazine used in the same condition as a
control had no detectable effect on adipocyte differentiation (Fig.
5C). Even at a concentration of 100 µg/ml (~450
µM), which severely inhibited cell proliferation of
nonconfluent 3T3-L1 preadipocyte, xylazine still had no adversary effect on 3T3-L1 preadipocyte differentiation (results not shown). It
was clear that cholesterol binding reagents,
-methylcyclodextrin and
filipin, blocked the IGF-1 receptor signal induced adipocyte differentiation, whereas nonspecific lipid binding reagent, xylazine, did not affect the differentiation process.

View larger version (76K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of cholesterol-binding reagents or
general lipid-binding reagent on hormonal induced 3T3-L1 adipocyte
differentiation. A, effect of -methylcyclodextrin on
3T3-L1 adipocyte differentiation. Postconfluent 3T3-L1 preadipocytes
were induced to differentiation in the presence of
-methylcyclodextrin as described under "Experimental
Procedures." The -methylcyclodextrin was used at concentrations
indicated. Day 8 cells were stained with Oil Red-O to reveal the
triglyceride droplets. Control, standard differentiation
induction. + -MCD, cells induced in the presence of
-methylcyclodextrin. 2.5 mM, 3.0 mM, and 4.0 mM,
-methylcyclodextrin concentrations. B, effect of filipin
on 3T3-L1 adipocyte differentiation. All of the experiments were
carried out in the same conditions as described for A. +Filipin, cells induced in the presence of filipin.
1.2 µg/ml, 1.35 µg/ml, and 1.5 µg/ml, filipin concentrations.
Control, standard differentiation induction. C,
effect of xylazine on 3T3-L1 adipocyte differentiation. All of the
experiments were carried out in the same conditions as described for
A. +Xylazine, cells induced in the presence of
xylazine. 5 µg/ml, 20 µg/ml, and 50 µg/ml, xylazine concentrations.
Control, standard differentiation induction.
|
|
During 3T3-L1 preadipocyte differentiation induction, growth-arrested
confluent cells were induced to reenter into a cell division process
called mitotic clonal expansion. This mitotic clonal expansion is
induced only by IGF-1 receptor signal (46). Since IGF-1 receptor
signal-induced adipocyte differentiation was blocked by lipid
rafts/caveolae disruption (Fig. 5), mitotic clonal expansion was also
inhibited in the similar condition. Our results indicated that the
presence of cholesterol binding reagents (
-methylcyclodextrin or
filipin) in the differentiation induction medium markedly decreased the
IGF-1 receptor signaling-induced mitotic clonal expansion (Fig.
6, B and D). At 4 mM
-methylcyclodextrin, mitotic clonal expansion was
completely blocked. However, exponentially growing 3T3-L1 preadipocytes
could still divide in the presence of
-methylcyclodextrin or
filipin, although the rate of cell division was slowed down, especially
at the high concentration (Fig. 6, A and C). This
result indicated that the inhibition of mitotic clonal expansion
during the 3T3-L1 preadipocyte differentiation process by
-methylcyclodextrin or filipin was due to the blockade of IGF-1 receptor signaling rather than the cell mitotic
machinery. In comparison, xylazine had a very similar effect on slowing
the proliferation rate of exponentially growing 3T3-L1 preadipocytes (Fig. 6E) but had no effect on IGF-1 receptor signal-induced
mitotic clonal expansion during the differentiation induction process (Fig. 6F). With these results, it was clear that cholesterol
binding reagents,
-methylcyclodextrin and filipin, could block both
IGF-1 receptor signal-induced adipocyte differentiation and mitotic clonal expansion, whereas the nonspecific lipid binding reagent, xylazine, had no effect on either adipocyte differentiation or mitotic
clonal expansion.

View larger version (57K):
[in this window]
[in a new window]
|
Fig. 6.
Effect of cholesterol-binding reagents or
general lipid-binding reagent on cell proliferation and mitotic clonal
expansion. A, effect of -methylcyclodextrin on cell
proliferation. Exponentially growing 3T3-L1 preadipocytes were treated
with -methylcyclodextrin at the indicated concentration (3.0 and 4.0 mM, respectively). The numbers shown here are
the average of two experiments. B, effect of
-methylcyclodextrin on mitotic clonal expansion during
differentiation induction. Cells were induced to differentiate as
described in Fig. 5A. Cell numbers at different days after
the induction were determined and normalized against that on day 0. The
results from three experiments were averaged. C, effect of
filipin on cell proliferation. Exponentially growing 3T3-L1
preadipocytes were treated with filipin at the indicated concentration
(1.0, 1.35, and 1.5 µg/ml, respectively). The numbers
shown here are the average of three experiments. D, effect
of filipin on mitotic clonal expansion during differentiation
induction. Cells were induced to differentiate as described in the
legend to Fig. 5B. Cell numbers at different days after the
induction were determined and normalized against that on day 0. The
results from three experiments were averaged. E, effect of
xylazine on cell proliferation. Exponentially growing 3T3-L1
preadipocytes were treated with xylazine at indicated concentration (5, 20, and 50 µg/ml, respectively). The numbers shown here are the
average of three experiments. F, effect of xylazine on
mitotic clonal expansion during differentiation induction. Cells were
induced to differentiate as described in the legend to Fig.
5C. Cell numbers at different days after the induction were
determined and normalized against that on day 0. The results from three
experiments were averaged.
|
|
Lipid Rafts/Caveolae Disruption Blocks Downstream
Signaling of IGF-1 Receptor but Not the Activation of Receptor
Itself--
Cellular responses (adipocyte differentiation and mitotic
clonal expansion) induced by IGF-1 receptor signal are long term effects: mitotic clonal expansion is induced in the first 48 h after hormonal stimulation, and adipocyte phenotype appears around 96 h after induction. Thus, it is difficult to analyze the
mechanism of lipid rafts/caveolae in IGF-1 receptor signaling by using
these cellular responses. Previously, we have reported that ERK1 and -2 were activated by IGF-1 receptor signaling within minutes after stimulation, and their activation was essential for mitotic clonal expansion (46). This activation of ERK1 and -2 by IGF-1 receptor signal
provides a feasible cellular response to analyze the function of lipid
rafts/caveolae in IGF-1 receptor signal transduction.
Since disruption of lipid rafts/caveolae blocked IGF-1 receptor
signal-induced adipocyte differentiation and mitotic clonal expansion,
IGF-1 receptor signaling might be blocked at the activation of IGF-1
receptor by ligand and/or the downstream signal transduction of the
activated IGF-1 receptor. If lipid rafts/caveolae are required for the
activation of IGF-1 receptor by its ligand, disruption of lipid
rafts/caveolae with cholesterol binding reagent will interfere with the
receptor activation by ligand and reduce the receptor
autophosphorylation. However, as shown in Fig.
7B, IGF-1 receptor
autophosphorylation stimulated by ligand (high level of insulin) was
not affected by the treatment of
-methylcyclodextrin. On the other
hand, disruption of lipid rafts/caveolae greatly affected the
downstream signal transduction of IGF-1 receptor. The activation of
ERK1 and -2 by IGF-1 receptor signal was significantly reduced by
-methylcyclodextrin treatment (Fig. 7C). These results suggested that in the IGF-1 receptor signaling process lipid
rafts/caveolae are required for IGF-1 receptor's downstream signal
transduction but not the activation of the receptor per
se.

View larger version (46K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of cholesterol-binding reagent,
-methylcyclodextrin, on IGF-1 receptor
signaling. The experiments were carried out as described under
"Experimental Procedures." A, samples before
immunoprecipitation were subjected to SDS-PAGE and Western blotted by
anti-IGF-1 receptor -subunit antibody to show total IGF-1 receptor.
Control, cells without -methylcyclodextrin treatment and;
+ -MCD, cells with -methylcyclodextrin treatment.
+Insulin, insulin stimulation; 0, 5,
10, and 30 min, stimulation time.
B, ligand-induced IGF-1 receptor autophosphorylation in the
presence or absence of -methylcyclodextrin. IGF-1 receptor was
immunoprecipitated from cell extracts as described under
"Experimental Procedures." Control, + -MCD,
+Insulin, and the time points (0, 1,
5, 10, and 30 min), same as
in A. IP, immunoprecipitation; IB,
Western blot; -IGF-1R , antibody against IGF-1 receptor
subunit; -P-Tyr, antibody against phosphotyrosine.
B, immunoprecipitation of lysis buffer as a blank control;
C, effect of -methylcyclodextrin treatment on ERK
activation by IGF-1 receptor or 1-methyl-3-isobutylxanthine.
Experiments were carried out as described under "Experimental
Procedures." The cells were treated in the same conditions as in
B. The ERK activation was analyzed by phosphorylation on the
activation site. Control, + -MCD,
+Insulin, and the time points (0, 1,
5, 10, and 30 min) are the
same as in B. +MIX, stimulation with
1-methyl-3-isobutylxanthine. -pERK, antibody against
tyrosine-phosphorylated ERK1 and -2; -ERK, antibody
against ERK1 and -2.
|
|
To rule out the possibility that
-methylcyclodextrin treatment
directly inhibits the kinases activating ERK1 and -2, not IGF-1
receptor signaling, the effect of
-methylcyclodextrin on ERK1 and -2 activation stimulated by 1-methyl-3-isobutylxantine was analyzed.
1-Methyl-3-isobutylxantine increases the intracellular cAMP
concentration by inhibiting cAMP phosphodiesterase, which is a plasma
membrane-independent process. Fig. 7C showed that
-methylcyclodextrin treatment had no effect on
1-methyl-3-isobutylxantine-stimulated ERK activation. The ERK enzyme
system was not adversely affected by
-methylcyclodextrin treatment.
Thus, it is clear that disruption of lipid rafts/caveolae by
cholesterol depletion only inhibited the membrane-dependent
IGF-1 receptor's downstream signaling and not the membrane-independent
intracellular cAMP signaling.
The phosphotyrosine protein band of smaller molecular
weight was insulin receptor
-subunit (Fig. 7B). It could
be detected by insulin receptor antibody (results not shown). This is
probably due to the insulin and IGF-1 hybrid receptor (52), which could be immunoprecipitated by IGF-1 receptor antibody.
 |
DISCUSSION |
In many ways, caveolae are lipid rafts enriched with structural
protein caveolins, which are the defining protein components in
caveolae. Although caveolins have three types, general types caveolin-1 and -2 and muscle-specific caveolin-3 (7), cells completely
lack caveolae in caveolin-1 knockout mice (53). This result from
caveolin-1-deficient mice has demonstrated the importance of caveolin-1
in the formation of caveolae and provided support for using caveolin-1
as an indicator for caveolae. By using immunofluorescence staining
(Fig. 1), sucrose density gradient centrifugation (Figs. 2 and 3), and
co-immunoprecipitation (Fig. 4), we have identified that IGF-1 receptor
was located in lipid rafts/caveolae in 3T3-L1 cells. To provide further
support, we have also shown that IGF-1 receptor and insulin receptor,
which has been indicated to be located in caveolae of adipocyte plasma
membrane (26, 51), were associated with the same membrane structures in
3T3-L1 adipocytes (Fig. 3B). These results provided strong
evidence that IGF-1 was located in lipid rafts/caveolae in 3T3-L1
preadipocytes and adipocytes.
It was consistently observed that, besides in caveolae, IGF-1 receptor
also appeared to be in the membrane structure slightly lighter than
caveolae. The peak of IGF-1 receptor and insulin receptor was in
fractions 6 and 7, whereas the peak of caveolin was in fractions 7 and
8 (Figs. 2 and 3B). However, in high level insulin-stimulated adipocytes, IGF-1 receptor, insulin receptor, and
caveolin were better correlated in density gradient separation (Fig.
3B). These results were observed in several independently repeated experiments (results not shown). Thus, it is likely that IGF-1
receptor was also located in the caveolin-free lipid rafts around the
caveolae and might further converge into caveolae upon ligand
stimulation. Currently, we are investigating this translocation induced
by ligand.
Although lipid rafts and caveolae are important in signal transduction
(1-7), their roles in IGF-1 receptor cellular signaling are not fully
understood. The identification of IGF-1 receptor in lipid
rafts/caveolae provided us an opportunity to study the role of lipid
rafts/caveolae in IGF-1 signaling. In 3T3-L1 preadipocytes, IGF-1
receptor signal is essential for inducing two cellular responses: adipocyte differentiation and mitotic clonal expansion. However, these
two cellular responses can be separately blocked by inhibitors without
affecting the other (41, 46). PD98059, an inhibitor of MEK-1, blocked
mitotic clonal expansion but not adipocyte differentiation, whereas
vanadate, a protein-tyrosine phosphatase inhibitor, only blocked
adipocyte differentiation. Since lipid rafts/caveolae are on the plasma
membrane, which is on the top of the IGF-1 receptor signal cascade,
disruption at the level of lipid rafts/caveolae will more likely block
all of the cellular responses induced by IGF-1 receptor signaling. Our
results clearly supported this hypothesis. Disruption of lipid
rafts/caveolae by cholesterol-binding reagents led to the blockade of
both cellular responses simultaneously (Figs. 5 and 6). Taken together,
these results composed a hierarchy of IGF-1 receptor signaling system
in 3T3-L1 cells. The signal generated by IGF-1 receptor requires the
assistance of lipid rafts/caveolae on plasma membrane to transmit into
the cell and activates different signal pathways, which lead to
adipocyte differentiation and mitotic clonal expansion, respectively.
It has been reported that
-methylcyclodextrin treatment does not
inhibit insulin receptor-induced ERK1 and -2 activation in primary rat
adipocytes (12). We observed similar results in 3T3-L1 adipocytes
(results not shown). However, in 3T3-L1 preadipocytes,
-methylcyclodextrin treatment dramatically inhibited IGF-1
receptor-induced ERK1 and -2 activation (Fig. 7). It should be noted
that 3T3-L1 adipocytes contain more cholesterol than preadipocytes, and
under the same
-methylcyclodextrin treatment, less caveolin-1 was
displaced from the low density centrifugation fractions in 3T3-L1 in
adipocytes (results not shown). Thus, it is likely that lipid
rafts/caveolae in adipocytes were not disrupted by
-methylcyclodextrin treatment as completely as in preadipocytes.
Although IGF-1 receptor may employ a different signal pathway from
insulin receptor to activate ERK1 and -2, we believe that the
discrepancy between 3T3-L1 adipocyte and preadipocyte in the activation
of ERK is more likely due to the incomplete disruption of lipid
rafts/caveolae in adipocyte.
Based on our present studies, only the downstream signal
transduction of the receptor required lipid rafts/caveolae (Fig. 7). A
possible function of lipid rafts/caveolae in IGF-1 receptor signaling
is to recruit intracellular signal molecules for the receptor.
Interestingly, studies of the phosphorylation of c-Crk, an endogenous
IGF-1 receptor tyrosine kinase substrate, suggest that physical contact
between IGF-1 receptor and c-Crk is essential for the phosphorylation
of the substrate by the receptor kinase (54, 55). IGF-1 receptor
tyrosine kinase can only phosphorylate c-Crk that has bound to the
receptor through its Src homology 2 domain. c-Crk protein with the Src
homology 2 domain deleted is not phosphorylated by IGF-1 receptor
tyrosine kinase. Recruitment of signal molecules by lipid
rafts/caveolae not only brings the downstream signal molecules into the
receptor but also allows the receptor to be in close contact with
signal molecules of many signal pathways for which lipid rafts/caveolae
act like a signaling hub.
Recent studies from caveolin-1 knockout mice indicate that the
caveolin-1-deficient mice show some abnormalities in adiposity (56).
The younger caveolin-1-deficient mice have a relatively intact
adipocyte tissue except in a few places like in mammary gland and
hypodermal fat layers and show similar body weight to their wild type
littermates. However, the older caveolin-1-deficient mice have much
smaller body sizes than their normal cohorts. This reduced body weight
in older caveolin-1-deficient mice is due to reduced adiposity. The
Cav-1 knockout mice also appear to be resistant to diet-induced
obesity. These results indicate a relatively normal fetal development
of adipose tissue in caveolin-1-deficient mice but a problem in
adulthood adipose tissue metabolism. In our present studies, lipid
rafts/caveolae appeared to be required for adipocyte differentiation
induction. Therefore, the caveolaeless knockout mice are probably
having problem in adulthood adipocyte differentiation.
Adulthood adipocyte differentiation has more and more been considered
as one of the leading causes in obesity, especially in hyperplastic
obesity (28, 45). New adipocytes can differentiated from the residual
preadipocytes in adipose tissue throughout the life span. The phenotype
of caveolin-1-deficient mice suggests that the lipid
rafts/caveolae-dependent adipocyte differentiation mechanism in 3T3-L1 cells might more closely resemble adipocyte differentiation in adult animal rather than in embryonic development. Further studies are needed to verify this hypothesis. Taken together with the strong evidence from Cav-1 knockout mice, our present study
has established the role of lipid rafts/caveolae in the adipocyte
differentiation process.