From the Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, and the Departments of § Medical Physiology and ¶ Anatomy, The Panum Institute, University of Copenhagen, Blegdamsvej 3, Copenhagen N 2200, Denmark
Received for publication, November 12, 2002, and in revised form, February 17, 2003
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ABSTRACT |
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Adipocytes play an important role in the
insulin-dependent regulation of organismal fuel metabolism
and express caveolae at levels as high or higher than any other cell
type. Recently, a link between insulin signaling and caveolae has been
suggested; nevertheless, adipocyte caveolae have been the subject of
relatively few studies, and their contents have been minimally
characterized. With the aid of a new monoclonal antibody, we developed
a rapid procedure for the immunoisolation of caveolae derived from the plasma membrane of adipocytes, and we characterized their protein content. We find that immunopurified adipocyte caveolae have a relatively limited protein composition, and they lack the raft protein,
flotillin, and insulin receptors. Immunogold labeling and electron
microscopy of the adipocyte plasma membrane confirmed the lack of
insulin receptors in caveolae. In addition to caveolins, the structural
components of caveolae, their major protein constituents, are the
semicarbazide-sensitive amine oxidase and the scavenger lipoprotein
receptor CD36. The results are consistent with a role for caveolae in
lipid flux in and of adipocytes.
Caveolae are 50-100-nm invaginations of the plasma membrane
(PM)1 which are formed by the
expression of one or more isoforms of caveolin, the protein that
produces their distinct structure (1). The membrane lipid composition
of caveolae is enriched in sphingolipids and cholesterol, and caveolae
represent a subtype of membrane lipid rafts, that is, subdomains of the
PM with specific lipid and protein compositions (2). The physiological
roles of caveolae remain uncertain (3), but they have been suggested to
participate in a large number of important cellular functions. These
include the formation of transcytotic/endocytic vesicles in endothelial cells (4, 5), the organization/localization of numerous transmembrane
signaling complexes in many cell types (6-8), and the regulation of
cellular cholesterol homeostasis (9-12). Given all of the important
roles for caveolae postulated above, it is somewhat surprising that
caveolin-1 knockout animals survive and are relatively normal (13, 14).
On the other hand, these animals do have vascular abnormalities,
particularly in the lung, and consequently, they have a reduced ability
to exercise. Interestingly, with age they show abnormalities in lipid
metabolism as a result of apparent adipocyte pathology (15). Indeed,
normal adipocytes have perhaps the highest content of caveolae in any
cell type. Estimates have been made that from 15 to 30% of the
adipocyte PM are caveolae (15-17), although why these structures are
so abundant in adipocytes is unknown.
It is now recognized that adipocytes play a complex and pivotal role in
organismal fuel metabolism as both recipients and generators of
endocrine/cytokine signals (18, 19). Indeed, Bergman (20) has
postulated that the actions of insulin on adipocytes are rate-limiting
for the regulation of overall fuel homeostasis by this hormone. For
this and other reasons, the possible role of lipid rafts and caveolae
on insulin signaling and regulated GLUT4 trafficking has been studied
extensively (for a recent review, see Ref. 21). As summarized by Bickel
(22), the published data are contradictory in that insulin receptors
and GLUT4 are reported to be localized in adipocyte caveolae by some
investigators and to be absent by others. Moreover and regardless of
the presence of insulin receptors there, lipid rafts that may include
caveolae have been suggested to be the locus of an important signaling complex downstream from the insulin receptor, linking it to GLUT4 translocation (22-24). Thus, the possible physiological role of caveolae in insulin action has generated considerable interest and activity.
As with the study of caveolae in any cell, technical issues of
isolation and purity may lie at the heart of the uncertainty regarding
the composition and physiological function of adipocyte caveolae.
Because caveolae are integral structures of the PM, methods needed to
be devised to separate them from the bulk PM. As reviewed in Ref. 6,
these methods include mechanical disruption (sonication/shearing)
and/or physicochemical treatment (brief extraction in Triton X-100 at
4 °C) followed by flotation in density gradients. Typically, these
protocols are lengthy, and their specificity and effectiveness are
questionable. Noncaveolar, detergent-resistant membrane rafts and
cytoskeleton aggregates may copurify with caveolae under some of these
conditions. An alternative approach is to coat the cell surface (of
endothelial cells) with cationized silica to stabilize the PM and
facilitate the detachment of caveolae (25). This procedure was improved
further with the introduction of an immunoisolation step using
anti-caveolin antibody (26, 27). As noted in the latter paper, the
speed of caveolae isolation can be a critical parameter, and
preparations obtained by immunoisolation show a more limited protein
composition for caveolae than those involving detergent resistance.
Here we describe a monoclonal antibody specific to caveolin-1 (7C8)
which can be immobilized on acrylic beads and used to immunoisolate
caveolae rapidly. We find that homogenization of adipocytes results in
a certain amount of caveolae being pinched off from the PM, and these
caveolae can be immunoisolated and characterized rapidly. We find
caveolae purified by this method to be devoid of GLUT4, the insulin
receptor, and flotillin. Indeed, and in agreement with previous freeze
fracture studies (28), adipocyte caveolae have a relatively limited
protein composition. In addition to the caveolins only two major
protein components of caveolae were identified: (a) the
semicarbazide-sensitive amine oxidase (SSAO), a very abundant adipocyte
protein (29) of unknown physiological significance (30); and
(b) the scavenger receptor CD36, which plays an important
role in mammalian fatty acid/lipid metabolism (31, 32). These results
plus those from the caveolin-1 knockout (15) are consistent with a
major function of adipocyte caveolae in lipid trafficking into and out
of these cells.
Antibodies and Western Blotting--
Monoclonal anti-caveolin-1
antibody (clone 7C8) was raised in our laboratory following our
published procedures used to obtain monoclonal antibodies against
intracellular membrane proteins (33, 34). The specificity of this
reagent was confirmed by its recognition in Western blots of proteins
corresponding in mass to caveolin-1 Subcellular Fractionation of Adipocytes--
The protocol was
adapted from Simpson et al. (36) as described previously
(37). Briefly, epididymal fat pads were removed from male
Sprague-Dawley rats (150-175 g) and transferred to KRP (12.5 mM HEPES, 120 mM NaCl, 6 mM KCl,
1.2 mM MgSO4, 1 mM
CaCl2, 0.6 mM Na2HPO4,
0.4 mM NaH2PO4, 2.5 mM
glucose, and 2% BSA (pH 7.4)) at 37 °C. Isolated adipocytes were
obtained by collagenase B (Roche Applied Science) treatment at 37 °C
for 45 min (38). After recovery from digestion for 45 min, cells were
stimulated or not with 20 nM insulin for 15 min. Hormonal
action was stopped with 2 mM KCN. Cells were then
transferred to HES (20 mM HEPES, 5 mM EDTA, 250 mM sucrose (pH 7.4)) and homogenized with a Teflon-glass tissue grinder. Subcellular fractions (PM, mitochondria and nuclei, heavy microsomes (HM), and light microsomes (LM)) were obtained by
differential centrifugation and resuspended in HES. LM could be
fractionated further (see Fig. 9) by sucrose velocity gradient (37).
Microsomes (0.3 mg of total protein) were loaded over 4.6 ml of a
10-35% (w/v) sucrose gradient in 20 mM HEPES, 5 mM EDTA and spun for 55 min at 280,000 × gmax. After centrifugation, fractions were
collected from the bottom of the tube. All buffers used with
subcellular fractions in this work contained a mixture of protease
inhibitors consisting of 1 µM aprotinin, 10 µM leupeptin, 1 µM pepstatin (American
Bioanalytical), and 5 mM benzamidine (Sigma).
Immunofluorescence--
The procedure described in Souza
et al. (39) was followed. Briefly, 3T3-L1 adipocytes at day
8 or 9 of differentiation were fixed in 2% paraformaldehyde for 10 min
at 25 °C, washed, and treated with primary antibody (2-8 µg/ml)
and respective secondary antibody labeled with Cy-3 or Cy-5 (Jackson
Immunoresearch) diluted 1:250. Staining of lipid droplets was achieved
with 1 µM Nile Red (Sigma). Fluorescence of dyes was
assessed by confocal microscopy.
Immunoprecipitation--
The PM fraction (50-100 µg of total
protein) suspended in PBS was solubilized with 60 mM octyl
glucoside for 2 h at 4 °C with constant agitation.
Insoluble material was removed by pelleting for 10 min in a
microcentrifuge. Monoclonal and polyclonal anti-caveolin antibodies or
nonspecific mouse and rabbit IgGs (5 µg) were incubated with the
supernatant overnight at 4 °C, then 20 µl of protein A beads
(Pierce) was added for 4 h. The supernatant with unbound proteins
was collected, and the beads were washed four times with octyl
glucoside in PBS buffer, rinsed once PBS, and eluted with SDS-PAGE
loading buffer containing 2% SDS.
Immunoadsorption of LM--
Protein A-purified 7C8 antibody as
well as nonspecific mouse IgG (Sigma) were immobilized to acrylic beads
(Reacti-gel GF 2000, Pierce) at ~1 mg of antibody/ml of resin,
according to instructions from the manufacturer. Beads were blocked
with 2% BSA in PBS for 2 h and washed with PBS. Microsomes
resuspended in PBS (containing 0.1% BSA for biotinylated samples) were
added at 5-20 µg of total protein/µl of resin for 16 h at
4 °C. The supernatant was recovered, and beads were washed with PBS.
Bound vesicular proteins were eluted sequentially with 1% Triton X-100
in PBS and sample buffer for PAGE containing 2% SDS.
Electron Microscopy--
LM were precleared with nonspecific IgG
beads for 16 h at 4 °C. The unbound fraction was mixed with 7C8
beads for 16 h at 4 °C. Beads were washed extensively with PBS,
and immunoadsorbed material was eluted with 100 µl of 0.2 M NaHCO3 (pH 11.0) in the presence of 0.1% BSA
for 30 min on ice. The supernatant was collected and the pH adjusted to
7.0 with 6 M HCl. The sample was fixed with 0.4%
OsO4 for 1 h on ice, applied to carbon-coated 300 mesh copper grid, and incubated 30 min at room temperature for adsorption. The grid was rinsed sequentially with water and 1% sodium
phosphotungstate (pH 7.4). Samples were analyzed on a Philips CM12
transmission electron microscope.
Immunogold electron microscopy was performed in the following fashion.
Adipocyte PM, adsorbed to EM grids with their cytoplasmic face exposed,
were prepared and labeled as described previously (17). The primary
monoclonal antibody against the C terminus of the insulin receptor
Vectorial Biotinylation of Membrane Proteins--
For cell
surface labeling, some modifications of the procedure described in Ref.
42 were made. Primary amines of BSA from KRP buffer were blocked with
acetic acid N-hydroxysuccinimide ester (Sigma) for 1 h
at 37 °C and the reagent removed by dialysis at 4 °C. Isolation
of adipocytes proceeded using KRP with blocked BSA until just before
biotinylation, when cells were washed with KRP without BSA.
Sulfosuccinimidobiotin (sulfo-NHS-biotin, Pierce) was added to
adipocytes at a concentration of 0.5 mg/ml and incubated for 2-15 min
in the presence or absence of insulin, according to specific
experimental design. After labeling, cells were treated with 50 mM Tris (pH 7.4) to quench unreacted biotin and 2 mM KCN, then the fractionation followed the regular
protocol. For biotinylation of isolated vesicles (either total LM or
vesicles enriched by velocity gradient (43), samples were treated with
0.5 mg/ml sulfo-NHS-biotin for 30 min at 37 °C. The reaction was
stopped with 100 mM Tris (pH 7.4) and vesicles recovered by
centrifugation (230,000 × gmax, 120 min).
Detection of biotinylated proteins transferred to Immun-Blot
polyvinylidene difluoride membranes (Bio-Rad) was performed with 100 ng/ml streptavidin-horseradish peroxidase conjugate (Pierce) prepared
in PBS-Tween with 2% BSA and chemiluminescent substrate.
Protein Microsequencing--
Sequence analysis was performed
under the supervision of Dr. William Lane at the Harvard Microchemistry
Facility by microcapillary reverse-phase high performance liquid
chromatography nanoelectrospray tandem mass spectrometry on a Finnigan
LCQ quadrupole ion trap mass spectrometer.
Characterization of Anti-caveolin Antibody, 7C8--
Fig.
1A shows that caveolin-1 Immunoprecipitated Caveolin Complexes from the Adipocyte PM Exclude
the Insulin Receptor--
Given that several groups have shown
association of the insulin receptor with caveolin both in cell-free
pulldowns (47) and by biochemical and morphological methods in
adipocytes (48-50), we wished to determine whether this was the case
in rat adipocyte PM. We used monoclonal 7C8 and polyclonal
anti-caveolin-1 antibodies as shown in Fig. 2 to immunoprecipitate
proteins from the rat adipocyte PM and then Western blotted with the
indicated antibodies. As has been shown in other cells (51),
caveolins-1 and -2 form a stable complex that can be immunoprecipitated
with both antibodies. None of these antibodies coimmunoprecipitate the
insulin receptor under the described conditions. We verified this
result with 7C8 using similar immunoprecipitation conditions except
that Triton X-100 was the detergent used (data not shown).
Interestingly, the caveolin complex immunoprecipitated with 7C8 or
caveolin-1 antibodies excluded flotillin (also, see Figs. 7 and 9).
Note that the lanes designated 7C8,
IP, show nonspecific bands that bracket the location of flotillin.
Microsomal Caveolin-rich Membranes Contain "Pinched Off"
Caveolae--
Although most of caveolin is in the PM fraction, ~10%
of the protein is found in internal membrane fractions (Fig.
1C and Refs. 37 and 52). Our original assumption was that
this represented caveolin-rich vesicles, perhaps Golgi-derived (53),
and we decided to immunoisolate and characterize these membranes.
Antibody 7C8 was immobilized on acrylic beads and was used to
immunoadsorb membranes from the LM fraction as shown in Fig.
3. After binding of membranes, proteins
were eluted sequentially in nondenaturing (1% Triton X-100) and
denaturing (2% SDS) conditions. Surprisingly, of the panel of eight
proteins tested that are postulated to be involved in aspects of
endocytosis/exocytosis, only caveolins-1 and -2 were immunoadsorbed
under these conditions. Proteins known to be markers of vesicular
trafficking in adipocytes such as SCAMPs and VAMPs (54) were absent as
was GLUT4. The reciprocal experiment of adsorbing GLUT4 was repeated
and confirmed earlier results (37) that caveolin is absent from GLUT4
vesicles (data not shown). A trans-Golgi network marker
(TGN38) and a recycling endosomal marker (transferrin receptor) also
did not colocalize with immunoadsorbed caveolin-rich membranes. In more
than 10 experiments, an average of 60% of caveolin-1 could be
immunoadsorbed, and increasing the amount of immobilized antibody
resulted in a maximum of 75% adsorption, presumably because of the
topography of the caveolin complexes which prevented their complete
immunoisolation. In any case, these results and those of the next two
figures support the notion that caveolin-rich vesicles found in
fractions enriched in intracellular vesicles represent caveolae pinched
off from the PM.
The immunoadsorbed material obtained as in the previous figure was
treated with high pH buffer (0.2 M sodium bicarbonate (pH 11)) for elution of intact membranes. Analysis by electron microscopy of the structures recovered from 7C8 beads showed them to be vesicular (Fig. 4) and identical to caveolae
isolated by other investigators using independent methodology (28).
These structures have a thickening of the membrane which may correspond
to caveolae coat, although the characteristic striations of caveolae
which can be revealed by rapid freeze etching studies (55) were absent.
Moreover, it is possible to identify regions that might correspond to
sealed openings of caveolae which would occur upon
homogenization/disruption of cells (Fig. 4). The range of vesicle
diameter, 30-130 nm, is broader than observed for native caveolae
(70-80 nm) but corresponds well to that observed for isolated caveolae
obtained by coating with cationized silica and/or Triton X-100
extraction (25, 26, 28).
If the structures shown in Fig. 4 represent pinched off caveolae, prior
to pinching/vesiculation, their lumen should be accessible to cell
surface labeling by vectorial reagents such sulfo-NHS-biotin, a
negatively charged molecule that cannot cross the hydrophobic barrier
of the lipid bilayer (42). Fig.
5A shows that labeling of
adipocytes for periods of 2-15 min followed by immunoadsorption of the
LM fraction with immobilized 7C8, elution, and detection with
streptavidin conjugates results in labeling of a relatively small
number of caveolar proteins. There is minimal if any change in labeling
pattern as a function of labeling time in this protocol. As seen in
Fig. 5B, exposure of adipocytes to insulin has no effect on
labeling, indicating that this hormone does not dramatically affect the
behavior and protein composition of caveolae. Fig. 5C
compares the labeling pattern of the bulk PM after Triton X-100 solubilization. The pattern of soluble and insoluble PM differs substantially from immunoadsorbed caveolin, indicating that we are not
simply immunadsorbing a vesiculated part of the bulk PM which happens
to have some caveolin/caveolae present.
Fig. 5D further shows that only cell surface proteins are
being labeled by this procedure. Here, the LM fraction, including pinched off caveolae, is labeled from the cytoplasmic side with sulfo-NHS-biotin before immunoadsorption. The very heavily labeled band
of 100 kDa in Fig. 5, A and B, which corresponds
to SSAO (Fig. 6), is not labeled at all
in this experiment because of its short cytoplasmic tail (see below).
Indeed, only the caveolins are heavily labeled, indicating that the
caveolae are sealed. Additional experiments where adipocytes were
labeled with a thiol-cleavable, cell-impermeant biotinylation reagent,
the PM isolated, solubilized, and adsorbed on immobilized avidin
followed by reduction and Western blotting, revealed that 100% of cell
surface SSAO could be labeled under these conditions. We observed no
labeled cortical actin or G Determination of Caveolae Protein Composition--
The results of
Fig. 5 led us to purify enough caveolae to determine what proteins in
addition to caveolin are associated with this structure. Eluates from
an immunoadsorption experiment were subjected to SDS-PAGE and silver
stained (Fig. 6A). The major stained bands include caveolins
at 22-24 kDa in both Triton and SDS eluates. Two additional bands were
observed in the Triton X-100 eluate, with apparent molecular
masses of 100 and 90 kDa. The two high molecular mass bands were
cut from the gel, digested with trypsin, and the peptides obtained were
sequenced by mass spectrometry. The sequence of 15 peptides derived
from p100 and 7 from p90 revealed these proteins to be, respectively,
SSAO and CD36. These are predicted to be transmembrane proteins with
large extracellular domains and very small intracellular domains.
Indeed, SSAO corresponds to the band heavily biotinylated by cell
surface labeling detected at approximately 97 kDa (Fig. 5, A
and B). However, there is no biotin-labeled band around the
molecular mass expected for CD36 (88 kDa) (Fig. 5, A and
B). Most likely, this is because the exceptionally heavy
glycosylation of this protein (56) sterically hinders it from reaction
with labeling reagents such as sulfo-NHS-biotin. The presence of SSAO
in caveolae was confirmed by Western blot (Fig. 6B). In this
experiment, approximately 30% of SSAO from LM was recovered along with
50% of caveolin. We were unable to detect CD36 by Western blotting
with a variety of commercial antibodies, but others have also reported
this protein to be localized in caveolae in other cell types (for
review, see Ref. 56). It was expected that SSAO and caveolin will not
completely colocalize in LM because some SSAO (18-24% of total) is
found in GLUT4-containing vesicles (29, 57), and caveolin and GLUT4
define independent compartments (Ref. 37 and this paper). SSAO and
GLUT4 may share early recycling pathways and then diverge because SSAO,
differently from GLUT4, is not translocated to the PM in response to
insulin stimulation (29, 57).
Dissociation of Caveolae and Insulin Signaling--
We could not
demonstrate the association of several markers for vesicular transport
with immunopurified caveolae (Fig. 3), nor could we detect the
association of caveolin with insulin receptors by coimmunoprecipitation
(Fig. 2) or by immunoisolation. However, flotillin has been implicated
to associate with caveolin by coimmunoprecipitation (46), but we are
unable to confirm this (Fig. 2). Flotillin has also been implicated in
bridging insulin signaling to GLUT4 trafficking (22, 24). Consequently,
we examined the effects of insulin on the association of insulin
receptors and flotillin with immunoadsorbed caveolae. As shown in Fig.
7 by Western blotting, the insulin
receptor is absent from caveolae isolated from both insulin-stimulated
and basal adipocytes (Fig. 7). As expected, GLUT4 is also absent from
these structures, and it changes its distribution upon insulin
stimulation, as does the insulin receptor, each in the expected
direction. Importantly, we see no association of flotillin with
caveolae in basal or insulin stimulated adipocytes (Fig. 7) (see also
Fig. 9) in contrast to previous results using gradients (58). There are
data supporting the presence of TC-10 in rafts/caveolae (22, 24). TC-10
is a rho family GTPase that regulates the actin-based cortical
cytoskeleton (59), remodeling of which has been suggested to play a
part in insulin-regulated GLUT4 translocation (60, 61). However, we
failed to observe caveolae-associated actin by Western blotting (Fig.
7B).
The localization of the insulin receptor in the PM of adipocytes was
also studied by electron microscopy of membranes from untreated cells.
PM were adhered to EM grids with the cytoplasmic face exposed and
labeled with mouse monoclonal antibodies against the C terminus of the
insulin receptor (Fig. 8A) or
caveolin (Fig. 8B), followed by gold-conjugated rabbit anti
mouse immunoglobulins. Labeling of the insulin receptor (long
arrows) was exclusively localized in the planar part of the PM and
not in caveolae (short arrows). The labeling density was 3.9 (±0.4) (mean ± S.E.) gold particles/µm2 of
membrane. Controls were prepared by competitive blockade of the primary
antibody by the addition of a C-terminal peptide of the receptor before
incubation with the membranes (data not shown), and the resultant
labeling density was 0.9 (±0.3) particles/µm2. Similarly
prepared membranes labeled for caveolin showed dense and specific
labeling of all caveolae (Fig. 8B).
The dissociation of caveolin-containing rafts from those with flotillin
is consistent with the results from fractionation of the LM by sucrose
velocity gradient (Fig. 9). Following
this procedure, the distribution of caveolin (caveolae) overlapped only
to a small degree with GLUT4, flotillin, and the insulin receptor.
Caveolin is enriched in fractions close to the top of the gradient
where membranes containing flotillin and the insulin receptor are
minimally present. On the other hand, these latter two proteins show
similar but distinct distribution profiles in insulin-stimulated
adipocytes. Thus, they may indeed colocalize to some degree to mediate
insulin-dependent signal transduction as proposed by
Saltiel, Pessin and colleagues (22, 24). On the other hand, we see only
traces of the insulin receptor from resting cells in
flotillin-containing fractions of the gradient, whereas, after insulin
stimulation there is a marked increase in the receptor with no apparent
changes in flotillin (Fig. 9). These results are not consistent with
the existence of a major insulin receptor-flotillin complex, although
we cannot rule out a transient association between these two
proteins.
We describe the simple and rapid isolation of caveolin-containing
membranes/caveolae from rat adipocyte LM using an antibody specific to
caveolin immobilized to acrylic beads. This procedure has advantages
over other methods (for review, see Ref. 6) in that it requires a
minimum number of manipulations and can be completed rapidly, speed
being a consideration to avoid possible artifacts such as the presence
of noncaveolar proteins (27). We present several lines of evidence
indicating that these immunologically purified membranes correspond to
pinched off caveolae from the PM. These are: (a) their
morphology by electron microscopy; (b) the ready labeling of
their proteins from the cell surface; (c) the presence of a
caveolin coat; and (d) the absence of vesicle trafficking
markers (Figs. 3-5). Morphological studies of adipocyte caveolae (17)
reveal that many of them have very narrow necks, and it is easy to
imagine that some of them can pinch off from the PM and vesiculate
during homogenization of adipocytes. Although caveolin has been
localized in the endocytic pathway (62, 63) and the Golgi network (53,
64) in some cell types, we see no evidence for this in rat adipocytes.
The rapid time course of cell surface labeling (Fig. 5) did not support
the possibility that caveolin-containing vesicles originate by
endocytosis. The prevalence of detached caveolae over recycling
endosomes/Golgi vesicles containing caveolin may reflect the proportion
of these two subpopulations in adipocyte microsomes, the former being
much more abundant than the latter. In any case, we also see a minimal effect of insulin on caveolae (Fig. 1C and Ref. 37),
suggesting that in the time course of our experiments, 15-30 min, rat
adipocyte caveolae are relatively static cell surface structures. This
conclusion is identical to that reported recently for caveolae in
several cultured cell lines as studied by microscopic procedures
(65).
A possible caveat that may apply to our study of adipocyte caveolae is
that our experimental approach yields from 5 to 10% of the total
cellular caveolin/caveolae. Thus, it cannot be excluded that what we
isolate is a subpopulation of caveolae with a particular protein
composition that is distinct from "average" PM caveolae. This
itself would be an interesting finding in any case, and it would still
support a role for these structures in lipid flux into and out of
adipocytes as discussed below. However, we favor the idea that what we
get is representative of the average caveolar environment because
numerous morphological studies do not support the concept of
heterogeneous caveolae populations. In any case, we are exploring
methods to increase the yield of caveolae from adipocytes to prove this point.
We were not able to find any association with immunopurified adipocyte
caveolae of some key molecules involved in insulin-sensitive glucose
transport in adipocytes, namely GLUT4, the insulin receptor, and
flotillin. The caveat noted above does not apply in the case of the
insulin receptor as studied by electron microscopy (Fig. 8). As noted
previously, there are published data both for and against the presence
of GLUT4 and insulin receptors in caveolae, and we will summarize this
evidence for each protein in turn and in light of our current results.
There is evidence at the electron microscopic level that GLUT4 is
de-enriched (66) or absent from caveolae (17, 55), or in contrast, it
is present and abundant in these structures (67, 68). It is known that
the presence of proteins in caveolae as determined morphologically can
be an artifact of fixation techniques and antibody clustering (69), but
nevertheless, these contradictory data are not possible to resolve at
face value. On the other hand, trafficking of GLUT4 in adipocytes
represents the movement of large amounts of membrane to and from the
cell interior to the cell surface and back again. The fact that we
(this study) and others (65) find caveolae to be quite static
structures in the time frame of vesicular traffic, 15-30 min, argues
against their playing a major role in this process. On the other hand,
there is evidence that clathrin-coated vesicles can serve this role for
GLUT4 in adipocytes (55, 70). GLUT4 association with caveolae has also
been suggested on the basis of detergent insolubility (44, 68), but
detergent insolubility implies association with lipid rafts, not
necessarily caveolae (2). Our conclusion is that the relatively rapid
movement of GLUT4 to and from the cell surface of adipocytes which
requires 20-30 min for a full cycle (71) is not compatible with
dynamics and composition of caveolae in these cells.
The insulin receptor has been detected in adipocyte caveolae through
immunostaining of adipocytes with gold particles (49). Furthermore, it
has been proposed that the receptor itself binds caveolin, based on
in vitro interaction data (47, 48). On the other hand,
Mastick et al. (72) found no evidence for insulin receptors
in caveolae from cultured adipocytes, although more recent study by
some of the same authors has reached the opposite conclusion. However,
we find no evidence for association of these proteins by
coimmunoprecipitation (Fig. 2), immunoadsorption (Fig. 7), and
immunogold electron microscopy (Fig. 8A), despite the abundance of caveolin in rat adipocytes (Fig. 8B). It has
been well established that the insulin receptor undergoes
ligand-dependent endocytosis (73) (shown here in Figs. 1
and 7) via clathrin-coated vesicles (74). Thus, the kinetics of insulin
receptor trafficking are similar to those of GLUT4, and the same
arguments put forward for GLUT4 also apply against a role for caveolae
in insulin receptor dynamics. A direct role for caveolae in insulin
signaling would therefore also seem unlikely, and in any case, it is
unnecessary in principle because the insulin receptor signals normally
in cells lacking caveolae (75).
Concerning flotillin, we cannot detect its interaction with caveolin by
coimmunoprecipitation, nor was it found in immunoisolated caveolae
(Fig. 7). These proteins also have a minimal colocalization in velocity
gradient centrifugation (Fig. 9). Thus, we suggest that caveolin and
flotillin are not ordinarily associated in the cell. The relationship
of the insulin receptor to flotillin and insulin signaling remains
unclear. Data supporting a role for flotillin in insulin signaling do
not include experiments showing a direct interaction of these two
proteins (22, 24). Our data only say that flotillin does not appear to
be a component of caveolae, in contradiction to what was implied in
these recent studies (22, 24) where immunofluorescence indicated some
degree of colocalization for these proteins. However, very recent
studies have suggested that this colocalization may be a result of the
formation in cultured adipocytes of large PM caves that are
representative of the bulk membrane composition and not isolated
caveolae (76). Moreover, insulin signaling occurs in hepatocytes where
caveolae are rare or absent but where association of the insulin
receptor with lipid rafts was suggested (75). Further studies will be
needed to address the role of flotillin in insulin signaling.
What we did find in adipocyte caveolae are the SSAO and the lipoprotein
scavenger receptor CD36. SSAO is found in many tissues, but it is
particularly abundant in caveolae-rich tissues including fat, lungs,
and muscle (29, 77). SSAO is estimated to represent 2% of total
protein in the PM of adipocytes (29), and accordingly it seems highly
likely that it must have an important function for those cells, but
what this function might be is not clear for any cell type (30). The
endogenous substrates for SSAO have not been determined, although it is
quite possible that oxidation of catecholamines by SSAO modulates
lipolysis in adipocytes. It was proposed that the generation of
hydrogen peroxide during amine oxidation by SSAO may originate
insulin-like effects such as GLUT4 translocation (57), although these
studies employed high concentrations of a nonphysiological amine. It
also remains unclear as to what percentage of SSAO is targeted to
caveolae, although this is likely to be a high percentage based on Fig.
6.
CD36 belongs to the class B scavenger receptor family and plays an
important role in lipid metabolism in mammals (31, 32). Previously,
this protein was proposed to localize in caveolae in lung endothelium,
first based on resistance to Triton solubilization (78) and later
confirmed by other methods (56). Here we confirm the presence of CD36
in adipocyte caveolae both by immunoisolation (Fig. 6) and
immunocytochemistry (data not shown). Together with CD36, another
member of the same family of scavenger receptors, the HDL receptor
SR-BI has also been proposed to localize in caveolae (56). The presence
of both proteins in caveolae and the putative trafficking of
intracellular lipoproteins containing caveolin (79) highlight the
possible importance of caveolae and caveolin in the traffic of lipids
in and out of the adipocytes.
We think it is of particular interest that SSAO and CD36 share the
structural feature that both are transmembrane proteins with very short
cytoplasmic tails comprising 5-8 amino acid residues. CD36 has two
predicted transmembrane domains located at the N and C termini of the
protein (80), and SSAO has a single predicted transmembrane domain at
the N terminus (29). This topology is in agreement with the absence of
biotin labeling for those proteins when performed from the cytoplasmic
face of caveolae (Fig. 5C). Caveolins were the proteins
almost exclusively labeled by such approach, and we interpret this as
indicating that caveolae lack transmembrane proteins with large
cytoplasmic domains. This observation is consistent with a
model where a type of net originating from caveolin oligomerization
(Fig. 10) covers the cytoplasmic
surface of caveolae. In that model, membrane proteins with small
cytoplasmic domains such as SSAO and CD36 would have free access to
caveolae, whereas proteins with large cytoplasmic domains
(e.g. transferrin receptor) would be excluded by steric
limitations. This model can be tested by transfection of the
appropriate chimeric proteins, and we are in the process of doing
so.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and -1
(see Fig.
1A) and by immunoprecipitation of caveolin with a commercial
antibody followed by Western blotting with 7C8 and by the reciprocal
experiment of reversing the roles of the two antibodies. Monoclonal
antibodies recognizing GLUT4 (33, 34) and SCAMPs (33, 34) have been
described previously. Polyclonal rabbit anti-peptide antibodies against
the insulin receptor were prepared as in Ref. 35. The following
antibodies were commercially acquired: anti-caveolin-1 (C13630),
anti-caveolin-2, anti-flotillin, and anti-TGN38 (from Transduction
Laboratories); anti-VAMP2 (from Synaptic Systems); anti-actin (from
Developmental Studies Hybridoma Bank, University of Iowa);
anti-transferrin receptor (from Zymed Laboratories); anti-CD36 (Cascade
Bioscience). Various researchers kindly provided sera against other
proteins: SSAO (Dr. Antonio Zorzano, University of Barcelona, Spain);
VAMP3 (Dr. Ronald Corley, Boston University School of Medicine);
insulin receptor (Dr. Ken Siddle, Cambridge University, UK). Primary
antibodies were detected in Western blots using secondary antibodies
conjugated to horseradish peroxidase (Sigma) diluted 1:3,000 and
chemiluminescent substrate (PerkinElmer Life Sciences).
-subunit (CT-1) was kindly provided by Dr. K. Siddle, Cambridge, UK.
The primary antibody against caveolin has been described earlier (40).
The secondary antibody (rabbit anti-mouse immunoglobulins, DAKO,
Denmark) was conjugated with 5-nm gold particles according to the
protocol of Slot and Geuze (41). The peptide used for control of the
specificity of insulin receptor labeling was comprised of the 15 C-terminal amino acids of the human insulin receptor and was obtained
from a commercial source.
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
and -1
are detected by Western blotting with 7C8, whereas a
commercial polyclonal antibody (no longer available) recognizes only
the
-form. These caveolin isoforms differ in the N terminus because
of alternate initiation sites for translation, methionines 1 and 32, respectively, for
and
(44), and thus 7C8 must recognize an
epitope between residue 32 and the C terminus of caveolin. Fig.
1B shows by immunofluorescence that caveolin detected by 7C8
is almost exclusively localized in the cell surface, as expected. Fig.
1C shows the labeling pattern in fractionated rat adipocytes
of caveolin (by 7C8), as well as that for various other proteins of
interest from resting and insulin-treated cells. As expected, insulin
causes a redistribution of intracellular (LM and HM) GLUT4 to the cell
surface (PM) (33). As we demonstrated previously by Western blotting
(45), the insulin receptor undergoes ligand-dependent
endocytosis, whereas caveolin shows a small
insulin-dependent decrease in the LM as a result of
apparent redistribution to the PM (37). The change in PM caveolin
cannot be accurately measured because of the large amount of caveolin
already at the cell surface. Interestingly, flotillin, reported to be
associated with caveolin by coimmunoprecipitation (46), has a different
distribution than caveolin in the LM and HM membrane fractions (Fig.
1C), does not coimmunoprecipitate with anti-caveolin
antibodies (Fig. 2), and has a different
sedimentation pattern than caveolin (see Fig. 9).
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Fig. 1.
Monoclonal antibody 7C8 recognizes
caveolin-1 in adipocyte PM. A, PM (50 µg of protein) from
resting adipocytes was obtained as described under "Materials and
Methods" and was analyzed by SDS-PAGE and Western blot with
monoclonal 7C8 and polyclonal antibody to caveolin-1. The positions of
caveolin-1 isoforms are indicated. Final detection was by
chemiluminescence. B, fixed and permeabilized adipocytes
(see "Materials and Methods") (day 9) were incubated with 8 µg/ml
antibody 7C8 and Nile Red and visualized by confocal microscopy.
C, membrane fractions from adipocytes (PM, HM, LM, and
cytoplasm (CYT)) from resting and insulin-treated (20 nM, 15 min) adipocytes were obtained as described under
"Materials and Methods." Equal proportions of each fraction were
subjected to SDS-PAGE, transferred to polyvinylidene difluoride
membranes, and Western blotted with the antibodies indicated (7C8 for
caveolin) prior to detection by chemiluminescence. For this and the
other figures, the data shown are representative of a minimum of three
independent experiments unless otherwise indicated.
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Fig. 2.
Caveolin does not bind insulin receptor or
flotillin. PM (70 µg of protein) from insulin-treated (100 nM, 15 min) and untreated adipocytes was immunoprecipitated
with 5 µg of monoclonal 7C8 (A) and 5 µg of rabbit
polyclonal anti-caveolin-1 (C13630) (B) as described under
"Materials and Methods." The appropriate mouse or rabbit
nonspecific IgGs were utilized in each case as controls. Equivalent
volumes of the supernatant (SN) from protein A beads with
unbound material and the SDS eluate (IP) were analyzed by
Western blot with the antibodies indicated (C13630 for caveolin-1)
(IR for the insulin receptor, also in Figs. 7 and 9), and
final detection was by chemiluminescence.
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Fig. 3.
Immunoadsorbed microsomal caveolin-containing
membranes lack markers of membrane protein trafficking. LM from
resting adipocytes (500 µg) were immunoadsorbed with 50 µl of 7C8-
and nonspecific IgG-coupled acrylic beads. Proteins were sequentially
eluted with Triton X-100 and SDS, and equivalent volumes of the eluates
and unbound material (SN) were subjected to SDS-PAGE
followed by Western blotting for the indicated proteins (TFR
for transferrin receptor), which were visualized by
chemiluminescence as in the previous figures.
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Fig. 4.
Immunoadsorbed microsomal caveolin-containing
membranes appear to be caveolae. LM from resting adipocytes (200 µg) were incubated with 50 µl of 7C8-coupled acrylic beads, eluted
at pH 11.0, and the eluate was fixed and stained and subjected to
electron microscopy as described under "Materials and Methods." The
bar indicates 100 nm. The representative images are from two
independent experiments.
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Fig. 5.
Vectorial biotinylation reveals
immunoadsorbed microsomal caveolin-containing membranes to be pinched
off caveolae. Isolated adipocytes were labeled with 0.5 mg/ml
sulfo-NHS-biotin for the times indicated in the absence of insulin.
Labeling was stopped by the addition of 50 mM Tris (pH
7.4). The cells were homogenized and fractions isolated as described
under "Materials and Methods." LM (500 µg) were immunoadsorbed on
7C8-coupled acrylic beads and eluted with SDS. After SDS-PAGE and
transfer, biotinylated proteins were detected by blotting with a
streptavidin-horseradish peroxidase conjugate and a chemiluminescent
substrate. B, adipocytes were exposed to 20 nM
insulin or not for 15 min. The biotinylation reagent was added 5 min
after the addition of insulin. The cells were processed and proteins
analyzed as in A except that membranes were also exposed to
beads coupled to a nonspecific IgG as a control. C, 50 µg
of PM from cells labeled for 15 min with sulfobiotin was treated with
1% Triton X-100 at 4 °C for 5 min. The samples were spun at
16,000 × gmax for 30 min, and equivalent
amounts of soluble and insoluble material were compared with
7C8-immunoadsorbed LM as in A and B. D, LM from nonstimulated adipocytes were fractionated by
sucrose velocity gradient (see "Materials and Methods"). The
caveolin-rich fractions were pooled and labeled with sulfo-NHS-biotin
for 30 min at 37 °C. Aliquots corresponding to 200 µg of starting
material (LM) were immunoadsorbed by 20 µl of 7C8- and nonspecific
IgG-coupled beads. Equivalent volumes of Triton X-100 and SDS eluates
were subjected to SDS-PAGE, transferred to polyvinylidene difluoride
membranes, blotted, and probed with streptavidin-horseradish peroxidase
conjugate as in A.
s in this experiment (data
not shown). These results and controls showing absence of cytoplasmic
protein labeling (42) support the fact that the biotinylation reaction
is vectorial and labels only cell surface-accessible proteins.
Interestingly and as shown in Fig. 5, A and B, we
always see significant cell surface labeling of caveolin in rat
adipocytes (band designated p20). This result is in
contradiction to accepted models for caveolin topology, which predict
no exposure to the extracellular milieu for this protein (7). It is
possible that this discrepancy may arise from reaction of caveolin with
reagent after cell homogenization, a possible consequence of using rat
adipocytes that require removal of fat before further analysis. The
controls described above argue against this, and in any case, because
SSAO cannot be labeled except from the cell surface, our interpretation
remains unchanged by the caveolin labeling results.
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Fig. 6.
SSAO and CD36 are major components of
adipocyte caveolae. A, LM from 3.4 mg of nonstimulated
adipocytes were subjected to a sucrose velocity gradient, caveolin-rich
fractions were pooled, divided into two equal aliquots, and
immunoadsorbed with 100 µl of beads coupled to either 7C8 or
nonspecific mouse IgG. The total amount of proteins recovered with 1%
Triton X-100 and half from SDS eluate were loaded onto a 6-15%
SDS-PAGE and silver stained. B, LM from 500 µg of
nonstimulated adipocytes were immunoadsorbed with 50 µl of 7C8 and
nonspecific IgG beads. Equivalent volumes of unbound material
(SN), Triton X-100, and SDS eluates were analyzed in Western
blot as in previous figures.
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Fig. 7.
Immunoadsorbed adipocyte caveolae lack
insulin receptors, flotillin, and GLUT4. LM from insulin-treated
(+) and -untreated ( ) adipocytes (500 µg) were immunoadsorbed with
50 µl of beads coupled to 7C8 or nonspecific IgG beads. Equivalent
volumes of unbound material (SN), Triton X-100, and SDS
eluates were subjected to SDS-PAGE and analyzed by Western blotting as
in the other figures.
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Fig. 8.
Immunogold electron microscopy shows insulin
receptors to be in the planar regions of adipocyte PM.
A, the electron micrograph (see "Materials and Methods")
shows a view of the cytoplasmic face of the adipocyte PM
immunogold-labeled for the C terminus of the insulin receptor
-subunit. All labeling (long arrows) is located in the
planar part of the membrane. Caveolae, clustered or single (short
arrows), are devoid of label. B, analogously prepared
adipocyte membrane immunogold-labeled for caveolin. Caveolae
(short arrows) are densely labeled, whereas the planar part
of the membrane is unlabeled. Specimens were negatively stained with
2% silicotungstate. Scale bars indicate 200 nm. The
experiment is representative of three independent studies
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Fig. 9.
Caveolin- and flotillin-containing membranes
do not colocalize in velocity gradient. LM (250 µg) from
insulin-treated (+) and -untreated ( ) adipocytes were fractionated by
sucrose velocity gradient, and equal volumes of the fractions were
analyzed by Western blot with the antibodies indicated.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 10.
Model for proteins in adipose
caveolae.
In summary, isolation and characterization of rat adipocyte caveolae
support their dissociation from insulin signaling in these cells.
Furthermore, we did not find other signaling molecules among the major
protein components of caveolae. Instead we found abundant CD36, a
surface receptor implicated in lipid metabolism, and SSAO of unknown
physiological role. Our results, together with other published data,
favor the hypothesis of adipocyte caveolae as cellular nodes for
control of lipid flux. Indeed, release of lipids from adipocytes has to
be tightly regulated because both an excess and a shortage of these
molecules can be harmful to the organism.
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ACKNOWLEDGEMENTS |
---|
We thank all researchers who kindly provided us with antibodies, Donald Gantz from Boston University School of Medicine for help with the electron microscopy analysis (Fig. 4), and Dr. Sandra Souza from the Human Nutrition Research Center at Tufts University for assistance with confocal analysis.
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FOOTNOTES |
---|
* This work was supported in part by National Institutes of Health Grants DK30425 and DK56935.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Recipient of a postdoctoral fellowship from the
Fundação de Amparo à Pesquisa do Estado de São Paulo.
To whom correspondence should be addressed: Dept. of
Biochemistry, Boston University School of Medicine, 715 Albany St.,
K412, Boston, MA 02118. E-mail: ppilch@bu.edu.
Published, JBC Papers in Press, March 11, 2003, DOI 10.1074/jbc.M211541200
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ABBREVIATIONS |
---|
The abbreviations used are: PM, plasma membrane(s); BSA, bovine serum albumin; GLUT4, glucose transporter isoform 4; HM, heavy microsome(s); LM, light microsome(s); NHS, N-hydroxysuccinimide; PBS, phosphate-buffered saline; SCAMP, secretory compartment-associated membrane protein; SSAO, semicarbazide-sensitive amine oxidase; VAMP, vesicle-associated membrane protein.
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