From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242
Received for publication, August 21, 2002, and in revised form, December 19, 2002
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ABSTRACT |
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It is well established that insulin stimulation
of glucose uptake requires the translocation of intracellular localized
GLUT4 protein to the cell surface membrane. This plasma
membrane-redistributed GLUT4 protein was partially co-localized with
caveolin as determined by confocal fluorescent microscopy but was fully
excluded from lipid rafts based upon Triton X-100 extractability.
Cholesterol depletion with methyl- One of the major acute actions of insulin is enhanced glucose
uptake in striated muscle and adipose tissue (1-3). This results from
the rapid translocation of the intracellular-sequestered GLUT41 glucose transporter
isoform to the plasma membrane (4, 5). The increase in plasma membrane
GLUT4 occurs due to a large increase in the rate of GLUT4 exocytosis
coupled with a smaller decrease in the rate of GLUT4 endocytosis (6,
7). Recent data suggest that two independent signal transduction
pathways are necessary for the full extent of insulin-stimulated GLUT4
translocation. In one case, the insulin receptor tyrosine
phosphorylates insulin receptor substrate-family proteins, resulting in
the activation of phosphatidylinositol 3-kinase and the generation of
phosphatidylinositol 3,4,5-triphosphate. Although less well defined,
the serine/threonine kinases phosphoinositide-dependent protein
kinase 1 and protein kinase B/Akt as well as protein kinase C Although substantial progress has been made in our understanding of the
GLUT4 exocytotic process, the mechanisms and pathways involved in GLUT4
endocytosis and recycling are poorly understood. Several studies have
indicated that GLUT4 is internalized through a
clathrin-dependent endocytic pathway. For example,
potassium depletion disrupts clathrin-coated pits and inhibits GLUT4
endocytosis (13). Inhibition of AP2 or dynamin function also prevents
endocytosis and results in the accumulation of GLUT4 at the cell
surface (14-16). Furthermore, morphological analysis demonstrates the
association of GLUT4 with clathrin-coated pits, and GLUT4 appears to
initially co-internalize with the transferrin receptor (17-19).
Whether or not GLUT4 partitions efficiently into caveolae-enriched
plasma membrane fractions remains controversial. Several studies report
that caveolin-enriched fractions contain the majority of GLUT4
(20-23), whereas others have not detected any association between
GLUT4 and caveolin (19, 24). Nevertheless, disruption of lipid raft
structure by cholesterol depletion effectively inhibits insulin-stimulated GLUT4 translocation and glucose uptake (23, 25, 26).
Furthermore, intracellular GLUT4 compartments were also observed to be
devoid of caveolin, suggesting that if GLUT4 is caveolin-associated at
the plasma membrane, after endocytosis GLUT4 must segregate from these
caveolin-enriched domains (27). One interpretation of these data is
that plasma membrane caveolin-enriched lipid raft microdomains may be
involved in the insulin-stimulated GLUT4 endocytic process. Consistent
with this hypothesis, cholesterol depletion has recently been reported
to inhibit GLUT4 endocytosis (23). However, this interpretation is
complicated because cholesterol depletion can also inhibit
clathrin-mediated endocytosis through a physical restraint on membrane
curvature (28, 29). Moreover, cholesterol depletion does not
distinguish between cell surface caveolae versus caveolae
localized to internal membrane compartments; thus it is not clear if
GLUT4 endocytosis has specifically been affected in these experiments.
To resolve some of these issues, we have examined the effect of
cholesterol extraction and expression of caveolin mutants on the
localization and rate of GLUT4 endocytosis in 3T3L1 adipocytes. Our
data demonstrate that increased expression of wild type caveolin accelerates the extent of GLUT4 translocation. In contrast, expression of a dominant-interfering caveolin mutant inhibited GLUT4 endocytosis without any significant effect on clathrin-dependent
internalization. However, the specific disruption of caveolin
organization only partially inhibited GLUT4 endocytosis, whereas
blockade of both clathrin and caveolin function resulted in a near
complete block of GLUT4 internalization. These data are consistent with
a model wherein caveolae and clathrin function together to mediate the efficient endocytosis of GLUT4.
Materials--
The Myc, HA, and clathrin antibodies were from
Santa Cruz Biotechnology (Santa Cruz, CA). Caveolin-2 antibody was from
Transduction Laboratories (Lexington, KY). Transferrin receptor
antibody was from Leinco Technologies, Inc. (Ballwin, MO). The
polyclonal antibody against GLUT4 (IAO2) and TC10 were obtained as
previously described (14, 30). Texas Red-conjugated transferrin was
from Molecular Probe (Eugene, OR). Fluorescent secondary antibodies
were purchased from Jackson Immunoresearch Laboratories (West Grove,
PA). Cholesterol, filipin III, methyl- Cell Culture and Transient Transfection by
Electroporation--
Murine 3T3L1 preadipocytes (American Type Tissue
Culture) were grown and differentiated as described previously (32).
Fully differentiated 3T3L1 adipocytes were electroporated with various cDNAs using a low voltage electroporation technique (0.15 V at 950 microfarads) as previously described (33). After transfection, the
cells were plated on collagen-coated coverslips and incubated for
18-24 h before analysis.
Immunofluorescence Microscopy--
Intact cell
immunofluorescence was performed by washing the cells once with
ice-cold PBS followed by fixation with 4% paraformaldehyde (Electron
Microscopy Sciences, Ft. Washington, PA) and 0.2% Triton X-100 in PBS
at room temperature for 10 min. The cells were then blocked with 5%
donkey serum. Plasma membrane sheets were prepared by the method of
Robinson et al. (19). Briefly, the membranes were fixed in
2% paraformaldehyde at room temperature for 20 min and blocked with
5% donkey serum. The cells or plasma membrane sheets were then
incubated with primary antibodies for 90 min at 37 °C and Texas Red-
or FITC-conjugated donkey secondary antibody for 2 h at room
temperature. The coverslips were mounted in Vectashield (Vector
Laboratories, Inc. Burlington, CA) and examined with 40× or 63× oil
immersion objectives using a Zeiss 510 confocal laser-scanning microscope.
Drug Treatment--
Fully differentiated 3T3L1 adipocytes were
pretreated with 10 mM methyl- GLUT4 Endocytosis Assay--
3T3L1 adipocytes co-transfected
with pcDNA3 vector, Myc-Cav1/WT, Myc-Cav1/S80E, or Myc-Cav1/S80A
and exofacial HA-tagged GLUT4 were insulin-stimulated for 30 min at
37 °C. Then the cells were chilled and incubated with HA monoclonal
antibody for 1 h at 4 °C to label the GLUT4 at the plasma
membrane, and cells were washed to remove insulin and excess HA
antibody as described previously (14). The cells were returned to
37 °C and incubated for various times to allow HA antibody-bound
GLUT4 to internalize. The reactions were stopped by washing once with
ice-cold PBS and fixing in 4% paraformaldehyde and 0.2% Triton X-100
in PBS at room temperature for 10 min. The cells were incubated with
Myc polyclonal antibody followed by Texas Red-anti-mouse and FITC-anti
rabbit secondary antibody. Translocation of HA antibody-bound GLUT4
from plasma membrane to the intracellular pool was examined by
immunofluorescence microscopy.
Transferrin Receptor Endocytosis--
3T3L1 adipocytes
overexpressing human transferrin receptor were pretreated with or
without 10 mM methyl- Cholera Toxin B Uptake--
Cholera toxin B uptake was examined
as described previously (35). Briefly, differentiated 3T3L1 adipocytes
were rinsed twice with Hanks' balanced salt solution and serum-starved
for 2 h. Then the cells were chilled and incubated with 4 µg/ml
FITC-labeled cholera toxin B for 30 min at 4 °C. The cells were
washed 4 times, returned to 37 °C, and incubated for 2.5 h.
Uptake of FITC-labeled cholera toxin B was examined by a confocal
laser-scanning microscope.
Triton X-100 Extraction and Immunoblots--
Plasma membrane
sheets of 3T3L1 adipocytes were extracted with a lysis buffer
containing with 25 mM Hepes, pH 7.4, 150 mM NaCl, 1% Triton X-100, 50 mM sodium fluoride, 10 mM sodium pyrophosphate, 1 mM sodium vanadate,
10 µg/ml aprotinin, 1 µg/ml pepstatin, 5 µg/ml leupeptin, and 1 mM phenylmethylsulfonyl fluoride for 15 min at 0 °C. The
samples were then centrifuged at 16,000 × g for 10 min, the supernatants were collected, and the pellets were washed once
with ice-cold PBS, re-centrifuged, and re-suspended in the lysis
buffer. The supernatants and re-suspended pellets were placed in
Laemmli sample buffer, and the proteins were separated in a 7.5-20%
gradient SDS-polyacrylamide gel, transferred to a polyvinylidene
difluoride-blotting membrane (Millipore Corp., Bedford, MA), and
analyzed for transferrin receptor, GLUT4, and caveolin by immunoblotting.
Plasma Membrane-localized GLUT4 Partially Associates with Large
Caveolin Clusters--
Previously, we and others have observed that
differentiated 3T3L1 adipocytes assemble large clusters of individual
caveolae (caveolae rosettes) that are discernible by confocal
fluorescent microscopy (25, 30, 36). Consistent with our previous
findings (30), the lipid raft-targeted small GTP-binding protein TC10 and caveolin co-localize with these plasma membrane structures (Fig.
1A, panels a-c).
However, the organization of these proteins is distinct from marker
proteins that do not associate in lipid raft microdomains such as the
transferrin receptor, which recycles from the plasma membrane through
clathrin-coated pits, and the clathrin coat protein itself (Fig.
1A, panels d-i). In the basal state, caveolin
displays its characteristic cluster organization that is apparent at
both low and high magnifications (Fig. 1B, panels
e and f). However, in the absence of insulin the amount of GLUT4 protein present in the plasma membrane is very low, and thus,
there is essentially no colocalization with caveolin (Fig. 1B, panels a, b, e,
f, i, and j). Insulin stimulation had
no significant effect on the organization of caveolin, but there was a
marked increase in the plasma membrane association of GLUT4 (Fig.
1B, panels c, d, g,
h, k, and l). Comparison of these
images indicated that GLUT4 was partially dispersed throughout the
plasma membrane. However, GLUT4 did display some co-localization with
the caveolin-positive ring structures, although not nearly as
pronounced as the TC10 marker.
Because the apparent association of GLUT4 with the large clusters of
organized caveolin was indeterminate, we assessed the association of
GLUT4 with lipid raft microdomains by cold Triton X-100 extraction
(Fig. 2). Total Triton X-100 extracts of
isolated plasma membrane sheets (lysates) demonstrated the presence of the transferrin receptor, GLUT4, and caveolin (Fig. 2, lane
1). After centrifugation, the resulting soluble fraction contained all three proteins, whereas the insoluble pellet fraction was completely devoid of the transferrin receptor and GLUT4 (Fig. 2,
lanes 2 and 3). Although a majority of the
caveolin 1 protein was found in the soluble fraction, there was a
substantial amount resistant to cold Triton X-100 extraction. As
expected, insulin stimulation demonstrated a large increase in
the amount of plasma membrane sheet-localized GLUT4 along with a
smaller increase in the translocation of the transferrin receptor (Fig.
2, lanes 4 and 5). Nevertheless, both the
transferrin receptor and GLUT4 remained completely cold Triton
X-100-soluble, whereas caveolin 1 was only partially extracted (Fig. 2,
lanes 5 and 6). Together, these data demonstrate
that GLUT4 is not an integral component of caveolin-containing lipid
raft microdomains in 3T3L1 adipocytes.
Cholesterol Depletion Inhibits GLUT4 Endocytosis--
Recently
several studies have reported that cholesterol extraction can inhibit
insulin-stimulated glucose uptake (23, 25, 26). To determine whether
this resulted from an increase in plasma membrane-localized GLUT4, we
next treated adipocytes with several agents known to alter lipid raft
microdomains through modification of cell surface cholesterol (Fig.
3). As expected, there was essentially no
detectable plasma membrane-associated GLUT4 protein in the basal state,
but after insulin stimulation GLUT4 was readily apparent (Fig.
3A, panels a and e). Treatment with
methyl-
To ensure that this was a specific effect of cholesterol depletion,
adipocytes were first treated with M
The increase in plasma membrane GLUT4 could result from either an
increase in exocytosis and/or decrease in endocytosis. Because previous
studies report that cholesterol extraction can inhibit endocytosis (28,
29), we determined the effect of M
Several studies report that cholesterol depletion can impair both lipid
raft- and non-lipid raft-dependent internalization (28,
29). To address this issue in our experimental system, we also
determined the effect of M Expression of Caveolin 1 Mutants Specifically Inhibits Lipid
Raft-dependent Endocytosis--
Caveolin 1 can undergo
phosphorylation at Ser-80 that results in the intracellular retention
of caveolin 1 and co-sequestration of caveolin 2 (37, 38). In turn, the
loss of cell surface caveolin 1 and two proteins prevents the formation
of caveolae structures and impairs caveolin-dependent
functions. Thus, to distinguish between caveolin-dependent
and non-caveolin-mediated endocytosis, we tested the ability of
caveolin 1 mutants to affect caveolin organization and function in
adipocytes (Fig. 6). The expressed wild
type caveolin 1 (Cav1/WT) protein was distributed in ring-like
clustered patterns on the plasma membrane that co-localized with
endogenous caveolin 2 (Fig. 6, panels a and d).
In contrast, expression of the caveolin 1 mutant (Cav1/S80E) that
mimics phosphorylation disrupted the organized plasma membrane caveolin
clusters, whereas the Cav1/S80A mutant was without any significant
effect (Fig. 6, panels b, c, e, and
f).
To determine whether Cav1/S80E impaired caveolin-dependent
endocytosis, we assessed the internalization of the cholera toxin B
subunit (Fig. 7). It has been well
established that the endocytosis of cholera toxin B occurs through
binding to the lipid raft/caveolae-associated glycolipid GM1
(39, 40, 53). Adipocytes transfected with the empty vector were first
incubated at 4 °C with cholera toxin B (CT-B) and then
warmed to 37 °C for 2. 5 h. Under these conditions, cholera
toxin B was effectively internalized in the empty vector control-transfected cells (Fig. 7A, panels a and
e). Similarly, expression of Cav1/S80A had no significant
effect on the intracellular accumulation of cholera toxin B (Fig.
7A, panels d and h). In contrast,
cells expressing Cav1/WT displayed a small increase in cholera toxin B
endocytosis, whereas Cav1/S80E had a reduction in the amount of
internalized cholera toxin B (Fig. 7A, panels b,
c, f, and g). Quantitation of the
number of cells displaying internalized cholera toxin B are presented
in Fig. 7B. In contrast to cholera toxin B, transferrin
receptor endocytosis was essentially identical in cells co-expressing
the empty vector, Cav1/WT, Cav1/S80E, and Cav1/S80A (Fig.
7C). These data demonstrate that unlike M
Having established a method to resolve clathrin- and
caveolin-dependent internalization, we next determined the
effect of caveolin expression on insulin-stimulated GLUT4 translocation (Fig. 8). In adipocytes co-transfected
with an empty vector and GLUT4-EGFP, insulin stimulated the
translocation of the GLUT4-EGFP reporter from intracellular storage
sites to the plasma membrane. Expression of Cav1/WT and Cav1/S80A had
no significant effect on either the basal state distribution or
insulin-stimulated GLUT4-EGFP translocation. In contrast, expression of
Cav1/S80E resulted in a marked increase in the basal accumulation of
GLUT4-EGFP at the plasma membrane that was not further stimulated by
insulin.
Analysis of GLUT4 endocytosis was then performed using the exofacial HA
epitope-tagged GLUT4 as previously described in Fig. 5. Compared with
vector-transfected cells, expression of Cav1/S80E significantly reduced
the extent of GLUT4 endocytosis (Fig. 9). Importantly, the Cav1/S80E inhibition was only partial, and the initial
phase of GLUT4 endocytosis appeared to be unaffected. In addition,
expression of Cav1/WT and Cav1/S80A appeared to enhance the extent of
GLUT4 endocytosis compared with the empty vector-transfected control
cells. Together these data demonstrate that the initial rate of GLUT4
endocytosis occurs through a clathrin-mediated process but that
caveolin function is necessary for the full extent of GLUT4
internalization.
Lipid rafts are membrane microdomains that are enriched in
cholesterol and glycosphingolipids that can form a relatively ordered and stable liquid phase within the more fluid, disordered structure of
phospholipid bilayers (41, 42). Adipocytes are one of the most abundant sources of a particular subtype of lipid raft called caveolae that accounts for a large amount of the plasma membrane surface (20, 24, 43). Caveolae are characterized by the presence of the
caveolin coat protein that forms Recently, several studies have also implicated caveolin-enriched lipid
raft microdomains in the insulin regulation of GLUT4 translocation (11,
12, 23, 25, 26, 30). The insulin receptor has been reported to
associate with caveolin and to generate a novel signaling cascade
involving the lipid raft recruitment and tyrosine phosphorylation of
the APS·Cbl·CAP complex (11, 47-50). This pathway appears
to function in concert with the insulin receptor tyrosine
phosphorylation and activation of the insulin receptor
substrate/phosphatidylinositol 3-kinase pathway in mediating the full
extent of GLUT4 translocation (12). Although controversial, after
insulin stimulation GLUT4 has been observed to co-localize with
caveolin by immunoelectron microscopy, fluorescent microscopy, sucrose
gradient flotation, and Triton X-100 extractability (20-23). Furthermore, cholesterol depletion and disruption of caveolae by M To more directly assess the specific role that caveolin/lipid rafts may
play in GLUT4 recycling, we took advantage of a caveolin 1 mutation
that mimics phosphorylation on serine 80 (Cav1/S80E). Previous studies
report that in the unphosphorylated state caveolin 1 assembles into
plasma membrane caveolae along with caveolin 2 (37, 38). However, when
phosphorylated on Ser-80, caveolin 1 is retained within the endoplasmic
reticulum in a complex with caveolin 2, and this phenotype is
recapitulated with the Cav1/S80E mutant. As expected, expression of
Cav1/S80E in adipocytes, but not Cav1/WT or Cav1/S80A, disrupted the
caveolae rosette organization and inhibited the internalization of
cholera toxin B, an established marker for lipid raft-localized
GM1-dependent endocytosis (40). More importantly, Cav1/S80E
had no significant effect on transferrin receptor internalization,
demonstrating that clathrin-mediated endocytosis was not perturbed
under these conditions. Thus, the expression of Cav1/S80E provided an
experimental tool to distinguish between caveolin- and
clathrin-dependent endocytic mechanisms.
Using this approach, an examination of GLUT4 distribution revealed an
insulin-independent plasma membrane accumulation of GLUT4 in cells
expressing Cav1/S80E but not Cav1/WT or Cav1/S80A. Furthermore,
Cav1/S80E reduced the extent of GLUT4 endocytosis, whereas Cav1/WT and
Cav1/S80A enhanced GLUT4 endocytosis. These data are consistent with
caveolin functioning in the GLUT4 endocytosis process. Importantly, the
effect of Cav1/S80E was only partial and primarily affected the extent
but not the initial rate of GLUT4 endocytosis. Similarly, Cav1/WT and
Cav1/S80A increased the extent but not the initial rate of GLUT4
endocytosis. These findings suggest that the initial rate of GLUT4
endocytosis occurs through a clathrin-dependent pathway and
accounts for greater than 50% of the cell surface GLUT4 protein. The
remaining 30-40% of the plasma membrane GLUT4 protein is either
slowly internalized through a caveolin-dependent vesicle
endocytosis or alternatively passes through a caveolin-organized
structural domain before entry into a clathrin-coated pit.
The hypothesis that GLUT4 internalization occurs via two distinct
and/or sequential pathways can reconcile several of the discrepancies
in the literature. Multiple studies utilize immunoelectron microscopy,
Triton X-100 extractability, and sucrose gradient flotation to assess
the co-localization of GLUT4 with caveolin and provide both evidence
for and against caveolin association (19-24, 51). These apparent
contradictory findings could result if different experimental
manipulations favored one GLUT4 population over the other. For example,
GLUT4 could exist in an equilibrium between caveolin and non-caveolin
regions of the plasma membrane and, therefore, may be concentrated in
one compartment depending upon the specific conditions used for
detergent extractions and/or fixation. Alternatively, the fraction of
GLUT4 that appears to undergo caveolin-dependent
endocytosis may not actually be within individual caveolae structures
but might be loosely associated around the periphery of the large
plasma membrane caveolin domains. Although we have no direct evidence
for this, we favor this latter possibility based upon several
observations. For example, proteins that are clearly
caveolin-associated (e.g. TC10) display a very strong and
distinct co-localization with the caveolin ring structures. In
contrast, GLUT4 is weakly co-localized near these domains and only
partially overlaps with caveolin. Furthermore, isolation of highly
purified adipocyte caveolae did not reveal the presence of GLUT4,
demonstrating that GLUT4 is not embedded within this type of lipid raft
structure.2 In addition,
recent electron microscopic analysis also suggests that insulin
recruits GLUT4 to large cave-like structures that contain caveolin in
addition to clathrin-coated pits, lipid raft, and non-lipid raft
markers (52). However, although GLUT4 appeared to be peripherally
associated with these large caveolin structures there was no specific
co-localization of GLUT4 within individual caveolae. This is consistent
with our cold Triton X-100 extraction results indicating that GLUT4 is
not directly embedded in lipid raft microdomains.
Thus, we propose a model in which GLUT4 is distributed between
non-caveolin regions of the plasma membrane and in peripheral association with large caveolin-containing domains. The non-caveolin regions recycle more rapidly and internalize through a
clathrin-dependent pathway, whereas the peripheral
caveolin-associated GLUT4 undergoes a slower redistribution into
clathrin-coated pit domains. Future studies are now necessary to
determine whether these two compartments are in equilibrium with each
other, to determine the pathways that account for the trafficking and
segregation of these two distinct GLUT4 populations, and to determine
how the higher order caveolin domains can modulate the functions of the
non-lipid raft regions of the plasma membrane.
-cyclodextrin, filipin, or
cholesterol oxidase resulted in an insulin-independent increase in the
amount of plasma membrane-localized GLUT4 that was fully reversible by cholesterol replenishment. This basal accumulation of cell surface GLUT4 occurred due to an inhibition of GLUT4 endocytosis. However, this
effect was not specific since cholesterol extraction also resulted in a
dramatic inhibition of clathrin-mediated endocytosis as assessed by
transferrin receptor internalization. To functionally distinguish
between caveolin- and clathrin-dependent endocytic processes, we took advantage of a dominant-interfering caveolin 1 mutant (Cav1/S80E) that specifically disrupts caveolae organization. Expression of Cav1/S80E, but not the wild type (Cav1/WT) or Cav1/S80A mutant, inhibited cholera toxin B internalization without any significant effect on transferrin receptor endocytosis. In parallel, Cav1/S80E expression increased the amount of plasma membrane-localized GLUT4 protein in an insulin-independent manner. Although Cav1/S80E also
decreased GLUT4 endocytosis, the extent of GLUT4 internalization was
only partially reduced (~40%). In addition, expression of Cav1/WT
and Cav1/S80A enhanced GLUT4 endocytosis by ~20%. Together, these
data indicate that the endocytosis of GLUT4 requires clathrin-mediated endocytosis but that the higher order structural organization of plasma
membrane caveolin has a significant influence on this process.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/
have been implicated in signaling events functioning downstream of
phosphatidylinositol 3-kinase (8-10). This pathway appears to be
spatially segregated from a parallel insulin receptor-signaling pathway
that results in the tyrosine phosphorylation of Cbl (11). In turn, Cbl
is recruited to plasma membrane lipid raft microdomains through the CAP
(Cbl-associated protein) adaptor protein that binds to both Cbl and the
caveolar protein flotillin (12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-cyclodextrin, and
FITC-labeled cholera toxin B were from Sigma. Cholesterol oxidase was
from Calbiochem. The Myc-tagged wild type caveolin 1 (Cav1/WT) cDNA
was mutated to replace serine 80 with glutamic acid (Cav1/S80E) or with
alanine (Cav1/S80A) via PCR and was cloned into pcDNA3 vector
(Invitrogen). GLUT4-EGFP cDNA was constructed as previously
described (31), and exofacial HA-tagged GLUT4 was generated by
inserting the sequence CTTAAGTACCCTTATGATGTGCCAGATTATGCCGCTAGCCTC into
the first exofacial loop of GLUT4 cDNA and cloning into pcDNA3
vector. Human transferrin receptor cDNA was purchased from American
Type Tissue Culture (Manassas, VA) and subcloned into pcDNA3 vector.
-cyclodextrin, 5 µg/ml
filipin III, or 2 units/ml cholesterol oxidase in serum starvation
medium for the indicated period and then stimulated by 100 nM insulin for 30 min. To examine the effect of cholesterol
recovery, cholesterol-methyl-
-cyclodextrin complexes were
synthesized as described previously (34). Briefly, 0.95 mg of
cholesterol was dissolved in 12.6 µl of isopropanol:CHCl3 (2:1) solution. Methyl-
-cyclodextrin (31.5 mg) was dissolved in
346.5 µl of double-distilled H2O and heated to 80 °C
with stirring. The cholesterol was added to methyl-
-cyclodextrin,
and the solution was stirred until clear. This solution contained 6.8 mM cholesterol. For use, complexes were diluted into serum
starvation medium to a final concentration of 0.2 mM. After
methyl-
-cyclodextrin treatment, the cells were incubated with 0.2 mM cholesterol-methyl-
-cyclodextrin complexes for the
indicated period.
-cyclodextrin for 30 min. Then the
cells were incubated with 5 µg/ml Texas Red-conjugated transferrin at
4 °C for 1 h to label the surface transferrin receptor followed
by washing the cells 4 times with ice-cold PBS. The cells were returned
to 37 °C and incubated for the indicated period to allow the
endocytosis of labeled transferrin receptor. Then transferrin receptor
endocytosis was examined by a confocal laser-scanning microscope.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
GLUT4 partially co-localizes with caveolin by
confocal immunofluorescent microscopy. A, 3T3L1
adipocyte plasma membrane sheets were examined for the distribution of
TC10 (panel a), the transferrin receptor (TfR;
panel d), clathrin (panel g), and caveolin 2 (panels b, e, and h) by confocal
immunofluorescent microscopy. The merged images are presented in
panels c, f, and i. B,
3T3L1 adipocytes were incubated in the absence (panels a,
b, e, f, i, and
j) and presence (panels c, d,
g, h, k, and l) of 100 nM insulin for 30 min. Plasma membrane sheets were prepared
and examined for the distribution of GLUT4 (panels a-d) and
caveolin 2 (panels e-h) by confocal immunofluorescent
microscopy. The merged images are presented in panels i-l.
The images in panels a, c, e,
g, i, and k were taken at 40×
magnification (zoom 2), and the images in panels b,
d, f, h, j, and
l were taken at 40× magnification (zoom 5). These are
representative images from five independent determinations.
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Fig. 2.
GLUT4 is not physically associated with
Triton X-100-insoluble plasma membrane lipid raft microdomains.
3T3L1 adipocytes were either left untreated (lanes 1-3) or
stimulated with 100 nM insulin (lanes 4-6) for
30 min before the preparation of plasma membrane sheets. The plasma
membranes were then scraped into a cold Triton X-100-containing buffer
(lane 1, lysate) and centrifuged to separate the soluble
(lane 2) from the insoluble (lane 3) fraction as
described under "Experimental Procedures." The samples were then
subjected to SDS-polyacrylamide gel electrophoresis and immunoblotted
for the presence of the transferrin receptor (TfR), GLUT4,
and caveolin 1 (Cav1). This is a representative immunoblot
from three independent determinations.
-cyclodextrin (M
CD) and cholesterol
(Chol) oxidase resulted in an increase in the cell surface
GLUT4 protein levels and did not display any further statistical
significant increase by insulin (Fig. 3A, panels
b, d, f, and h). Although filipin also increased the basal level of plasma membrane-associated GLUT4, insulin was still capable of inducing a further stimulation (Fig. 3A, panels c and g). These results
were quantitated by determining the relative fluorescent intensity of
plasma membrane sheets from three independent experiments (Fig.
3B).
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Fig. 3.
Cholesterol depletion results in an
insulin-independent accumulation of cell surface GLUT4 protein.
A, 3T3L1 adipocytes were incubated in the absence
(panels a and e) or presence of 10 mM
M CD (panels b and f), 5 µg/ml filipin
(panels c and g), or 2 µg/ml cholesterol
oxidase (Chol Oxidase, d and h) for 30 min. The cells were then treated without (panels a-d) or
with (panels e-h) 100 nM insulin for 30 min.
Plasma membrane sheets were prepared and examined for the distribution
of GLUT4 by confocal fluorescent microscopy. These are representative
images from three independent determinations. B, the
relative extent of GLUT4 translocation was quantified by determining
the relative fluorescent intensity of the plasma membrane sheets.
CD and subsequently repleted
with cholesterol (Fig. 4). As is readily
apparent, M
CD treatment resulted in the cell surface appearance of
GLUT4 that was fully reversed by re-introduction of cholesterol in a
time-dependent manner (Fig. 4, panels a-f).
Together, these data demonstrate that cholesterol depletion results in
the accumulation of GLUT4 protein at the plasma membrane.
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Fig. 4.
Cholesterol repletion reverses the
insulin-independent plasma membrane accumulation of GLUT4 after
cholesterol depletion. 3T3L1 adipocytes were incubated in the
absence (panel a) or presence of 10 mM M CD
(panel b) for 30 min. The cells that were M
CD-treated
where then incubated with a cholesterol-M
CD complex for 10 (panel c), 30 (panel d), 60 (panel e),
and 120 (panel f) min before the preparation of plasma
membrane sheets and examined for the distribution of GLUT4 by confocal
fluorescent microscopy. These are representative images from three
independent determinations. SFM, serum-free
medium.
CD on GLUT4 and transferrin
receptor internalization (Fig. 5).
Adipocytes were transfected with an exofacial HA epitope-tagged GLUT4
and stimulated with insulin to induce GLUT4 translocation and
accumulation of the HA epitope on the plasma membrane. After HA
antibody labeling at 4 °C, the insulin was removed by extensive
washing, and the cells were then warmed to 37 °C for various times.
As is apparent, the HA-GLUT4 was strongly labeled at the cell surface
with essentially no interior labeling at 4 °C (Fig. 5A,
panels a and e). In untreated cells, there was a
rapid time-dependent internalization of GLUT4 that
concentrated in small compartments beneath the plasma membrane and in
the perinuclear region (Fig. 5A, panels b-f).
Pretreatment of the cells with M
CD resulted in a marked reduction in
the time-dependent appearance of intracellular-localized
GLUT4 (Fig. 5A, panels f-h). It should be noted
that although M
CD treatment decreased the rate of GLUT4
internalization, it was not completely inhibited. This is better
exemplified by plotting the number of cells displaying internalized
GLUT4 after insulin removal (Fig. 5B).
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[in a new window]
Fig. 5.
Cholesterol depletion with
M CD inhibits both GLUT4 and transferrin
receptor internalization. A, 3T3L1 adipocytes were
transfected with the exofacial epitope-tagged HA-GLUT4 cDNA and,
after overnight expression, were incubated in the absence (panels
a-d) or presence of 10 mM M
CD (panels
e-h) for 30 min. The cells were then treated with 100 nM insulin for 30 min, cooled to 4 °C, and incubated
with an HA epitope-specific antibody for 60 min. After extensive
washing to remove the insulin, the cells were incubated for 0 (panels a and e), 15 (panels b and
f), 30 (panels c and g), and 60 (panels d and h) min at 37 °C. The cells were
then fixed and subjected to confocal fluorescent microscopy using a
Texas Red anti-HA secondary antibody. B, the number of cells
displaying a perinuclear localization of the exofacial-labeled HA-GLUT4
was plotted as a function of time. These data were obtained from the
counting of 40 cells per experiment from 3 independent determinations.
C, in parallel, adipocytes were incubated in the absence or
presence of M
CD, cooled to 4 °C, and labeled with Texas
Red-conjugated transferrin. The cells were then washed and warmed to
37 °C, and the number of cells displaying internalized trans- ferrin receptor (TfR) was determined. These data were
obtained from the counting of 80 cells per experiment from 2 independent determinations. W/O,
with/without.
CD treatment on the internalization of the
transferrin receptor (Fig. 5C). After cell surface labeling at 4 °C and warming to 37 °C, the exofacial labeled transferrin receptor internalized with a similar rate and extent as GLUT4 in
untreated cells. Similarly, the rate and extent of internalized transferrin receptor was substantially reduced in cells pretreated with
M
CD. Thus, the impairment of GLUT4 endocytosis by cholesterol depletion accounts for its accumulation at the plasma membrane. However, these data do not distinguish whether GLUT4 endocytosis occurs
through either a clathrin-dependent and/or lipid
raft-dependent mechanism.
View larger version (52K):
[in a new window]
Fig. 6.
Expression of the caveolin 1 S80E mutant
(Cav1/S80E) disrupts caveolin organization in 3T3L1 adipocytes.
3T3L1 adipocytes were transfected with the cDNAs encoding Myc
epitope-tagged Cav1/WT, Cav1/S80E, and Cav1/S80E mutants. Twenty-four h
later, the cells were fixed immunolabeled with a caveolin 2 antibody
(panels a-c) and the Myc antibody (panels d-f),
and the bottom of the cells in contact with the coverslips were
examined by confocal fluorescent microscopy. These are representative
images obtained from three independent determinations.
CD, Cav1/S80E
specifically inhibits caveolin-dependent endocytosis without affecting clathrin-dependent endocytosis.
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Fig. 7.
Expression of Cav1/S80E inhibits cholera
toxin B but not transferrin receptor internalization.
A, 3T3L1 adipocytes were transfected with the empty vector
(pcDNA3) or vector encoding Myc epitope-tagged Cav1/WT, Cav1/S80E,
and Cav1/S80E mutants. Twenty-four h later, the cells were cooled to
4 °C, incubated for 30 min with 4 µg/ml FITC-labeled cholera toxin
B (CT-B), and warmed to 37 °C for 2.5 h. The cells
were then fixed and subjected to confocal fluorescent microscopy for
the presence of the Myc epitope and FITC-cholera toxin B
internalization. These are representative images obtained from two
independent determinations. B, the amount of internalized
cholera toxin B was quantified by counting the number of cells
displaying intracellular labeling relative to control
vector-transfected cells. These data were obtained from the counting of
a total of 80 cells from 2 independent experiments. C, in
parallel, the transfected cells were cooled to 4 °C and labeled with
Texas Red-labeled transferrin and warmed to 37 °C for the times
indicated. TfR, transferrin receptor. These data were
obtained from the counting of 40 cells per experiment from 3 independent determinations.
View larger version (22K):
[in a new window]
Fig. 8.
Expression of Cav1/S80E results in the
insulin-independent accumulation of GLUT4 at the plasma membrane.
3T3L1 adipocytes were co-transfected with the GLUT4-EGFP cDNA and
either the empty vector (pcDNA3) or vectors encoding Myc
epitope-tagged Cav1/WT, Cav1/S80E, and Cav1/S80E mutants. Twenty-four h
later, the cells were incubated in the absence (open bars)
or presence (closed bars) of 100 nM insulin for
30 min at 37 °C. The cells were then fixed and subjected to confocal
fluorescent microscopy for the presence of the Myc epitope and the
localization of GLUT4-EGFP. These data were obtained from the counting
of 40 cells per experiment from 4 independent determinations.
View larger version (29K):
[in a new window]
Fig. 9.
Expression of Cav1/S80E inhibits, whereas
expression of Cav1/WT and Cav1/S80A stimulates, GLUT4 endocytosis.
3T3L1 adipocytes were co-transfected with the HA-GLUT4 cDNA and
either the empty vector (pcDNA3) or vectors encoding Myc
epitope-tagged Cav1/WT, Cav1/S80E, and Cav1/S80E mutants. Twenty-four h
later, the cells were incubated with 100 nM insulin for 30 min at 37 °C and then cooled to 4 °C. The cells were then
incubated with the HA antibody for 60 min and extensively washed to
remove the insulin and unbound antibody. The cells were warmed to
37 °C for the times indicated, and confocal fluorescent microscopy
was performed for the presence intracellular localization of the HA and
Myc epitopes. These data were obtained from the counting of 40 cells
per experiment from 3 independent determinations.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
- or flask-shaped invaginations
(44). In adipocytes, many of these individual caveolae are organized
into large ring-like clusters (caveolae-rosettes) that are visible by
light microscopy (25, 30, 36). Numerous studies demonstrate that lipid
raft domains play an important role in the spatial compartmentalization
of various signaling proteins, effector functions, and for specific
membrane-trafficking events (45, 46). For example, caveolae are
involved the specific assembly of receptor and non-receptor tyrosine
kinase-signaling complexes as well as trimeric and small GTP-binding
proteins. In addition, these domains are involved in
potocytosis, transcytosis, clathrin-independent endocytosis, and
bacterial entry (42, 45).
CD
treatment has been found to block insulin-stimulated GLUT4 translocation and glucose uptake (23, 25, 26). However, the
interpretation of these latter findings is difficult because M
CD can
also prevent clathrin-mediated endocytosis as well as the insulin
receptor activation and recruitment of APS·Cbl·CAP-signaling complex (23, 25, 26, 28, 29). Indeed, our data also demonstrated that
cholesterol depletion with M
CD, filipin, and cholesterol oxidase all
increased the plasma membrane content of GLUT4 in an
insulin-independent manner. These data are consistent with other
studies demonstrating that inhibition of endocytosis results in an
accumulation of GLUT4 at the plasma membrane. However, although M
CD
markedly inhibited GLUT4 endocytosis, it was also a potent inhibitor of
transferrin receptor internalization, an established marker for
clathrin-mediated endocytosis. Thus, this type of pharmacological
approach does not necessarily distinguish between caveolin- and
non-caveolin-dependent events.
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ACKNOWLEDGEMENT |
---|
We thank Amanda Kalen for care and maintenance of 3T3L1 adipocytes.
![]() |
FOOTNOTES |
---|
* This work was supported by National Institutes of Health Research Grants DK33823, DK59291, and DK55811.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.
To whom correspondence should be addressed. Tel.: 319-335-7823;
Fax: 319-335-7886; E-mail: Jeffrey-Pessin@uiowa.edu.
Published, JBC Papers in Press, December 20, 2002, DOI 10.1074/jbc.M208563200
2 P. Pilch, personal communication.
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ABBREVIATIONS |
---|
The abbreviations used are:
GLUT4, insulin-responsive glucose transporter;
Cav1, caveolin 1;
MCB, methyl-
-cyclodextrin;
PBS, phosphate-buffered saline;
CAP, Cbl-associated protein;
HA, hemagglutinin;
WT, wild type;
FITC, fluorescein isothiocyanate;
EGFP, enhanced green fluorescent
protein.
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