From the Department of Physiology and Biophysics, The University of Iowa, Iowa City, Iowa 52242
Received for publication, October 23, 2000, and in revised form, January 30, 2001
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ABSTRACT |
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Various studies have demonstrated that the
platelet-derived growth factor (PDGF) receptor in adipocytes can
activate PI 3-kinase activity without affecting insulin-responsive
glucose transporter (GLUT4) translocation. To investigate this
phenomenon of receptor signaling specificity, we utilized single cell
analysis to determine the cellular distribution and signaling
properties of PDGF and insulin in differentiated 3T3L1 adipocytes. The
insulin receptor was highly expressed in a large percentage of the cell
population (>95%) that also expressed caveolin 2 and GLUT4 with very
low levels of the PDGF receptor. In contrast, the PDGF receptor was only expressed in ~10% of the differentiated 3T3L1 cell population with relatively low levels of the insulin receptor, caveolin 2, and
GLUT4. Consistent with this observation, insulin stimulated the
phosphorylation of Akt in the caveolin 2- and GLUT4-positive cells, whereas PDGF primarily stimulated Akt phosphorylation in the
caveolin 2- and GLUT4-negative cell population. Furthermore, transfection of the PDGF receptor in the insulin receptor-, GLUT4-, and
caveolin 2-positive cells resulted in the ability of PDGF to stimulate
GLUT4 translocation. These data demonstrate that differentiated 3T3L1
adipocytes are not a homogeneous population of cells, and the lack of
PDGF receptor expression in the GLUT4-positive cell population accounts
for the inability of the endogenous PDGF receptor to activate GLUT4 translocation.
One of the most important biological actions of insulin is to
increase glucose uptake into striated muscle and adipose tissue (1-4).
In the basal state, the insulin-responsive glucose transporter (GLUT4)1 protein is primarily
localized in various intracellular compartments with relatively low
levels in the plasma membrane (5-8). Insulin triggers a large increase
in the rate of GLUT4 vesicle exocytosis and a small decrease in the
rate of GLUT4 internalization by endocytosis, resulting in a large
increase in cell surface GLUT4 protein and subsequent increase in
glucose uptake.
Currently, the molecular details of insulin signaling leading to GLUT4
translocation remain highly speculative and, in certain aspects,
controversial. However, substantial data have established that the
activation of the type IA PI 3-kinase and subsequent generation of PI
3,4,5-trisphosphate are essential for the insulin stimulation of GLUT4
translocation. For example, multiple studies using various
pharmacological inhibitors, dominant-interfering mutants, expression of
a phosphatidylinositol 5'-phosphatase, and expression of a
constitutively active catalytic subunit are all consistent with a
necessary PI 3-kinase activity for insulin-stimulated GLUT4
translocation (9-16).
However, despite the general agreement that PI 3-kinase activity is
necessary for insulin-stimulated glucose uptake, a clear demonstration
of a sufficient role for PI 3-kinase has not been forthcoming. Indeed,
several lines of evidence suggest that one or more PI
3-kinase-independent signals may be required for insulin-stimulated GLUT4 translocation. For example, two naturally occurring insulin receptor mutations were unable to induce GLUT4 translocation and glucose uptake yet were fully capable of activating PI 3-kinase (17).
Activation of the PI 3-kinase through integrin or interleukin 4 receptors had only a small stimulation of GLUT4 translocation and
glucose uptake, which was markedly enhanced in the presence of insulin
(18, 19). In this regard, the addition of a cell-permeable PI
3,4,5-trisphosphate analog had no effect on glucose uptake but fully
stimulated glucose transport in the presence of insulin despite a
complete inhibition of PI 3-kinase activity (20). More recently, a
second insulin receptor-dependent signaling pathway has
been identified that requires the recruitment of the CAP-Cbl complex to plasma membrane lipid raft microdomain (21).
Although it is likely that other signaling cascades, in addition to the
activation of the PI 3-kinase pathway are required for this process,
several studies have reported that activation of the PI 3-kinase by
PDGF is sufficient to induce GLUT4 translocation and/or glucose uptake
to a similar extent as insulin (22-24). In contrast, other studies
have not detected a significant PDGF-stimulated GLUT4 translocation in
3T3L1 adipocytes (25-28).
To explore the potential basis for this difference in PDGF
responsiveness, we have utilized single cell analysis to determine the
distribution of PDGF and insulin receptors with respect to GLUT4
translocation-responsive versus nonresponsive 3T3L1
adipocyte cell populations.
Materials--
The monoclonal phosphotyrosine (PY20), polyclonal
caveolin 1, and monoclonal caveolin 2 antibodies were purchased from
Transduction Laboratories (Lexington, KY), and the phospho-Akt was
purchased from New England Biolabs (Beverly, MA). The monoclonal PDGF
receptor Cell Culture and Transient Transfection by
Electroporation--
3T3L1 adipocytes (American Type Culture
Collection, Manassas, VA) were grown and differentiated as described
previously (30). Briefly, 9-10-day postdifferentiation adipocytes were
electroporated and 18 h later serum-starved for 2 h in
Dulbecco's modified Eagle's medium containing 25 mM
glucose and 0.1% bovine serum albumin. The cells were left untreated
or stimulated with either insulin (100 nM) or PDGF (20 or
50 ng/ml) for the time indicated at 37 °C.
Immunoblotting--
3T3L1 cells were washed twice with ice-cold
PBS, and cell extracts were prepared by solubilization in lysis buffer
(25 mM HEPES, pH 7.4, 1% Nonidet P-40, 10% glycerol, 50 mM NaF, 10 mM NaP2O4,
137 mM NaCl, 1 mM
Na3VO4, 10 µg/ml aprotinin, 1 µg/ml pepstatin, 5 µg/ml leupeptin, and 1 mM
phenylmethylsulfonyl fluoride). Clarified whole cell lysates were
separated on 8% SDS-polyacrylamide gels, transferred to polyvinylidene
difluoride membranes (Millipore Corp., Bedford, MA), and subjected to
Western blotting.
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. (31). 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 mouse monoclonal and rabbit polyclonal antibodies as
indicated for 90 min at 37 °C. Primary antibodies were detected with
Texas Red dye-conjugated donkey anti-mouse antibody and FITC-conjugated
donkey anti-rabbit 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
on a Zeiss LSM510 confocal laser-scanning microscope.
Oil Red O Staining--
Oil Red O staining for lipid was
performed on cells that were fixed with 4% paraformaldehyde, 0.2%
Triton X-100 in PBS for 10 min and washed in PBS. The cells were then
incubated with 0.6% (w/v) Oil Red O solution dissolved in 50%
dimethyl sulfoxide for 1 h at room temperature. The cells were
then blocked with 5% donkey serum and followed by incubation with
primary rabbit polyclonal antibodies as indicated and secondary
fluorescein isothiocyanate-conjugated donkey anti-rabbit antibody as
described above. The Oil Red O staining was detected using the same
filters as that for Texas Red on the confocal laser-scanning microscope.
There are two isoforms of the PDGF receptor (PDGFR
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
subunit antibody was obtained from Biogenesis (Sandown,
NH), and fluorescent secondary antibodies were purchased from Jackson Immunoresearch Laboratories (West Grove, PA). All other antibodies were
purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA) except
for the polyclonal antibody against rat GLUT4 (IAO2), which was
generated as previously described (29). The human PDGF
receptor was
kindly provided by Dr. Andrius Kazlauskas (Harvard Medical School,
Boston, MA).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
and
PDGFR
) that can homo- and heterodimerize to generate three
functional PDGF receptors (32-34). In addition, there are three PDGF
ligand chains (PDGF-A, PDGF-B, and PDGF-C) that can form at least four functional ligands (PDGF-AA, PDGF-BB, PDGF-AB, and PDGF-CC). The PDGF-BB ligand can interact and activate all three PDGF receptor isoforms, whereas the PDGFR-
homodimer is the universal receptor capable of interacting with all the known ligands. To determine which
functional PDGF receptor(s) are present in differentiated 3T3L1
adipocytes, we performed phosphotyrosine immunoblots from control and
insulin-, PDGF-AA-, and PDGF-BB-stimulated cells (Fig. 1). As expected, insulin stimulation
resulted in a marked increase in tyrosine phosphorylation of IRS1 and
the insulin receptor
subunit (Fig. 1, lanes 1 and 2). In contrast, PDGF-AA had no effect on tyrosine
phosphorylation, whereas PDGF-BB induced the tyrosine phosphorylation
of the PDGF receptor (Fig. 1, lanes 3 and
4). Based upon the ligand specificity of the PDGF receptors,
these data indicate that differentiated adipocytes express the
PDGF
and/or PDGF
receptors.
View larger version (56K):
[in a new window]
Fig. 1.
Differentiated 3T3L1 adipocytes are
responsive to insulin and PDGF-BB but not to PDGF-AA.
Differentiated 3T3L1 adipocytes were either left untreated
(C, lane 1) or incubated with 100 nM insulin (I, lane 2), 20 ng/ml PDGF-AA (AA, lane 3), or 20 ng/ml PDGF-BB (BB, lane 4) for 10 min
at 37 °C. Whole cell extracts were prepared, and 20 µg were
subjected to phosphotyrosine (PY20) immunoblotting as described under
"Experimental Procedures."
Caveolin is a major protein component of caveolae structures and plays
an important role in the assembly of a signaling cascade leading to
GLUT4 translocation (21, 35-38). Caveolae are also highly induced
during adipocyte differentiation and can occupy up to 20% of the cell
surface (39, 40). Thus, the expression of caveolin is not only a marker
for adipocyte differentiation but only the caveolin-positive
cell population is expected to display insulin-responsive GLUT4
translocation. As expected, the majority of the insulin
receptor-positive adipocytes were found to co-express caveolin 2 (Fig.
2A, a-c). However,
there was a small percentage of cells that had relatively low levels of
caveolin 2. These cells also had relatively low levels of insulin
receptor. Some of these cells had a fibroblast-like morphology, but
others were more rounded, typical of adipocytes. In any case, only a small percentage of the cells were positive for the PDGF receptor (Fig.
2A, d). Importantly, these PDGF receptor-positive
cells had very low levels of caveolin 2 expression (Fig. 2A,
d-f). GLUT4 expression was also relatively high in the same
cell population that co-expressed caveolin 2 (Fig. 2A,
g-i).
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We and others have recently observed that caveolin can be detected as torus or donut-shaped structures in isolated plasma membranes from 3T3L1 adipocytes (21, 36). As previously observed, the insulin receptor was found to partially co-localize in these donut-shaped caveolin 2-positive structures in isolated plasma membrane sheets (Fig. 2B, a-c). In contrast, the plasma membrane sheets that we detected containing the PDGF receptor were essentially devoid of these caveolin 2 structures (Fig. 2B, d-f).
To more directly compare the distribution of the insulin and PDGF
receptors, we next examined their cellular distribution by
immunofluorescence. Similar to that observed in Fig. 2, the majority of
the cell population was insulin receptor-positive; however, a cell
field that was relatively abundant in PDGF receptors is shown in Fig.
3A. In any case, the insulin
and PDGF receptors were clearly distributed in distinct cell
populations with relatively little overlap (Fig. 3A,
a-c).
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To confirm this apparent cellular heterogeneity in receptor distribution, we next compared the distribution of the insulin and PDGF receptors in comparison with Oil Red O staining as a marker for fully differentiated adipocytes (Fig. 3B). As expected, the majority of the cell population displayed strong Oil Red O staining in large spherical globules diagnostic for the accumulation of lipid droplets (Fig. 3B, d-f). In the absence of any primary antibody, there was a low level of labeling throughout the field (Fig. 3B, a and g). However, all the immunoreactive insulin receptor positive cells were also positive for Oil Red O staining (Fig. 3B, b, e, and h). The arrow indicates that the signal cell in this field that was negative for the insulin receptor was also negative for the presence of lipid. In contrast, to the insulin receptor, the cells positive for the PDGF receptor had a more fibroblastic appearance and were devoid of lipid droplets (Fig. 3B, c, f, and i). These data demonstrate that the insulin receptor-containing cells are primary fully differentiated adipocytes, whereas the small population of PDGF receptor-positive cells are morphologically distinct from differentiated adipocytes, at least in terms of lipid storage.
To examine the signaling properties of these apparently distinct cell
subtypes, we utilized single cell analysis to compare the responsive
cell populations to Akt phosphorylation. In the basal state, the
phosphoserine Akt antibody displayed a low level of immunoreactivity in
the entire cell population visualized (Fig. 4, a-c). Insulin stimulation
resulted in the vast majority of insulin receptor-positive cells
displaying an increase in phospho-Akt immunofluorescence (Fig. 4,
d-f). Although there was an increase in phospho-Akt
immunofluorescence throughout the cell, it was primarily localized at
the cell surface membrane, indicative of Akt translocation to the
plasma membrane (41-44). In contrast, PDGF stimulation resulted in
only a fraction of the cell population undergoing Akt phosphorylation
(Fig. 4, g-i). These cells had a more central localization
of phospho-Akt and had relatively lower levels of insulin receptor
immunoreactivity. Thus, taken together, these data demonstrate that the
insulin and PDGF receptor are distributed in distinct cell populations
of differentiated 3T3L1 adipocytes. Furthermore, insulin and PDGF
responsiveness in these cell populations is specifically defined by the
presence of these receptor subtypes.
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Although in our differentiated 3T3L1 adipocytes the PDGF receptor is
found in a relatively small subset of cells that also has low levels of
the insulin receptor, caveolin 2, and GLUT4, other studies have
reported that PDGF receptor activation can result in GLUT4
translocation (22-24). Therefore, to determine if the PDGF receptor
has the capacity to induce GLUT4 translocation when present in the
appropriate cell context, we next transfected the human PDGFR into
differentiated 3T3L1 adipocytes (Fig. 5). Using a low voltage electroporation technique that primarily results in
the transfection/expression of adipocytes and excludes fibroblasts (45), the human PDGF receptor was found to be expressed in insulin receptor-, caveolin-, and GLUT4-positive adipocytes (Fig.
5A, a-i). In the basal state, neither the
untransfected nor human PDGF receptor-transfected adipocytes displayed
significant immunoreactivity with the phospho-Akt antibody (Fig.
5B, a-c). Insulin stimulation resulted in the
phosphorylation of Akt in both the untransfected and human PDGF
receptor-transfected cells (Fig. 5B, d-f).
However, the insulin stimulation of Akt phosphorylation and
translocation to the plasma membrane was somewhat reduced in the PDGF
receptor-expressing cells. Quantitation of the number of cells
displaying phospho-Akt rim fluorescence indicated that 79% of the
non-PDGF receptor-expressing adipocytes were insulin-responsive, which
was reduced to 45% in the human PDGF receptor-expressing cell
population (Fig. 5C). In any case, expression of the human
PDGF receptor resulted in a PDGF stimulation of phospho-Akt
immunoreactivity similar to that of insulin (Fig. 5B,
g-i). Under these conditions, 78% of the human PDGF
receptor-expressing adipocytes displayed phospho-Akt at the plasma
membrane (Fig. 5C).
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The ability of the expressed PDGF receptor to activate Akt in the
insulin-responsive cell population provided an assay to determine if
the PDGF receptor could couple to GLUT4 translocation. As typically
observed (46, 47), expression of a GLUT4-EGFP fusion protein resulted
in a perinuclear and small cytoplasmic vesicle distribution (Fig.
6A, b). Insulin
stimulation resulted in a strong continuous cell surface GLUT4-EGFP
fluorescence characteristic of GLUT4 translocation (Fig. 6A,
d). Since few cells expressed the endogenous PDGF receptor
(Fig. 6A, a, c, and e),
PDGF stimulation had a very minor effect on GLUT4-EGFP translocation
(Fig. 6A, f). Quantitation of these data
demonstrated that in the basal state ~15% of the cells displayed a
plasma membrane GLUT4-EGFP rim fluorescence (Fig. 6B).
Insulin stimulation resulted in ~73% of the adipocytes displaying
GLUT4-EGFP translocation, whereas PDGF stimulation resulted in only
24% of the cells translocating GLUT4-EGFP (Fig. 6B). In
cell overexpressing the human PDGF receptor, the number of cells
displaying GLUT4 cell surface labeling in the basal state decreased to
7% (Fig. 6A, g and h, and
B). Although insulin was still capable of inducing GLUT4
translocation, the number of cells was reduced to 40% (Fig.
6A, i and j) and (Fig. 6B).
These data are consistent with the reduction in insulin-stimulated phospho-Akt activation in the cell population transfected with the
human PDGF receptor (Fig. 5). Nevertheless, PDGF was now capable of
inducing GLUT4-EGFP translocation to a similar extent as insulin (Fig.
6A, k and h, and (Fig. 6B).
Thus, these data demonstrate that expression of the PDGF receptor in
the appropriate cell context will result in the coupling of the PDGF
receptor to GLUT4 translocation. Importantly, however, the expression
of the PDGF receptor itself has only a partial stimulatory effect and
appears to suppress the full extent of insulin stimulation.
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In summary, there is a substantial controversy about whether the PDGF
receptor can couple to the translocation of GLUT4 in adipocytes
(22-28). Our data demonstrate that in well differentiated 3T3L1
adipocytes, the PDGF receptor is primarily expressed in a distinct
minor cell population that expresses low levels of the insulin
receptor, caveolin, and GLUT4. This findings are consistent with
earlier studies demonstrating that the PDGF receptor mRNA and
protein are markedly decreased during adipocyte differentiation (48,
49). Thus, in our cell context, PDGF receptors are unable to couple to
GLUT4 translocation. The observation that activation of the endogenous
PDGF receptor can induce GLUT4 translocation by some investigators
probably reflects cells that are in an earlier stage of adipocyte
differentiation. Similarly, it has been reported that overexpression of
the PDGF and epidermal growth factor receptors can result in
GLUT4 translocation (22, 24, 50). Consistent with these data, we have
also observed that overexpression of the PDGF receptor can couple to
GLUT4 translocation. However, it is important to note that the PDGF
receptor is substantially less effective than insulin under these
conditions. Together, these data underscore the heterogeneity in the
differentiated 3T3L1 adipocyte cell population and clearly demonstrate
the need to take this diversity into account when analyzing the signal transduction and regulatory pathways controlling GLUT4 translocation.
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ACKNOWLEDGEMENTS |
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We thank Dr. Andrius Kazlauskas for providing the human PDGF receptor cDNA and Diana Boeglin for technical assistance.
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FOOTNOTES |
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* This work was supported by National Institutes of Health Grants DK33823 and DK25295.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.
Supported by a mentor-based postdoctoral fellowship from the
American Diabetes Association.
§ To whom all correspondence should be addressed. Tel.: 319-335-7823; Fax: 319-335-7886; E-mail: Jeffrey-Pessin@uiowa.edu.
Published, JBC Papers in Press, February 5, 2001, DOI 10.1074/jbc.M009684200
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ABBREVIATIONS |
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The abbreviations used are: GLUT4, insulin-responsive glucose transporter; EGFP, enhanced green fluorescence protein; PI, phosphatidylinositol; PDGF, platelet-derived growth factor; PDGFR, PDGF receptor; PBS, phosphate-buffered saline.
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