Calmodulin Antagonists Inhibit Insulin-Stimulated GLUT4 (Glucose Transporter 4) Translocation by Preventing the Formation of Phosphatidylinositol 3,4,5-Trisphosphate in 3T3L1 Adipocytes
Chunmei Yang,
Robert T. Watson,
Jeffrey S. Elmendorf,
David B. Sacks and
Jeffrey E. Pessin
Department of Physiology and Biophysics (C.Y., R.T.W., J.S.E.,
J.E.P.) The University of Iowa Iowa City, Iowa 52242
Department of Pathology (D.B.S.) Brigham and Womens
Hospital and Harvard Medical School Boston, Massachusetts 02115
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ABSTRACT
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It has been previously reported that calmodulin
plays a regulatory role in the insulin stimulation of glucose
transport. To examine the basis for this observation, we examined the
effect of a panel of calmodulin antagonists that demonstrated a
specific inhibition of insulin-stimulated glucose transporter 4 (GLUT4)
but not insulin- or platelet-derived growth factor (PDGF)-stimulated
GLUT1 translocation in 3T3L1 adipocytes. These treatments had no effect
on insulin receptor autophosphorylation or tyrosine phosphorylation of
insulin receptor substrate 1 (IRS1). Furthermore, IRS1 or
phosphotyrosine antibody immunoprecipitation of phosphatidylinositol
(PI) 3-kinase activity was not affected. Despite the marked insulin and
PDGF stimulation of PI 3-kinase activity, there was a near complete
inhibition of protein kinase B activation. Using a fusion protein of
the Grp1 pleckstrin homology (PH) domain with the enhanced green
fluorescent protein, we found that the calmodulin
antagonists prevented the insulin stimulation of phosphatidylinositol
3,4,5-trisphosphate [PI(3,4,5)P3] formation in vivo.
Similarly, although PDGF stimulation increased PI 3-kinase activity in
in vitro immunoprecipitation assays, there was also no
significant formation of PI(3,4,5)P3 in vivo. These
data demonstrate that calmodulin antagonists prevent insulin-stimulated
GLUT4 translocation by inhibiting the in vivo production of
PI(3,4,5)P3 without directly affecting IRS1- or
phosphotyrosine-associated PI 3-kinase activity. This phenomenon is
similar to that observed for the PDGF stimulation of 3T3L1 adipocytes.
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INTRODUCTION
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It is well established that insulin stimulation results in
increased glucose uptake in adipose and muscle cells primarily from the
recruitment of the glucose transporter 4 (GLUT4) protein to the cell
surface (1, 2, 3, 4, 5, 6). This event is triggered by the initial activation and
autophosphorylation of the insulin receptor tyrosine-specific protein
kinase and subsequent tyrosine phosphorylation of downstream effectors,
most notably insulin receptor substrates 1 and 2 (IRS1 and IRS2)
(7, 8, 9). The tyrosine phosphorylation of IRS1/2 provides docking sites
for the SH2 domains of several effectors and in particular, the p85
regulatory subunit of the phosphatidylinositol 3-kinase (PI 3-kinase)
(9). Although numerous studies have clearly demonstrated that PI
3-kinase function is necessary for insulin-stimulated GLUT4
translocation, the identification of the subsequent targets remains
controversial with evidence both for and against the involvement of
protein kinase B (PKB/Akt) and the atypical protein kinase C (PKC)
isoforms
and
(10, 11, 12, 13, 14, 15, 16).
Recently, several studies have implicated a calmodulin-dependent step
in regulated exocytosis of synaptic vesicles in neurons and in
vacuole fusion in yeast (17, 18, 19). Although it is generally
believed that there is no specific regulatory role for calcium in the
metabolic actions of insulin, it has been reported that calmodulin
antagonists inhibit insulin-stimulated glucose transport activity in
adipocytes and skeletal muscle (20, 21, 22, 23). Furthermore, calmodulin is
capable of being phosphorylated by the insulin receptor both in
vitro and in vivo and was found to directly interact
with IRS1 and the PI 3-kinase in vitro (24, 25, 26). However,
the signaling steps and potential mechanisms by which calmodulin
antagonists inhibit insulin-stimulated glucose uptake have not been
investigated. In this manuscript, we demonstrate that a panel of
calmodulin antagonists specifically inhibit insulin-stimulated GLUT4
but not GLUT1 translocation. This apparently occurs through an
inhibition of PI(3, 4, 5)P3 formation in vivo but does not
result from the inhibition of PI 3-kinase activity as determined in
phosphotyrosine and IRS1 immunocomplex assays in vitro. The
effect of these antagonists is similar to that of platelet-derived
growth factor (PDGF) stimulation, which also appears to have no effect
on PI 3-kinase activity when analyzed in immunoprecipitates but which
is also unable to induce the formation of PI(3, 4, 5)P3 in
vivo.
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RESULTS
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Calmodulin Antagonists Inhibit Insulin-Stimulated GLUT4
Translocation
Previous studies have observed that pretreatment of 3T3L1
adipocytes with calmodulin antagonists inhibited insulin-stimulated
glucose transport activity (20). To determine whether this was due to
the effect of the inhibitors on GLUT4 translocation, 3T3L1 adipocytes
were pretreated with a panel of calmodulin inhibitors (Fig. 1
). As typically observed, insulin
stimulation resulted in the translocation of GLUT4 to the cell surface
membrane as detected by increased GLUT4 immunofluorescence in isolated
plasma membrane sheets (Fig. 1
, A and B, panels 1 and 8). Preincubation
of the 3T3L1 adipocytes with trifluoperazine (TFP) had little effect on
the basal state level of plasma membrane-localized GLUT4 but markedly
inhibited insulin-stimulated GLUT4 translocation in a dose-dependent
manner (Fig. 1A
, panels 2, 3, 9, and 10). To further examine the
specificity for calmodulin in this process, we next examined the effect
of the highly selective inhibitor W13. Pretreatment with W13 also
resulted in an inhibition of insulin-stimulated GLUT4 translocation
(Fig. 1A
, panels 6, 7, 13, and 14). In contrast, the less effective
structural analog W12 had only a marginal inhibition of
insulin-stimulated GLUT4 translocation (Fig. 1A
, panels 4, 5, 11, and
12). Similarly, the calmodulin-specific antagonists Orphiobolin A
(Orph) and W7 were both potent inhibitors of GLUT4 translocation
whereas W5, the less effective structural analog of W7, had essentially
no effect (Fig. 1B
, panels 114).

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Figure 1. Calmodulin Antagonists Inhibit Insulin-Stimulated
GLUT4 Translocation
A, 3T3L1 adipocytes were left untreated (panels 1 and 8) or pretreated
for 20 min with either 40 µM or 70 µM
trifluoperazine (TFP; panels 2, 3, 9, and 10), W12 (panels 4, 5, 11,
and 12) and W13 (panels 6, 7, 13, and 14). B, 3T3L1 adipocytes were
left untreated (panels 1 and 8) or pretreated for 20 min with either 25
µM or 50 µM Orphiobolin A (Orph; panels 2,
3, 9, and 10), 40 µM or 70 µM W5 (panels 4,
5, 11, and 12) and W7 (panels 6, 7, 13, and 14). The cells were then
incubated in the absence (panels 17) or presence (panels 814) of
100 nM insulin for 30 min at 37 C. Plasma membrane sheets
were prepared and processed for GLUT4 immunofluorescence as described
in Materials and Methods. These are representative
fields of plasma membrane sheets obtained from five independent
experiments.
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Calmodulin Antagonists Have No Effect on Insulin- or
PDGF-Stimulated GLUT1 Translocation
In addition to GLUT4, 3T3L1 adipocytes express the GLUT1 glucose
transporter isoform, which also displays insulin-stimulated
translocation to the plasma membrane (27). As expected, insulin
stimulation resulted in an increased amount of GLUT1 protein at the
plasma membrane (Fig. 2
, panels 1 and 8).
Pretreatment of 3T3L1 adipocytes with trifluoperazine had no effect on
the amount of basal or insulin-stimulated plasma membrane-associated
GLUT1 protein (Fig. 2
, panels 2 and 9). Similarly, none of the other
calmodulin antagonists or control structural analogs had any effect on
either the basal or insulin-stimulated translocation of GLUT1 (Fig. 2
, panels 37, 1014).

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Figure 2. Calmodulin Antagonists Do Not Inhibit
Insulin-Stimulated GLUT1 Translocation
3T3L1 adipocytes were left untreated (panels 1 and 8) or pretreated for
20 min with 70 µM trifluoperazine (TFP; panels 2 and 9),
70 µM W12 (panels 3 and 10), 70 µM W13
(panels 4 and 11), 50 µM Orphiobolin A (Orph; panels 5
and 12), 70 µM W5 (panels 6 and 13) and 70
µM W7 (panels 7 and 14). The cells were then incubated in
the absence (panels 17) or presence (panels 814) of 100
nM insulin for 30 min at 37 C. Plasma membrane sheets were
prepared and processed for GLUT1 immunofluorescence as described in
Materials and Methods. These are representative fields
of plasma membrane sheets obtained from four independent experiments.
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To examine the specificity of the calmodulin antagonists on the
signaling events elicited by another growth factor receptor, we
examined the ability of the PDGF tyrosine kinase receptor to induce
GLUT4 and GLUT1 translocation (Fig. 3
).
Previously, several studies have observed that PDGF and/or epidermal
growth factor stimulation can result in GLUT4 translocation while other
studies have reported the opposite, i.e. PDGF does not
induce GLUT4 translocation in 3T3L1 adipocytes (28, 29, 30). Although
controversial, we have also observed that PDGF stimulation does not
induce the translocation of GLUT4 whereas in the same population of
cells insulin was an effective stimulator (Fig. 3A
, panels 13). As
previously observed, pretreatment of the cells with W13 prevented the
insulin stimulation of GLUT4 translocation (Fig. 3A
, panels 46).
However, both insulin and PDGF were capable of inducing GLUT1
translocation (Fig. 3B
, panels 13). Furthermore, W13 pretreatment was
unable to inhibit either the insulin or PDGF stimulation of GLUT1
translocation (Fig. 3B
, panels 46). Similar results were also
obtained with W7 and trifluoperazine treatments (data not shown).
Together, these data demonstrate that these calmodulin antagonists are
effective inhibitors of insulin-stimulated GLUT4 translocation but do
not block general exocytosis, at least for those pathways mediating
GLUT1 translocation.

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Figure 3. Calmodulin Antagonist Inhibit Insulin-Stimulated
GLUT4 Translocation but Not Insulin- or PDGF-Stimulated GLUT1
Translocation
3T3L1 adipocytes were either untreated (panels 13) or pretreated for
20 min with 70 µM W13 (panels 46). The cells then
incubated in the absence (panels 1 and 4) or in the presence of 100
nM insulin (panels 2 and 5) or 3 nM PDGF
(panels 3 and 6) for 30 min at 37 C. Plasma membrane sheets were then
prepared and processed for either GLUT4 (A) or GLUT1 (B)
immunofluorescence as described in Materials and
Methods. These are representative fields of plasma membrane
sheets obtained from four independent experiments.
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Insulin Receptor Autophosphorylation and IRS1 Tyrosine
Phosphorylation Are Not Affected by Calmodulin Antagonists
To investigate the insulin-mediated signaling step(s) inhibited by
the calmodulin antagonists, we first examined both insulin receptor
autophosphorylation and IRS1 tyrosine phosphorylation by
phosphotyrosine immunoblotting (Fig. 4
).
As typically observed, insulin stimulation resulted in the tyrosine
phosphorylation of the approximately 185-kDa IRS1 protein and the
approximately 95-kDa insulin receptor ß-subunit (Fig. 4
, lanes 1 and
2). Pretreatment of 3T3L1 adipocytes with the nonfunctional structural
analog W12 had no effect on the insulin stimulation of either the
insulin receptor or IRS1 tyrosine phosphorylation (Fig. 4
, lanes 3 and
4). Similarly, pretreatment with the specific calmodulin antagonist,
W13, also did not affect the insulin stimulation of insulin receptor or
IRS1 tyrosine phosphorylation (Fig. 4
, lanes 5 and 6). Identical
results were obtained when we examined the effects of trifluoperazine,
W5, and W7 (data not shown).

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Figure 4. Calmodulin Antagonists Do Not Affect Insulin
Receptor Autophosphorylation or IRS1 Tyrosine Phosphorylation
3T3L1 adipocytes were either untreated (lanes 1 and 2) or pretreated
for 20 min with 70 µM W12 (lanes 3 and 4) or 70
µM W13 (lanes 5 and 6). The cells were then incubated in
the absence (lanes 1, 3, and 5) or in the presence of 100
nM insulin (lanes 2, 4, and 6) for 5 min at 37 C.
Whole-cell detergent lysates were generated and immunoblotted using the
PY20 phosphotyrosine antibody as decribed in Materials and
Methods. This is a representative immunoblot from five
independent experiments.
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Immunoprecipitation of IRS Protein-Associated PI 3-Kinase Activity
Is Not Affected by Calmodulin Antagonists
Previous studies have established an essential requirement
for PI 3-kinase activity for insulin-stimulated GLUT4 translocation
(10, 11). In addition, the majority of insulin-stimulated PI 3-kinase
is associated with the IRS proteins (31). As typically observed, there
was a marked increase in the amount of IRS1-immunoprecipitated PI
3-kinase activity after insulin stimulation (Fig. 5A
, lanes 1 and 2). Neither W12 nor W13
pretreatment had any effect on the subsequent insulin stimulation of PI
3-kinase in the IRS1 immunoprecipitates (Fig. 5A
, lanes 3 and 4). Since
the PDGF receptor does not tyrosine phosphorylate the IRS proteins but
instead directly associates with the PI 3-kinase after activation, we
examined PI 3-kinase activity in phosphotyrosine immunoprecipitates
(Fig. 5B
). As expected, both insulin and PDGF treatment resulted in a
strong increase in the amount of PI 3-kinase activity associated with
tyrosine-phosphorylated proteins (Fig. 5B
, lanes 13). Consistent with
the IRS1 immunoprecipitation results, W12 and W13 had no effect on the
insulin stimulation of PI 3-kinase activity in the phosphotyrosine
immunoprecipitates (Fig. 5B
, lanes 4 and 5). Similarly,
trifluoperazine, W5, and W7 were also ineffective in inhibiting
insulin-stimulated PI 3-kinase activity in phosphotyrosine
immunoprecipitates (data not shown). Together, these data demonstrated
that the calmodulin antagonists do not inhibit the insulin-stimulated
association of the PI 3-kinase with IRS proteins. In addition, although
PDGF does not stimulate GLUT4 translocation, it is perfectly capable of
inducing the association of PI 3-kinase activity with the PDGF
receptor.

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Figure 5. Calmodulin Antagonists Have No Effect on
Insulin-Stimulated Immunoprecipitable PI 3-Kinase Activity
A, 3T3L1 adipocytes were either untreated (lanes 1 and 2) or pretreated
for 20 min with 70 µM W12 (lane 3) or 70 µM
W13 (lane 4). The cells were then incubated in the absence (lane 1) or
in the presence of 100 nM insulin (lanes 24) for 5 min at
37 C. Whole cell detergent lysates were prepared, immunoprecipitated
with the IRS1 antibody and assayed for the presence of PI 3-kinase
activity as described under Materials and Methods. B,
3T3L1 adipocytes were either untreated (lanes 13) or pretreated for
20 min with 70 µM W12 (lane 4) or 70 µM W13
(lane 5). The cells were then incubated in the absence (lane 1) or in
the presence of 3 nM PDGF (lane 2) or 100 nM
insulin (lanes 35) for 5 min at 37 C. Whole-cell detergent lysates
were prepared, immunoprecipitated with the PT66 phosphotyrosine
antibody, and assayed for the presence of PI 3-kinase activity as
described in Materials and Methods. IP,
Immunoprecipitation; PIP, phosphatidylinositol phosphate; Ori, origin;
C, control; I, insulin; P, PDGF. These are representative results from
four independent experiments.
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Calmodulin Antagonists Inhibit Insulin Stimulation of PKB
Activation
One major downstream target of the PI 3-kinase is the
serine/threonine kinase PKB (13). This kinase becomes activated upon
the binding of the PKB PH domain to PI(3, 4, 5)P3 and by the subsequent
phosphorylation on serine 473 and threonine 308 by the
phosphoinositide-dependent protein kinases, PDK1 and PDK2 (13, 32, 33).
We therefore next examined the phosphorylation of PKB to assess PDK
activation and production of PI(3, 4, 5)P3 in vivo (Fig. 6
). Insulin stimulation resulted in a
marked increase in serine 473 phosphorylation compared with the control
unstimulated cells (Fig. 6A
, lanes 1 and 3). Pretreatment with the
noneffective calmodulin antagonist W12 did not affect the subsequent
insulin stimulation of PKB serine 473 phosphorylation (Fig. 6A
, lanes 4
and 5). However, W13 markedly blunted the insulin-stimulated PKB serine
473 phosphorylation (Fig. 6A
, lanes 6 and 7). The insulin stimulation
of serine 473 phosphorylation in the presence of W13 was similar to the
weak phosphorylation induced by PDGF treatment (Fig. 6A
, lane 2).
Similarly, the insulin-stimulated phosphorylation of threonine 308 was
completely inhibited by pretreatment with W13 but was unchanged in the
presence of W12 (Fig. 6B
, lanes 47). Furthermore, PDGF was also
completely ineffective in stimulating PKB threonine 308 phosphorylation
(Fig. 6B
, lanes 13).

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Figure 6. Calmodulin Antagonists Inhibit Insulin-Stimulated
Phosphorylation of PKB
A, 3T3L1 adipocytes were either untreated (lanes 13) or pretreated
for 20 min with 70 µM W12 (lanes 4 and 5) or 70
µM W13 (lanes 6 and 7). The cells were then incubated in
the absence (lanes 1, 4, and 6) or in the presence of 3 nM
PDGF (lane 2) or 100 nM insulin (lanes 3, 5, and 7) for 5
min at 37 C. Whole-cell detergent lysates were prepared and
immunoblotted with the phosphoserine 473-specific (A) and
phosphothreonine 308-specific (B) PKB antibodies. These are
representative immunoblots from five independent experiments.
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Insulin-Stimulated PI(3, 4, 5)P3 Formation in Vivo Is
Inhibited by Calmodulin Antagonists
Since PKB is an established target of PI 3-kinase activation,
these data suggest that either there was an inhibition of PDK1/2
activities and/or a block of PI(3, 4, 5)P3 formation in vivo.
Previous studies have demonstrated that the pleckstrin homology (PH)
domain of Grp1 has a high degree of specificity and affinity for
PI(3, 4, 5)P3 (34, 35). Therefore, to assess the in vivo
production of PI(3, 4, 5)P3, we took advantage of this property and
generated a fusion protein consisting of the enhanced green fluorescent
protein (EGFP) fused to the PH domain of Grp1 (EGFP-PH/Grp1). In the
absence of insulin, expression of EGFP-PH/Grp1 resulted in its
predominant localization into the nucleus with a smaller amount
distributed throughout the cell cytoplasm (Fig. 7A
, panel 1). The accumulation of the
EGFP-PH/Grp1 fusion protein in the nucleus is a property of EGFP
in 3T3L1 adipocytes as expression of just EGFP itself also
results in a predominant nuclear localization (data not shown). In any
case, insulin stimulation resulted in the accumulation of the
EGFP-PH/Grp1 fusion protein at the cell surface membrane indicative of
PI(3, 4, 5)P3 formation at the plasma membrane (Fig. 7A
, panel 3). In
contrast, PDGF stimulation was unable to induce a significant increase
in PI(3, 4, 5)P3, at least as detected by the EGFP-PH/Grp1 fusion protein
(Fig. 7A
, panel 2). Although insulin was fully capable of stimulating
the plasma membrane accumulation of PI(3, 4, 5)P3 in the presence of W12,
this was substantially inhibited by the specific calmodulin antagonist
W13 (Fig. 7A
, panels 4 and 5). In addition, the apparent extent of
plasma membrane fluorescence was substantially reduced even in those
cells that still displayed a translocation of the EGFP-PH/Grp1 fusion
protein.

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Figure 7. Calmodulin Antagonists Inhibit Insulin-Stimulated
Formation of PI(3 4 5 )P3 in Intact 3T3L1 Adipocytes
A, 3T3L1 adipocytes were transfected with the EGFP-PH/Grp1 cDNA.
After 24 h, the cells were either untreated (panels 13) or
pretreated for 20 min with 70 µM W12 (panel 4) or 70
µM W13 (panel 5). The cells were then incubated in the
absence (panel 1) or in the presence of 3 nM PDGF (panel 2)
or 100 nM insulin (panels 35) for 30 min at 37 C. The
cells were then fixed in 2% paraformaldehyde and visualized by
confocal fluorescence microscopy as described in Materials
and Methods. B, Quantitation of the number of
cells displaying EGFP-PH/Grp1 cell surface fluorescence was determined
from counting of 50 cells from 3 independent experiments. Each
bar represents the average number of cells displaying
cell surface fluorescence ±SD.
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Quantitation of these data demonstrated that in the basal state
approximately 18% of the transfected 3T3L1 adipocyte cell population
displayed a cell surface EGFP-PH/Grp1 fluorescence that was not
significantly different after PDGF stimulation (Fig. 7B
). In contrast,
insulin stimulation resulted in greater than 80% of the cells with a
strong cell surface fluorescence. Although W12 pretreatment resulted in
a small decrease in the number of cells displaying an
insulin-stimulated plasma membrane fluorescence (60%), this was
reduced to less than 40% by preincubation of the cells with W13. Thus,
these data demonstrate that the calmodulin antagonists do not directly
inhibit PI 3-kinase activity but instead prevent the in vivo
formation of PI(3, 4, 5)P3, thereby accounting, at least in part, for the
lack of insulin-stimulated PKB activation and GLUT4 translocation.
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DISCUSSION
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It is well established that insulin stimulation results in
increased glucose uptake in striated muscle and adipose tissue through
a mechanism that requires the translocation of intracellular
compartmentalized GLUT4 protein to the plasma membrane (1, 2, 3, 4, 5, 6). However,
the signal transduction pathway(s) responsible for this event is poorly
understood. At present, only two signaling proteins are universally
accepted as essential for this process, the insulin receptor itself and
the Type 1 PI 3-kinase (7, 10, 11). In attempts to identify additional
regulatory proteins involved in this process, several laboratories have
examined the effect of various inhibitors of signaling pathways. In
this regard, it has recently been reported that calmodulin directly
interacts with the PI 3-kinase and can increase PI 3-kinase activity
in vitro (26). Although numerous studies have established
that insulin does not regulate intracellular calcium concentrations,
several studies have suggested a potential role for calmodulin in
insulin action (20, 21, 22, 23, 24). For example, insulin has been reported to
induce the tyrosine phosphorylation of calmodulin (24). In addition,
treatment of both adipocytes and muscle with calmodulin antagonists
inhibited insulin-stimulated glucose transport activity (20, 21, 22, 23). More
recently, a role for calmodulin in membrane trafficking events has been
suggested, including vacuole fusion in yeast, exocytosis of synaptic
vesicles, endocytosis of the serotonin 5-HT1A
receptor, and the recycling of the transferrin receptor (17, 18, 19, 36, 37). Based upon these data, we examined the effect of a series of
calmodulin antagonists on the GLUT4 and GLUT1 vesicle translocation
process. Our data demonstrate that specific calmodulin antagonists
abrogate insulin-stimulated GLUT4 translocation. However, it is
important to recognize that although effects of these agents have the
appropriate dose and specificity as calmodulin antagonists, they do not
directly prove the involvement of calmodulin per se.
Nevertheless, the effect of these antagonists is relatively specific
for GLUT4 trafficking as they had no effect on insulin or PDGF
stimulation of GLUT1 translocation. Thus, whether these agents are
functioning through calmodulin or an as yet unidentified effector
protein, they appear to specifically block an essential step in the
GLUT4 translocation process.
To address this issue, we have determined that these calmodulin
antagonists have no significant effect on insulin receptor
autophosphorylation or tyrosine phosphorylation of IRS1. In contrast,
these agents prevented the activation of PKB by inhibiting its
insulin-stimulated serine/threonine phosphorylation. This is consistent
with a necessary role of calmodulin in the serum stimulation of PKB
activity in neuroblastoma cells (38). Surprisingly, however, despite
the inhibition of insulin-stimulated PKB phosphorylation, there was no
effect on the association of the PI 3-kinase with the IRS proteins or
on the catalytic activity of the coimmunoprecipitated PI 3-kinase. This
phenomenon was similar to that observed for PDGF stimulation of 3T3L1
adipocytes, which resulted in the in vitro induction of PI
3-kinase without any significant effect on PKB phosphorylation.
To further examine this discrepancy, we took advantage of the recently
documented selectivity of specific PH domains for PI(3, 4, 5)P3 (34, 35, 39, 40, 41, 42). In this assay system, EGFP-PH fusion proteins have been
successfully used to monitor the in vivo formation of
PI(3, 4, 5)P3 (35, 39, 41, 42). In particular the PH domain of Grp1 has
demonstrated that insulin stimulation results in the predominant
production of PI(3, 4, 5)P3 at the plasma membrane of 3T3L1 adipocytes
(35). Thus, based upon the selective affinity of the Grp1 PH domain for
PI(3, 4, 5)P3, we have observed that the calmodulin antagonists prevented
a significant increase in PI(3, 4, 5)P3 formation in vivo.
Similarly, PDGF stimulation also was apparently ineffective in inducing
the formation of PI(3, 4, 5)P3 at the plasma membrane. Although it
remains possible that EGFP-PH/Grp1 reporter system is unable to detect
specific PI(3, 4, 5)P3 subcompartments and/or displays affinity for an as
yet identified product, the simplest interpretation of these data is
that the calmodulin antagonists and PDGF stimulation uncouples PI
3-kinase activity from the steady-state accumulation of PI(3, 4, 5)P3
in vivo.
There are two possible mechanisms that can account for these
findings. First, the calmodulin antagonists could induce the activation
of a phosphatidylinositol phosphate phosphatase such as SHIP, thereby
rapidly reducing any increase in PI(3, 4, 5)P3. Although formally
possible, this is highly unlikely as PDGF would also have to
sufficiently activate this phosphatase to prevent any measurable
increase in PI(3, 4, 5)P3 formation. Alternatively, the calmodulin
antagonists could alter the subcellular targeting of the PI 3-kinase,
making it inaccessible to its substrate, PI(4, 5)P2. We favor this
hypothesis as calmodulin appears to be responsible for the appropriate
intracellular targeting of a number of molecules including the
localization of p21Cip1 to the nucleus,
calcium-calmodulin-dependent protein kinase II to postsynaptic
densities, and Rad to the cell cytoskeleton (43, 44, 45). In this regard,
PDGF recruits the PI 3-kinase to the PDGF receptor, whereas insulin
targets the PI 3-kinase to the IRS proteins (9, 46, 47). This
difference in PI 3-kinase targeting may reflect its signaling function
in vivo without any intrinsic change in catalytic activity.
In support of this model, it has been observed that the
insulin-stimulated tyrosine phosphorylated IRS1 protein becomes
localized to a low-density microsome cytoskeleton-enriched fraction
(48). Similarly, insulin has also been reported to induce the
redistribution of the PI 3-kinase to a similar low-density microsome
fraction (48, 49). This is in marked contrast to PDGF, which targets
the PI 3-kinase directly to the plasma membrane (48). Thus, we
speculate that PDGF receptor activation in 3T3L1 adipocytes sequesters
the PI 3-kinase into a subcellular compartment that is substrate
inaccessible. Similarly, treatment of these cells with the calmodulin
antagonists could prevent the appropriate subdomain targeting of the PI
3-kinase, thereby preventing the formation of PI(3, 4, 5)P3.
In any case, our data demonstrate that calmodulin antagonists
specifically prevent insulin-stimulated GLUT4 translocation by
inhibiting the in vivo formation of PI(3, 4, 5)P3. Similarly,
the inability of PDGF to stimulate PI(3, 4, 5)P3 production also accounts
for its ineffectiveness to induce GLUT4 translocation. At present the
mechanism(s) that apparently uncouples PI 3-kinase activity as
determined by in vitro kinase assays from PI(3, 4, 5)P3
production in vivo is an important issue that may provide
the basis for receptor signaling specificity in adipocytes.
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MATERIALS AND METHODS
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Materials
Trifluoperazine, W12, W13, W5, W7, and the phosphotyrosine
antibody (PT66) were purchased from Sigma(St. Louis, MO).
Orphiobolin A was obtained from Calbiochem (La Jolla, CA),
and the rabbit polyclonal antibody to GLUT1 was a generous gift from
Dr. Michael Mueckler (Washington University, St. Louis, MO.). The GLUT4
antibody (IA02) was isolated as described previously (50). The
phosphotyrosine (PY20H) and IRS1 antibodies were purchased from
Transduction Laboratories, Inc. (Lexington, KY) and
Santa Cruz Biotechnology, Inc. (Santa Cruz, CA),
respectively. The phosphoserine- and threonine-specific PKB antibodies
were from New England Biolabs, Inc. (Beverly, MA).
Lissamine rhodamine-conjugated donkey antirabbit IgG was purchased from
Jackson ImmunoResearch Laboratories, Inc. (West Grove,
PA), and Vectashield was obtained from Vector Laboratories, Inc. (Burlingame, CA). The EGFP-PH/Grp1 plasmid was generated by
cloning the PH domain of Grp1 (kindly provided by Dr. Michael Czech,
University of Massachusetts Medical Center) into the HindIII
and BamHI sites of pEGFP-C2 vector (CLONTECH Laboratories, Inc., Palo Alto, CA).
Cell Culture
3T3L1 adipocytes (American Type Culture Collection,
Manassas, VA) were cultured in DMEM containing 25 mM
glucose and 10% calf serum at 37 C in an 8% CO2
atmosphere. At confluence, cells were differentiated by incubation in
medium containing 25 mM glucose, 10% FBS, 1 µg/ml
insulin, 1 mM dexamethasone, and 0.5 mM
isobutyl-1-methylxanthine. After 4 days the medium was changed to DMEM,
25 mM glucose, and 10% FBS. Cells were routinely used at
1012 days post differentiation. Before use, cells were washed two
times with PBS and serum-starved in DMEM with 0.1% BSA for at least
2 h.
Plasma Membrane Sheet Assay and Confocal Microscopy
Preparation of plasma membrane sheets from 3T3L1 adipocytes was
performed by the method of Robinson et al. (51). Briefly,
after growth factor treatment, cells were washed in ice-cold PBS and
incubated for 30 sec in ice-cold 0.5 mg/ml
poly-L-lysine in PBS. The cells were then swollen
in 1/3x KHMgE buffer (1x concentration, 70 mM
KCl, 30 mM HEPES, pH 7.5, 5
mM MgCl2, 3
mM EGTA) by three rinses. The swollen cells were
placed in 1x KHMgE buffer with 1 mM
dithiothreitol and 0.1 mM
phenylmethylsulfonyl fluoride and sonicated for 2 sec using a microtip
at setting 4.8 on a 550 Sonic Dismembrator (Fisher Scientific, Hampton, NH). The bound membrane sheets were
fixed for 20 min in 2% paraformaldehyde (Electron Microscopy Sciences,
Fort Washington, PA), quenched in 100 mM
glycine/PBS for 15 min at room temperature, and washed three times in
PBS. The sheets were then blocked for 30 min at room temperature in 5%
donkey serum/PBS and incubated for 1 h with either a 1:100
dilution of GLUT4 antibody or 1:500 dilution of GLUT1 antibody. The
sheets were then washed three times with PBS and incubated for 1 h
with a 1:100 dilution of the lissamine rhodamine-conjugated donkey
antirabbit antibody. After incubation with the secondary antibody, the
sheets were washed three more times in PBS, coverslipped with
Vectashield, and viewed on a Bio-Rad Laboratories, Inc.
(Richmond, CA) laser confocal microscope.
Immunoblotting
3T3L1 cell lysates were prepared from six-well dishes of
adipocytes that had been treated with calmodulin inhibitors and growth
factors or left untreated. Cells were washed twice with ice-cold PBS,
scraped into lysis buffer (25 mM HEPES, pH 7.4, 100
mM NaCl, 1 mM EGTA, 1% NP40, 50 mM
NaF, 2 mM
Na4P2O7,
1 mM Na3VO4, 10
µg/ml aprotinin, 5 µg/ml leupeptin, 1 µg/ml pepstatin, and 1
mM phenylmethylsulfonyl fluoride), and incubated with
rotation for 20 min at 4 C. Insoluble material was removed by
microcentrifugation for 10 min at 4 C. The lysates were then subjected
to reducing SDS-PAGE (8% acrylamide) and transferred to polyvinylidene
fluoride membrane (Millipore Corp., Bedford, MA).
The membranes were immunoblotted with either the phosphotyrosine
antibody or the PKB antibodies.
Immunoprecipitation and PI 3-Kinase Assay
Whole-cell detergent lysates were immunoprecipitated for 2
h at 4 C with either a phosphotyrosine antibody conjugated to agarose
(PT-66) or the IRS1 antibody followed by 1 h incubation with
protein A+-agarose. The immunoprecipitated lipid
kinase activity was determined as described by Turinsky et
al. (52). Briefly, the immunoprecipitates were incubated with 40
µCi of [
-32P]ATP plus 20 µg of
phosphatidylinositol (Avanti Polar Lipids, Birmingham, AL) for 15 min
at room temperature. The radiolabeled phospholipid product was spotted
onto silica plates (Analtech, Newark, DE), subjected to TLC, and
visualized by autoradiography.
Transient Transfection
Differentiated 3T3L1 adipocytes were transiently transfected by
a modification of the electroporation method described previously (53).
Briefly, fully differentiated adipocytes were electroporated (0.16 kV
and 950 microfarads) with 50 µg of the EGFP-PH/Grp1 plasmid DNA per
cuvette. After electroporation, the adipocytes were replated on
collagen-coated tissue culture plates and allowed to recover for
24 h before use.
 |
ACKNOWLEDGMENTS
|
---|
We wish to thank Dr. Kenneth Coker and Diana Boeglin for their
assistance in this study.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Jeffrey E. Pessin, Department of Physiology and Biophysics, University of Iowa, Iowa City, Iowa 52242-1109.
This work was supported by research grants DK-33823, DK-49871, and
DK-25295 from the NIH (J.E.P.) and a grant from the American Diabetes
Association (D.B.S.). J.S.E. was the recipient of Postdoctoral
Fellowship Training Grant 398234 from the Juvenile Diabetes
Foundation.
Received for publication October 19, 1999.
Revision received November 23, 1999.
Accepted for publication December 1, 1999.
 |
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