(Received for publication, January 30, 1996)
From the
Phosphatidylinositol (PI) 3-kinase is hypothesized to be a
signaling element in the acute redistribution of intracellular GLUT4
glucose transporters to the plasma membrane in response to insulin.
However, some receptors activate PI 3-kinase without causing GLUT4
translocation, suggesting specific cellular localization may be
critical to this PI 3-kinase function. Consistent with this idea,
complexes containing PI 3-kinase bound to insulin receptor substrate 1
(IRS-1) in 3T3-L1 adipocytes are associated with intracellular
membranes (Heller-Harrison, R., Morin, M. and Czech, M. (1995) J.
Biol. Chem. 270, 24442-24450). We report here that in
response to insulin, activated complexes of IRS-1PI 3-kinase can
be immunoprecipitated with anti-IRS-1 antibody from detergent extracts
of immunoadsorbed GLUT4-containing vesicles prepared from 3T3-L1
adipocytes. The targeting of PI 3-kinase to rat adipocyte
GLUT4-containing vesicles using vesicles prepared by sucrose velocity
gradient ultracentrifugation was also demonstrated. Insulin treatment
caused a 2.3-fold increase in immunoreactive p85 protein in these
GLUT4-containing vesicles while anti-p85 immunoprecipitates of PI
3-kinase activity in GLUT4-containing vesicle extracts increased to a
similar extent. HPLC analysis of the GLUT4 vesicle-associated PI
3-kinase activity showed insulin-mediated increases in PI 3-P, PI
3,4-P
, and PI 3,4,5-P
when PI, PI 4-P, and PI
4,5-P
were used as substrates. Our data demonstrate that
insulin directs the association of PI 3-kinase with GLUT4-containing
vesicles in 3T3-L1 and rat adipocytes, consistent with the hypothesis
that PI 3-kinase is involved in the insulin-regulated movement of GLUT4
to the plasma membrane.
One of the major physiological effects of insulin is its ability to stimulate glucose uptake in adipose tissue and muscle(1, 2) . The acute stimulation of glucose transport rates in response to insulin is associated with the rapid translocation of GLUT4 transporters to the cell surface from an intracellular location, most likely within the tubulovesicular endosomal membrane system (for reviews see Refs. 3 and 4). Rapid appearance of GLUT4 on the cell surface in response to insulin has been well documented utilizing a variety of techniques including biochemical cell fractionation(1, 2) , photoaffinity labeling (5, 6, 7) , trypsin cleavage(8) , immunoelectron microscopy(9, 10) , and antibody binding assays of expressed epitope-tagged transporters(11, 12) . However, it is still not known how early events initiated by insulin binding to its cell surface receptor lead to increased glucose uptake.
One early step in the
insulin signaling pathway is the activation of PI 3-kinase ()secondary to the phosphorylation of IRS-1 on tyrosine
residues (for reviews see (13, 14, 15, 16) ). Phosphotyrosine
residues specifically found within multiple YXXM motifs on
IRS-1 have been demonstrated to bind Src homology 2 (SH2) domains
present on the p85 regulatory subunit of PI 3-kinase following insulin
stimulation(17, 18, 19) . The binding of p85
to IRS-1 activates the p110 catalytic subunit of PI 3-kinase which
catalyzes the phosphorylation of phosphoinositides at the D3 position
of the inositol ring(17, 20) . A key role for
3`-phosphoinositides in membrane trafficking is suggested by studies on
yeast PI 3-kinase, denoted VPS34, which is required for the proper
delivery of soluble hydrolases to the yeast vacuole(21) .
Similarly, mutant platelet-derived growth factor and colony stimulating
factor receptors lacking binding sites for PI 3-kinase are defective in
their sorting to a degradative pathway(22, 23) . That
PI 3-kinase may also participate in insulin-dependent membrane
trafficking of GLUT4 is suggested by the demonstration that inhibitors
of PI 3-kinase activity abolish insulin-mediated translocation of GLUT4
to the plasma membrane in both 3T3-L1 and rat
adipocytes(24, 25) . However, activation of PI
3-kinase by insulin cannot fully account for the effects of the enzyme
on glucose transport since other receptor signaling systems activate PI
3-kinase activity to the same extent without causing translocation of
GLUT4(26) . A critical aspect of this function of PI 3-kinase
may therefore be its directed localization to the specialized
intracellular membranes that sequester GLUT4.
The above
considerations have led us to hypothesize that insulin receptor
activation causes the rapid delivery of IRS-1PI 3-kinase
complexes to intracellular GLUT4-enriched membranes where
3`-phosphoinositides may accumulate and function in the
membrane-associated events which accompany the translocation of GLUT4
to the cell surface. The high concentration of tyrosine-phosphorylated
IRS-1 present in intracellular low density microsomes (27, 28) and our recent finding that membrane
association of IRS-1 can be regulated by insulin in 3T3-L1 adipocytes (29) reinforce this hypothesis. Two separate studies have
reported that GLUT4-containing vesicles prepared from rat adipocytes do
not contain PI 3-kinase, IRS-1, or insulin
receptors(27, 30) . However, the insulin stimulations
in these studies were for 10 min or longer. In the present studies, we
tested the possibility that rapid association of activated
IRS-1
PI 3-kinase complexes with the GLUT4-containing vesicles
might occur. Our data demonstrate that insulin-activated PI 3-kinase
indeed specifically associates with GLUT4-containing vesicles of both
3T3-L1 adipocytes and rat adipocytes. These results are consistent with
the hypothesis that PI 3-kinase and its reaction products play a
functional role in the movement of intracellular GLUT4 to the cell
surface in response to insulin.
In experiments designed to demonstrate the specificity of the immunoadsorption of GLUT4-containing vesicles (Fig. 2), 1 mM GLUT4 COOH-terminal peptide was included in Buffer B during the incubation of anti-GLUT4 IgG with Protein A-Sepharose. The beads were washed with Buffer B, and then antibody-bound beads in Buffer B containing 1 mM peptide were added to the precleared low density microsomes and allowed to incubate for 24 h. The supernatants were collected and subjected to immunoblot analysis. The beads were washed in Buffer B, and immunoadsorbed vesicles were solubilized in Buffer B containing 1% Nonidet P-40. The detergent-solubilized vesicles were then immunoprecipitated with anti-IRS-1 and subjected to a PI 3-kinase assay as described (32) using exogenous PI as substrate followed by thin layer chromatography (33) .
Figure 2:
Insulin increases IRS-1-bound PI 3-kinase
activity in GLUT4-containing vesicles. Low density microsomes were
prepared from 3T3-L1 adipocytes treated with (+) or
without(-) 100 nM insulin for 3.5 min. 1.5 mg of total
protein were then incubated in the presence of anti-GLUT4 IgG bound to
Protein A-Sepharose with (+) or without(-) 1 mM GLUT4 COOH-terminal peptide. A, immunoadsorbed
GLUT4-containing vesicles were solubilized, immunoprecipitated with
anti-IRS-1 IgG, and the immunoprecipitates were subjected to a PI
3-kinase assay as described under ``Experimental
Procedures.'' B, spots corresponding to PI 3-P on the
thin layer plate were quantitated using a Betascope. The data presented
are the average values from three independent experiments. C,
the supernatants from the immunoadsorption of low density microsomes
with anti-GLUT4 IgG bound to Protein A-Sepharose in the presence
(+) or absence(-) of 1 mM GLUT4 COOH-terminal
peptide were collected, and equal amounts of protein (25 µg) were
resolved by SDS-PAGE on a 10% gel and electrophoretically transferred
to nitrocellulose for 2 h at 200 mA. The filter was blocked and
subsequently incubated with anti-GLUT4 followed by I-Protein A. Also shown are the low density microsome
fractions (25 µg/lane) for this
experiment.
3T3-L1 adipocytes were stimulated with insulin for times
ranging from 1-15 min. Immunoblot analysis of low density
microsome fractions and plasma membrane fractions prepared from these
cells using anti-GLUT4 (Fig. 1A) demonstrates that
GLUT4 decreases in the low density microsome fraction and increases in
the plasma membrane fraction in a time-dependent manner, as previously
published(36) . A slight increase in GLUT4 (30%) can be
consistently detected in the plasma membrane fraction as early as 1
min, and further increases are observed at later time points, as also
reported for rat adipocytes(28) .
Figure 1:
Immunoadsorption of GLUT4-containing
vesicles from 3T3-L1 adipocytes. Low density microsome (LDM)
and plasma membrane (PM) fractions were prepared from 3T3-L1
adipocytes treated with 100 nM insulin for the times
indicated. A, protein (25 µg) from each fraction was
resolved by SDS-PAGE on a 10% gel and was electrophoretically
transferred to nitrocellulose for 2 h at 200 mA. The filter was blocked
and subsequently incubated with anti-GLUT4 followed by I-Protein A. The band corresponding to GLUT4 is
designated with an arrowhead. B, low density microsomes were
prepared from 3T3-L1 adipocytes treated with 100 nM insulin
for the times indicated. 125 µg of total protein were then
incubated in the presence of anti-GLUT4 IgG or nonimmune rabbit IgG
bound to Protein A-Sepharose as described under ``Experimental
Procedures.'' Immunoadsorbed vesicles were reduced and alkylated
prior to solubilization in sample buffer and resolution by SDS-PAGE and
were then subjected to immunoblot analysis with anti-GLUT4 followed by
I-Protein A as detailed above. The band corresponding to
GLUT4 is designated with an arrowhead.
GLUT4-containing vesicles were prepared from basal and insulin-stimulated 3T3-L1 adipocytes by immunoadsorption from the low density microsome fraction using anti-GLUT4 IgG. Results presented in Fig. 1B demonstrate that this method of preparing GLUT4-containing vesicles is highly specific in that no detectable GLUT4 protein is observed in the immunoprecipitates when nonimmune IgG is utilized to immunoadsorb vesicles instead of anti-GLUT4 IgG. GLUT4 protein is recovered in the supernatants from the nonimmune IgG immunoadsorption reactions while there is little detectable GLUT4 protein in the anti-GLUT4 supernatants (data not shown). Approximately 85% of the GLUT4-containing vesicles are immunoadsorbed with our anti-GLUT4 IgG following the initial clearing of low density microsomes with Protein A-Sepharose alone. Also shown in Fig. 1B is the decrease in GLUT4-containing vesicles in the low density microsome fractions with prolonged exposure to insulin.
In order to determine
whether activated complexes of IRS-1PI 3-kinase are associated
with GLUT4-containing vesicles, anti-GLUT4 immunoadsorbed vesicles were
solubilized in detergent-containing buffer, immunoprecipitated with
anti-IRS-1 IgG, and the anti-IRS-1 immunoprecipitates were subjected to
a PI 3-kinase assay. Fig. 2(A, lanes 1 and 3) shows that relative to basal conditions, increased PI
3-kinase activity is co-immunoprecipitated with IRS-1 from
GLUT4-containing vesicles upon a 3.5-min insulin stimulation. We also
tested whether the association of IRS-1
PI 3-kinase complexes with
the GLUT4-containing vesicles was specific by including a GLUT4
COOH-terminal peptide which specifically binds the anti-GLUT4 IgG
during the vesicle immunoadsorption incubation. As shown in Fig. 2(A, lanes 2 and 4), the
presence of GLUT4 peptide during immunoadsorption of GLUT4-containing
vesicles significantly inhibited IRS-1-associated PI 3-kinase activity.
The average values from three independent experiments showed the PI
3-kinase activity associated with GLUT4 vesicles increased 630%
relative to basal activity, and this increased activity was diminished
to 233% in the presence of GLUT4 peptide (Fig. 2B). In
all three experiments, the insulin effect on PI 3-kinase activity was
2.2-fold or greater relative to basal activity. In order to confirm the
effectiveness of this peptide in blocking immunoadsorption of
GLUT4-containing vesicles, equal amounts of the supernatants from the
immunoadsorption of the low density microsome fractions with anti-GLUT4
IgG in the absence or presence of COOH-terminal peptide were
immunoblotted for GLUT4. Fig. 2C shows that GLUT4
peptide significantly inhibits immunoadsorption of the GLUT4-containing
vesicles by the anti-GLUT4 IgG. The peptide-mediated decreases in PI
3-kinase activity (Fig. 2B) and immunoadsorption of
GLUT4-containing vesicles as measured by densitometric scanning of the
GLUT4 protein bands (Fig. 2C) were 63% and 68%,
respectively. These results demonstrate that the association of
IRS-1
PI 3-kinase complexes with GLUT4-containing vesicles is
highly specific.
We next tested whether an insulin-mediated stimulation of PI 3-kinase activity also occurs in GLUT4-containing vesicles from rat adipocytes. GLUT4-containing vesicles from freshly isolated rat adipocytes were prepared by fractionating low density microsomes in a sucrose velocity gradient, a procedure recently published for the purification of GLUT4-containing vesicles(34, 37) . As observed by Kandror et al.(34) and as depicted in Fig. 3, GLUT4-containing vesicles from rat adipocytes show a distinct sedimentation distribution and sediment faster than most structures present in the low density microsome fraction. Insulin treatment of rat adipocytes (3.5 min) caused a demonstrable reduction in the amount of GLUT4-containing vesicles in the low density microsome fraction, presumably due to their translocation and fusion with the plasma membrane, as well as a reproducible shift (see inset of Fig. 3) in the sedimentation of GLUT4-containing vesicles when compared with vesicles from control cells.
Figure 3: GLUT4-containing vesicles isolated from rat adipocytes by sucrose velocity gradient ultracentrifugation. Low density microsome fraction (1.5 mg of protein) from basal and insulin-treated (3.5 min) rat adipocytes were centrifuged in a 28-ml 10-35% sucrose gradient as described(34) . Inset shows immunoblot analysis of fractions using anti-GLUT4 followed by horseradish peroxidase-anti-rabbit. Graph shows the overall distribution of GLUT4 in basal (closed triangles) and insulin-treated adipocytes (open triangles) and total protein for basal adipocytes (closed circles).
As shown in panel A of Fig. 4, insulin treatment (3.5 min) caused a 1.6-fold increase in immunoreactive p85 protein in the low density microsome fraction while a 2.3-fold increase in the level of p85 was observed in the GLUT4-containing vesicles. PI 3-kinase activities measured in anti-p85 immunoprecipitates of the low density microsome fraction and GLUT4-containing vesicles were increased to corresponding levels (Fig. 4B). However, only a fraction of the total PI 3-kinase activity present in the low density microsome fraction was in the GLUT4-containing vesicle preparation since equal amounts of protein were used in the assays (Fig. 4B). The PI 3-kinase activity associated with both the low density microsome fraction and the GLUT4-containing vesicles was abolished by 100 nM wortmannin (data not shown). Similar results were obtained by preparing GLUT4-containing vesicles from basal and insulin-stimulated 3T3-L1 adipocytes using the sucrose velocity gradient procedure. A 4.8-fold increase in anti-p85 immunoprecipitated PI 3-kinase activity was observed in the GLUT4-containing vesicles from insulin-stimulated 3T3-L1 adipocytes when compared with vesicles from control cells (data not shown).
Figure 4: Insulin-stimulated PI 3-kinase associates with GLUT4-containing vesicles from rat adipocytes. GLUT4-containing vesicles were prepared from unstimulated or insulin-stimulated (3.5 min) rat adipocytes by sucrose velocity gradient ultracentrifugation. A, protein (25 µg) from each preparation was resolved by SDS-PAGE on a 7.5% gel and was electrophoretically transferred to nitrocellulose for 8 h at 150 mA. The filter was blocked, incubated with anti-p85 PI 3-kinase and then horseradish peroxidase-anti-rabbit followed by detection by chemiluminescence. Bands corresponding to p85 were quantitated using a scanning densitometer. B, GLUT4-containing vesicles (25 µg) were solubilized, immunoprecipitated with anti-p85 PI 3-kinase, and the immunoprecipitates were then subjected to PI 3-kinase assay as described under ``Experimental Procedures.'' Spots corresponding to PI 3-P on the thin layer plate were quantitated using a Betascope. The data presented for A and B are the average values from three independent experiments ± S.E.
GLUT4-containing vesicles prepared from control and
insulin-stimulated rat adipocytes were also assayed directly for PI
3-kinase activity using PI, PI 4-P, and PI 4,5-P as
substrates (Fig. 5). The HPLC profiles of the deacylated
polyphosphoinositides from the assay reaction demonstrate
insulin-stimulated increases of approximately 6-fold, 3-fold, and
3.3-fold in PI 3-P, PI 3,4-P
, and PI 3,4,5-P
,
respectively (Fig. 5C). The presence of 100 nM wortmannin in the assay completely abolished the
insulin-stimulated increases in polyphosphoinositides associated with
the GLUT4-containing vesicles (Fig. 5B). Large amounts
of PI 4-P, which were not stimulated by insulin treatment, were also
generated confirming PI 4-kinase is present in the GLUT4-containing
vesicles(30) . In addition, when GLUT4-containing vesicles
prepared by the sucrose velocity gradient procedure were immunoadsorbed
with anti-GLUT4 and then assayed for PI 3-kinase activity, HPLC
analysis demonstrated a 2-fold increase in insulin-stimulated PI 3-P
(data not shown).
Figure 5:
Effect
of insulin on PI 3-kinase activity in GLUT4-containing vesicles. A, GLUT4-containing vesicles prepared from control (solid
line) and insulin-treated rat adipocytes (dotted line)
were assayed for PI 3-kinase activity by incubating the vesicles with
PI, PI 4-P, and PI 4,5-P in the presence of
[
-
P]ATP. Shown are the HPLC profiles of
P-labeled deacylated polyphosphoinositides (gPIs). B, same as above but assay was performed in
the presence of 100 nM wortmannin. C, graph shows the
effect of insulin on PI 3-P, PI 3,4-P
, and PI
3,4,5-P
. The data shown are from 3 independent experiments
with different vesicle preparations ±
S.E.
The association of activated PI 3-kinase with
intracellular membranes enriched in GLUT4 reported here combined with
other data presented in this study and our previous work (29) suggests the following hypothesis: membrane-bound IRS-1
becomes tyrosine-phosphorylated upon insulin stimulation, associates
with PI 3-kinase, and is internalized on endosomal vesicles. A
population of these IRS-1PI 3-kinase complexes are delivered to
the intracellular storage pool of GLUT4-containing vesicles. The
products of PI 3-kinase catalytic action in GLUT4-containing vesicles
cause budding, fusion, or movement of these membranes which regulates
GLUT4 translocation to the plasma membrane. The data in this study do
not exclude the possibility that other mechanisms are involved in
mediating increased PI 3-kinase activity in GLUT4-containing membranes
in response to insulin nor do they address the mechanism by which PI
3-kinase and its 3` phosphoinositide products trigger GLUT4
translocation, an important question for future investigation. However,
a conceptual framework for the hypothetical activation of
ADP-ribosylation factor-regulated vesicular fusion events by
phosphoinositides is currently being
developed(38, 39) .
Recent data obtained from other laboratories also support the hypothesis that activated PI 3-kinase is involved in regulating GLUT4 trafficking in adipocytes. For example, IRS-1 antisense constructs introduced into rat adipocytes transfected with epitope-tagged GLUT4 decreased the sensitivity to insulin of GLUT4 translocation to the cell surface(40) . When IRS-1 was co-expressed in these cells, this effect was abolished. The specific insulin-mediated localization of PI 3-kinase to the specialized compartment that sequesters GLUT4 might also explain why activation of PI 3-kinase by other receptor signaling systems do not cause GLUT4 translocation(26) . Taken together, the data presented in this study and the findings of others are consistent with the hypothesis that PI 3-kinase is an essential component in the insulin signaling pathway leading to glucose uptake and GLUT4 translocation.