Aldolase Mediates the Association of F-actin with the
Insulin-responsive Glucose Transporter GLUT4*
Aimee W.
Kao
§,
Yoichi
Noda¶§,
John H.
Johnson¶,
Jeffrey E.
Pessin
, and
Alan R.
Saltiel¶
From the
Department of Physiology and Biophysics,
University of Iowa, Iowa City, Iowa 52242, ¶ Department of Cell
Biology, Parke-Davis Pharmaceutical Research Division, Ann Arbor,
Michigan 48105, and the Department of Physiology, University of
Michigan School of Medicine, Ann Arbor, Michigan 48109
 |
ABSTRACT |
To identify potential proteins interacting with
the insulin-responsive glucose transporter (GLUT4), we generated fusion
proteins of glutathione S-transferase (GST) and the final
30 amino acids from GLUT4 (GST-G4) or GLUT1 (GST-G1). Incubation of
these carboxyl-terminal fusion proteins with adipocyte cell extracts
revealed a specific interaction of GLUT4 with fructose 1,6-bisphosphate
aldolase. In the presence of aldolase, GST-G4 but not GST-G1 was able
to co-pellet with filamentous (F)-actin. This interaction was prevented by incubation with the aldolase substrates, fructose 1,6-bisphosphate or glyceraldehyde 3-phosphate. Immunofluorescence confocal microscopy demonstrated a significant co-localization of aldolase and GLUT4 in
intact 3T3L1 adipocytes, which decreased following insulin stimulation.
Introduction into permeabilized 3T3L1 adipocytes of fructose
1,6-bisphosphate or the metabolic inhibitor 2-deoxyglucose, two agents
that disrupt the interaction between aldolase and actin, inhibited
insulin-stimulated GLUT4 exocytosis without affecting GLUT4
endocytosis. Furthermore, microinjection of an aldolase-specific antibody also inhibited insulin-stimulated GLUT4 translocation. These
data suggest that aldolase functions as a scaffolding protein for GLUT4
and that glucose metabolism may provide a negative feedback signal for
the regulation of glucose transport by insulin.
 |
INTRODUCTION |
The insulin-responsive glucose transporter GLUT4 is expressed
primarily in adipose tissue, skeletal, and cardiac muscle (1-4). Under
basal conditions, GLUT4 slowly recycles between poorly defined intracellular compartments and the plasma membrane with the vast majority sequestered in these intracellular storage sites. Insulin stimulates a large increase in the rate of GLUT4 exocytosis concomitant with a smaller decrease in the rate of GLUT4 endocytosis (5-7). The
overall insulin-induced changes in GLUT4 trafficking kinetics result in
a 10-20-fold increase in the number of cell surface GLUT4 proteins
that accounts for the majority of insulin-stimulated increases in
glucose transport activity (8, 9).
Recently, several laboratories have begun to examine the subcellular
distribution of GLUT4 to identify the mechanism responsible for the
intracellular sequestration of the GLUT4 protein. Steady-state and
kinetic analysis of various expressed GLUT4 chimeric proteins have
indicated that both the amino- and carboxyl-terminal domains are
important in GLUT4 internalization from the plasma membrane (10-15).
In particular, the carboxyl-terminal dileucine motif (SLL) was found to
substantially alter GLUT4 trafficking kinetics and steady-state
localization (16, 17). Although a specific GLUT4 sequence responsible
for intracellular localization has yet to be identified, the presence
of a carboxyl-terminal retention signal is consistent with the
observation that expression of the GLUT4 carboxyl-terminal domain
results in the translocation of the endogenous GLUT4 protein to the
plasma membrane (18). In addition, the presence of a GLUT4
carboxyl-terminal binding protein can also account for the apparent
increase in GLUT4 carboxyl-terminal antibody immunoreactivity following
insulin stimulation (19, 20).
To address these issues, we have used GST fusion proteins to identify
and characterize GLUT4 carboxyl-terminal specific binding proteins. In
this manuscript, we demonstrate that the carboxyl-terminal domain of
GLUT4 can specifically associate with the bifunctional glycolytic
enzyme fructose-1,6-bisphosphate aldolase. This in vitro
interaction appears to be physiologically important as glycolytic intermediates that disrupt aldolase-actin interactions also inhibit insulin-stimulated GLUT4 translocation.
 |
EXPERIMENTAL PROCEDURES |
cDNA Constructs--
DNA fragments encoding the
carboxyl-terminal sequences of GLUT1 (G1),
IASGFRQGGASQSAKTPEELPHPLGADSQV; GLUT4 (G4),
SATFRRTPSLLEQEVKPSTELEYLGPDEND; and GLUT4* (G4*),
SATFRRTPSASEQEVKPSTELEYLGPDEND were provided by Dr. Morris Birnbaum and
subcloned into pGEX-KG vector. The resultant glutathione
S-transferase
(GST)1 fusion proteins were
purified by glutathione affinity chromatography as described by the
manufacturer (Amersham Pharmacia Biotech).
GST Fusion Protein Pull-down Experiments--
3T3L1 fibroblasts
were differentiated into adipocytes as described (21). In labeling
experiments, 3T3L1 adipocytes were incubated with 10 µCi/ml
Tran35S label (ICN) for 24 h before isolation of cell
extracts in 50 mM Hepes, pH 7.4, 1% Triton X-100, 10%
glycerol. Insoluble material was separated by microcentrifugation and
extracts precleared by incubation with glutathione-Sepharose beads for
1 h at 48 °C followed by a second 1 h incubation with the
GST fusion protein. The beads were then washed three times with 50 mM Hepes, pH 7.4, 0.1% Triton-100, 10% glycerol, bound
proteins eluted using Laemmli sample buffer and samples subjected to
SDS-PAGE, and autoradiography or aldolase immunoblotting. For large
scale preparation of the GST-GLUT4 binding proteins, rat epididymal fat
pads were isolated from 50 rats. Cell extracts were incubated with the
GST fusion proteins, eluants transferred onto polyvinylidene difluoride
membrane and N-terminal sequences determined using an automated protein
sequencer at the University of Michigan Protein Core Facility.
Immunoblotting--
Immunoblotting was performed as described
(22) using an aldolase antibody (kindly provided by Dr. Jurgen
Bereiter-Hahn), IAO2 GLUT4-specific antibody (22) or clathrin heavy
chain antibody (Transduction Laboratories).
F-actin/Aldolase/GLUT4 Co-pelleting Experiments--
Aldolase
and GST fusion protein solutions were dialyzed against 10 mM imidazole, 10 mM KCl, 1 mM
MgCl2, 0.1 mM dithiothreitol, pH 6.8, overnight
at 48 °C and precleared by centrifugation at 45,000 rpm (TLA100
rotor, Beckman). 200 µg of rabbit skeletal muscle F-actin (Sigma)
purified as described (23) and 100 µl of the 20-µl aldolase
solution were mixed for 30 min at room temperature following 5-10
strokes in a pestle homogenizer. The mixture was centrifuged and 300 µg/ml GST fusion protein was added to the pellet and suspended with a
small plastic homogenizer rod. After incubation for 30 min at room
temperature, the mixture was again centrifuged, and the supernatants
and pellets subjected to SDS-PAGE. Proteins were detected by Coomassie
Brilliant Blue staining. Fructose 1,6-bisphosphate (FBP) or
glyceraldehyde 3-phosphate (G3P) were added to both the aldolase and
the GST fusion proteins at a concentration of 50 µM as indicated.
Whole Cell Immunofluorescence Double Labeling--
3T3L1
adipocytes on glass coverslips were serum-starved for 3 h then
left untreated or stimulated for 30 min with 100 nM
insulin. Cells were fixed and permeabilized with 2% paraformaldehyde,
0.2% Triton X-100 in phosphate-buffered saline for 10 min and quenched in 100 mM glutamine/phosphate-buffered saline for 15 min.
Cells were then washed and sequentially incubated in 5% donkey serum, 1% bovine serum albumin/phosphate-buffered saline blocking buffer, 1:200 IA02 anti-GLUT4 and 1:100 anti-aldolase (Biogenesis), and 12.5 µg/ml FITC-conjugated donkey anti-rabbit and 12.5 µg/ml Texas Red-conjugated donkey anti-goat antibodies (Jackson ImmunoResearch Laboratories). Coverslips were mounted onto glass slides with Vectashield mounting medium (Vector Labs) and viewed with a Bio-Rad laser confocal.
Plasma Membrane Sheet Preparation--
Plasma membrane sheets
were prepared from 3T3L1 adipocytes as described (24). For
2-deoxyglucose (2DG) treatment of intact 3T3L1 adipocytes, cells were
serum starved and placed into glucose-free Dulbecco's modified
Eagle's medium containing 5 mM pyruvate and 5 mM glutamine with or without 5 mM 2DG for 5 min
before insulin stimulation. For fluorescence microscopy, the membrane
sheets were fixed with 2% formaldehyde and sequentially incubated with 5% donkey serum, a 1:100 dilution of a rabbit anti-insulin regulatable glucose transporter antibody (East Acres) and 6.25 µg/ml of
LRSC-conjugated donkey anti-rabbit antibody (Jackson ImmunoResearch
Laboratories). For immunoblotting experiments, cells were stimulated
with 20 nM insulin for 20 min, membrane sheets were
isolated and solubilized in a modified lysis buffer (22) with 0.05%
Triton X-100. A protein assay was performed, and lysates were placed
into Laemmli sample buffer without boiling.
Streptolysin-O Permeabilization--
The 3T3L1 adipocytes were
permeabilized with Streptolysin-O (SLO; Murex Diagnostics Limited) as
described (25) with minor modifications. Briefly, adipocytes were
washed 3 times with an intracellular buffer (20 mM HEPES,
pH 7.2, 140 mM L-glutamic acid, 5 mM EGTA, 7.5 mM MgCl2, 5 mM NaCl, 2 mM CaCl2) and then
incubated for 5 min at 378 °C in intracellular buffer with 0.8 IU/ml
Streptolysin-O and 10 mM MgATP. For immunofluorescence
studies, cells were washed in buffer (10 mM MgATP, 1 mM dithiothreitol, and 0.1% bovine serum albumin) and
treated for 5 min with either 5 mM 2DG, 25 mM
FBP, or other aldolase substrates as indicated before insulin stimulation.
Microinjection--
3T3L1 adipocytes were placed into L-15
medium and microinjected using an Eppendorf Micromanipulator 5171 and
Transjector 5246. Injection buffer consisted of 100 mM KCl
and 5 mM sodium phosphate, pH 7.2, with 1.0 mg/ml
FITC-dextran (Sigma), 1.2 mg/ml maltose-binding protein (MBP)-Ras
CAAX, and 2.5 mg/ml either aldolase antibody (Biogenesis) or
pre-immune serum (Research Genetics). Microinjected cells were placed
in serum-free Dulbecco's modified Eagle's medium containing 0.1%
bovine serum albumin for 1.5 h, treated with or without 20 nM insulin for 20 min, and plasma membrane sheets isolated. Sheets were incubated with a 1:1,000 dilution of sheep anti-MBP antibody (generously provided by Dr. Morris Birnbaum) and 1:400 dilution of IAO2 GLUT4 antibody, followed by incubation in 12.5 mg/ml
FITC-conjugated donkey anti-sheep and Cy5-conjugated donkey anti-rabbit
antibodies (Jackson ImmunoResearch Laboratories). In each experiment,
20-40 injected cells positive for FITC-MBP were scored for
GLUT4-specific immunofluorescence by two individuals blinded to
treatment. The results of two or three independent experiments were
analyzed using the analysis program InStat 2.0.
 |
RESULTS AND DISCUSSION |
To identify proteins that interact with the GLUT4
carboxyl-terminal domain, we expressed the GLUT1 and GLUT4
carboxyl-terminal 30 amino acids as GST fusions, GST-G1 and GST-G4,
respectively. An additional fusion construct of the GLUT4 carboxyl
terminus was generated containing a substitution of two residues shown to be important in GLUT4 endocytosis, leucines 488 and 489, with alanine and serine (GST-G4*), respectively (15-17). Incubation of GST
and GST-G4 with extracts from [35S]methionine-labeled
3T3L1 adipocytes resulted in the binding of multiple proteins. However,
there were two faint bands at 40 and 43 kDa that appeared to
specifically precipitate with GST-G4 and GST-G4* but not with GST or
GST-G1 (Fig. 1A). Pretreatment of 3T3L1 adipocytes with insulin before isolation of the cell extracts
had no significant effect on the in vitro association of the
40 and 43 kDa protein with GST-G4 and GST-G4* (Fig. 1A).

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Fig. 1.
GLUT4 carboxyl terminus interacts
specifically with aldolase in 3T3L1 and primary rat adipocyte
extracts. A, extracts from
[35S]methionine-labeled 3T3L1 adipocytes were prepared
from unstimulated or insulin-stimulated cells and incubated with GST,
GST-G4, GST-G1, or GST-G4*. Precipitated samples were resolved by
SDS-PAGE and subjected to autoradiography. B, GST, GST-G4,
GST-G1, or GST-G4* were incubated with or without extracts prepared
from primary isolated rat adipocytes. Following precipitation, samples
were resolved by SDS-PAGE and visualized by Coomassie Blue staining.
C, 3T3L1 adipocyte extracts were precipitated with GST,
GST-G4, GST-G1, and GST-G4* and subjected to immunoblotting with an
aldolase-specific antibody.
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|
Cell extracts were also prepared from primary isolated rat adipocytes
and incubated with the GST fusion proteins (Fig. 1B). Similar to 3T3L1 adipocytes, Coomassie Blue staining of these precipitates demonstrated that the 40- and 43-kDa proteins were specifically precipitated with GST-G4 and GST-G4* but not by GST or
GST-G1. Although other more intense bands were detected on the
Coomassie Blue stained gel, these were also seen in the absence of
lysate and were therefore nonspecific. We could not obtain a defined
amino acid sequence following purification of the 43-kDa protein;
however, the 40-kDa protein was partially sequenced and identified as
fructose 1,6-bisphosphate aldolase. To confirm that the 40-kDa protein
that bound GST-G4 and GST-G4* was aldolase, immunoblotting with an
aldolase-specific antibody was performed (Fig. 1C).
Incubation of cell extracts with GST-G4 and GST-G4* resulted in the
co-precipitation of a 40-kDa protein that demonstrated specific
immunoreactivity with an aldolase antibody. In contrast, no
immunoreactive proteins were detected in the GST or GST-G1 precipitates. Furthermore, as observed in the
[35S]methionine-labeled 3T3L1 adipocytes, cell extracts
from insulin-stimulated cells also precipitated aldolase
immunoreactivity in the presence of GST-G4 and GST-G4* but not GST or
GST-G1. These data demonstrate that the 40-kDa protein that
specifically binds the carboxyl-terminal domain of GLUT4 is fructose
1,6-bisphosphate aldolase.
Aldolase is an unusual protein in that it not only possesses enzymatic
activity but also plays a structural role in the assembly of the actin
cytoskeleton (26). The interaction of aldolase with actin can be
modulated by aldolase substrates and products (27, 28). We therefore
sought to determine the allosteric effect of these and related
molecules on the binding of aldolase to GST-G4 (Fig.
2). Dose-response relationships
demonstrated that 1 mM FBP, a concentration similar to the
Km of the enzyme for this substrate, completely
inhibited the binding of aldolase to GST-G4 in vitro (Fig.
2A). The aldolase reaction product, G3P, also inhibited
GST-G4 binding to aldolase, although at a significantly higher
concentration, 10 mM (Fig. 2B). In contrast, the
aldolase product dihydroxyacetone phosphate, as well as two other
structurally related sugars, fructose 1-phosphate and fructose
6-phosphate, had no effect on the interaction between GST-G4 and
aldolase (Fig. 2, C, D, and E).

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Fig. 2.
GLUT4 carboxyl terminus interaction with
aldolase is specifically inhibited by fructose 1,6-bisphosphate and
glyceraldehyde 3-phosphate. Cell extracts from 3T3L1 adipocytes
were incubated with GST-G4 in the presence of 0.1, 0.5, 1, or 10 µM FBP (A) or G3P (B).
Alternatively, the incubations were conducted in the presence of 10, 50, or 100 µM dihydroxyacetone phosphate
(DHAP) (C), fructose 1-phosphate (F1P)
(D) or fructose 6-phosphate (F6P) (E). Following
precipitation, the samples were subjected to Western blotting using an
aldolase-specific antibody.
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|
Previous studies have demonstrated that aldolase is associated with the
actin cytoskeleton and can cross-link actin fibers (27, 29). Because
the actin cytoskeleton has also been implicated in the regulation of
GLUT4 trafficking (30, 31), we next examined the ability of aldolase to
function as a molecular scaffold linking GLUT4 to F-actin (Fig.
3). F-actin was pre-incubated with
aldolase, followed by a second incubation with GST-G1 or GST-G4. When
the actin was pelleted by centrifugation, GST-G4 was found to be
specifically associated with actin in the presence of aldolase (Fig. 3,
lane 1). GST-G4 did not co-pellet with actin in the absence
of aldolase, suggesting that the GLUT4 protein binds to actin
indirectly (Fig. 3, lane 5). Likewise, the aldolase·GST-G4
complex was not pelleted by centrifugation in the absence of actin
(Fig. 3, lane 7). GST-G1 did not associate with actin in
either the absence or presence of aldolase (Fig. 3, lanes 4 and 6). As previously observed in Fig. 2, FBP and G3P can
inhibit the association of aldolase with GST-G4. FBP and G3P were also
able to prevent the formation of the F-actin·aldolase·GST-G4
ternary complex (Fig. 3, lanes 2 and 3). These
data indicate that in vitro, aldolase can function as a
molecular scaffold linking the carboxyl-terminal domain of GLUT4 with
filamentous actin.

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Fig. 3.
Actin, aldolase, and GLUT4 carboxyl terminus
form a ternary complex that is disrupted by FBP or G3P. F-actin
was resuspended in a precleared aldolase solution and then pelleted by
centrifugation. The pellet was then resuspended in GST-G4 or GST-G1
containing buffer. FBP or G3P were included in both aldolase and fusion
protein solutions as indicated. Polymerized actin was isolated by
microcentrifugation, the pellets resolved by SDS-PAGE, and proteins
visualized by Coomassie Blue staining.
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To determine whether aldolase and GLUT4 were co-localized in
vivo, double immunofluorescence labeling was performed. 3T3L1 adipocytes were treated with or without insulin, fixed, and incubated with aldolase and GLUT4-specific antibodies followed by labeling with
complimentary fluorescent-conjugated secondary antibodies (Fig.
4). Under basal conditions, the membrane
bound GLUT4 protein was found primarily in a peri-nuclear distribution
and in small vesicles scattered throughout the cytoplasm (Fig. 4,
panel A). Following insulin stimulation, GLUT4 moved to the
cell surface generating a "rim"-like fluorescence (Fig. 4,
panel D). Conversely, the soluble protein aldolase stained
in a diffusely cytosolic pattern although it was also concentrated in
the perinuclear region of the cell (Fig. 4, panels B and
E). When their respective labels are superimposed, GLUT4 and
aldolase exhibited a high degree of co-localization (Fig. 4,
panels C and F), suggesting an in vivo association between aldolase and the GLUT4-containing compartments.

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Fig. 4.
Aldolase and GLUT4 are partially co-localized
in 3T3L1 adipocytes. Serum-starved 3T3L1 adipocytes were
stimulated without (panels A-C) or with
(panels D-F) 100 nM insulin. Cells
were then fixed and incubated with GLUT4 and aldolase-specific primary
antibodies and FITC or Texas Red (TR)-conjugated secondary
antibodies as indicated. Cells were visualized by laser confocal
microscopy. Bar, 110 µm.
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|
To further investigate the in vivo significance of these
findings, we took advantage of the glycolytic inhibitor 2DG, which induces the dissociation of perinuclear aldolase from the actin cytoskeleton (32). In adipocytes, the insulin-induced translocation of
GLUT4 to the plasma membrane can be detected by the fluorescent detection of a GLUT4 antibody on the cytoplasmic face of isolated plasma membrane sheets. Pretreatment of 3T3L1 adipocytes with 5 mM 2DG slightly reduced the amount of basal plasma membrane GLUT4 immunofluorescence (Fig.
5A, panels 1 and
3). In contrast, pretreatment with 2DG markedly attenuated
the insulin-stimulated translocation of GLUT4 to the cell surface (Fig.
5A, panels 2 and 4). The 2DG
inhibition of insulin-stimulated GLUT4 translocation occurred in a
dose-dependent manner with an EC50 of
approximately 100 mM (data not shown). This inhibition of
GLUT4 translocation was also observed in GLUT4 immunoblots of isolated
plasma membranes without any effect on the plasma membrane association
of the clathrin heavy chain (Fig. 5B).

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Fig. 5.
2DG and FBP inhibit insulin-stimulated GLUT4
translocation. Panel A, 3T3L1 adipocytes
were pre-incubated with or without 5 mM 2DG. The cells were
then treated with insulin followed by isolation of plasma membrane
sheets and immunofluorescence microscopy using a GLUT4-specific
antibody. Panel B, 3T3L1 adipocytes were
incubated as above and the samples immunoblotted with a GLUT4 or
clathrin antibody. C, 3T3L1 adipocytes were permeabilized
with SLO, pre-incubated in the absence or presence of 25 mM
FBP and then treated as in panel A. Panel D,
permeabilized 3T3L1 adipocytes were pre-incubated with FBP and then
treated as in panel B. Bar, 110 µm.
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The blockade of insulin-stimulated GLUT4 translocation by 2DG could be
due to either an inhibition of exocytosis or an increase in
endocytosis. To determine whether 2DG affected the rate of GLUT4
endocytosis, we stimulated cells with insulin, removed the insulin by
washing with an acidic buffer, and then assayed plasma membrane GLUT4
at various times after insulin removal in the presence of 2DG.
Disappearance of GLUT4 from the cell surface after insulin removal was
not inhibited by 2DG treatment but rather seemed to be slightly
enhanced (data not shown). This small increase in the rate of GLUT4
endocytosis in the presence of 2DG likely reflected the inhibition of
further GLUT4 exocytosis following insulin removal. This effect was
similar to that reported for wortmannin, which also inhibits GLUT4
exocytosis but appears to enhance endocytosis due to the inhibition of
residual exocytosis (33). These data demonstrate that the rate of GLUT4
endocytosis was not significantly affected by 2DG pretreatment and is
consistent with an inhibition of GLUT4 exocytosis.
Although cells were incubated with glutamine and pyruvate as
alternative energy sources during 2DG treatment, cellular ATP levels
were found to decrease by 40% (data not shown). Because endocytosis
and exocytosis may have different sensitivities to ATP depletion, the
inhibition of insulin-stimulated GLUT4 exocytosis could have been due
to this decrease in ATP. To ensure adequate cellular ATP levels while
directly assessing the uncoupling of aldolase from GLUT4 in
vivo, 3T3L1 adipocytes were permeabilized with SLO in the presence
of 10 mM MgATP (Fig. 5, C and D).
Under these experimental conditions, cellular metabolism did not
deplete local concentrations of ATP (data not shown). In the
SLO-permeabilized adipocytes, insulin induced an increase in the
translocation of GLUT4 to the plasma membrane (Fig. 5C,
panels 1 and 2). As in the intact cells,
preincubation with 2DG prevented GLUT4 translocation (data not shown).
In addition, although preincubation with FBP slightly enhanced the
basal amount of GLUT4 associated with the plasma membrane, FBP
pretreatment markedly inhibited the insulin-stimulated translocation of
GLUT4 (Fig. 5C, panels 3 and 4). It
should be noted that the small increase in plasma membrane-associated
GLUT4 following SLO permeabilization has also been previously observed by others (25). In agreement with the immunofluorescence analysis, immunoblotting of isolated plasma membranes demonstrated that FBP
preincubation partially increased the basal plasma membrane-associated GLUT4 and inhibited the insulin-stimulated increase without affecting the plasma membrane association of the clathrin heavy chain (Fig. 5D).
To examine the metabolite specificity for the inhibition of
insulin-stimulated GLUT4 translocation, we compared the effects of
several aldolase substrates, products and related sugars on GLUT4
translocation (data not shown). Consistent with their effect on
aldolase-actin binding, pretreatment of SLO-permeabilized adipocytes with dihydroxyacetone phosphate, fructose 1-phosphate, fructose 6-phosphate, or glucose had no significant effect on insulin-stimulated GLUT4 translocation compared with control cells. To further confirm a
specific requirement for aldolase in insulin-stimulated GLUT4 translocation, we next microinjected cells with control or aldolase antibodies and assessed single cell GLUT4 translocation (Fig. 6). Specific identification of the
microinjected cells was accomplished by co-injection with a
carboxyl-terminal domain of Ras fused to the MBP as a plasma membrane
marker. In unstimulated cells, microinjection of either pre-immune IgG
or the aldolase IgG had no effect on GLUT4 translocation, which
remained at low basal levels (Fig. 6A, panels
1-4). Microinjection of pre-immune IgG did not inhibit the insulin-stimulated translocation of GLUT4 (Fig. 6B,
panels 1 and 2). In contrast, the
aldolase-specific IgG reduced the extent of GLUT4 translocation only in
the cells that were microinjected but not in the surrounding
nonmicroinjected cells (Fig. 6B, panels 3 and
4). Quantitation of these data demonstrated that insulin stimulated GLUT4 translocation in 49% of cells microinjected with pre-immune IgG and 29% of cells microinjected with aldolase-specific IgG (Fig. 6C). Together, these data provide compelling
evidence for a specific functional role of fructose 1,6-bisphosphate
aldolase in the insulin stimulation of GLUT4 translocation in
adipocytes.

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Fig. 6.
Microinjection of an aldolase antibody
inhibits insulin-stimulated GLUT4 translocation. 3T3L1 adipocytes
were microinjected with MBP-Ras and either pre-immune serum or an
aldolase antibody. Cells were left unstimulated (A) or
stimulated (B) with insulin. Isolated plasma membrane sheets
were incubated with MBP or GLUT4 antibodies followed by FITC or
Cy5-conjugated secondary antibodies. Microinjected cells
(arrowheads) were identified by FITC anti-MBP-specific
immunofluorescence, whereas the membrane-localized GLUT4 was assessed
by Cy5 anti-GLUT4-specific immunofluorescence. Bar, 30 µm.
C, microinjected 3T3L1 adipocytes were scored for
GLUT4-specific immunofluorescence. The percentage of injected cells
demonstrating GLUT4 at the cell surface (GLUT4 positive
sheets) are shown for control ( ) and insulin-stimulated ( )
cells. Asterisks (*) indicate that the percentage of GLUT4 positive
cells following insulin stimulation was significantly different between
those injected with anti-aldolase antibody compared with pre-immune
serum (p < 0.05). Results are the average of two
(control) or three (insulin-stimulated) independent experiments.
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Previous studies have demonstrated that aldolase exhibits functional
duality. In addition to its enzymatic activity, this protein plays a
structural role in the binding and polymerization of actin (28, 29,
34). Depolymerization of the actin cytoskeleton with cytochalasin D or
latrunculin B has been shown to attenuate insulin-stimulated GLUT4
translocation, suggesting that an intact actin cytoskeleton is required
for insulin-stimulated GLUT4 translocation (30, 31). Consistent with
this model, our data demonstrate that aldolase, a functional tetramer,
may also serve as a scaffolding protein linking GLUT4 and hence GLUT4
vesicles, to the actin cytoskeleton. In in vitro
experiments, aldolase not only specifically interacted with the
carboxyl-terminal domain of GLUT4, but linked GLUT4 to polymerized
actin. This interaction was disrupted by specific substrates of
aldolase that inhibit GLUT4-aldolase or aldolase-actin interactions.
These specific substrates not only prevented aldolase-actin interaction
but also prevented GLUT4-aldolase binding in vitro and in a
manner consistent with their ability to interfere with insulin-stimulated GLUT4 translocation in vivo. Furthermore,
aldolase and GLUT4 exhibited an overlapping subcellular distribution,
and microinjection of an aldolase-specific antibody reduced the
insulin-stimulated translocation of GLUT4. Together, these data
strongly suggest that aldolase plays a critical role in the dynamic
association of GLUT4 vesicles with the actin cytoskeleton. The ability
of aldolase substrates to disrupt the actin·aldolase·GLUT4 complex, as well as GLUT4 vesicle trafficking, suggests that the metabolism of
glucose may provide a negative feedback signal to prevent further exocytosis of GLUT4 vesicles and potentially contribute to the mechanism of glucotoxicity.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Morris Birnbaum for providing
glucose transporter, MBP-Ras cDNAs, and MBP antibody, Dr. Jurgen
Bereiter-Hahn for supplying aldolase antibody, Dr. Jeffrey Elmendorf
for assistance in analyzing microinjection results and Dr. Konstantin
Kandror for helpful discussion and critical reading of the manuscript.
 |
FOOTNOTES |
*
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.
§
These authors contributed equally to the work.
To whom correspondence should be addressed: Parke-Davis
Pharmaceutical Research Div., Warner-Lambert Co., 2800 Plymouth Rd., Ann Arbor, MI 48105. Tel.: 734-622-3960; Fax: 734-622-5668; E-mail: alan.saltiel{at}wl.com.
 |
ABBREVIATIONS |
The abbreviations used are:
GST, glutathione
S-transferase;
PAGE, polyacrylamide gel electrophoresis;
FITC, fluorescein isothiocyanate;
2DG, 2-deoxyglucose;
MBP, maltose-binding protein;
FBP, fructose 1,6-bisphosphate;
G3P, glyceraldehyde 3-phosphate;
SLO, Streptolysin-O.
 |
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