Laboratory of Molecular Cardiology, Diabetes Research Institute, D-40225 Düsseldorf, Germany
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ABSTRACT |
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Ventricular
cardiomyocytes and cardiac tissue of lean and genetically obese
(fa/fa) Zucker rats were used 1) to study the role of the p85 regulatory subunit isoforms p85 and p85
for insulin signaling through the phosphatidylinositol (PI) 3-kinase pathway, and 2) to elucidate the implications of these
mechanisms for cardiac insulin resistance. Western blot analysis of
cardiomyocyte lysates revealed expression of p85
and p85
but no
detectable amounts of the splice variants of p85
. Essentially no
p85
subunit of PI 3-kinase was found to be associated with insulin
receptor substrate (IRS)-1 or IRS-2 in basal and insulin-stimulated (5 min) cardiomyocytes. Instead, insulin produced a twofold increase in
p85
associated with IRS-1, leading to a three- to fourfold increase
in p85
-associated PI 3-kinase activity. This response was
significantly reduced in obese animals. Comparable results were
obtained in the intact heart after in vivo stimulation. In GLUT-4-containing vesicles, an increased abundance (3.7 ± 0.7-fold over basal) of p85
was observed after insulin stimulation
of lean animals, with no significant effect in the obese group. No p85
could be detected in GLUT-4-containing vesicles. Recruitment of
the p110 catalytic subunit of PI 3-kinase and a twofold increase in
enzyme activity in GLUT-4-containing vesicles by insulin was observed
only in lean rats. We conclude that, in the heart, p85
recruits PI
3-kinase activity to GLUT-4 vesicles, whereas p85
represents the
main regulator of IRS-1- and IRS-2-mediated PI 3-kinase activation.
Furthermore, multiple defects of PI 3-kinase activation, involving both
the p85
and the p85
adaptor subunits, may contribute to cardiac
insulin resistance.
GLUT-4-containing vesicles; obesity; insulin resistance; cardiac muscle
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INTRODUCTION |
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IT IS NOW WELL RECOGNIZED that phosphatidylinositol (PI) 3-kinase operates as an essential signal transduction element in most, if not all, of the cellular processes regulated by insulin in its target tissues (for review, see Refs. 1, 7, 40). In addition, PI 3-kinase is activated by a variety of growth factor tyrosine kinase receptors and thus regulates multiple and diverse cellular functions, including mitogenesis (10), membrane ruffling (25), cytoskeletal organization and cell motility (45), membrane trafficking (39), and insulin-stimulated glucose transport and glycogen synthesis (4, 20, 42). Class 1 PI 3-kinase is a heterodimeric enzyme consisting of a p110 catalytic subunit and a p85 regulatory subunit (1). The latter exerts adaptor functions by interacting with tyrosine-phosphorylated cognate motifs via two src homology 2 (SH2) domains, finally leading to an increased catalytic activity of the PI 3-kinase complex (32). The diversity of cellular functions controlled by PI 3-kinase requires a high level of specificity for the activation of this enzyme. This process, however, remains poorly understood.
One level of specificity relates to the tissue-specific expression of a
considerable array of different adaptor subunits encoded by at least
three different genes, termed p85, p85
, and p55
(40). The p85
gene generates two truncated splice
variants, p55
and p50
, that lack the SH3 and several other
NH3-terminal domains (2, 19). Isoform p50
was found to be highly expressed in human skeletal muscle
(38) and exhibited the most pronounced effect of insulin
on recruitment into antiphosphotyrosine immunoprecipitates (38). Furthermore, targeted disruption of the p85
gene
resulted in increased insulin sensitivity, most probably resulting from the isoform switch to the p50
variant (43). It is
therefore generally thought that the p85
regulatory subunit and its
splice variants play a pivotal role in insulin signaling. The function of the p85
isoform, which has a 62% amino acid homology with p85
(34), has remained controversial. In an earlier study, Baltensperger et al. (3) reported that p85
does not
mediate marked activation of PI 3-kinase, whereas in skeletal muscle
this isoform contributes significantly to the activation process
(38). An even higher association of p85
with insulin
receptor substrate (IRS)-1 compared with p85
was found in L6
myoblasts (13), and similar but distinct insulin receptor
signaling complexes of the two isoforms were observed in rat hepatoma
cells (41). These data suggest that the different adaptor
subunit variants might play a prominent role in controlling downstream
insulin signaling in a highly tissue-specific manner.
A second level of specificity relates to the compartmentalization of the IRS/PI 3-kinase signaling complexes (18). It is well known that insulin induces the recruitment of PI 3-kinase activity to intracellular membranes, whereas growth factors like platelet-derived growth factor (PDGF) shift this enzyme to the plasma membrane (33, 49). This difference is thought to explain the inability of PDGF to activate glucose transport despite increasing PI 3-kinase activity (33). A compelling hypothesis is that insulin recruits PI 3-kinase activity to the intracellular GLUT-4-containing vesicles, thus being directly linked to the process of GLUT-4 translocation (16). Three groups have confirmed the presence of PI 3-kinase activity in GLUT-4-containing vesicles after insulin stimulation of 3T3-L1 adipocytes (12, 16, 48); however, others (5) have failed to detect this colocalization. Furthermore, in skeletal muscle, PI 3-kinase activity was shown to be absent from GLUT-4-containing vesicles (26, 27). So far, no study has addressed the question of whether adaptor subunits may differentially recruit PI 3-kinase activity to different cellular compartments, certainly representing an additional, yet unexplored, level of specificity of PI 3-kinase signaling.
In the present investigation, we have analyzed and compared the
involvement of different adaptor subunit isoforms in cardiac insulin
signaling 1) at early steps by measuring the interaction with IRS proteins, and 2) at downstream reactions by
monitoring the recruitment of PI 3-kinase to GLUT-4-containing
vesicles. Furthermore, we have extended this analysis to
insulin-resistant, genetically obese (fa/fa) Zucker rats. In
our recent studies on this animal model, we have shown a failure of
insulin-regulated recruitment of cardiac GLUT-4 (46) and
an altered response of IRS-1-associated PI 3-kinase activity
(24); however, the relative contribution of the different
adaptor subunits to these defects is unknown. It is well established
that cardiac insulin resistance plays an important role in a variety of
disease states (8) and that the perpetual contractile
activity of the myocardium may require highly specialized signaling
pathways within the dynamic context of regulators of cardiac function
(8). The present study shows that, in cardiac muscle, the
p85 subunit recruits PI 3-kinase activity to GLUT-4-containing
vesicles independent of IRS proteins, whereas p85
mediates
substantial activation of PI 3-kinase at the level of IRS-1. Both
reactions are impaired in obesity, suggesting that multiple alterations
of PI 3-kinase activation contribute to insulin resistance.
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MATERIALS AND METHODS |
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Chemicals.
125I-labeled protein A (30 mCi/mg) and
[-32P]ATP (6,000 Ci/mmol) were purchased from Amersham
(Braunschweig, Germany). Reagents for SDS-PAGE were supplied by
Pharmacia and Sigma (Deisenhofen, Germany). Insulin (Actrapid HM, 100 units/ml) was supplied by Novo (Mainz, Germany). Protein G
agarose and protein A-trisacryl were purchased from Pierce (Rockford,
IL). The polyclonal GLUT-4 antiserum was obtained from a rabbit
injected with a peptide corresponding to the COOH-terminal 12 amino
acids of GLUT-4 (TELEYLGPDEND) coupled to keyhole limpet hemocyanin
(Eurogentec, Belgium). The anti-IRS-1 antibody was kindly provided by
Dr. J. A. Maassen (Leiden, The Netherlands). Rabbit polyclonal
antiserum, raised against a glutathione S-transferase fusion
protein corresponding to the amino-terminal SH2-domain of human p85
(p85
NSH2), was kindly supplied by Dr. P. R. Shepherd (London, UK). Rabbit polyclonal antiserum raised against a
glutathione S-transferase (GST) fusion protein corresponding
to the SH3-domain of p85
was generously provided by Dr. T. Asano
(Tokyo, Japan). Monoclonal anti-p85
PI 3-kinase antibody and the
monoclonal horseradish peroxidase-conjugated anti-phosphotyrosine
antibody (RC20) were purchased from Transduction Laboratories
(Lexington, KY). Horseradish peroxidase-conjugated goat anti-rabbit and
goat anti-mouse IgG antibodies were from Promega (Mannheim, Germany).
Polyclonal anti-p110
PI 3-kinase antibody was from Santa Cruz
Biotechnology (Santa Cruz, CA); polyclonal anti-p85 PI 3-kinase
antibody (p85PAN), Jurkat cell lysate, and EGF-stimulated
A431 cell lysate were obtained from UBI (Lake Placid, NY). All other
chemicals were of the highest grade commercially available.
Isolation of cardiomyocytes and membrane preparation from whole
heart.
Genetically obese (fa/fa) male Zucker rats
(12-16 wk old, 480-520 g) were kindly provided by Dr. L. Herberg (Düsseldorf, Germany). Blood samples were collected for
determinations of glucose and insulin as outlined previously
(9). Ca2+-tolerant cardiac myocytes were
isolated by perfusion of adult rat heart with collagenase as detailed
previously (24). The final cell suspension was incubated
for 60 min until further use in HEPES buffer [130 mM NaCl, 4.7 mM KCl,
1.2 mM KH2PO4, 25 mM HEPES, 5 mM glucose, 2%
(wt/vol) BSA, pH 7.4, equilibrated with oxygen] containing
MgSO4 and CaCl2 (final concentrations, 1 mM) at
37°C in a rotating water bath shaker. Cell viability was judged by
determination of the percentage of rod-shaped cells and averaged 90-97% under all incubation conditions. Plasma and microsomal membranes from cardiac tissue were prepared as recently described by us
(37). Briefly, cardiac tissue was removed and homogenized at 4°C in a buffer containing 10 mM Tris · HCl, 0.1 mM
phenylmethylsulfonyl fluoride (PMSF), 1 mM
Na3VO4, and 2.6 mM dithiothreitol (DTT) by
application of an Ultraturrax for 60 s. Homogenization was continued by 10 strokes in a glass-Teflon homogenizer followed by
3 × 3 strokes in a tight-fitting Potter-Elvehjem homogenizer. After centrifugation at 3,000 g, the supernatant was
centrifuged at 200,000 g for 90 min to pellet the crude
membrane fraction and obtain the cytosol (supernatant). Further
purification was achieved by applying the pellet to a discontinuous
gradient consisting of 0.57, 0.72, 1.07, and 1.43 M sucrose buffer and
centrifugation at 40,000 g for 16 h. Membranes were
harvested from each sucrose layer and stored at 70°C. Protein was
determined using a modification of the Bio-Rad protein assay with BSA
as a standard. Ouabain-sensitive Na+/K+-ATPase
and Ca2+/K+-ATPase were used as marker enzymes
for sarcolemma and microsomal membranes, respectively, and were
determined as described (37). Membranes recovered from the
0.72 M sucrose layer were enriched five- to sevenfold in the activity
of the Na+/K+-ATPase and were considered as a
plasma membrane fraction (PM), whereas membranes obtained in the 1.07 M
sucrose layer showed a 0.5-fold activity of this marker enzyme when
compared with the homogenate. Furthermore,
Ca2+/K+-ATPase increased fourfold in this
fraction. The 1.07 M membrane fraction was henceforth termed the
microsomal membrane fraction (MM). For the insulin-stimulated studies,
rats received a tail vein injection of regular insulin (4 units/100 g),
and hearts were removed 20 min later.
Immunoadsorption of GLUT-4-containing vesicles. GLUT-4-enriched membrane vesicles were prepared essentially as previously described (47). Briefly, microsomal membranes were incubated for 14 h with protein G agarose at 4°C in PBS, pH 7.4, containing PMSF (0.1 mM), DTT (2.6 mM), EDTA (1 mM), and BSA (0.4%). After centrifugation, GLUT-4 antiserum was added to the supernatant, and the membranes were sonicated for 20 s. Control membranes were treated identically, except that the preimmune rabbit serum was added. After a 5-h incubation at 4°C, the membranes were pelleted, resuspended, and incubated for additional 16 h with protein-G agarose. After centrifugation, the beads were washed four times with PBS, and the vesicle proteins were eluted with Laemmli sample buffer (29) for SDS-PAGE or solubilized in a PBS-buffer (pH 7.4) containing PMSF (0.1 mM), DTT (2.6 mM), EDTA (1 mM), Na3VO4 (1 mM), and 2% Triton X-100 for immunoprecipitation.
Immunoprecipitation. Isolated cardiomyocytes or cardiac tissue of basal and insulin-stimulated lean and obese Zucker rats was homogenized at 4°C in a buffer containing 50 mM Tris, 150 mM NaCl, 20 mM NaF, 10 mM EDTA, 1 mM Na3VO4, 0.3 mM PMSF, 10 mM benzamidine, 15 µM pepstatin, and Triton X-100 (1% vol/vol), pH 7.4, by application of an Ultraturrax for 30 s. Homogenization was continued by 10 strokes in a glass-Teflon homogenizer followed by 9 strokes in a tight-fitting Potter-Elvehjem homogenizer. After incubation for 2 h, the suspension was centrifuged at 16,000 g for 2 min, and the supernatant was further incubated with a mixture of protein G agarose and protein A-trisacryl beads for 2 h at 4°C. After centrifugation, the supernatant was subjected to immunoprecipitation.
For immunoprecipitation of IRS-1, the IRS-1 antibody was preadsorbed to a mixture of protein G agarose and protein A-trisacryl beads for 2 h at 4°C. The adsorbed antibody was then added to the precleared lysate and incubated for 16 h at 4°C with gentle rotation. After centrifugation, the immunopellet was washed three times with complete lysis buffer and twice with PBS. For immunoprecipitation of PI 3-kinase from GLUT-4 vesicles with either anti-p85 or anti-p85Immunoblotting. Protein samples were separated by SDS/PAGE by use of 8-18% gradient gels and were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Germany). Membranes were blocked for 90 min in PBS, pH 7.4, containing 0.05% Tween 20 and 10% milk powder, or, for phosphotyrosine detection, for 60 min in 10 mM Tris, pH 7.5, 50 mM NaCl, 0.1% Tween 20, and 1% BSA. Thereafter, membranes were incubated for 16 h at 4°C with dilutions from 1:250 to 1:1,000 of the different antibodies. After extensive washing with blocking buffer without milk powder or BSA, membranes were incubated for 2 h with 125I-protein A (0.3 µCi/ml) or with horseradish peroxidase-conjugated goat anti-rabbit or goat anti-mouse IgG. Membranes were again extensively washed and either visualized by enhanced chemiluminescence on a LumiImager work station or visualized and quantified on a FUJIX BAS 1000 bioimaging analyzer (Fuji, Japan). Significance of reported differences was evaluated using the null hypothesis and t-statistics for unpaired data.
Assay of PI 3-kinase activity.
PI 3-kinase activity was measured as described previously
(24). Briefly, 50 µl of a reaction mixture containing
0.2 mg/ml PI, 20 mM HEPES, pH 7.2, 0.4 mM EGTA, 0.4 mM
Na2HPO4, and 10 mM MgCl2 with or
without wortmannin (1 µM) were added to the immunoprecipitates. The
kinase buffer was incubated with the immunoprecipitates for 5 min at
room temperature, and the reaction was started by addition of
[-32P]ATP (40 µM and 0.2 µCi/µl). After 20 min,
the reaction was stopped by the addition of 30 µl of 4 N HCl and 130 µl of chloroform-methanol (1/1). The organic phase was extracted and
spotted on a silica gel thin-layer chromatography plate (Merck,
Darmstadt, Germany) and was developed in chloroform-methanol-25%
NH4OH-water (43:38:5:7, vol/vol). Plates were dried and
subsequently visualized.
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RESULTS |
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Expression of p85 subunit variants in cardiac muscle and
interaction with IRS proteins.
Isoforms p85 and p85
represent two widely expressed subunits of
PI 3-kinase, whereas the splice variants of p85
exhibit a more
tissue-specific expression (40). To determine the pattern of cardiac p85 expression, we used 1) a polyclonal
anti-p85
NSH2 antiserum raised against a
GST-fusion protein corresponding to the amino-terminal SH2-domain of
human p85
, which antiserum readily detects the p50 and p55 truncated
splice variants of p85
but does not crossreact with the
-isoform
(38); 2) an antiserum raised against the
SH3-domain of p85
with exclusive specificity for this isoform
(19); and 3) a broad specific polyclonal
p85-antibody (p85PAN). As shown in Fig.
1A, the p85
signal is
clearly visible in cardiomyocyte lysates, whereas essentially no splice
variants with the expected molecular mass of 48-55 kDa were
observed (Fig. 1A, left lane). This observation
is additionally supported by detection with the p85PAN
antiserum. The p85
isoform was detected with a slightly higher molecular mass compared with p85
. We therefore conclude that p85
and p85
must be considered the major regulatory subunits of PI
3-kinase in the heart.
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Recruitment of p85 isoforms and PI 3-kinase activity to
GLUT-4-containing vesicles in response to insulin.
We then addressed the question of whether the two p85 adaptor isoforms
might be involved in the recruitment of PI 3-kinase activity to
intracellular compartments, potentially in an isoform-specific fashion.
This was of particular interest, because only p85 associates to
IRS-1 in the heart (see above) and because IRS-1 is assumed to mediate
the downstream targeting of PI 3-kinase to GLUT-4-containing vesicles
(16). To examine the colocalization of PI 3-kinase and
GLUT-4 and to evaluate possible alterations at this level of insulin
signaling in the insulin-resistant state, lean and obese Zucker rats
were treated with insulin, and GLUT-4-containing vesicles from cardiac
muscle were obtained as outlined in MATERIALS AND METHODS.
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DISCUSSION |
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Class 1 PI 3-kinase represents a key element of the
insulin-signaling cascade; however, this enzyme is activated by many
receptor and nonreceptor tyrosine kinases mediating a variety of
effects different from insulin (1, 7, 10, 36, 40).
Therefore, the question of specificity of insulin-regulated PI 3-kinase
activation remains a key step to an improved understanding of insulin
action. Two apparently independent characteristics determine the
specificity of PI 3-kinase activation. First, a panel of regulatory
adaptor subunit isoforms with a tissue-specific expression exists
(40); second, the compartmentalization of the PI 3-kinase
signaling complexes plays a crucial role in directing insulin action to the downstream effector sites (18, 33, 49). In the present study, we have analyzed these processes in isolated cardiomyocytes and
cardiac tissue after in vivo stimulation with insulin. We now present
for the first time conclusive evidence for a functionally different
involvement of the p85 and the p85
adaptor subunit in insulin
signaling, pointing to an additional level of specificity in the
insulin-PI 3-kinase pathway.
Two widely expressed isoforms of the PI 3-kinase adaptor subunit,
p85 and p85
, have been described (34), and these
proteins were also detected in adult cardiomyocytes. However, the
truncated splice variants of p85
, p50 and p55, were below the
detection level in cardiac tissue. This differs substantially from
skeletal muscle, in which the p50 variant exhibits an even higher
expression than p85
(38). It is worth noting that we
have used the p85
NSH2 antiserum of Ref. 38,
and that this antiserum does not recognize any splice
variants of p85
in 3T3-L1 adipocytes (33), a cell line
not expressing p85
(3). The tissue-specific expression of the adaptor subunit variants supports the notion (40)
of individual functions of these proteins, most probably in a
cell-specific context. The present study demonstrates such a functional
difference between the p85
and the p85
isoform in the heart, in
that 1) only p85
associates to IRS-1 and IRS-2 in
response to insulin, 2) p85
mediates a significant
activation of PI 3-kinase, and 3) only p85
is recruited
to GLUT-4-containing vesicles.
The two isoforms of p85 share 62% amino acid homology
(34) and form stable complexes with the catalytic p110
subunit of PI 3-kinase (15). Several functional
differences between p85 and p85
have been reported. Thus
triggering of the CD3 antigen complex in T-cells was found to induce a
rapid serine phosphorylation of p85
, whereas p85
remained
unaffected (35). Furthermore, Giorgino et al.
(13) reported a selective upregulation of p85
expression in L6 myoblasts, and similar but distinct insulin receptor signaling complexes of the two isoforms were observed in rat hepatoma cells (41). We show here that, in the heart,
insulin-regulated activation of IRS-1-associated PI 3-kinase is
mediated by p85
, whereas p85
does not interact with IRS-1 or
IRS-2. The latter finding may be explained by the interaction of p85
with a yet-unidentified phosphoprotein, pp200, which was observed in
contraction- and insulin-stimulated cardiomyocytes (44).
In their earlier study using transfected cell lines, Baltensperger et
al. (3) hypothesized that PI 3-kinase is recruited to
IRS-1 irrespective of the isoform of the regulatory subunit and that,
in a second step, the intrinsic activity increases when associated with
p85
, whereas p85
was found to be insulin insensitive. We report
here a completely different situation in a primary cell, showing that
p85
mediates a substantial activation of PI 3-kinase, in agreement
with recent results in skeletal muscle (38). We therefore
conclude that, in the heart, the p85
adaptor subunit represents an
essential element of insulin signaling with a function different from
that of p85
.
A second key finding of the present investigation consists of the
observation that insulin stimulation results in a significant recruitment of PI 3-kinase activity to GLUT-4-containing vesicles and
that this activity is associated with the p85 regulatory subunit of
the enzyme. The p85
adaptor subunit could not be detected in the
GLUT-4-containing vesicles despite its association with IRS-1 and
IRS-2. We therefore conclude that the regulatory subunit serves to
mediate the assembly of PI 3-kinase and GLUT-4-vesicles in this
downstream-signaling complex of insulin action. Our data fit with a
scenario in which the two isoforms of the p85 adaptor subunit of PI
3-kinase mediate diversification of cardiac insulin signaling in that
1) p85
recruits PI 3-kinase activity to the GLUT-4-vesicles and thus participates in GLUT-4 translocation, and
2) p85
serves to activate PI 3-kinase upstream of the
translocation process. This latter reaction will lead to the
stimulation of protein kinase B (PKB)/Akt, which is known to regulate
cardiac glycolysis (6), protein synthesis, and gene
expression (1).
Although it is well accepted that nearly all the insulin-stimulated PI 3-kinase activity is located on intracellular membranes (33, 49), the issue of PI 3-kinase colocalizing to GLUT-4 has remained controversial (5, 12, 16, 26, 27, 48). Heller-Harrison et al. (16) reported that, in response to insulin, activated complexes of IRS-1 · PI 3-kinase can be immunoprecipitated with anti-IRS-1 antibody from detergent extracts of immunoadsorbed GLUT-4-containing vesicles prepared from 3T3-L1 adipocytes. They also showed that insulin treatment caused a two- to threefold increase in the amount of the regulatory subunit p85 and the enzymatic activity of PI 3-kinase in GLUT-4-containing vesicles prepared from these cells, in excellent agreement with our observations in the heart. Klip's group [Wang et al. (48)] confirmed the presence of PI 3-kinase activity in GLUT-4-containing vesicles obtained from 3T3-L1 adipocytes and suggested that actin filaments might be involved in the relocalization of PI 3-kinase to GLUT-4 vesicles. However, in skeletal muscle, PI 3-kinase was shown to be absent from GLUT-4-containing vesicles (26, 27), although it is generally believed that GLUT-4-containing vesicles from these two tissues are very similar (21). The present study on cardiac muscle clearly shows that, in this muscle tissue, the PI 3-kinase can be found in GLUT-4-containing vesicles in response to insulin. As outlined by Kristiansen et al. (26), this may be explained by the tissue-dependent copurification of cytoskeletal elements in the GLUT-4 vesicle preparation, since the cytoskeletal network may participate in binding and directing PI 3-kinase to the GLUT-4 vesicles. The precise function of PI 3-kinase activity in the GLUT-4-containing vesicles and its relationship to GLUT-4 translocation is yet unknown. One possibility relates to the recent report that Akt-2 associates to GLUT-4-containing vesicles and phosphorylates their component proteins in response to insulin (28). In this context, the production of phosphatidylinositol 3,4,5-trisphosphates and phosphatidylinositol 3,4-bisphosphates by PI 3-kinase may serve as binding sites for Akt (11). It is worth noting that we also observe the insulin-dependent recruitment of Akt-2 to our GLUT-4 vesicle preparation obtained from cardiac muscle (22), using the same in vivo protocol as described here for the recruitment of PI 3-kinase. Certainly, additional mechanisms, like binding of phosphoinositides to proteins that mediate intracellular vesicle trafficking, must also be considered. Glucose-regulated protein-1 and cytohesin-1, two proteins that bind phosphoinositides through a pleckstrin homology domain and display a region of high sequence similarity to the yeast Sec7 protein, represent recently identified members of this family (23, 30).
The data obtained in insulin-resistant Zucker rats lend further
support to the notion that PI 3-kinase activity within or in
association with the GLUT-4-containing vesicles may serve to trigger
the trafficking process. The complete lack of PI 3-kinase activity in
GLUT-4-containing vesicles of obese rats and the inability of insulin
to recruit p85 to the vesicles correlate with our earlier
observations of a complete block of insulin-stimulated GLUT-4
translocation in cardiac muscle of these animals (46) and
makes it likely that the defective relocalization of PI 3-kinase to
GLUT-4 vesicles represents an essential element of the
insulin-resistant state. The molecular basis of this defect remains
undefined; however, it must be different from defects at the level of
IRS-1 and IRS-2, because cardiac p85
does not interact with these
proteins. Earlier studies had shown a defect in PI 3-kinase activation
by insulin in muscle tissue of obese rodents (17) and
patients (14); however, these defects were attributed to a
diminished tyrosine phosphorylation of IRS-1 and are located upstream
of GLUT-4 translocation. As described here and in our earlier study
(24), we do not see any alteration in the
insulin-regulated tyrosine phosphorylation of IRS-1 in cardiac tissue
of obese Zucker rats. Instead, we have reported the
hyperphosphorylation of IRS-1 on serine/threonine leading to a
disturbed PI 3-kinase activation in cardiomyocytes of obese Zucker rats
(24). Consistently, we show here a reduced IRS-1/p85
PI
3-kinase activity in this animal model, most probably resulting from a
defective recruitment of PI 3-kinase to IRS-1.
In summary, the present study shows that, in cardiac muscle, the two
regulatory subunit isoforms of PI 3-kinase, p85 and p85
, have
different functional implications for mediating downstream insulin
signaling. Recruitment of p85
and PI 3-kinase activity to
GLUT-4-containing vesicles in response to insulin and the complete loss
of this effect in the insulin resistance of obesity make it likely that
an adaptor isoform-specific compartmentalization of PI 3-kinase
represents an additional level of specificity in insulin signaling.
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ACKNOWLEDGEMENTS |
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This work was supported by the Ministerium für Wissenschaft und Forschung des Landes Nordrhein-Westfalen, the Bundesministerium für Gesundheit, the Deutsche Forschungsgemeinschaft (SFB 351 C2), EU COST Action B5, and BIOMED Concerted Action 3084. D. M. Ouwens is supported by the Royal Netherlands Academy of Sciences (Casimir-Ziegler Fellowship) and by the Netherlands Organization for Scientific Research (NWO GB-MW).
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FOOTNOTES |
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Address for reprint requests and other correspondence: J. Eckel, Diabetes Research Institute, Auf'm Hennekamp, D-40225 Düsseldorf, Germany (E-mail: eckel{at}uni-duesseldorf.de).
Current address of I. Uphues: Boehringer Ingelheim Pharma KG, 88397 Biberach, Germany; current address of D. M. Ouwens: Dept. of Molecular Cell Biology, Leiden University Medical Center, 2333 AL Leiden, The Netherlands.
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.
Received 14 February 2000; accepted in final form 19 September 2000.
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