Phosphatidylinositol 3-Kinase Redistribution Is Associated With Skeletal Muscle Insulin Resistance in Gestational Diabetes Mellitus
Jianhua Shao1,
Hiroshi Yamashita1,
Liping Qiao1,
Boris Draznin2, and
Jacob E. Friedman1
1 Departments of Pediatrics, Biochemistry and Molecular Genetics, and
2 Research Service, Veterans Affairs Medical Center, University of Colorado Health Sciences Center, Denver, Colorado
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ABSTRACT
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Insulin resistance during pregnancy provokes gestational diabetes mellitus (GDM); however, the cellular mechanisms for this type of insulin resistance are not well understood. We evaluated the mechanisms(s) for insulin resistance in skeletal muscle from an animal model of spontaneous GDM, the heterozygous C57BL/KsJ-db/+ mouse. Pregnancy triggered a novel functional redistribution of the insulin-signaling environment in skeletal muscle in vivo. This environment preferentially increases a pool of phosphatidylinositol (PI) 3-kinase activity associated with the insulin receptor, away from insulin receptor substrate (IRS)-1. In conjunction with the redistribution of PI 3-kinase to the insulin receptor, there is a selective increase in activation of downstream serine kinases Akt and p70S6. Furthermore, we show that redistribution of PI 3-kinase to the insulin receptor increases insulin-stimulated IRS-1 serine phosphorylation, impairs IRS-1 expression and its tyrosine phosphorylation, and decreases the ability of IRS-1 to bind and activate PI 3-kinase in response to insulin. Thus, the pool of IRS-1associated PI 3-kinase activity is reduced, resulting in the inability of insulin to stimulate GLUT4 translocation to the plasma membrane. These defects are unique to pregnancy and suggest that redistribution of PI 3-kinase to the insulin receptor may be a primary defect underlying insulin resistance in skeletal muscle during gestational diabetes.
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INTRODUCTION
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Gestational diabetes mellitus (GDM), defined as impaired glucose tolerance with first recognition during pregnancy, affects 28% of all pregnancies and is associated with a high risk of type 2 diabetes in subsequent years as well as diabetes and obesity in offspring. Although insulin resistance is a universal finding in pregnancy and GDM, the cellular mechanisms for this type of insulin resistance are not well understood. Women with GDM have more pronounced insulin resistance than women with normal glucose tolerance during pregnancy (1), which provokes ß-cell failure and leads to impaired glucose tolerance (2). Insulin-stimulated glucose disposal declines 4060% from early to late pregnancy (1,3). However, the levels of GLUT4 in skeletal muscle, the major site of whole-body glucose disposal, are unchanged in women with GDM (1,3,4). In human GDM, a primary defect in insulin action involves impaired glucose transport in both skeletal muscle and adipose tissue (5,6). In addition, there is impaired insulin receptor tyrosine kinase activity, which contributes to worsening of insulin resistance in GDM (6,7).
The insulin-signaling events that regulate glucose transport are the subject of intense investigation. Most available data support the hypothesis that activation of the p85/p110-type phosphatidylinositol (PI) 3-kinase through its recruitment to phosphotyrosine sites on insulin receptor substrates (insulin receptor substrate [IRS]-1 and IRS-2) is required for glucose transporter translocation in response to insulin (8,9). In addition to IRS-1 and IRS-2, recent evidence has also shown that the p85 subunit of PI 3-kinase can bind directly to the autophosphorylated insulin receptor to recruit PI 3-kinase signaling to the insulin receptor (10,11,12). The PI 3-kinase associated with the insulin receptor may have serine kinase as well as lipid kinase activity (13,14,15). However, the physiological significance of this pool of PI 3-kinase activity to regulate glucose transport remains uncertain. Interestingly, insulin-resistant human GDM subjects demonstrate increased p85
expression in skeletal muscle during late gestation (6), and reducing the p85
regulatory subunit of PI 3-kinase in transgenic mice appears to improve insulin sensitivity (16). However, the role of increased p85
in the insulin resistance of GDM is unclear. Also unknown is whether insulin signaling downstream from PI 3-kinase leading to GLUT4 translocation is inhibited in skeletal muscle during GDM.
Recent evidence suggests that serine phosphorylation of IRS-1 reduces its ability to act as an insulin receptor substrate and may play a significant role in insulin resistance at the level of glucose uptake in diabetes (17,18,19,20,21). Hyper-Ser/Thr phosphorylation of IRS-1 impairs insulin-induced tyrosine phosphorylation of IRS-1, PI 3-kinase activation, and glucose uptake (17,18,22). Moreover, persistent IRS-1 Ser/Thr phosphorylation induces IRS-1 degradation (15,20,22). The potential serine kinases involved in SERIRS-1 serine phosphorylation include GSK3 (23), JNK1 (24), PKC (18), and, more recently, aPKC (25,26) and Akt (27). In addition, several studies indicate that Ser/Thr phosphorylation and degradation of IRS-1 are mediated by a rapamycin-sensitive pathway (PI 3-kinase/Akt/m-TOR/p70S6 kinase), independent of ras/MAP kinase (15,20,22). IRS-1 is extensively phosphorylated on serine residues in response to treatment with tumor necrosis factor (TNF)-
and hyperinsulinemia (21,28), and this can inhibit insulin receptor tyrosine kinase activity (29). In human GDM subjects, IRS-1 tyrosine phosphorylation is decreased in skeletal muscle, due in part to reduced IRS-1 levels (6). Thus, increased serine kinase activity could account for the inhibition of IRS-1 tyrosine phosphorylation as well as its degradation and the insulin resistance of glucose transport found in skeletal muscle during GDM.
The goal of the present study was to determine the potential mechanisms(s) for insulin resistance to glucose uptake in skeletal muscle from an animal model of spontaneous GDM, the C57BL/KsJ-db/+ mouse (30,31). Here, we provide evidence that GDM triggers a novel functional redistribution of the insulin-signaling environment in skeletal muscle of the intact animal. PI 3-kinase activity associated with an insulin receptor was increased threefold in GDM mice compared with nonpregnant controls, consistent with increased expression and binding of p85
to the insulin receptor. Total insulin-stimulated PI 3-kinase activity was significantly greater in GDM skeletal muscle and was associated with a two- to fivefold increase in 473Ser-Akt and p70S6 kinase activation, whereas basal and insulin-stimulated IRS-1 serine phosphorylation was significantly higher by two- to threefold over that of controls. Consistent with this result, insulin-stimulated coupling between IRS-1 and p85
was inhibited, and skeletal muscle GLUT4 translocation to plasma membranes was severely decreased in GDM. These results suggest that GDM triggers a novel redistribution of PI 3-kinase away from IRS-1 to the insulin receptor. This functional redistribution increases the PI 3-kinase/Akt/p70S6 kinase signaling pathway, while at the same time downregulating IRS-1 expression and tyrosine phosphorylation and reducing IRS-1associated PI 3-kinase activity. Together, these defects result in insulin resistance at the level of GLUT4 translocation in skeletal muscle during GDM.
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RESEARCH DESIGN AND METHODS
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Materials.
Insulin radioimmunoassay kits were purchased from Linco Research (St. Louis, MO). Bovine serum albumin (BSA) and the protease inhibitors aprotinin and leupetin were purchased from Boehringer Mannheim (Indianapolis, IN). Myelin basic protein (MBP), phenylmethylsulfonyl fluoride (PMSF), the glucose assay kit, and all other reagent grade chemicals were from Sigma (St. Louis, MO). GLUT4 and phosph-Serine antibodies were obtained from Chemicon International (Temecula, CA). Anti-Akt, phosph-Akt (Ser473), p70 S6 kinase, phosph-p70 S6 (Thr421/Ser424), and phosph-IRS-1 (Ser612) antibodies were from Cell Signaling Technology (Beverly, MA). Polyclonal antibodies to both protein kinase C (PKC)-
and PKC
(against the COOH-terminus) were from Santa Cruz Biotechnology (Santa Cruz, CA). Human recombinant insulin was from Sigma. Secondary horseradish peroxidase (HRP)-conjugated antibody, protein A Sepharose, and chemiluminescence reagents (ECL kit) were obtained from Amersham Life Science (Arlington, IL). The polyvinylidene difluoride (PVDF) membrane, electrophoresis equipment, Western blotting reagents, and protein assay kits were from Bio-Rad (Hercules, CA). Anti-p85
, insulin receptor ß-subunit, and IRS-1 polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phosphotyrosine and anti-p85
monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY). [
-32P]ATP was obtained from NEN Life Science (Boston, MA).
Experimental animals.
Female C57BL/KsJ-db/+ (db/+) mice and their wild-type littermates C57BL/KsJ-+/+ (+/+) were purchased from the Jackson Laboratory (Bar Harbor, ME) at 8 weeks of age. They were maintained on a 12-h light/dark cycle in a temperature-controlled room and given free access to commercial mouse food and water. At 6080 days of age, female mice were placed individually together with +/+ males, and mating was confirmed by the presence of a copulatory plug the next morning, designated day 0 of gestation. All procedures were approved by the University of Colorado Animal Care and Use Committee.
Glucose tolerance test in the mouse model.
Glucose tolerance tests were performed in mice fasted for 6 h before testing. Conscious unrestrained mice were injected intraperitoneally with glucose (2 g/kg body wt), and blood was sampled from the tail at 0, 30, and 60 min after glucose injection. Then, 100 µl whole blood was collected from the tail using heparinized capillary tubes. The blood sample was allowed to clot on ice and was centrifuged for 20 min at 13,000 rpm at 4°C. The serum was frozen at -80°C until assayed for glucose and insulin. Serum glucose was measured by calorimetric glucose oxidase assay. Basal insulin concentration was measured using commercial radioimmunoassay enzyme-linked immunosorbent kits for mice. Assays were conducted in duplicate, and the intra-assay coefficient of variation was <5%.
Insulin challenge test in the mouse model.
Insulin tolerance tests were performed after a 6-h fast. Conscious unrestrained mice were injected intraperitoneally with insulin (0.75 units/kg body wt), and blood was sampled from the tail at 15, 30, and 60 min after glucose injection. The glucose concentration in whole blood was measured immediately using a blood glucose meter.
Acute insulin stimulation in vivo and tissue collection.
At day 18 of pregnancy, selected mice were anesthetized with ketamine (150 mg/kg) and acepromazine (5 mg/kg), abdominal cavities were opened, and the portal veins were exposed. Then, 300 mg gastrocnemius muscle from one hindlimb was rapidly removed and immediately frozen in liquid nitrogen. A maximal bolus of insulin (10 units/kg body wt) was then injected into the portal vein as described previously (30). A gastrocnemius muscle biopsy from the opposite limb was excised 5 min after injection and immediately frozen. The samples were stored at 80°C until analysis.
Total and plasma membrane GLUT4 immunoblotting.
A total of 300 mg muscle tissue from mouse was homogenized in 2 ml ice-cold lysis buffer (50 mmol/l Tris-HCl, pH 7.4, 1% NP-40, 0.25% sodium deoxycholate, 150 mmol/l NaCl, 1 mmol/l EGTA, 1 mmol/l PMSF, 1 µg/ml aprotinin, 5 µg/ml leupeptin, 1 mmol/l Na3VO4, and 1 mmol/l NaF) using a Polytron PTA 20S generator (Brinkman Instruments, Westbury, NY) at maximum speed for 30 s. The homogenates were allowed to sit on ice and solubilized for 30 min, followed by centrifugation at 350,000g for 60 min at 4°C. The supernatant was collected and stored at -20°C as total protein samples. To determine GLUT4 content, aliquots of homogenate protein (75 µg) and plasma membrane protein (40 µg) were separated using polyacrylamide (SDS-PAGE) gels. The proteins were transferred to PVDF membranes using a semi-dry transfer apparatus. The membrane blocking, probing, and visualization procedure was the same as described in the next paragraph, except that the GLUT4 antibody was used. For the preparation of plasma membranes,
200 mg frozen hindlimb muscle was prepared as described previously by us and others (32). Briefly, muscle was homogenized in a buffer consisting of 255 mmol/l sucrose, 100 mmol/l Tris/HCl, pH 7.6, and 0.2 mmol/l EDTA and centrifuged at 3,400g for 20 min. The pellets were resuspended, and total and plasma membrane fractions were separated by centrifugation at 68,000g for 16 h with a sucrose gradient.
Phosphorylation and Western blotting.
To determine the level of p85
, Akt, and p70S6k phosphorylation, 50 µg protein from pre- and post-insulinstimulated samples were subjected to 7 or 10% SDS-PAGE. After transferring and blocking, the membrane was incubated with Phospho-Akt (Ser473) or Phosph-p70S6k (Thr421/Ser424) antibody (1:1,000 in Tris-buffered saline with Tween [TBS-T] with 1% BSA; Cell Signaling Technology) overnight at 4°C. The membrane was detected with enhanced chemiluminescence (ECL) (Amersham Pharmacia Biotech, Arlington Heights, IL) according to the manufacturers instructions. For tyrosine phosphorylation of p85
, 500 µg protein of crude homogenate was immunoprecipitated with 5 µg anti-phosphotyrosine antibody (PY20; Transduction Laboratories) overnight at 4°C. The samples were mixed with 20 µl protein A Sepharose (50% slurry; Pharmacia Biotech, Uppsala, Sweden) for 2 h at 4°C. The immunoprecipitates were washed four times with 1 ml TBS with 0.2 mmol/l sodium orthovanadate, resuspended in 20 µl Laemmli sample buffer, and boiled for 5 min. Samples were resolved by 7% SDS-PAGE gel and transferred to PVDF membrane using a Mini Trans-Blot Transfer cell (Bio-Rad, Hercules, CA). The membrane was blocked with 5% nonfat milk in TBS-T for 1 h at room temperature and incubated with anti-p85
antibody (Upstate Biotechnology) overnight at 4°C. The membrane was washed three times in TBS-T and incubated with anti-rabbit IgG-HRP secondary antibody (1:2,000 dilution in TBS-T) for 1 h at room temperature. Membranes were washed again as above, and ECL detection reagents were added for 1 min and immediately exposed to X-ray film. For IRS-1associated p85
, 500 µg total protein was immunoprecipitated with antiIRS-1 antibody and subjected to SDS-PAGE gel as described above. The membrane was probed with anti-p85
antibody overnight at 4°C. To determine the total protein levels of IRS-1, Akt, p85
/p70S6k, and PKC
/
, 50 µg muscle tissue homogenate protein was treated with Laemmli sample buffer, boiled for 5 min, and resolved on a 7 or 10% denaturing SDS-PAGE gel and transferred to PVDF membranes. Membranes were blocked with 5% nonfat milk (Bio-Rad) in TBS-T for 1 h at room temperature. The membrane was washed three times with TBS-T and probed with a monoclonal anti-p85 antibody (1:2,000 dilution in TBS-T; Transduction Laboratories), anti-Akt antibody (1:1,000 dilution in TBS-T; New England Biolabs), or anti-PKC
/
antibody (1:3,000 dilution in TBS-T; Santa Cruz Biotechnology). The bands were visualized with ECL and exposed to Kodak Biomax film (Eastman Kodak, Rochester, NY). The bands were quantified using a SciScan 500 (USB, Cleveland, OH) with 50 µg of a normal mouse skeletal muscle protein sample to control for gel-to-gel variation.
PKC
/
activity assay.
The isoform-specific enzyme activity was measured as described previously (33,34) using MBP as a substrate. Briefly, 200 µg protein was immunoprecipitated with 0.4 µg anti-PKC
/
polyclonal antibody. After an overnight incubation at 4°C, immunoprecipitates were collected with protein A Sepharose as described above. The beads were washed twice with buffer A (50 mmol/l MOPS-HCl [pH 7.5], 0.5% [vol/vol] Triton X-100, 10% [vol/vol] glycerol, 0.1% [wt/vol] BSA, 5 mmol/l EDTA, 5 mmol/l EGTA, 20 mmol/l NaF, 50 mmol/l ß-glycerophosphate, 2 mmol/l sodium orthovanadate, 2 mmol/l dithiothreitol, 1 µg/ml leupeptin, and 2 mmol/l PMSF), once with buffer A containing 1 mol/l NaCl, and once with buffer B (20 mmol/l Tris-HCl (pH 7.5), 5 mmol/l MgCl2, and 1 mmol/l EGTA. The precipitates were then incubated for 15 min at 30°C with 0.4 µCi [
-32P]ATP in 25 µl enzyme assay buffer that contained 35 mmol/l Tris-HCl (pH 7.5), 10 mmol/l MgCl2, 0.5 mmol/l EGTA, 0.1 mmol/l CaCl2, 40 µmol/l unlabeled ATP, and 20 µg MBP in each reaction.
PI 3-kinase activity assay.
PI 3-kinase activity was determined as previously described (35). Briefly, 500 µg protein was immunoprecipitated with antiIRS-1, p85
, or insulin receptor antibody overnight at 4°C. Immunoprecipitates were washed three times with PBS containing 1% NP-40 and 0.1 mmol/l Na3VO4, followed by three times with 100 mmol/l Tris-HCl, pH 7.5, containing 500 mmol/l LiCl and 0.1 mmol/l Na3VO4, and two times with 10 mmol/l Tris-HCl, pH 7.5, 100 mmol/l NaCl, 1 mmol/l EDTA, and 0.1 mmol/l Na3VO4. To evaluate PI 3-kinase activity, PI and [
-32P]ATP (10 µCi/reaction) were added to the immunoprecipitate at room temperature for 10 min. Reactions were stopped with 20 µl 8 N HCl and 160 µl chloroform-methanol (1:1) and centrifuged. The lipid-containing organic phase was resolved on oxalate-coated thin-layered chromatography plates, developed in chloroform/methanol/water/ammonium hydroxide (60:47:11.3:2), and lipid species were visualized by autoradiography with an intensifying screen at 80°C.
IRS-1 serine phosphorylation.
For the total serine phosphorylation assay, protein was immunoprecipitated with an antiIRS-1 antibody as above. The immunoprecipitates were resolved by 7% SDS-PAGE gel and transferred to the membrane, followed by incubation with anti-phosphoserine antibody.
Statistical analysis.
Data are expressed as means ± SE. Statistical differences were determined by analysis of variance or by Students t test. P < 0.05 was considered statistically significant.
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RESULTS
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Impaired glucose tolerance in a spontaneous GDM mouse model.
During pregnancy, mice heterozygous for the leptin receptor (db/+) gain more weight, develop glucose intolerance, and become severely insulin resistant compared with nonpregnant control mice (30,31). Figure 1A shows the results of a glucose-tolerance test carried out in female C57BL/KsJ-db/+ (db/+) mice and their wild-type C57BL/KsJ-+/+ (+/+) littermates before conception and on day 17 of gestation. The glucose concentration in the db/+ pregnant mice was 3250% greater at 30 and 60 min compared with either db/+ nonpregnant controls or +/+ pregnant mice, indicative of impaired glucose tolerance. Fasting plasma insulin concentration was elevated sixfold in the GDM mice (data not shown), whereas fasting glucose was unchanged. Peripheral insulin sensitivity was assessed on day 18 of gestation using an insulin challenge test (Fig. 1B). As expected, insulin-induced reduction in plasma glucose in db/+ mice was severely impaired by 66% at 15 min (P < 0.05) and by 38% at 60 min (P < 0.05) compared with nonpregnant controls, indicative of marked insulin resistance. Pregnant +/+ mice were also insulin resistant compared with +/+ nonpregnant controls. However, GDM mice were significantly more insulin resistant than pregnant +/+ mice (P < 0.05).
Impaired insulin-stimulated GLUT4 translocation in mice with GDM.
To determine how pregnancy triggers severe insulin resistance in GDM mice, we analyzed insulin signal transduction in GDM mice compared with their nonpregnant db/+ controls. First, to determine the effect of insulin on GLUT4 translocation, we measured GLUT4 protein levels on plasma membranes in skeletal muscle before and after insulin injection in vivo in nonpregnant db/+ control and db/+ pregnant GDM mice (Fig. 2). Table 1 shows the 5'nucleotidase activity in whole homogenate and skeletal muscle plasma membrane fraction before and after insulin. The activities were similar between groups, suggesting the recovery of total and fractionated plasma membranes was qualitatively similar in control and GDM mice. After insulin stimulation, plasma membrane GLUT4 reached a maximal level (twofold greater) 5 min after treatment in control mice (P < 0.05). In contrast, in GDM mice, the basal plasma membrane GLUT4 in muscle was significantly lower (by 20%), and insulin-stimulated GLUT4 in plasma membranes was significantly reduced (by 50%) (P < 0.001). No further increase in plasma membrane GLUT4 was noted in later time points after insulin stimulation in GDM mice. Total GLUT4 levels in whole-muscle homogenates were similar in control and GDM mice (data not shown). Thus, total GLUT4 and the membranes associated with GLUT4 were unchanged, but plasma membrane GLUT4 was lower in the GDM mice.

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FIG. 2. Reduced basal and insulin-stimulated translocation of GLUT4 to plasma membranes in skeletal muscle from GDM mice. After overnight fasting, mice were anesthetized, and portal veins were exposed. A skeletal muscle biopsy was removed before and 5 min after insulin bolus injection. The plasma membrane (PM) protein was prepared as described in RESEARCH DESIGN AND METHODS. The protein was subjected to the 10% SDS-PAGE gel. The transferred membranes were blocked and probed with anti-GLUT4 antibody. The bands were visualized by ECL. Values are the mean ± SE of the densitometry values expressed in arbitrary units as compared with the values obtained in the internal control protein, which was used in each gel for the variation of a different gel. There is a representative blot of at least three independent assays. *P < 0.05 vs. basal or insulin-stimulated muscle from GDM mice, respectively.
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GDM decreases insulin-stimulated IRS-1 tyrosine phosphorylation but increases IRS-1 serine phosphorylation.
To investigate whether reduced insulin sensitivity in GDM mice involved decreased insulin signaling through IRS-1, we measured tyrosine phosphorylation of IRS-1 before and after insulin injection (Fig. 3A). IRS-1 tyrosine phosphorylation was threefold lower in GDM mice compared with controls after insulin stimulation. This was partially due to a 35% reduction in IRS-1 protein levels in GDM mice (Fig. 3B, P < 0.05). However, even after normalization for decreased IRS-1 protein content, the tyrosine phosphorylation per unit of IRS-1 was still significantly lower (by twofold) in GDM mice than in controls (P < 0.05). Because GDM mice demonstrated lower IRS-1 tyrosine phosphorylation, we sought to determine whether there was a change in basal and insulin-stimulated serine phosphorylation of IRS-1. As shown in Fig. 3C, insulin increased IRS-1 serine phosphorylation one- to twofold in control mice, whereas in GDM mice, basal and insulin-stimulated serine phosphorylation was significantly higher by two- to threefold over that of controls (P < 0.05).

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FIG. 3. GDM decreases IRS-1 tyrosine phosphorylation but increases serine phosphorylation. A: Skeletal muscle tissue samples were collected before and 5 min after insulin stimulation. Protein was immunoprecipitated with antiIRS-1 antibody. The immunoprecipitates were separated through 7% SDS-PAGE and blotted with antiphospho-tyrosine antibody. B: A total of 50 µg protein was separated by 7% SDS-PAGE and immunoblotted with antiIRS-1 antibody. C: IRS-1 was immunoprecipitated before and after insulin stimulation and blotted with anti-phosphoserinespecific antiserum. *P < 0.01 vs. control.
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Redistribution of p85 to the insulin receptor away from IRS-1 in GDM mice.
To investigate the consequences of increased IRS-1 serine phosphorylation in GDM, we measured the association of the p85
subunit of PI 3-kinase in IRS-1 immunoprecipitates (Fig. 4A). Insulin stimulated a fourfold increase in p85
in IRS-1 immunoprecipitates from control animals. However, this association was significantly reduced by twofold in GDM skeletal muscle (P < 0.05).
However, despite reduced IRS-1 coupling with p85 in GDM mice, insulin stimulated the amount of phosphotyrosine associated p85
by twofold greater in GDM mice compared with controls (Fig. 4C, P < 0.05). This result was accompanied by a 34% increase (P < 0.05) in p85
protein levels in the GDM mice (Fig. 4D). Complete depletion of IRS-1 after immunoprecipitation was confirmed by blotting the supernatants with IRS-1 antibody, which demonstrated that no detectable IRS-1 was present.
To address the apparent paradox of increased total p85
protein and its increased tyrosine phosphorylation (yet reduced IRS-1 association with p85
), we investigated whether p85
was binding to the insulin receptor in postIRS-1 immunoprecipitates. Figure 4B shows that before immunodepletion of IRS-1, insulin stimulated an increase in the pool of p85
associated with insulin receptors in control mice. In GDM animals, however, the basal levels of p85
associated with the insulin receptor in postIRS-1 immunoprecipitates was increased by 50% compared with controls (P < 0.05), and insulin treatment stimulated the association further by 1.7-fold over that of control mice (P < 0.05). This significant increase and redistribution of p85 to the insulin receptor was confirmed in IRS-1 immunodepleted extracts, as shown in Fig. 4E.
Redistribution of PI 3-kinase activity in GDM skeletal muscle.
To determine whether increased p85
binding to the insulin receptor observed in GDM mice was associated with greater PI 3-kinase activity, we measured the total (p85
) associated PI 3-kinase activity and the distribution of PI 3-kinase associated with IRS-1, IRS-2, and the insulin receptor (Fig. 5). Insulin increased the total PI 3-kinase activity in mice by 45% in GDM mice versus controls (P < 0.05). In contrast, IRS-1associated PI 3-kinase activity was reduced by greater than threefold in GDM mice (P < 0.05). However, we found that the level of PI 3-kinase activity in insulin receptor immunoprecipitates was increased threefold in GDM mice (P < 0.01). This result was consistent with increased p85 binding, suggesting that GDM was associated with redistribution of the PI 3-kinase pool away from IRS-1 to the insulin receptor. The levels of IRS-2associated PI 3-kinase activity were quite low and remained unchanged in GDM mice (results not shown).

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FIG. 5. Redistribution of PI 3-kinase activity in GDM skeletal muscle to the insulin receptor away from IRS-1. Protein (500 µg) was immunoprecipitated with anti-p85 antibody to pull down total PI 3-kinase (A), or immunoprecipitated with antiIRS-1 (B), or in postIRS-1 immunoprecipitates using an insulin receptor antibody (C). PI 3-kinase activity was measured as described in RESEARCH DESIGN AND METHODS. The bar graph shows quantification of PI 3-kinase activity in the autoradiograms from six samples in each group. PIP3, phosphatidyl-inositol-3,4,5-triphosphate. Values are means ± SE. *P < 0.01 vs. control.
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GDM increases Akt expression and insulin-stimulated activation.
To determine whether insulin signaling downstream from PI 3-kinase was enhanced in GDM skeletal muscle, we measured Akt expression/phosphorylation in response to insulin (Fig. 6A). Insulin increased 473Ser-Akt phosphorylation in a time-dependent manner, and phosphorylation reached a maximal level 5 min after injection (data not shown). The basal Akt phosphorylation was not different between GDM and control mice. However, insulin increased 473Ser-Akt phosphorylation 3-fold in control mice and 6.5-fold in GDM mice (Fig. 6A). Akt protein content was increased twofold in GDM compared with control mice (Fig. 6B, P < 0.05). After normalizing for increased Akt protein, maximal insulin-stimulated phosphorylation of Akt was twofold greater in GDM mice than in control mice (P < 0.01).

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FIG. 6. Increased Akt protein levels and phosphorylation in skeletal muscle of GDM mice. All mice were fasted overnight, and skeletal muscle samples obtained before and after insulin injection were collected as described in RESEARCH DESIGN AND METHODS. An equivalent amount of protein was subjected to SDS-PAGE gel and Western blotted with an antibody that recognizes phosphorylated Ser473-Akt (A) or an antibody that recognizes both Akt1 and Akt2 (B). The autoradiograms in A and B are each representative of three different gels. Values are means ± SE for six mice per group. *P < 0.01 vs. control.
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Enhanced activation of p70 S6 kinase in GDM mice.
To determine whether events downstream from Akt were also enhanced in GDM, we determined the expression/phosphorylation of p70 S6 kinase in skeletal muscle (Fig. 7). There was no difference in total p70S6k protein level between controls and GDM mice. Insulin increased p70S6k phosphorylation twofold in control mice. However, in GDM mice the basal level of serine phosphorylation was increased twofold (P < 0.05), and the insulin-stimulated p70S6k phosphorylation was nearly fivefold greater compared with controls (P < 0.001).

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FIG. 7. Enhanced activation of p70 S6 kinase in GDM mice. A: p70 S6 kinase protein expression, and basal and insulin-stimulated activation in skeletal muscle from GDM mice. A total of 50 µg protein was separated by 10% SDS-PAGE. After transferring to the membrane, the total protein expression was detected by immunoblotting with the anti-p70S6k antibody. p70 S6 kinase activation was measured by a special antibody that only binds with Thr421/Ser424 phosphorylated p70S6k. *P < 0.001 vs. control.
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PKC
/
protein expression and kinase activity.
Atypical PKC
has also been proposed to mediate serine phosphorylation of IRS-1 downstream from PI 3-kinase (25,26). We therefore investigated the PKC
/
protein level and activity in skeletal muscle tissue from control and GDM mice. A representative autoradiogram shows the protein levels of PKC
/
in control and GDM mice (Fig. 8). There was no difference in PKC
/
content in skeletal muscle between control and GDM animals (P > 0.05). PKC
/
activity was measured with the MBP substrate. Because the anti-PKC
/
antibody cross-reacts with both PKC
and PKC
, the result represents the total PKC
/
kinase activity. As reported previously (33), insulin activated PKC
/
in skeletal muscle. The insulin-stimulated increase in PKC
/
activity measured using MBP as a substrate was 2.5-fold after 5 min of insulin stimulation. However, there was no difference in basal or insulin-stimulated PKC
/
activity between control and GDM mice.
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DISCUSSION
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In the present study, we show that pregnancy triggers a novel functional redistribution of a pool of PI 3-kinase to the insulin receptor away from IRS-1 in skeletal muscle from GDM mice. This binding of p85 to the insulin receptor may couple the insulin receptor to PI 3-kinase activity, thereby inducing serine phosphorylation of IRS-1, and may be a primary cause for the insulin resistance of pregnancy. Pregnancy-induced insulin resistance is usually compensated for by increased insulin secretion, allowing glucose tolerance to remain in the normal range. However, in GDM, insulin resistance is usually present before gestation and worsened further during pregnancy, thereby provoking moderate to severe glucose intolerance for the first time during gestation.
To our knowledge, this is the first study that has identified the insulin receptor as a major source of PI 3-kinase activity in an animal model of insulin resistance. The increase in the insulin receptorassociated pool of PI 3-kinase removes PI 3-kinase from the IRS-1associated pool responsible for GLUT4 translocation and glucose transport. In contrast, the insulin receptorassociated pool of PI 3-kinase is sufficient to stimulate downstream signaling events, including the activation of Akt, p70S6 kinase, and IRS-1 serine phosphorylation. Despite the increase in total PI 3-kinase activity, IRS-1associated PI 3-kinase was severely impaired. Consistent with this result, insulin-stimulated coupling between IRS-1 and p85
was inhibited, and GLUT4 translocation was markedly decreased. These results suggest that pregnancy triggered a redistribution of two pools of PI 3-kinase in skeletal muscle, resulting in an increase in insulin-stimulated serine kinase activity and marked insulin resistance at the level of GLUT4 translocation in vivo.
The mechanisms that trigger the preferential redistribution of PI 3-kinase activity to the insulin receptor are unknown; however, several possibilities exist. First, the tyrosine kinase activity of the insulin receptor itself could play a role in attracting the p85 association with the insulin receptor. Studies suggest that the phosphorylation status of the insulin receptor regulates sorting between the plasma membrane and endosomes, and this process plays a major role in the p85 content associated with the insulin receptor (12,36). Our previous studies in human GDM suggest that pregnancy induces a reversible serine phosphorylation of the insulin receptor in skeletal muscle (7). It is therefore possible that serine phosphorylation in GDM alters key tyrosine residues important for p85 binding and/or resorting of the insulin receptor that could prolong the p85insulin receptor association. Second, because IRS-1 protein levels were downregulated and p85
increased in GDM mice, it is possible that a proportion of p85 may have been directed to the plasma membrane in proximity to the insulin receptor. Consistent with this possibility, adenovirus-mediated overexpression of a membrane-targeted form of PI 3-kinase has been shown to induce chronic activation of PI 3-kinase, leading to an increase in Akt, p70 S6 kinase, and rapamycin-sensitive IRS-1 serine phosphorylation and its accelerated degradation in 3T3L1 adipocytes (15,22). However, it is also possible that the redirected pool of PI 3-kinase associated with the insulin receptor was secondary to the serine/threonine phosphorylation of IRS-1 that inhibits p85
binding to IRS-1. Third, it is possible that growth factors associated with pregnancy (i.e., placental growth hormone, prolactin, and estrogen) may play a role in directing p85 binding to the plasma membrane. It is well established that these growth factors can stimulate the p85 regulatory subunit of PI 3-kinase without increasing glucose transport activity. Given that insulin-stimulated GLUT4 translocation is defective in GDM, it is possible that increased total PI 3-kinase activity could integrate signals from insulin and other growth factors on processes important during gestation other than glucose transport (such as protein synthesis or anti-apoptosis). All three of these possibilities remain to be tested.
A second important finding in the present study was that insulin resistance in GDM is associated with increased serine kinase activity downstream from PI 3-kinase. Insulin signaling from PI 3-kinase subsequently diverges into at least two independent pathwaysan Akt pathway and a PKC
/
pathwayand the latter pathway also contributes, at least in part, to insulin-stimulated glucose transport (37,38,39). The level of insulin-stimulated Ser473-Akt phosphorylation, a substrate for PI 3-kinase, was increased two- to threefold in muscles from GDM mice in response to insulin. Because Akt is in the pathway for insulin signal transduction and regulates GLUT4 translocation and glucose transport, as reported previously (40,41,42,43,44), the impaired insulin-stimulated GLUT4 translocation observed in the present study seems paradoxical. However, Akt lies in the crossroad of multiple cellular signaling pathways and is also activated by IGF-I, prolactin, and other growth factors without increasing glucose transport (45,46). Conceivably, the increase in Akt expression and activity we observed could be due to a synergism of insulin together with the hormones of pregnancy. Recent studies have also shown that activation of Akt1 and Akt2 in vivo are much less impaired than activation of PI 3-kinase in insulin-resistant states, and the mechanisms for divergent alterations in insulin action on Akt1 and Akt2 activities in tissues of insulin-resistant obese rats probably involve small changes in Akt2 expression and activation and may not play a major role in the muscle insulin resistance, at least in obesity (47).
Recent studies have also shown that the atypical PKC (aPKC) isoforms
and
are phosphorylated by insulin via PI 3-kinase and are involved in regulating insulin-stimulated glucose transport in tissue culture cells (33,38,48). Decreased aPKC activity is associated with insulin resistance (49,50,51), and compounds that restore insulin-stimulated glucose uptake in adipose tissues from diabetic animals can increase aPKC expression without altering PI 3-kinase activity (51,52). The levels of PKC
/
expression and insulin-stimulated PKC
/
activity were normal in insulin-resistant muscle from GDM mice, suggesting that aPKCs are not responsible for impaired GLUT4 translocation in GDM. Alternatively, recent studies suggest that increases in atypical PKC
activity actually dissociate the insulin receptor and IRS-1 and impair IRS-1associated PI 3-kinase activity by increasing IRS-1 serine phosphorylation (25,26). However, it is not known whether PKC
phosphorylates IRS-1 directly or whether PKC
effects are mediated by a downstream effector of PKC
. p70S6 kinase is another potential candidate for IRS-1 serine phosphorylation and is activated by PKC
. Recent evidence suggests that IRS-1 serine/threonine phosphorylation and degradation during chronic hyperinsulinemia is prevented by rapamycin, which inhibits p70S6 kinase and partially prevents the reduction in phosphotyrosine content (22). Consistent with these studies, our results show basal and insulin-stimulated p70S6 kinase activities were up to fivefold higher in skeletal muscle from GDM mice, and the levels of basal and insulin-stimulated IRS-1 serine phosphorylation were increased two- to fourfold. Our results suggest that the increased p70S6 kinase in skeletal muscle in vivo may contribute to the serine phosphorylation of IRS-1. However, because this was a whole animal study, we cannot verify the mechanism of the increased p70S6 kinase activity in vivo using rapamycin. However, other in vitro studies have found that increased PI 3-kinase/Akt pathway is responsible for increasing activation of p70S6 kinase (15,22,27), and our results show the total PI 3-kinase and Akt activities are higher in GDM.
In different insulin-resistant states, reduced glucose transport in skeletal muscle is closely associated with impaired GLUT4 translocation rather than a change in total GLUT4 protein levels (8). It has been suggested from studies in rats and cells that increased IRS-1 Ser/Thr phosphorylation inhibits PI 3-kinase activity and GLUT4 translocation; however, the factors responsible for serine kinase activity have been difficult to demonstrate under physiological conditions in vivo. IRS-1 has >70 potential Ser/Thr phosphorylation sites. Delahaye et al. (53) reported that phosphorylation of serine sites 612, 632, 662, and 731 on IRS-1, which are adjacent to YXXM binding motifs for PI 3-kinase, negatively regulate IRS-1 binding to p85
(54). We also investigated the phosphorylation of serine 612, which is downstream from PKC (18). Using an antiphosph-612Ser-IRS-1 antibody, there was no difference between control and GDM mice (J.S., J.E.F., unpublished data). Rui et al. (21) recently demonstrated that IRS-1 was phosphorylated on 307Ser during hyperinsulinemia in human and mouse skeletal muscle and in 3T3L1 cells in response to TNF-
and hyperinsulinemia. We found the ability of insulin to stimulate IRS-1 on 307Ser was not altered in skeletal muscle of the GDM mice (M. White, L. Rui, J.E.F., unpublished data). However, TNF-
mediated serine phosphorylation of IRS-1 has also been shown to occur on 636Ser and 639Ser, induced by an mTOR pathway (55).
Although our results cannot be specific to the exact serine sites in IRS-1, we speculate that the increased PI 3-kinase pool associated with the insulin receptor is responsible for the increase in Akt/p70S6 kinase signaling during pregnancy to trigger serine phosphorylation of IRS-1, thereby reducing IRS-1 tyrosine phosphorylation and its association with p85 (Fig. 9). Our results suggest that PI 3-kinase/Akt is increased leading to p70S6 kinase activation in GDM (Fig. 9, solid arrow), whereas in the IRS-1 and GLUT4 translocation pathway, Akt has little effect (Fig. 9, broken arrow). With regard to PKC, there was no effect on GLUT4 translocation in GDM mice (Fig. 9, broken arrow). Rather, the compartment for insulin receptorPI 3-kinase p70S6 kinase activation (Fig. 9, solid arrow) is favored in GDM mice. We speculate that insulin signals these downstream proteins in compartments other than those used for GLUT4 translocation in GDM mice, resulting in increased insulin-stimulated IRS-1 serine phosphorylation and reduced binding to the p85 subunit of PI 3-kinase. Thus, the normal pool of PI 3-kinase associated with IRS-1 is reduced, resulting in decreased GLUT4 translocation and severe insulin resistance to glucose disposal in vivo. This novel pathway may play a critical role in the insulin resistance that provokes gestational diabetes.
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ACKNOWLEDGMENTS
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This research was supported by National Institutes of Health grant no. NIH-HD11089 to J.E.F.
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FOOTNOTES
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Address correspondence and reprint requests to Jacob E. Friedman, University of Colorado Health Sciences Center, 4200 E. Ninth Ave., Denver, CO 80262. E-mail: jed.friedman{at}uchsc.edu.
Received for publication 6 July 2001 and accepted in revised form 24 September 2001.
BSA, bovine serum albumin; GDM, gestational diabetes mellitus; ECL, enhanced chemiluminescence; HRP, horseradish peroxidase; IRS, insulin receptor substrate; MBP, myelin basic protein; PI, phosphatidylinositol; PKC, protein kinase C; PMSF, phenylmethylsulfonyl fluoride; PVDF, polyvinylidene difluoride; TBS-T, Tris-buffered saline with Tween; TNF, tumor necrosis factor.
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