Phosphatidylinositol 3-Kinase Redistribution Is Associated With Skeletal Muscle Insulin Resistance in Gestational Diabetes Mellitus

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)-λ and PKCζ (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-p85α, insulin receptor β-subunit, and IRS-1 polyclonal antibodies were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-phosphotyrosine and anti-p85α monoclonal antibodies were obtained from Transduction Laboratories (Lexington, KY). [γ-³²P]ATP was obtained from NEN Life Science (Boston, MA).

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 60–80 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 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 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.

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.

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.

To determine the level of p85α, Akt, and p70S6k phosphorylation, 50 μg protein from pre- and post-insulin–stimulated 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 manufacturer’s instructions. For tyrosine phosphorylation of p85α, 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-p85α 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-1–associated p85α, 500 μg total protein was immunoprecipitated with anti–IRS-1 antibody and subjected to SDS-PAGE gel as described above. The membrane was probed with anti-p85α antibody overnight at 4°C. To determine the total protein levels of IRS-1, Akt, p85α/p70S6k, and PKCλ/ζ, 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-PKCλ/ζ 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.

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-PKCλ/ζ 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 β-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 [γ-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 was determined as previously described (35). Briefly, 500 μg protein was immunoprecipitated with anti–IRS-1, p85α, 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 [γ-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.

For the total serine phosphorylation assay, protein was immunoprecipitated with an anti–IRS-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.

Data are expressed as means ± SE. Statistical differences were determined by analysis of variance or by Student’s t test. P < 0.05 was considered statistically significant.

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 32–50% 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).

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.

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).

To investigate the consequences of increased IRS-1 serine phosphorylation in GDM, we measured the association of the p85α subunit of PI 3-kinase in IRS-1 immunoprecipitates (Fig. 4A). Insulin stimulated a fourfold increase in p85α 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 p85α 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 p85α 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 p85α protein and its increased tyrosine phosphorylation (yet reduced IRS-1 association with p85α), we investigated whether p85α was binding to the insulin receptor in post–IRS-1 immunoprecipitates. Figure 4B shows that before immunodepletion of IRS-1, insulin stimulated an increase in the pool of p85α associated with insulin receptors in control mice. In GDM animals, however, the basal levels of p85α associated with the insulin receptor in post–IRS-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.

To determine whether increased p85α binding to the insulin receptor observed in GDM mice was associated with greater PI 3-kinase activity, we measured the total (p85α) 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-1–associated 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-2–associated PI 3-kinase activity were quite low and remained unchanged in GDM mice (results not shown).

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).

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).

Atypical PKCζ has also been proposed to mediate serine phosphorylation of IRS-1 downstream from PI 3-kinase (25,26). We therefore investigated the PKCλ/ζ protein level and activity in skeletal muscle tissue from control and GDM mice. A representative autoradiogram shows the protein levels of PKCλ/ζ in control and GDM mice (Fig. 8). There was no difference in PKCλ/ζ content in skeletal muscle between control and GDM animals (P > 0.05). PKCλ/ζ activity was measured with the MBP substrate. Because the anti-PKCλ/ζ antibody cross-reacts with both PKCλ and PKCζ, the result represents the total PKCλ/ζ kinase activity. As reported previously (33), insulin activated PKCλ/ζ in skeletal muscle. The insulin-stimulated increase in PKCλ/ζ 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 PKCλ/ζ activity between control and GDM mice.

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 receptor–associated pool of PI 3-kinase removes PI 3-kinase from the IRS-1–associated pool responsible for GLUT4 translocation and glucose transport. In contrast, the insulin receptor–associated 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-1–associated PI 3-kinase was severely impaired. Consistent with this result, insulin-stimulated coupling between IRS-1 and p85α 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 p85–insulin receptor association. Second, because IRS-1 protein levels were downregulated and p85α 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 p85α 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 pathways—an Akt pathway and a PKCλ/ζ pathway—and 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 λ and ζ 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 λ/ζ expression and insulin-stimulated PKC λ/ζ 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 PKCζ activity actually dissociate the insulin receptor and IRS-1 and impair IRS-1–associated PI 3-kinase activity by increasing IRS-1 serine phosphorylation (25,26). However, it is not known whether PKCζ phosphorylates IRS-1 directly or whether PKCζ effects are mediated by a downstream effector of PKCζ. p70S6 kinase is another potential candidate for IRS-1 serine phosphorylation and is activated by PKCζ. 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 p85α (54). We also investigated the phosphorylation of serine 612, which is downstream from PKC (18). Using an anti–phosph-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-α 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-α–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 receptor–PI 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.

This research was supported by National Institutes of Health grant no. NIH-HD11089 to J.E.F.