Maternal insulin resistance is essential for efficient provision of glucose to the fetus. Although elevation of placental hormones is known to relate to the development of insulin resistance, the precise underlying mechanism of maternal insulin resistance is unknown. Therefore, we examined the molecular mechanisms of progesterone causing insulin resistance in 3T3-L1 adipocytes. Progesterone at 10−4 M, but not 10−5 M, reduced the amount of IRS-1. As a result, insulin-induced phosphorylation of IRS-1, the association of IRS-1 with p85α, and subsequent phosphorylation of Akt1 and -2 was decreased moderately by 10−4 M progesterone. Subsequently, insulin-induced translocation of GLUT4 to the plasma membrane evaluated by immunostaining on the plasma membrane sheet by confocal laser microscope was also decreased by 10−4 M progesterone. In contrast, 2-[3H]deoxyglucose (2DG) uptake was markedly inhibited by both 10−5 and 10−4 M progesterone in a dose-dependent manner. Surprisingly, 2DG uptake elicited by adenovirus-mediated expression of constitutive-active mutant of PI 3-kinase (myr-p110) and Akt (myr-Akt) was suppressed by progesterone. Interestingly, insulin-induced tyrosine phosphorylation of Cbl and activation of TC10 were inhibited by progesterone at 10−5 M. These results indicate that progesterone is implicated in insulin resistance during pregnancy by inhibiting the PI 3-kinase pathway at the step of 1) IRS-1 expression and 2) distal to Akt and 3) by suppressing the PI 3-kinase-independent pathway of TC10 activation by affecting Cbl phosphorylation.
- gestational diabetes
- insulin resistance
a variety of hormones secreted from the placenta play important roles in maintaining the gestational environment during pregnancy (22, 25). Production of these hormones increases gradually as the gestational age progresses (22, 25). The possible involvement of these hormones in the occurrence of insulin resistance during pregnancy has been suggested (1, 18, 23, 27, 29). We have reported previously that high concentrations of estradiol induced insulin resistance via membrane estrogen receptor (ER)-mediated activation of JNK and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1) in 3T3-L1 adipocytes (23). Human placental lactogen (hPL), human placental growth hormone (hPGH), and prolactin, in addition to the elevation of TNFα-associated insulin resistance in late pregnancy, are also known to induce insulin resistance (1, 18, 27, 29).
Progesterone is an essential sex steroid produced by the placenta during pregnancy to maintain gestation. Although serum progesterone concentration is at the range of 10−7 M in the luteal phase, it increases and reaches to 10−6 M in late gestational periods (35). Since most sex hormones are accumulated and pooled in adipose tissues, the steroid content of adipose tissue is known to be ∼10-fold higher than that in the general circulation (6). On the basis of these, the estimated high concentration of progesterone may affect insulin sensitivity in adipocytes. In fact, enlarged adipocytes and decreased insulin sensitivity were observed in surgical postmenopausal monkeys treated with estrogen and medroxyprogesterone acetate (34). Female progesterone receptor (PR)-knockout mice showed lower fasting glucose levels and higher insulin levels than control mice (26). Although this evidence implicates that progesterone facilitates insulin resistance states (26, 34), the underlying molecular mechanism by which progesterone induces insulin resistance is largely unknown.
It has been well established that activation of phosphatidylinositol (PI) 3-kinase and subsequent activation of Akt through the IRS signaling pathway are essential for insulin-stimulated GLUT4 translocation (31). In addition, another insulin-signaling pathway responsible for the translocation of GLUT4 is known in adipocytes (4, 14, 40). TC10 located in the lipid raft microdomain is activated via tyrosine phosphorylation of Cbl, following insulin receptor association with Cbl-associated protein (CAP) and adaptor protein containing a PH and SH2 domain (APS) (14). This Cbl/TC10 pathway plays a crucial role in insulin-induced GLUT4 translocation and glucose uptake in adipocytes (14).
To clarify the involvement of progesterone in the pathophysiology of insulin resistance during pregnancy, we examined the molecular mechanism by which progesterone affects insulin's metabolic signaling, leading to glucose uptake in 3T3-L1 adipocytes. Here, we show that progesterone causes insulin resistance by multiple mechanisms. Progesterone suppressed the PI 3-kinase pathway by promoting IRS-1 degradation and suppressed the subsequent phosphorylation of Akt. In addition, progesterone inhibited GLUT4 translocation and glucose uptake in a step distal to Akt phosphorylation. Furthermore, progesterone inhibited TC10 activation by suppressing insulin-induced Cbl phosphorylation.
MATERIALS AND METHODS
Human recombinant insulin was provided by Novo Nordisk Pharmaceutical (Copenhagen, Denmark). A polyclonal anti-insulin receptor β-subunit (IRβ) antibody, a monoclonal anti-p85α antibody, a monoclonal anti-Akt1 antibody, and a monoclonal anti-phospho-tyrosine (PY99) antibody were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A polyclonal anti-IRS-1 antibody and a polyclonal anti-Akt2 antibody were from Millipore (Billerica, MA). A polyclonal anti-Ser307 phosphospecific IRS-1 antibody, a polyclonal anti-Ser636/639 phosphospecific IRS-1 antibody, and a polyclonal anti-Ser473 phosphospecific Akt antibody were from Cell Signaling Technology (Beverly, MA). A monoclonal anti-GLUT4 antibody (1F8) was from Abcam (Cambridge, UK). A monoclonal FITC-conjugated anti-mouse IgG antibody was from Jackson ImmunoResearch Laboratories (West Grove, PA). Progesterone, RU-486, and ICI-182,780 were obtained from Sigma (St. Louis, MO). Enhanced chemiluminescence reagents were from GE Healthcare UK (Buckinghamshire, UK). Dulbecco's modified Eagle's medium (DMEM) was from GIBCO-BRL Japan (Tokyo, Japan). All other reagents were of analytical grade and purchased from Sigma or Wako Pure Chemical Industries (Osaka, Japan).
Cell culture and differentiation of 3T3-L1 adipocytes.
3T3-L1 fibroblasts were grown and passaged in DMEM supplemented with 10% newborn calf serum. Cells at 2–3 days postconfluence were used for differentiation. The differentiation medium contained 10% fetal calf serum (FCS), 250 nM dexamethasone, 0.5 mM isobutyl methylxanthine, and 500 nM insulin. After 3 days, the differentiation medium was replaced with postdifferentiation medium containing 10% FCS and 500 nM of insulin. After an additional 3 days, postdifferentiation medium was replaced with DMEM supplemented with 10% FCS.
Adenoviral expression of myr-p110 and myr-Akt.
Adenovirus vectors encoding constitutively active forms of bovine p110α and rat Akt1 by adding the Src myristration signal sequence at the NH2 terminus (myr-p110 and myr-Akt) were described previously (17, 19, 39). A LacZ virus encoding β-galactosidase was used as a control. Myr-p110 or myr-Akt was transiently expressed in differentiated 3T3-L1 adipocytes by means of adenovirus-mediated gene transfer, as described previously (39). In brief, a multiplicity of infection of 20–40 plaque-forming units/cell was used to infect 3T3-L1 adipocytes, with the virus being left on the cells for 16 h prior to removal. Subsequent experiments were conducted 24–48 h after initial addition of the virus. The efficiency of adenovirus-mediated gene transfer was ∼95%.
Immunoprecipitation and Western blotting.
Western blotting was performed as described previously (33, 37, 38). In brief, 3T3-L1 adipocytes grown in six-well multiplates were serum starved for 16 h with or without various inhibitors, followed by incubation with progesterone for the indicated times. The cells were then treated with 17 nM insulin at 37°C for the specified times. The cell lysates or immunoprecipitates were separated by SDS-PAGE, transferred onto membranes, and immunoblotted. Densitometric analysis was conducted directly from the blotted membrane by utilizing LAS-4000 lumino-image analyzer system (Fujifilm, Tokyo, Japan).
Measurement of insulin-induced 2-[3H]deoxyglucose uptake.
3T3-L1 adipocytes grown in six-well multiplates were serum starved overnight and further incubated in KRP-HEPES buffer containing 1% BSA for 3 h at 37°C with or without various concentrations of progesterone. The cells were subsequently stimulated with various concentrations of insulin. Following 15 min of insulin treatment, 0.1 μCi of 2-[3H]deoxyglucose (2DG) was added for 4 min. The reaction was stopped by the addition of 10 μM cytochalasin B. The cells were washed three times with PBS and solubilized with 0.2 mM SDS-0.2 N NaOH. The radioactivity incorporated into the cells was measured by liquid scintillation counting.
Immunodetection of plasma membrane sheets.
Plasma membrane (PM) sheets from 3T3-L1 adipocytes were prepared by modification of previous reports (12, 15). Briefly, cells grown on coverslips were serum starved for 16 h and were subjected to insulin stimulation for 15 min. Cells were then placed on ice, washed twice in ice-cold PBS, and incubated three times with ice-cold hypotonic swelling buffer containing 23 mM KCl, 10 mM HEPES, 5 mM MgCl2, and 1 mM EGTA, pH 7.5. The cells were sonicated in buffer containing 70 mM KCl, 30 mM HEPES, 5 mM MgCl2, 3 mM EGTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 μM pepstatin A, 1 μM leupeptin, and 1 mM dithiothreitol, pH 7.5. PM sheets were fixed with 4% (vol/vol) paraformaldehyde in PBS for 15 min, and the reaction was quenched with 0.1 M glycine in PBS for 10 min. PM sheets were then blocked with PBS containing 5% BSA for 10 min and incubated with anti-GLUT4 antibody for 16 h at 4°C and for 45 min with secondary FITC-conjugated anti-mouse IgG antibodies at room temperature. Coverslips were washed twice with PBS and mounted using Dako mounting solution (Dako Japan, Tokyo, Japan).
Confocal microscopy image acquisition and quantification of GLUT4 on PM sheets.
Images were obtained using Leica TCS SP5 laser scanning confocal microscopy (Leica Microsystems Japan, Tokyo, Japan) by argon laser (excitation 488 nm) at room temperature using an ×40 objective lens at the same gain setting, unless indicated otherwise. The gain detector was set using cells or PM sheets labeled with only secondary antibody, and images were analyzed using Leica Application Suit software. PM sheets were individually outlined, and pixel intensity within each sheet was determined; the results are expressed as intensity per unit area. Background intensity was determined using areas outside of PM lawns and was subtracted from each reading. At least 300 PM sheets were examined for each experimental condition.
Preparation of crude PM fraction and measurement of TC10 activity.
TC10 activity was measured as described previously (3). Fully differentiated 3T3-L1 adipocytes grown in 10-cm dishes (3 dishes/point) were treated with 10−4 M progesterone for 16 h. After insulin stimulation for 5 min, cells were washed with cold HEPES-EDTA-sucrose buffer containing 20 mM HEPES, 1 mM EDTA, 255 mM sucrose, 1 mM PMSF, 10 μg/ml aprotinin, and 10 μM leupeptin, pH 7.4. Cells were scraped and homogenized in a Dounce homogenizer for 20 strokes. Homogenates were centrifuged at 800 g for 10 min at 4°C, and the postnuclear supernatant was collected. Postnuclear supernatant was ultracentrifuged at 19,000 g for 20 min. The pellets were resuspended in 1.0 ml of binding buffer containing 25 mM Tris, 1 mM DTT, 10 mM MgCl2, 40 mM NaCl, and 0.5% Nonidet P-40 (NP-40), pH 7.5, and subjected to pulldown assay.
To measure in vivo levels of activated GTPase, p21-activated kinase (PAK1) pulldowns using GST-PAK1 binding domain (GST-PBD) conjugated to agarose were performed. A lipid raft-enriched plasma membrane fraction was resuspended in 1.0 ml of binding buffer in the presence of 7 μg of GST-PBD-agarose (Cytoskeleton, Denver, CO) for 1 h at 4°C. The beads were washed three times with washing buffer containing 25 mM Tris, 1 mM DTT, 10 mM MgCl2, 40 mM NaCl, and 1% NP-40, pH 7.5, and once with washing buffer without NP-40. The beads were resuspended in 20 μl of SDS sample buffer, subjected to 15% SDS-PAGE, and analyzed by immunoblotting with anti-TC10 antibody.
Data are represented as means ± SE. P values were determined by one-way ANOVA with Student-Newman-Keuls multiple comparison test. P < 0.05 was considered significant.
Progesterone inhibited insulin-induced tyrosine phosphorylation of IRS-1 by degradation via PR.
Although progesterone has been shown to be implicated in insulin resistance (18, 27), the underlying molecular mechanism is largely unknown. We examined the effect of progesterone on insulin signaling in 3T3-L1 adipocytes. Insulin-induced tyrosine phosphorylation of IRβ was not affected by treatment with progesterone at any concentration (Fig. 1). In contrast, the expression of IRS-1 was decreased significantly to 65.6 ± 7.7% by treatment with 10−4 M progesterone (Fig. 2A). Consistently, insulin-induced tyrosine phosphorylation of IRS-1 was decreased to 64.4 ± 9.7% at 10−4 M (Fig. 2B). The reduction of IRS-1 was abolished by pretreatment with RU-486, a potent antagonist of PR and glucocorticoid receptor, whereas the knockdown of glucocorticoid receptor did not affect progesterone-induced reduction of IRS-1 expression (data not shown). In addition, pretreatment with ICI-182,780, a potent antagonist of ER, had no effect of progesterone on the decreased phosphorylation and expression of IRS-1 (Fig. 2C), suggesting that progesterone-induced degradation of IRS-1 is mediated through PR. Furthermore, phosphorylation of IRS-1 at Ser307 and Ser636/639 residues was reduced by treatment with progesterone, indicating that these phosphorylations do not appear to be related to the reduction of IRS-1 (Fig. 2, D and E).
Progesterone suppressed insulin-induced association of IRS-1 with p85α subunit of PI 3-kinase and phosphorylations of Akt1 and -2.
We next examined the association of IRS-1 with the p85α subunit of PI 3-kinase. Insulin-induced IRS-1/p85α association was apparently decreased to 56.6 ± 3.5% by treatment with progesterone at 10−4 M (Fig. 3A). In contrast to the report with hPGH (1), the amount of p85α was not altered by treatment with progesterone at any concentration tested (Fig. 3B). As a result, insulin-induced phosphorylation of Akt1, Akt2, and p44/42 MAP kinase was reduced significantly to 71.6 ± 4.6, 44.6 ± 14.0 (Fig. 4, A and B), and 48.3 ± 4.1% (data not shown), respectively, by treatment with progesterone at 10−4 M. The amount of Akt1, Akt2, and p44/42 MAP kinase was not altered by treatment with progesterone (data not shown).
Effects of progesterone on insulin-, myr-p110-, and myr-Akt-induced glucose uptake.
Since progesterone suppressed insulin-induced phosphorylation of Akt, we examined the effect of progesterone on insulin-induced [3H]2DG uptake. The dose-dependent effect of progesterone on insulin-induced 2DG uptake is shown in Fig. 5A. Although the suppression of insulin signaling was apparent only at 10−4 M progesterone in the steps of IRS-1, the association of IRS-1 with p85α, and phosphorylation of Akt, insulin-induced 2DG uptake was more markedly suppressed at 10−5 and 10−4 M progesterone. Insulin-stimulated (17 nM) glucose uptake was reduced to 51.0 ± 3.3 and 16.7 ± 3.1%, respectively, at 10−5 and 10−4 M progesterone.
Since the degree of inhibition of glucose uptake was more profoundly affected than Akt phosphorylation, we examined the effect of progesterone on 2DG uptake induced by a constitutive active mutant of PI 3-kinase and Akt. Interestingly, both myr-p110- and myr-Akt-induced stimulation of 2DG uptake were also significantly suppressed by treatment with 10−4 M progesterone (Fig. 5, B and C). These results indicate that progesterone also inhibited insulin-induced glucose uptake at a step downstream of Akt.
Effect of progesterone on insulin-induced GLUT4 translocation to the plasma membrane.
We further examined whether progesterone affected insulin-induced GLUT4 translocation to the PM or subsequent steps for glucose uptake. We prepared a PM sheet and examined insulin-induced GLUT4 translocation to the PM by analyzing immunostained GLUT4 using a confocal laser microscope (Fig. 6). The intensity of GLUT4 at the PM sheet was significantly elevated 3.3-fold by 17 nM insulin stimulation for 15 min (Fig. 6, A and C). Insulin-induced recruitment of GLUT4 to the PM sheet was significantly suppressed 1.5-fold by treatment with 10−4 M progesterone. These results indicate that progesterone inhibits insulin-induced glucose uptake by suppression of GLUT4 translocation. The amount of GLUT4 (Fig. 6B) and GLUT1 (data not shown) was not altered by treatment with 10−4 M progesterone for 16 h.
Effect of progesterone on insulin-induced tyrosine phosphorylation of Cbl and activation of TC10.
Since the Cbl/TC10 pathway in adipose tissue has been shown to be important for insulin-induced GLUT4 translocation leading to glucose uptake (14), we investigated the effect of progesterone on the pathway. Importantly, insulin-induced tyrosine phosphorylation of Cbl was suppressed significantly by progesterone treatment without affecting expression levels of Cbl (Fig. 7, A and B). Progesterone at 10−5 and 10−4 M reduced insulin-induced tyrosine phosphorylation of Cbl to 69.4 ± 11.4 and 20.8 ± 6.5%, respectively.
Finally, we examined directly the effect of progesterone on insulin-induced activation of TC10. When treated with insulin, TC10 activity increased 7.5-fold over the basal level (Fig. 7C). Consistent with the results of Cbl phosphorylation, progesterone markedly suppressed the increasing effect of insulin on TC10 activity. It was increased only 2.9-fold by insulin in the presence of progesterone at 10−4 M.
The hormonal and metabolic environment changes markedly to adapt to fetal growth during pregnancy (22, 25). To effectively supply glucose nutrition for fetal development, maternal insulin resistance occurs and is remarkable after midgestation (29). On the other hand, the prevalence of gestational diabetes mellitus (GDM) doubled from 1994 to 2002, and further increment is predicted, in accord with the marked increment of patients with type 2 diabetes throughout the world (5). GDM is associated with not only the subsequent onset of type 2 diabetes in mothers (20) but also fetal malformation and an increased prevalence of type 2 diabetes in later life (2). Therefore, elucidation of the underlying mechanism causing maternal insulin resistance is of particular importance to establish appropriate preventive and therapeutic approaches.
Among various placental hormones, placental growth hormone has been shown to suppress insulin action by upregulation of p85α in 3T3-L1 adipocytes and provides a potential explanation for insulin resistance in pregnancy (1). In addition, progesterone is one of the candidates possibly implicated in the insulin resistance during pregnancy. Epidemiological studies have revealed that administration of progestin for contraceptive usage is associated with increased incidence of type 2 diabetes (13). A hyperglycemic hyperinsulinemic clamp study showed that progesterone administration for 8 wk reduced whole body glucose uptake in humans (13). Concerning the role of progesterone in insulin secretion, female progesterone receptor knockout mice demonstrated a reduced fasting blood glucose level with elevation of serum insulin by increased β-cell proliferation (26), indicating the possible relation of progesterone with β-cell proliferation and/or function. However, the precise mechanism by which progesterone causes insulin resistance is so far unknown.
We clarified the molecular mechanism by which progesterone inhibited insulin's metabolic action at diverse steps in insulin signaling in 3T3-L1 adipocytes. First, progesterone apparently decreased the expression of IRS-1 and subsequent reduction of insulin-induced tyrosine phosphorylation of IRS-1, p85α/IRS-1 association, and phosphorylation of Akt1 and -2. Degradation of IRS-1 is one of the main mechanisms that causes insulin resistance when exposed to proinflammatory cytokines (10), chronic insulin stimulation (28, 32), free fatty acids (7, 10), and another steroid hormone, glucocorticoid (30). Degradation is known to be associated with the phosphorylation of IRS-1 on the serine residue (10). In this regard, progesterone did not enhance the phosphorylation of IRS-1 at Ser307 and Ser636/639 residues known to be implicated in the subsequent degradation of IRS-1 (Fig. 2, D and E), although we cannot rule out the possibility that other Ser residues of IRS-1 are involved in degradation. It is also possible that the activation of protein tyrosine phosphatases is implicated in the decreased tyrosine phosphorylation of IRS-1, whereas the expression of at least two tyrosine phosphatases, protein tyrosine phosphatase 1B and leukocyte common antigen-related protein phosphatase, was also unaltered by progesterone treatment (data not shown). However, the impairment at the step of IRS-1 does not appear to be crucial, since both myr-p110- and myr-Akt-induced stimulation of 2DG uptake were suppressed significantly by treatment with progesterone.
Second, our results indicate that progesterone inhibited the metabolic signaling of insulin at a step distal to Akt. The inhibitory effects of progesterone were more evident on glucose uptake rather than on the degradation of IRS-1 and phosphorylation of Akt. In fact, glucose uptake induced by adenoviral-mediated expression of either the constitutive active mutant of PI 3-kinase or Akt was again suppressed significantly by progesterone treatment. We further clarified that the step involved is proximal to GLUT4 translocation, since insulin-induced GLUT4 translocation was significantly perturbed by progesterone (Fig. 6). Therefore, progesterone appears to inhibit insulin signaling between Akt and GLUT4 translocation.
Third, we found that progesterone inhibited insulin-induced tyrosine phosphorylation of Cbl and activation of TC10 (Fig. 7). The fundamental role of the Cbl/TC10 pathway in glucose metabolism in the whole body is still controversial; however, several studies indicate the tissue-specific importance of the Cbl/TC10 pathway in glucose uptake in adipose tissue, especially on the basis of studies in 3T3-L1 adipocytes (4, 14, 40). Suppression of Cbl phosphorylation was reported in several insulin-resistant conditions. Reduced phosphorylation accompanied with the decreased expression of insulin receptor was reported in white adipose tissue of ob/ob mice (11). Since progesterone did not affect the expression of insulin receptor, we assumed that inhibition of Cbl phosphorylation might be due to the decrease in APS and/or CAP expression (21); however, these expression levels were not altered by progesterone treatment (data not shown). Notably, progesterone at 10−5 M markedly suppressed and at 10−4 M almost completely suppressed the phosphorylation of Cbl, and the dose-dependent effect was very closely correlated with the extent of progesterone-induced inhibition of glucose uptake. Therefore, we consider that this mechanism may be most profoundly implicated in the pathogenesis of insulin resistance induced by progesterone in adipocytes. In addition, our results obtained by treatment with progesterone at 10−5 M, at least in part, may have physiological relevance in insulin resistance in adipocytes during pregnancy since the estimated concentration of progesterone in adipose tissue appears to reach around 10−5 M in late gestational periods based on previous reports (6, 35).
Insulin regulates glucose uptake into cells by inducing GLUT4 redistribution from intracellular storage sites to the plasma membrane (12). In addition, the translocation step of GLUT4 to the vicinity of the plasma membrane is regulated by a mechanism through microtubes for rolling, fusion, or docking of GLUT4 vesicles with actin rearrangement (9, 12). Since progesterone inhibited the Cbl/TC10 pathway and the Akt pathway, we examined which step of GLUT4 redistribution was dominantly affected by progesterone. The present study clearly indicates that progesterone remarkably suppressed insulin-induced GLUT4 translocation without affecting total protein amounts of GLUT4 (Fig. 6). These results are consistent with previous reports that the expression of GLUT4 was unchanged in the adipose tissue of ovariectomized rats with progesterone administration (36) and that insulin-induced GLUT4 translocation was impaired in omental adipocytes from patients with GDM (8).
Progesterone is a member of the steroid family, and its classical nuclear PRA and PRB produced from a single gene is expressed in humans (16). Recently, other types of membrane PRs have been identified (24). Although the functions of these new members of PR are currently under investigation, these PRs located at the plasma membrane are presumed to mediate rapid, nongenomic actions of progesterone (16). In the present study, progesterone-induced reduction of IRS-1 and suppression of Cbl phosphorylation were observed after 16 h of treatment (data not shown), and the inhibitory effects of progesterone were abolished by pretreatment with RU-486 (Fig. 2C), a specific inhibitor of conventional PR that does not interact with membrane PRs (16). Therefore, the progesterone-induced insulin resistance observed in the present study appears to be mediated through conventional PR.
In summary, the present study clarified the molecular mechanism by which progesterone inhibits insulin-induced GLUT4 translocation and glucose uptake in 3T3-L1 adipocytes. Namely, 1) progesterone suppressed the PI 3-kinase-mediated pathway, leading to Akt phosphorylation by reducing the expression of IRS-1; 2) progesterone further inhibited the metabolic signaling of insulin at a step distal to Akt phosphorylation; and, moreover, 3) progesterone decreased insulin-induced phosphorylation of Cbl and activation of TC10 located at a lipid raft.
This work was supported in part by a grant from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to T. Sasaoka).
The authors have nothing to disclose.
We thank Dr. Tomoichiro Asano (University of Hiroshima, Japan) and Dr. Wataru Ogawa (University of Kobe, Japan) for kindly providing myr-p110 and myr-Akt adenovirus, respectively. We also thank Dr. Manabu Ishiki (University of Toyama, Japan) for excellent technical instruction and Natsumi Matsumoto (University of Toyama, Japan) for excellent technical assistance.
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