Altered Subcellular Distribution of Estrogen Receptor Is Implicated in Estradiol-Induced Dual Regulation of Insulin Signaling in
http://www.100md.com
《内分泌学杂志》
Departments of Obstetrics and Gynecology (K.N., S.S.) and Clinical Pharmacology (T.W., S.H., H.T., T.S.) and First Department of Medicine (K.F., M.I., M.K.), University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
Abstract
We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. E2 enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10–8 M, but inhibited these effects at 10–5 M. A concentration of 10–5 M E2 enhanced insulin-induced phosphorylation of IRS-1 at Ser307, which was abolished by treatment with a c-Jun NH2-terminal kinase inhibitor. In addition, the effect of E2 was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable E2, E2-BSA, did not affect the insulin-induced phosphorylation of Akt at 10–8 M, but inhibited it at 10–5 M. Furthermore, E2 decreased the amount of estrogen receptor at the plasma membrane at 10–8 M, but increased it at 10–5 M. In contrast, the subcellular distribution of estrogen receptor was not altered by the treatment. These results indicate that E2 affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of E2 inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser307 via a c-Jun NH2-terminal kinase-dependent pathway, and that the subcellular redistribution of estrogen receptor in response to E2 may explain the dual effect of E2.
Introduction
HUMAN PREGNANCY IS associated with hyperinsulinemia and a progressive decline in insulin sensitivity (1). Thus, a number of clinical studies indicate that insulin resistance occurs in late pregnancy (2, 3). The cellular changes in insulin resistance during late pregnancy have been ascribed to altered levels of placental-derived hormones, including estrogen, progesterone, and human placental lactogen (4). Among these sex steroids, high concentrations of estrogen are known to inhibit insulin-stimulated glucose uptake in cultured cells (5). In addition, increased levels of TNF- have been associated with insulin resistance in late pregnancy (6). Thus, estrogen and TNF- produced in the placenta appear to play a key role in insulin resistance (5, 6). Although the mechanism of insulin resistance caused by TNF- has been studied intensively, the mechanism by which estrogen induces insulin resistance is unknown (6). In contrast, physiological levels of estrogen are known to protect against insulin resistance in women (7). Along this line, clinical studies suggest that after menopause women have an increased risk of developing diabetes (8, 9). Therefore, estrogen appears to control glucose homeostasis in a concentration-specific manner. The insulin signaling system leading to glucose uptake has been studied intensively in 3T3-L1 adipocytes (10).
The binding of insulin to the -subunit of the insulin receptor results in autophosphorylation and activation of the -subunit (11, 12, 13). The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS-1) at tyrosine residues (11, 12, 13). Tyrosine-phosphorylated IRS-1 binds to the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), leading to the activation of the p110 catalytic subunit (11, 12, 13). PI3-kinase generates PI triphosphate from PI-bisphosphate, leading to the phosphorylation and activation of Akt (11, 12, 13). Akt is known to be an important mediator of the insulin-induced uptake of glucose (14). The cellular mechanisms by which estrogen affects the insulin signaling system are unknown.
Estrogen binds receptors of two subtypes: estrogen receptor , first identified in 1986, and estrogen receptor , cloned subsequently (15, 16). Estrogen receptors are expressed not only in the reproductive organs of males and females, but also in nonreproductive tissues, including fat tissues (17, 18). It has been shown that the effect of estrogen is mediated by the binding of estrogen receptors in the cytosol, resulting in translocation and transcriptional activation in the nucleus (19). In addition, recent reports suggest a nontranscriptional action mediated by the estrogen receptor (20). Along these lines, estrogen receptors are distributed in the plasma membrane in addition to the cytosol and nucleus (21). However, the role of estrogen receptors at specific cellular locations in the regulation of insulin signaling is unknown.
In the present study we studied the effect of the main estrogen, estradiol (E2), on insulin signaling leading to glucose uptake in 3T3-L1 adipocytes. In addition, to clarify the molecular mechanism by which E2 affects the insulin signaling system, we investigated the isoform and subcellular distribution of the estrogen receptor mediating the effect of E2.
Materials and Methods
Materials
Recombinant human insulin was provided by Lilly Research Laboratories (Indianapolis, IN). Recombinant human TNF- was supplied by Dainippon Pharmaceutical Co. (Osaka, Japan). 17-Estradiol (E2) and E2-BSA were purchased from Sigma-Aldrich Corp. (St. Louis, MO). A complete estrogen receptor antagonist, ICI182,780, was obtained from Tocris (Ellisville, MO). The Jun NH2-terminal kinase (JNK) inhibitor, SP600125, was purchased from BIOMOL (Plymouth Meeting, PA). 2-[1,2-3H]Deoxyglucose (2-DOG) was obtained from DuPont NEN Life Science Products (Boston, MA). A monoclonal antiphosphotyrosine antibody (PY20) was purchased from Transduction Laboratories (Lexington, KY). A polyclonal anti-IRS-1 antibody, a polyclonal anti-Ser307 phosphospecific IRS-1 antibody, a polyclonal anti-Ser612 phosphospecific IRS-1 antibody, a polyclonal anti-Ser636 phosphospecific IRS-1 antibody, a monoclonal anti-p85 antibody, a polyclonal anti-Ser473 phosphospecific Akt antibody, a polyclonal anti-Thr308 phosphospecific Akt antibody, and a polyclonal anti-Akt antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). A polyclonal antiinsulin receptor antibody, horseradish peroxidase-conjugated antimouse and antirabbit IgG antibodies, a polyclonal antiestrogen receptor antibody, and a polyclonal antiestrogen receptor antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phenol red-free DMEM and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). Protein G-Sepharose was purchased from Pharmacia Biotech (Uppsala, Sweden). Reagents for electrophoresis were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). All other reagents were of analytical grade and purchased from Sigma-Aldrich Corp. or Wako Pure Chemical Industries (Osaka, Japan).
Cell culture and treatment
3T3-L1 fibroblasts were grown and passaged in phenol red-free DMEM supplemented with 10% donor calf serum. Cells, 2 d after confluence, were used for differentiation. The differentiation medium contained 10% charcoal-stripped FBS (CS-FBS), 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1 μM insulin. After charcoal stripping, E2 and estrone concentrations in serum decreased from 0.48 to less than 0.07 pM and from 0.36 to less than 0.12 pM, respectively. After 3 d, the differentiation medium was replaced with postdifferentiation medium containing 10% CS-FBS and 800 nM insulin. After 3 more days, the postdifferentiation medium was replaced with phenol red-free DMEM supplemented with 10% CS-FBS. The medium was changed every 3 d until the cells were used for experiments. Fourteen to 16 d after the induction of differentiation, more than 95% of the cells had morphological and biochemical properties of adipocytes. E2 dissolved in ethanol and TNF- in PBS were added to the cell culture medium, in which 106 cells were contained/plate, without FBS for 16 h.
2-DOG uptake
3T3-L1 adipocytes derived from differentiation of 3T3-L1 fibroblasts were grown in six-well multiplates and were serum-starved for 3 h. The cells were stimulated with 17 nM insulin for 15 min in Krebs-Ringer-phosphate-HEPES buffer containing 10 mM HEPES, 131.2 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, and 2.5 mM NaH2PO4, and 1% BSA (pH 7.4). 2-[3H]DOG (0.1 mM; 0.4 kBq/well) was added for 4 min. The reaction was stopped by the addition of 10 μM cytochalasin B. The cells were washed three times with ice-cold PBS and solubilized with 0.2% sodium dodecyl sulfate-0.2 N NaOH (22, 23). The radioactivity incorporated into the cells was measured by liquid scintillation counting. The results were corrected for nonspecific uptake determined by 2-[3H]DOG uptake in the presence of 10 μM cytochalasin B. Nonspecific binding was less than 10% of the total uptake.
Subcellular fractionation
The cells were washed twice with PBS and once with HEPES sucrose (HES) buffer containing 255 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM Na3VO4, 2 μg/ml aprotinin, and 50 ng/ml okadaic acid (pH 7.4). The cells were then immediately homogenized by 20 strokes with a motor-driven homogenizer in HES buffer containing 20 mM HEPES, 255 mM sucrose, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 2 μg/ml aprotinin, and 50 ng/ml okadaic acid (pH 7.4) at 4 C. The homogenates obtained from two 10-cm diameter dishes per condition were subjected to subcellular fractionation to isolate the plasma membrane, cytosol, and nucleus as described previously (24). In brief, the homogenates were centrifuged at 19,000 x g for 20 min. The resulting supernatant was centrifuged at 250,000 x g for 90 min. The remaining supernatant was centrifuged at Centricon-30 (Amicon, Inc., Beverly, MA) and used as cytosol. The pellet obtained from the initial spin was resuspended in HES buffer, layered onto a 1.12-M sucrose cushion, and centrifuged at 100,000 x g in a swing rotor for 60 min. A white fluffy band at the interface was collected, resuspended in HES buffer, and centrifuged at 40,000 x g for 20 min, yielding a pellet of plasma membrane. A viscous brown pellet was collected, yielding a pellet of nucleus. The purities of the nuclear and cytosolic preparations were determined by examining histone as a nuclear marker and calpain as a cytosolic marker. The samples were adjusted to a final protein concentration of 1–3 mg/ml, which was measured by the Bradford method, and stored at –80 C before use.
Immunoprecipitation and Western blotting
The cells or cell preparations were lysed in a buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na3VO4, 50 nM sodium fluoride, 10 μg/ml aprotinin, and 1 μM leupeptin (pH 7.4) for 30 min at 4 C. The lysates were centrifuged to remove insoluble materials. The supernatants (100 μg protein) were immunoprecipitated with antibodies for 16 h at 4 C. The immunoprecipitates or total cell lysates were then separated by 7.5% or 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) using a Bio-Rad Transblot apparatus. The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% BSA or 5% nonfat milk (pH 7.5) for 2 h at 20 C. They were then probed with antibodies for 2 h at 20 C or for 16 h at 4 C. After the membranes had been washed in a buffer containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.5), blots were incubated with a horseradish peroxidase-linked secondary antibody and subjected to enhanced chemiluminescence detection using ECL reagent according to the manufacturer’s instruction (Amersham Biosciences, Indianapolis, IN) (25, 26). In each experiment, the intensity of the band derived from control cells was averaged and assigned a value of 1 arbitrary unit, and the intensities of all treated groups were expressed relative to this value.
Statistical analysis
Experiments were separately conducted at least four times, and data are presented as the mean ± SE. P values were determined by ANOVA and Scheffe test. P < 0.05 was considered statistically significant.
Results
Effect of E2 on insulin-stimulated glucose uptake
We first examined the concentration-dependent effect of E2 on insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes (Fig. 1A). Insulin at 17 nM stimulated 2-DOG uptake by 6.5 ± 0.6-fold in 3T3-L1 adipocytes. Low concentrations of E2 further increased uptake. Insulin-induced 2-DOG uptake was increased 18.3 ± 3.6% by treatment with 10–8 M E2 compared with that without E2 treatment. Conversely, concentrations of E2 above 10–7 M inhibited insulin-stimulated 2-DOG uptake. After treatment with 10–5 M E2, insulin-induced 2-DOG uptake was decreased by 25.8 ± 3.7% compared with that without E2 treatment. Insulin stimulated 2-DOG uptake in a concentration-dependent manner (Fig. 1B). Treatment with 10–8 M E2 significantly enhanced 0.17, 1.7, and 17 nM insulin-induced 2-DOG uptake, whereas 10–5 M E2 apparently inhibited 1.7 and 17 nM insulin-stimulated glucose uptake. E2 treatment alone for up to 16 h did not stimulate glucose uptake, and the amount of glucose transporter 4 was not altered by treatment with any concentration of E2 (data not shown).
Effect of E2 on early steps of insulin signaling
Because E2 affected insulin-stimulated glucose uptake, the possible involvement of E2 in the early steps of insulin signaling was examined. Insulin-induced tyrosine phosphorylation of the insulin receptor was not affected by treatment with E2 at any concentration (Fig. 2A). Interestingly, insulin-induced tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the p85 subunit of PI3-kinase were promoted by treatment with a physiological concentration of E2. Thus, insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-1/p85 association were increased by 41.7 ± 3.4% and 22.9 ± 5.9%, respectively, after treatment with 10–8 M E2. In contrast, they were inhibited by treatment with concentrations of E2 above 10–7 M. At 10–5 M, E2 inhibited the insulin-induced tyrosine phosphorylation of IRS-1 and IRS-1/p85 association by 53.7 ± 3.7% and 25.9 ± 3.4%, respectively (Fig. 2, B and C). Amounts of protein among samples were confirmed to be equal by immunoblotting with antiinsulin receptor antibody (Fig. 2D), anti-IRS-1 antibody (Fig. 2E), and anti-p85 antibody (Fig. 2F).
Effect of E2 on insulin-induced phosphorylation of Akt
Akt lies downstream of PI3-kinase and is important for insulin’s stimulation of glucose uptake (23, 27, 28). Because Akt is activated by phosphorylation at Ser473 and Thr308, we examined the effect of E2 on the insulin-induced phosphorylation of Akt (Fig. 3, A and B). Consistent with the results of insulin-induced tyrosine phosphorylation of IRS-1 and IRS-1/p85 association, insulin-induced phosphorylation of Akt was increased by treatment with low concentrations of E2 and decreased by treatment with high concentrations of E2. Insulin-induced phosphorylation of Akt at Ser473 and Thr308 was enhanced 22.0 ± 3.3% and 30.1 ± 3.2%, respectively, by treatment with 10–8 M E2, whereas it was inhibited 44.7 ± 3.5% and 50.8 ± 3.6%, respectively, by treatment with 10–5 M E2. Amounts of protein among samples were confirmed to be equal by immunoblotting with anti-Akt antibody (Fig. 3C).
Effect of combined presence of E2 and TNF- on insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt
TNF- together with estrogen are secreted from the placenta and are known to be involved in insulin resistance in the late phases of pregnancy, especially in preeclampsia (29, 30). The effect of combined presence of E2 (10–6 M) and TNF- on the insulin-induced tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt was examined. Insulin-induced tyrosine phosphorylation of IRS-1 was inhibited 24.6 ± 2.9% and 35.7 ± 3.6% by treatment with E2 and TNF-, respectively (Fig. 4A). Insulin signaling was maximally inhibited by treatment with 50 ng/ml TNF-. Treatment with both E2 and TNF- further inhibited insulin signaling by 61.3 ± 4.2%. Similarly, treatment with E2, TNF-, or both inhibited the insulin-induced phosphorylation of Akt by 21.8 ± 3.3%, 34.9 ± 3.0%, and 65.4 ± 3.3%, respectively (Fig. 4B). These results indicate that TNF- combined with a submaximal concentration of E2 further inhibit insulin signaling, leading to glucose uptake.
Effect of E2 on insulin-induced serine phosphorylation of IRS-1
E2 affected insulin signaling at least at the level of tyrosine phosphorylation of IRS-1. Because tyrosine phosphorylation of IRS-1 is modulated by serine phosphorylation of IRS-1 (31), we examined the effect of E2 on the insulin-induced serine phosphorylation of IRS-1 (Fig. 5). The insulin-induced phosphorylation of IRS-1 at Ser307 was increased by treatment with E2 in a concentration-dependent manner. The extent of phosphorylation at Ser307 was increased 73.9 ± 4.1% by treatment with 10–5 M E2 (Fig. 5A). In contrast, the insulin-induced phosphorylation of IRS-1 at Ser612 (Fig. 5B) and Ser636 (Fig. 5C) was not altered by treatment with E2. These results indicate that a high concentration of E2 inhibits insulin signaling at the level of IRS-1 at least in part by increasing the phosphorylation of IRS-1 at Ser307.
Effect of a JNK inhibitor on the alteration of insulin signaling caused by E2 treatment
Because IRS-1 is known to be phosphorylated by JNK at Ser307 (32), we examined the influence of a JNK inhibitor, SP600125, on the effect of E2. The enhancement of the insulin-induced phosphorylation at Ser307 caused by 10–5 M E2 was abrogated by treatment with the JNK inhibitor (Fig. 6A). As a result, inhibition of the insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 6B) and serine phosphorylation of Akt (Fig. 6C) caused by E2 treatment was restored by pretreatment with the JNK inhibitor.
Effects of ICI182,780 and E2-BSA on E2-induced alteration of insulin signaling
We employed a specific estrogen receptor antagonist, ICI182,780, to examine whether the stimulatory effect of E2 on insulin signaling seen at 10–8 M is mediated by estrogen receptor (Fig. 7A). Pretreatment with 10 μM ICI 182,780 abrogated the enhancement of insulin-induced tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt caused by 10–8 M E2. Pretreatment with 1 μM ICI182,780 had the same effect. Similarly, we examined the effect of ICI182,780 to clarify whether the inhibitory effect of E2 at 10–5 M on insulin signaling is mediated by the estrogen receptor. Pretreatment with ICI182,780 at 10 μM, but not at 1 μM, restored the inhibition of insulin-stimulated tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt caused by 10–5 M E2. These results indicate that the estrogen receptor is implicated in the effects of both concentrations of E2 on insulin signaling.
The estrogen receptor is located on the plasma membrane in addition to the cytosol and nucleus (21). To examine the impact of membrane-based estrogen receptors, a membrane-impermeable E2, E2-BSA, was employed. Insulin-induced tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt were not enhanced by 10–8 M E2-BSA in contrast to E2 (Fig. 7B). However, higher concentrations of E2-BSA inhibited these actions, similar to the results obtained with E2. Thus, 10–5 M E2-BSA inhibited the insulin-induced tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt by 54.9 ± 3.5% (data not shown) and 49.4 ± 4.1%, respectively (Fig. 7B). These results indicate that estrogen receptors at the plasma membrane are implicated in the inhibitory effect of E2 at high concentrations, but are not involved in the enhancing effect of E2 at low concentrations.
Effect of E2 treatment on subcellular distribution of estrogen receptors and
We also investigated the subcellular redistribution of the estrogen receptor in response to E2. Treatment with 10–8 M E2 reduced the amount of estrogen receptor at the plasma membrane by 17.1 ± 2.9%, but increased the amount in the nucleus by 90.7 ± 4.4%. The amount of estrogen receptor in the cytosol was not apparently changed by E2 treatment. In contrast, treatment with 10–5 M E2 increased the amount of estrogen receptor at the plasma membrane by 71.3 ± 4.1%, but decreased it in the cytosol and nucleus by 36.3 ± 3.8% and 47.3 ± 3.4%, respectively (Fig. 8, A–C). In contrast, the amounts of estrogen receptor at the plasma membrane, in the cytosol, and in the nucleus were not apparently altered by treatment with any concentration of E2 (Fig. 8, D–F). In addition, insulin treatment did not appear to alter the subcellular distribution of estrogen receptors.
Discussion
Our results demonstrated that treatment with a physiological concentration of E2 (10–8 M) enhanced the insulin-induced uptake of glucose, whereas treatment with a high concentration of E2 (10–5 M) inhibited it. These results clearly indicate that E2 controls glucose metabolism in a concentration-specific manner. These results are consistent with clinical reports that the incidence of type 2 diabetes increases in women after menopause, and that insulin resistance occurs in women during late pregnancy (4, 7, 8). Taken together, the present results strongly indicate that physiological concentrations of E2 enhance insulin sensitivity, whereas high concentrations of E2 inhibit insulin-induced glucose metabolism. Part of the E2 may be converted to estrone by 17-hydroxysteroid dehydrogenase (33). We cannot rule out the possibility that the effect of E2 is at least in part derived from estrone in 3T3-L1 adipocytes.
Treatment with E2 at any concentration did not affect the insulin-induced tyrosine phosphorylation of the insulin receptor. In contrast, the insulin-induced tyrosine phosphorylation of IRS-1, the association of IRS-1 with p85, and the phosphorylation of Akt were modulated by E2. Along with the insulin-induced glucose uptake, they were enhanced by 10–8 M E2 treatment and inhibited by 10–5 M E2 treatment. These results clearly indicate that E2 affects insulin signaling, leading to glucose uptake at least in part at the step of insulin-induced tyrosine phosphorylation of IRS-1. Although a previous report identified the decrease as the cause of 10–7 M E2-induced insulin resistance in 3T3-L1 adipocytes (5), 10–7 M E2 did not significantly affect insulin signaling, and the amount of IRS-1 was not changed by the treatment in our experiment. The difference may arise from the experimental conditions employed. In this regard, we employed fully differentiated 3T3-L1 adipocytes 14–16 d after differentiation, whereas the previous study used the cells 8–12 d after the induction of differentiation (5). By contrast, our results are consistent with reports indicating the possible mechanisms of insulin resistance in pregnant women (2, 34). Insulin-induced tyrosine phosphorylation of IRS-1 is reported to be decreased in the skeletal muscle of pregnant women compared with that in nonpregnant women (2). The reduction was even greater in pregnant women with type 2 diabetes (2). Therefore, it is possible that the effect of E2 at the level of tyrosine phosphorylation of IRS-1 is involved at least in part in the insulin resistance seen in pregnant women. It is worth noting that the cause of insulin resistance in pregnancy appears to be complex. The role of E2 in other target tissues of insulin in addition to fat cells and the roles of other hormones including progesterone need to be clarified.
Serine phosphorylation of IRS-1 is known to be a mechanism in the attenuation of insulin signaling, resulting in inhibition of tyrosine phosphorylation of IRS-1 (31). Phosphorylation of IRS-1 at Ser307, Ser612, and Ser636 is known to cause insulin resistance in 3T3-L1 adipocytes (35, 36). Interestingly, phosphorylation of IRS-1 at Ser307, but not at Ser612 or Ser636, is induced by treatment with E2 in a concentration-dependent manner. These results indicate that high concentrations of E2 inhibit insulin signaling at least in part via phosphorylation of IRS-1 at Ser307. Serine 307 of IRS-1 is known to be the site of phosphorylation caused by JNK, which is a crucial mediator of insulin resistance (32). JNK1 activity is abnormally elevated in the state of insulin resistance, and an absence of JNK1 results in enhanced insulin signaling in mice and cultured cells (37). In the present study, inhibition of JNK activity by a JNK inhibitor abrogated the E2-induced inhibition of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt. These results strengthen our idea that E2-induced JNK activation is implicated in the phosphorylation of IRS-1 at Ser307, followed by inhibition of the insulin-induced tyrosine phosphorylation of IRS-1 and phosphorylation of Akt.
TNF- is known to be a key factor causing insulin resistance by inhibiting insulin-induced tyrosine phosphorylation of IRS-1 (38, 39). Because TNF- is also secreted from the placenta and has been implicated in insulin resistance in pregnancy, we examined the combined effects of E2 and TNF- on insulin signaling. Because the data indicated that TNF- combined with a submaximal concentration of E2 further inhibited insulin signaling, both agents appear to be involved in insulin resistance during late pregnancy.
ICI182,780 is known to be an antagonist of the estrogen receptor (40). Because our results showed that pretreatment with ICI182,780 abrogated the modulation of insulin-stimulated Akt phosphorylation caused by both 10–8 and 10–5 M E2, the effect of E2 on insulin signaling appears to be mediated by the estrogen receptor. Studies have revealed the existence of two receptor subtypes, estrogen receptors and (15, 16). Although several functional differences have been reported between these subtypes, a number of studies have demonstrated that estrogen binds to the estrogen receptor in the cytosol, and that its effect is elicited via estrogen receptors translocated to the nucleus, resulting in transcriptional activation (19, 20, 21). In addition to being located in the cytosol and, to a lesser extent, the nucleus, the estrogen receptor is known to exist on the plasma membrane (41, 42). Recent studies indicate a nontranscriptional role for the estrogen receptor in the mediation of estrogen signaling (20, 43). However, the role of estrogen receptors in the specific cellular localization during insulin signaling is totally unknown. The present study showed that membrane-impermeable E2 (E2-BSA) at 10–5 M inhibited the insulin-induced tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt, whereas they were not enhanced by E2-BSA at 10–8 M. These results indicate that E2’s effect at high concentrations, but not at physiological concentrations, appears to be mediated by the estrogen receptors on the plasma membrane. Importantly, estrogen receptor , but not estrogen receptor , was redistributed upon treatment with E2. Thus, E2 at a physiological concentration (10–8 M) elicited the redistribution of estrogen receptor from plasma membrane to nucleus, whereas a high concentration of E2 (10–5 M) induced redistribution from cytosol and nucleus to plasma membrane. Taken together, estrogen receptor at the plasma membrane appears to be involved in the inhibitory effect of 10–5 M E2 on insulin signaling. The possible importance of estrogen receptor at the plasma membrane is strengthened by the fact that E2 affects the membrane-proximal signaling of the insulin receptor located at the plasma membrane. These results are consistent with recent reports that the PI3-kinase/Akt signaling cascade is a downstream target of nonnuclear estrogenic signaling (44, 45). The lack of an effect of E2 on the cellular distribution of estrogen receptor may be due to the low levels of estrogen receptor in 3T3-L1 adipocytes (46).
In summary, our results indicate that 1) E2 affects the metabolic action of insulin in a concentration-specific manner; 2) high concentrations of E2 inhibit insulin signaling at the level of IRS-1 by modulating the phosphorylation of IRS-1 at Ser307 via a JNK-dependent pathway; and 3) estrogen receptor translocated to the plasma membrane in response to E2 is implicated in E2’s inhibitory effect in 3T3-L1 adipocytes.
Footnotes
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to T.S., M.K., and S.S.); the 21st Century Center of Excellence (COE) program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to S.S.); and a grant for Research on Sensory and Communicative Disorders from the Ministry of Health, Labor, and Welfare, Japan (to T.S.).
First Published Online November 3, 2005
Abbreviations: 2-DOG, 2-Deoxyglucose; CS-, charcoal-stripped; E2, 17-estradiol, estradiol; FBS, fetal bovine serum; HES, HEPES sucrose; IRS-1, insulin receptor substrate-1; JNK, c-Jun NH2-terminal kinase; PI, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonylfluoride.
Accepted for publication October 21, 2005.
References
Yamashita H, Shao J, Friedman JE 2000 Physiologic and molecular alterations in carbohydrate metabolism during pregnancy and gestational diabetes mellitus. Clin Obstet Gynecol 43:87–98
Friedman JE, Ishizuka T, Shao J, Huston L, Highman T, Catalano P 1999 Impaired glucose transport and insulin receptor tyrosine phosphorylation in skeletal muscle from obese women with gestational diabetes. Diabetes 49:1807–1814
Ryan EA, O’Sullivan MJ, Skylar JS 1985 Insulin action during pregnancy: studies with the euglycemic clamp technique. Diabetes 34:380–389
Ryan EA, Enns L 1988 Role of gestational hormones in the induction of insulin resistance. J Clin Endocrinol Metab 67:341–347
Collison M, Campbell IW, Salt IP, Dominiczak AF, Connell JMC, Lyall H, Gould GW 2000 Sex hormones induce insulin resistance in 3T3–L1 adipocytes by reducing cellular content of IRS proteins. Diabetologia 43:1374–1380
Kirwan JP, Haugel-de Mouzon S, Lepercq J, Challier J-C, Huston-Presley L, Friedman JE, Kalhan SC, Catalano PM 2002 TNF- is a predictor of insulin resistance in human pregnancy. Diabetes 51:2207–2213
Brussaard HE, Gevers-Leuven JA, Fllich M, Kluft C, Krans HMJ 1997 Short-term oestrogen replacement therapy improves insulin resistance, lipids and fibrinolysis in postmenopausal women with NIDDM. Diabetologia 40:843–849
Hernandez-Ono A, Monter-Carreola G, Zamora-Gonzalez J, Cardoso-Saldana G, Posadas-Sanchez R, Torres-Tamayo M, Posadas-Romero C 2002 Association of visceral fat with coronary risk factors in a population-based sample of postmenopausal women. Int J Obes Relat Metab Disord 26:33–39
Brochu M, Starling RD, Tchernof A, Matthews DE, Garcia-Rubi E, Poehlman ET 2000 Visceral adipose tissue is an independent correlate of glucose disposal in older obese postmenopausal women. J Clin Endocrinol Metab 85:2378–2384
Inoue G, Cheatham B, Emkey R, Kahn CR 1998 Dynamics of insulin signaling in 3T3–L1 adipocytes: differential compartmentalization and trafficking of insulin receptor substrate (IRS)-1 and IRS-2. J Biol Chem 273:11548–11555
Czech MP, Corvera S 1999 Signaling mechanisms that regulate glucose transport. J Biol Chem 274:1865–1868
Saltiel AR, Kahn CR 2001 Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414:799–806
Virkamki A, Ueki K, Kahn CR 1999 Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 103:931–943
Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA 1996 Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15:6541–6551
Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134–139
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930
Enmark E, Gustafsson J-A 1999 Oestrogen receptors–an overview. J Intern Med 246:133–138
Mendelsohn ME 2002 Protective effects of estrogen on the cardiovascular system. Am J Cardiol 89(Suppl):12E–18E
Hager GL, Lim CS, Elbi C, Baumann CT 2000 Trafficking of nuclear receptors in living cells. J Steroid Biochem Mol Biol 74:249–254
Moss RL, Gu Q, Wong M 1997 Estrogen nontranscriptional signaling pathway. Rec Prog Horm Res 52:33–70
Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 9:404–410
Kitamura T, Ogawa W, Sakaue H, Hino Y, Kuroda S, Tanaka M, Matsumoto M, Maeda T, Konishi H, Kikkawa U, Kasuga M 1998 Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol Cell Biol 18:3708–3717
Ueki K, Yamamoto-Honda R, Kaburagi Y, Yamauchi T, Tobe K, Burgering BMT, Coffer PJ, Komuro I, Akanuma Y, Yazaki Y, Kadowaki T 1998 Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem 273:5315–5322
Takano A, Usui I, Haruta T, Kawahara J, Uno T, Iwata M, Kobayashi M 2001 Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin. Mol Cell Biol 21:5050–5062
Ishihara H, Sasaoka T, Hori H, Wada T, Hirai H, Haruta T, Langlois WJ, Kobayashi M 1999 Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling. Biochem Biophys Res Commun 260:265–272
Wada T, Sasaoka T, Funaki M, Hori H, Murakami S, Ishiki M, Haruta T, Asano T, Ogawa W, Ishihara H, Kobayashi M 2001 Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3–L1 adipocytes via its 5'-phosphatase catalytic activity. Mol Cell Biol 21:1633–1646
Burgering BM, Coffer PJ 1995 Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599–602
Cong L-N, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, Quon MJ 1997 Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11:1881–1890
Chen HL, Yang YP, Hu XL, Yelavarthi KK, Fishback JL, Hunt JS 1991 Tumor necrosis factor mRNA and protein are present in human placental and uterine cells at early and late stages of gestation. Am J Pathol 139:327–335
Innes KE, Wimsatt JH, McDuffie R 2001 Relative glucose tolerance and subsequent development of hypertension in pregnancy. Obstet Gynecol 97:905–910
Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF 2002 Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J Biol Chem 277:1531–1537
Aguirre V, Uchida T, Yenush L, Davis R, White MF 2000 The c-Jun NH2-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser307. J Biol Chem 275:9047–9054
Corbould AM, Judd SJ, Rodgers RJ 1998 Expression of types 1, 2, and 3 17-hydroxysteroid dehydrogenase in subcutaneous abdominal and intra-abdominal adipose tissue of women. J Clin Endocrinol Metab 83:187–194
Shao J, Catalano PM, Yamashita H, Ruyter I, Smith S, Youngren J, Friedman JE 2000 Decreased insulin receptor tyrosine kinase activity and plasma cell membrane glycoprotein-1 overexpression in skeletal muscle from obese women with gestational diabetes mellitus (GDM): evidence for increased serine/threonine phosphorylation in pregnancy and GDM. Diabetes 49:603–610
Rui L, Aguirre V, Kim JK, Shulman GI, Lee A, Corbould A, Dunaif A, White MF 2001 Insulin/IGF-1 and TNF- stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest 107:181–189
Gual P, Gremeaux T, Gonzalez T, Le Marchand-Brustel Y, Tanti J-F 2003 MAP kinases and mTOR mediate insulin-induced phosphorylation of insulin receptor substrate-1 on serine residues 307, 612, and 632. Diabetologia 46:1532–1542
Hirosumi J, Tuncman G, Chang L, Grgün CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS 2002 A central role for JNK in obesity and insulin resistance. Nature 420:333–336
Peraldi P, Hotamisligil GS, Buurman WA, White MF, Spiegelman BM 1996 Tumor necrosis factor (TNF)- inhibits insulin signaling through stimulation of the p55 TNF receptor and activation of sphingomyelinase. J Biol Chem 271:13018–13022
Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF, Spiegelman BM 1996 IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF- and obesity-induced insulin resistance. Science 271:665–668
Wakeling AE, Dukes M, Bowler J 1991 A potent specific pure antiestrogen with clinical potential. Cancer Res 51:3867–3873
Pietras RJ, Nemere I, Szego CM 2001 Steroid hormone receptors in target cell membranes. Endocrine 14:417–427
Ho KJ, Liao JK 2002 Nonnuclear actions of estrogen. Arterioscler Thromb Vasc Biol 22:1952–1961
Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER and ER expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319
Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK 2000 Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407:538–541
Simoncini T, Rabkin E, Liao JK 2003 Molecular basis of cell membrane estrogen receptor interaction with phosphatidylinositol 3-kinase in endothelial cells. Arterioscler Thromb Vasc Biol 23:198–203
Rodriguez-Cuenca S, Monjo M, Proenza AM, Roca P 2005 Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu. Am J Physiol 288:E200–E207(Kiyofumi Nagira, Toshiyasu Sasaoka, Tsut)
Abstract
We investigated the mechanisms by which estrogen alters insulin signaling in 3T3-L1 adipocytes. Treatment with 17-estradiol (E2) did not affect insulin-induced tyrosine phosphorylation of insulin receptor. E2 enhanced insulin-induced tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), IRS-1/p85 association, phosphorylation of Akt, and 2-deoxyglucose uptake at 10–8 M, but inhibited these effects at 10–5 M. A concentration of 10–5 M E2 enhanced insulin-induced phosphorylation of IRS-1 at Ser307, which was abolished by treatment with a c-Jun NH2-terminal kinase inhibitor. In addition, the effect of E2 was abrogated by pretreatment with a specific estrogen receptor antagonist, ICI182,780. Membrane-impermeable E2, E2-BSA, did not affect the insulin-induced phosphorylation of Akt at 10–8 M, but inhibited it at 10–5 M. Furthermore, E2 decreased the amount of estrogen receptor at the plasma membrane at 10–8 M, but increased it at 10–5 M. In contrast, the subcellular distribution of estrogen receptor was not altered by the treatment. These results indicate that E2 affects the metabolic action of insulin in a concentration-specific manner, that high concentrations of E2 inhibit insulin signaling by modulating phosphorylation of IRS-1 at Ser307 via a c-Jun NH2-terminal kinase-dependent pathway, and that the subcellular redistribution of estrogen receptor in response to E2 may explain the dual effect of E2.
Introduction
HUMAN PREGNANCY IS associated with hyperinsulinemia and a progressive decline in insulin sensitivity (1). Thus, a number of clinical studies indicate that insulin resistance occurs in late pregnancy (2, 3). The cellular changes in insulin resistance during late pregnancy have been ascribed to altered levels of placental-derived hormones, including estrogen, progesterone, and human placental lactogen (4). Among these sex steroids, high concentrations of estrogen are known to inhibit insulin-stimulated glucose uptake in cultured cells (5). In addition, increased levels of TNF- have been associated with insulin resistance in late pregnancy (6). Thus, estrogen and TNF- produced in the placenta appear to play a key role in insulin resistance (5, 6). Although the mechanism of insulin resistance caused by TNF- has been studied intensively, the mechanism by which estrogen induces insulin resistance is unknown (6). In contrast, physiological levels of estrogen are known to protect against insulin resistance in women (7). Along this line, clinical studies suggest that after menopause women have an increased risk of developing diabetes (8, 9). Therefore, estrogen appears to control glucose homeostasis in a concentration-specific manner. The insulin signaling system leading to glucose uptake has been studied intensively in 3T3-L1 adipocytes (10).
The binding of insulin to the -subunit of the insulin receptor results in autophosphorylation and activation of the -subunit (11, 12, 13). The activated insulin receptor phosphorylates insulin receptor substrate-1 (IRS-1) at tyrosine residues (11, 12, 13). Tyrosine-phosphorylated IRS-1 binds to the p85 subunit of phosphatidylinositol 3-kinase (PI3-kinase), leading to the activation of the p110 catalytic subunit (11, 12, 13). PI3-kinase generates PI triphosphate from PI-bisphosphate, leading to the phosphorylation and activation of Akt (11, 12, 13). Akt is known to be an important mediator of the insulin-induced uptake of glucose (14). The cellular mechanisms by which estrogen affects the insulin signaling system are unknown.
Estrogen binds receptors of two subtypes: estrogen receptor , first identified in 1986, and estrogen receptor , cloned subsequently (15, 16). Estrogen receptors are expressed not only in the reproductive organs of males and females, but also in nonreproductive tissues, including fat tissues (17, 18). It has been shown that the effect of estrogen is mediated by the binding of estrogen receptors in the cytosol, resulting in translocation and transcriptional activation in the nucleus (19). In addition, recent reports suggest a nontranscriptional action mediated by the estrogen receptor (20). Along these lines, estrogen receptors are distributed in the plasma membrane in addition to the cytosol and nucleus (21). However, the role of estrogen receptors at specific cellular locations in the regulation of insulin signaling is unknown.
In the present study we studied the effect of the main estrogen, estradiol (E2), on insulin signaling leading to glucose uptake in 3T3-L1 adipocytes. In addition, to clarify the molecular mechanism by which E2 affects the insulin signaling system, we investigated the isoform and subcellular distribution of the estrogen receptor mediating the effect of E2.
Materials and Methods
Materials
Recombinant human insulin was provided by Lilly Research Laboratories (Indianapolis, IN). Recombinant human TNF- was supplied by Dainippon Pharmaceutical Co. (Osaka, Japan). 17-Estradiol (E2) and E2-BSA were purchased from Sigma-Aldrich Corp. (St. Louis, MO). A complete estrogen receptor antagonist, ICI182,780, was obtained from Tocris (Ellisville, MO). The Jun NH2-terminal kinase (JNK) inhibitor, SP600125, was purchased from BIOMOL (Plymouth Meeting, PA). 2-[1,2-3H]Deoxyglucose (2-DOG) was obtained from DuPont NEN Life Science Products (Boston, MA). A monoclonal antiphosphotyrosine antibody (PY20) was purchased from Transduction Laboratories (Lexington, KY). A polyclonal anti-IRS-1 antibody, a polyclonal anti-Ser307 phosphospecific IRS-1 antibody, a polyclonal anti-Ser612 phosphospecific IRS-1 antibody, a polyclonal anti-Ser636 phosphospecific IRS-1 antibody, a monoclonal anti-p85 antibody, a polyclonal anti-Ser473 phosphospecific Akt antibody, a polyclonal anti-Thr308 phosphospecific Akt antibody, and a polyclonal anti-Akt antibody were obtained from Upstate Biotechnology, Inc. (Lake Placid, NY). A polyclonal antiinsulin receptor antibody, horseradish peroxidase-conjugated antimouse and antirabbit IgG antibodies, a polyclonal antiestrogen receptor antibody, and a polyclonal antiestrogen receptor antibody were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Phenol red-free DMEM and fetal bovine serum (FBS) were obtained from Invitrogen Life Technologies, Inc. (Gaithersburg, MD). Protein G-Sepharose was purchased from Pharmacia Biotech (Uppsala, Sweden). Reagents for electrophoresis were obtained from Bio-Rad Laboratories, Inc. (Hercules, CA). All other reagents were of analytical grade and purchased from Sigma-Aldrich Corp. or Wako Pure Chemical Industries (Osaka, Japan).
Cell culture and treatment
3T3-L1 fibroblasts were grown and passaged in phenol red-free DMEM supplemented with 10% donor calf serum. Cells, 2 d after confluence, were used for differentiation. The differentiation medium contained 10% charcoal-stripped FBS (CS-FBS), 0.5 mM 3-isobutyl-1-methylxanthine, 1 μM dexamethasone, and 1 μM insulin. After charcoal stripping, E2 and estrone concentrations in serum decreased from 0.48 to less than 0.07 pM and from 0.36 to less than 0.12 pM, respectively. After 3 d, the differentiation medium was replaced with postdifferentiation medium containing 10% CS-FBS and 800 nM insulin. After 3 more days, the postdifferentiation medium was replaced with phenol red-free DMEM supplemented with 10% CS-FBS. The medium was changed every 3 d until the cells were used for experiments. Fourteen to 16 d after the induction of differentiation, more than 95% of the cells had morphological and biochemical properties of adipocytes. E2 dissolved in ethanol and TNF- in PBS were added to the cell culture medium, in which 106 cells were contained/plate, without FBS for 16 h.
2-DOG uptake
3T3-L1 adipocytes derived from differentiation of 3T3-L1 fibroblasts were grown in six-well multiplates and were serum-starved for 3 h. The cells were stimulated with 17 nM insulin for 15 min in Krebs-Ringer-phosphate-HEPES buffer containing 10 mM HEPES, 131.2 mM NaCl, 4.7 mM KCl, 1.2 mM MgSO4, 2.5 mM CaCl2, and 2.5 mM NaH2PO4, and 1% BSA (pH 7.4). 2-[3H]DOG (0.1 mM; 0.4 kBq/well) was added for 4 min. The reaction was stopped by the addition of 10 μM cytochalasin B. The cells were washed three times with ice-cold PBS and solubilized with 0.2% sodium dodecyl sulfate-0.2 N NaOH (22, 23). The radioactivity incorporated into the cells was measured by liquid scintillation counting. The results were corrected for nonspecific uptake determined by 2-[3H]DOG uptake in the presence of 10 μM cytochalasin B. Nonspecific binding was less than 10% of the total uptake.
Subcellular fractionation
The cells were washed twice with PBS and once with HEPES sucrose (HES) buffer containing 255 mM sucrose, 20 mM HEPES, 1 mM EDTA, 1 mM phenylmethylsulfonylfluoride (PMSF), 1 mM Na3VO4, 2 μg/ml aprotinin, and 50 ng/ml okadaic acid (pH 7.4). The cells were then immediately homogenized by 20 strokes with a motor-driven homogenizer in HES buffer containing 20 mM HEPES, 255 mM sucrose, 1 mM EDTA, 1 mM PMSF, 1 mM Na3VO4, 2 μg/ml aprotinin, and 50 ng/ml okadaic acid (pH 7.4) at 4 C. The homogenates obtained from two 10-cm diameter dishes per condition were subjected to subcellular fractionation to isolate the plasma membrane, cytosol, and nucleus as described previously (24). In brief, the homogenates were centrifuged at 19,000 x g for 20 min. The resulting supernatant was centrifuged at 250,000 x g for 90 min. The remaining supernatant was centrifuged at Centricon-30 (Amicon, Inc., Beverly, MA) and used as cytosol. The pellet obtained from the initial spin was resuspended in HES buffer, layered onto a 1.12-M sucrose cushion, and centrifuged at 100,000 x g in a swing rotor for 60 min. A white fluffy band at the interface was collected, resuspended in HES buffer, and centrifuged at 40,000 x g for 20 min, yielding a pellet of plasma membrane. A viscous brown pellet was collected, yielding a pellet of nucleus. The purities of the nuclear and cytosolic preparations were determined by examining histone as a nuclear marker and calpain as a cytosolic marker. The samples were adjusted to a final protein concentration of 1–3 mg/ml, which was measured by the Bradford method, and stored at –80 C before use.
Immunoprecipitation and Western blotting
The cells or cell preparations were lysed in a buffer containing 30 mM Tris, 150 mM NaCl, 10 mM EDTA, 1% Nonidet P-40, 1 mM PMSF, 1 mM Na3VO4, 50 nM sodium fluoride, 10 μg/ml aprotinin, and 1 μM leupeptin (pH 7.4) for 30 min at 4 C. The lysates were centrifuged to remove insoluble materials. The supernatants (100 μg protein) were immunoprecipitated with antibodies for 16 h at 4 C. The immunoprecipitates or total cell lysates were then separated by 7.5% or 15% SDS-PAGE and transferred onto polyvinylidene difluoride membranes (Millipore Corp., Bedford, MA) using a Bio-Rad Transblot apparatus. The membranes were blocked in a buffer containing 50 mM Tris, 150 mM NaCl, 0.1% Tween 20, and 2.5% BSA or 5% nonfat milk (pH 7.5) for 2 h at 20 C. They were then probed with antibodies for 2 h at 20 C or for 16 h at 4 C. After the membranes had been washed in a buffer containing 50 mM Tris, 150 mM NaCl, and 0.1% Tween 20 (pH 7.5), blots were incubated with a horseradish peroxidase-linked secondary antibody and subjected to enhanced chemiluminescence detection using ECL reagent according to the manufacturer’s instruction (Amersham Biosciences, Indianapolis, IN) (25, 26). In each experiment, the intensity of the band derived from control cells was averaged and assigned a value of 1 arbitrary unit, and the intensities of all treated groups were expressed relative to this value.
Statistical analysis
Experiments were separately conducted at least four times, and data are presented as the mean ± SE. P values were determined by ANOVA and Scheffe test. P < 0.05 was considered statistically significant.
Results
Effect of E2 on insulin-stimulated glucose uptake
We first examined the concentration-dependent effect of E2 on insulin-stimulated 2-DOG uptake in 3T3-L1 adipocytes (Fig. 1A). Insulin at 17 nM stimulated 2-DOG uptake by 6.5 ± 0.6-fold in 3T3-L1 adipocytes. Low concentrations of E2 further increased uptake. Insulin-induced 2-DOG uptake was increased 18.3 ± 3.6% by treatment with 10–8 M E2 compared with that without E2 treatment. Conversely, concentrations of E2 above 10–7 M inhibited insulin-stimulated 2-DOG uptake. After treatment with 10–5 M E2, insulin-induced 2-DOG uptake was decreased by 25.8 ± 3.7% compared with that without E2 treatment. Insulin stimulated 2-DOG uptake in a concentration-dependent manner (Fig. 1B). Treatment with 10–8 M E2 significantly enhanced 0.17, 1.7, and 17 nM insulin-induced 2-DOG uptake, whereas 10–5 M E2 apparently inhibited 1.7 and 17 nM insulin-stimulated glucose uptake. E2 treatment alone for up to 16 h did not stimulate glucose uptake, and the amount of glucose transporter 4 was not altered by treatment with any concentration of E2 (data not shown).
Effect of E2 on early steps of insulin signaling
Because E2 affected insulin-stimulated glucose uptake, the possible involvement of E2 in the early steps of insulin signaling was examined. Insulin-induced tyrosine phosphorylation of the insulin receptor was not affected by treatment with E2 at any concentration (Fig. 2A). Interestingly, insulin-induced tyrosine phosphorylation of IRS-1 and the association of IRS-1 with the p85 subunit of PI3-kinase were promoted by treatment with a physiological concentration of E2. Thus, insulin-stimulated tyrosine phosphorylation of IRS-1 and IRS-1/p85 association were increased by 41.7 ± 3.4% and 22.9 ± 5.9%, respectively, after treatment with 10–8 M E2. In contrast, they were inhibited by treatment with concentrations of E2 above 10–7 M. At 10–5 M, E2 inhibited the insulin-induced tyrosine phosphorylation of IRS-1 and IRS-1/p85 association by 53.7 ± 3.7% and 25.9 ± 3.4%, respectively (Fig. 2, B and C). Amounts of protein among samples were confirmed to be equal by immunoblotting with antiinsulin receptor antibody (Fig. 2D), anti-IRS-1 antibody (Fig. 2E), and anti-p85 antibody (Fig. 2F).
Effect of E2 on insulin-induced phosphorylation of Akt
Akt lies downstream of PI3-kinase and is important for insulin’s stimulation of glucose uptake (23, 27, 28). Because Akt is activated by phosphorylation at Ser473 and Thr308, we examined the effect of E2 on the insulin-induced phosphorylation of Akt (Fig. 3, A and B). Consistent with the results of insulin-induced tyrosine phosphorylation of IRS-1 and IRS-1/p85 association, insulin-induced phosphorylation of Akt was increased by treatment with low concentrations of E2 and decreased by treatment with high concentrations of E2. Insulin-induced phosphorylation of Akt at Ser473 and Thr308 was enhanced 22.0 ± 3.3% and 30.1 ± 3.2%, respectively, by treatment with 10–8 M E2, whereas it was inhibited 44.7 ± 3.5% and 50.8 ± 3.6%, respectively, by treatment with 10–5 M E2. Amounts of protein among samples were confirmed to be equal by immunoblotting with anti-Akt antibody (Fig. 3C).
Effect of combined presence of E2 and TNF- on insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt
TNF- together with estrogen are secreted from the placenta and are known to be involved in insulin resistance in the late phases of pregnancy, especially in preeclampsia (29, 30). The effect of combined presence of E2 (10–6 M) and TNF- on the insulin-induced tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt was examined. Insulin-induced tyrosine phosphorylation of IRS-1 was inhibited 24.6 ± 2.9% and 35.7 ± 3.6% by treatment with E2 and TNF-, respectively (Fig. 4A). Insulin signaling was maximally inhibited by treatment with 50 ng/ml TNF-. Treatment with both E2 and TNF- further inhibited insulin signaling by 61.3 ± 4.2%. Similarly, treatment with E2, TNF-, or both inhibited the insulin-induced phosphorylation of Akt by 21.8 ± 3.3%, 34.9 ± 3.0%, and 65.4 ± 3.3%, respectively (Fig. 4B). These results indicate that TNF- combined with a submaximal concentration of E2 further inhibit insulin signaling, leading to glucose uptake.
Effect of E2 on insulin-induced serine phosphorylation of IRS-1
E2 affected insulin signaling at least at the level of tyrosine phosphorylation of IRS-1. Because tyrosine phosphorylation of IRS-1 is modulated by serine phosphorylation of IRS-1 (31), we examined the effect of E2 on the insulin-induced serine phosphorylation of IRS-1 (Fig. 5). The insulin-induced phosphorylation of IRS-1 at Ser307 was increased by treatment with E2 in a concentration-dependent manner. The extent of phosphorylation at Ser307 was increased 73.9 ± 4.1% by treatment with 10–5 M E2 (Fig. 5A). In contrast, the insulin-induced phosphorylation of IRS-1 at Ser612 (Fig. 5B) and Ser636 (Fig. 5C) was not altered by treatment with E2. These results indicate that a high concentration of E2 inhibits insulin signaling at the level of IRS-1 at least in part by increasing the phosphorylation of IRS-1 at Ser307.
Effect of a JNK inhibitor on the alteration of insulin signaling caused by E2 treatment
Because IRS-1 is known to be phosphorylated by JNK at Ser307 (32), we examined the influence of a JNK inhibitor, SP600125, on the effect of E2. The enhancement of the insulin-induced phosphorylation at Ser307 caused by 10–5 M E2 was abrogated by treatment with the JNK inhibitor (Fig. 6A). As a result, inhibition of the insulin-induced tyrosine phosphorylation of IRS-1 (Fig. 6B) and serine phosphorylation of Akt (Fig. 6C) caused by E2 treatment was restored by pretreatment with the JNK inhibitor.
Effects of ICI182,780 and E2-BSA on E2-induced alteration of insulin signaling
We employed a specific estrogen receptor antagonist, ICI182,780, to examine whether the stimulatory effect of E2 on insulin signaling seen at 10–8 M is mediated by estrogen receptor (Fig. 7A). Pretreatment with 10 μM ICI 182,780 abrogated the enhancement of insulin-induced tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt caused by 10–8 M E2. Pretreatment with 1 μM ICI182,780 had the same effect. Similarly, we examined the effect of ICI182,780 to clarify whether the inhibitory effect of E2 at 10–5 M on insulin signaling is mediated by the estrogen receptor. Pretreatment with ICI182,780 at 10 μM, but not at 1 μM, restored the inhibition of insulin-stimulated tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt caused by 10–5 M E2. These results indicate that the estrogen receptor is implicated in the effects of both concentrations of E2 on insulin signaling.
The estrogen receptor is located on the plasma membrane in addition to the cytosol and nucleus (21). To examine the impact of membrane-based estrogen receptors, a membrane-impermeable E2, E2-BSA, was employed. Insulin-induced tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt were not enhanced by 10–8 M E2-BSA in contrast to E2 (Fig. 7B). However, higher concentrations of E2-BSA inhibited these actions, similar to the results obtained with E2. Thus, 10–5 M E2-BSA inhibited the insulin-induced tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt by 54.9 ± 3.5% (data not shown) and 49.4 ± 4.1%, respectively (Fig. 7B). These results indicate that estrogen receptors at the plasma membrane are implicated in the inhibitory effect of E2 at high concentrations, but are not involved in the enhancing effect of E2 at low concentrations.
Effect of E2 treatment on subcellular distribution of estrogen receptors and
We also investigated the subcellular redistribution of the estrogen receptor in response to E2. Treatment with 10–8 M E2 reduced the amount of estrogen receptor at the plasma membrane by 17.1 ± 2.9%, but increased the amount in the nucleus by 90.7 ± 4.4%. The amount of estrogen receptor in the cytosol was not apparently changed by E2 treatment. In contrast, treatment with 10–5 M E2 increased the amount of estrogen receptor at the plasma membrane by 71.3 ± 4.1%, but decreased it in the cytosol and nucleus by 36.3 ± 3.8% and 47.3 ± 3.4%, respectively (Fig. 8, A–C). In contrast, the amounts of estrogen receptor at the plasma membrane, in the cytosol, and in the nucleus were not apparently altered by treatment with any concentration of E2 (Fig. 8, D–F). In addition, insulin treatment did not appear to alter the subcellular distribution of estrogen receptors.
Discussion
Our results demonstrated that treatment with a physiological concentration of E2 (10–8 M) enhanced the insulin-induced uptake of glucose, whereas treatment with a high concentration of E2 (10–5 M) inhibited it. These results clearly indicate that E2 controls glucose metabolism in a concentration-specific manner. These results are consistent with clinical reports that the incidence of type 2 diabetes increases in women after menopause, and that insulin resistance occurs in women during late pregnancy (4, 7, 8). Taken together, the present results strongly indicate that physiological concentrations of E2 enhance insulin sensitivity, whereas high concentrations of E2 inhibit insulin-induced glucose metabolism. Part of the E2 may be converted to estrone by 17-hydroxysteroid dehydrogenase (33). We cannot rule out the possibility that the effect of E2 is at least in part derived from estrone in 3T3-L1 adipocytes.
Treatment with E2 at any concentration did not affect the insulin-induced tyrosine phosphorylation of the insulin receptor. In contrast, the insulin-induced tyrosine phosphorylation of IRS-1, the association of IRS-1 with p85, and the phosphorylation of Akt were modulated by E2. Along with the insulin-induced glucose uptake, they were enhanced by 10–8 M E2 treatment and inhibited by 10–5 M E2 treatment. These results clearly indicate that E2 affects insulin signaling, leading to glucose uptake at least in part at the step of insulin-induced tyrosine phosphorylation of IRS-1. Although a previous report identified the decrease as the cause of 10–7 M E2-induced insulin resistance in 3T3-L1 adipocytes (5), 10–7 M E2 did not significantly affect insulin signaling, and the amount of IRS-1 was not changed by the treatment in our experiment. The difference may arise from the experimental conditions employed. In this regard, we employed fully differentiated 3T3-L1 adipocytes 14–16 d after differentiation, whereas the previous study used the cells 8–12 d after the induction of differentiation (5). By contrast, our results are consistent with reports indicating the possible mechanisms of insulin resistance in pregnant women (2, 34). Insulin-induced tyrosine phosphorylation of IRS-1 is reported to be decreased in the skeletal muscle of pregnant women compared with that in nonpregnant women (2). The reduction was even greater in pregnant women with type 2 diabetes (2). Therefore, it is possible that the effect of E2 at the level of tyrosine phosphorylation of IRS-1 is involved at least in part in the insulin resistance seen in pregnant women. It is worth noting that the cause of insulin resistance in pregnancy appears to be complex. The role of E2 in other target tissues of insulin in addition to fat cells and the roles of other hormones including progesterone need to be clarified.
Serine phosphorylation of IRS-1 is known to be a mechanism in the attenuation of insulin signaling, resulting in inhibition of tyrosine phosphorylation of IRS-1 (31). Phosphorylation of IRS-1 at Ser307, Ser612, and Ser636 is known to cause insulin resistance in 3T3-L1 adipocytes (35, 36). Interestingly, phosphorylation of IRS-1 at Ser307, but not at Ser612 or Ser636, is induced by treatment with E2 in a concentration-dependent manner. These results indicate that high concentrations of E2 inhibit insulin signaling at least in part via phosphorylation of IRS-1 at Ser307. Serine 307 of IRS-1 is known to be the site of phosphorylation caused by JNK, which is a crucial mediator of insulin resistance (32). JNK1 activity is abnormally elevated in the state of insulin resistance, and an absence of JNK1 results in enhanced insulin signaling in mice and cultured cells (37). In the present study, inhibition of JNK activity by a JNK inhibitor abrogated the E2-induced inhibition of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt. These results strengthen our idea that E2-induced JNK activation is implicated in the phosphorylation of IRS-1 at Ser307, followed by inhibition of the insulin-induced tyrosine phosphorylation of IRS-1 and phosphorylation of Akt.
TNF- is known to be a key factor causing insulin resistance by inhibiting insulin-induced tyrosine phosphorylation of IRS-1 (38, 39). Because TNF- is also secreted from the placenta and has been implicated in insulin resistance in pregnancy, we examined the combined effects of E2 and TNF- on insulin signaling. Because the data indicated that TNF- combined with a submaximal concentration of E2 further inhibited insulin signaling, both agents appear to be involved in insulin resistance during late pregnancy.
ICI182,780 is known to be an antagonist of the estrogen receptor (40). Because our results showed that pretreatment with ICI182,780 abrogated the modulation of insulin-stimulated Akt phosphorylation caused by both 10–8 and 10–5 M E2, the effect of E2 on insulin signaling appears to be mediated by the estrogen receptor. Studies have revealed the existence of two receptor subtypes, estrogen receptors and (15, 16). Although several functional differences have been reported between these subtypes, a number of studies have demonstrated that estrogen binds to the estrogen receptor in the cytosol, and that its effect is elicited via estrogen receptors translocated to the nucleus, resulting in transcriptional activation (19, 20, 21). In addition to being located in the cytosol and, to a lesser extent, the nucleus, the estrogen receptor is known to exist on the plasma membrane (41, 42). Recent studies indicate a nontranscriptional role for the estrogen receptor in the mediation of estrogen signaling (20, 43). However, the role of estrogen receptors in the specific cellular localization during insulin signaling is totally unknown. The present study showed that membrane-impermeable E2 (E2-BSA) at 10–5 M inhibited the insulin-induced tyrosine phosphorylation of IRS-1 (data not shown) and serine phosphorylation of Akt, whereas they were not enhanced by E2-BSA at 10–8 M. These results indicate that E2’s effect at high concentrations, but not at physiological concentrations, appears to be mediated by the estrogen receptors on the plasma membrane. Importantly, estrogen receptor , but not estrogen receptor , was redistributed upon treatment with E2. Thus, E2 at a physiological concentration (10–8 M) elicited the redistribution of estrogen receptor from plasma membrane to nucleus, whereas a high concentration of E2 (10–5 M) induced redistribution from cytosol and nucleus to plasma membrane. Taken together, estrogen receptor at the plasma membrane appears to be involved in the inhibitory effect of 10–5 M E2 on insulin signaling. The possible importance of estrogen receptor at the plasma membrane is strengthened by the fact that E2 affects the membrane-proximal signaling of the insulin receptor located at the plasma membrane. These results are consistent with recent reports that the PI3-kinase/Akt signaling cascade is a downstream target of nonnuclear estrogenic signaling (44, 45). The lack of an effect of E2 on the cellular distribution of estrogen receptor may be due to the low levels of estrogen receptor in 3T3-L1 adipocytes (46).
In summary, our results indicate that 1) E2 affects the metabolic action of insulin in a concentration-specific manner; 2) high concentrations of E2 inhibit insulin signaling at the level of IRS-1 by modulating the phosphorylation of IRS-1 at Ser307 via a JNK-dependent pathway; and 3) estrogen receptor translocated to the plasma membrane in response to E2 is implicated in E2’s inhibitory effect in 3T3-L1 adipocytes.
Footnotes
This work was supported in part by a Grant-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (to T.S., M.K., and S.S.); the 21st Century Center of Excellence (COE) program from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (to S.S.); and a grant for Research on Sensory and Communicative Disorders from the Ministry of Health, Labor, and Welfare, Japan (to T.S.).
First Published Online November 3, 2005
Abbreviations: 2-DOG, 2-Deoxyglucose; CS-, charcoal-stripped; E2, 17-estradiol, estradiol; FBS, fetal bovine serum; HES, HEPES sucrose; IRS-1, insulin receptor substrate-1; JNK, c-Jun NH2-terminal kinase; PI, phosphatidylinositol 3-kinase; PMSF, phenylmethylsulfonylfluoride.
Accepted for publication October 21, 2005.
References
Yamashita H, Shao J, Friedman JE 2000 Physiologic and molecular alterations in carbohydrate metabolism during pregnancy and gestational diabetes mellitus. Clin Obstet Gynecol 43:87–98
Friedman JE, Ishizuka T, Shao J, Huston L, Highman T, Catalano P 1999 Impaired glucose transport and insulin receptor tyrosine phosphorylation in skeletal muscle from obese women with gestational diabetes. Diabetes 49:1807–1814
Ryan EA, O’Sullivan MJ, Skylar JS 1985 Insulin action during pregnancy: studies with the euglycemic clamp technique. Diabetes 34:380–389
Ryan EA, Enns L 1988 Role of gestational hormones in the induction of insulin resistance. J Clin Endocrinol Metab 67:341–347
Collison M, Campbell IW, Salt IP, Dominiczak AF, Connell JMC, Lyall H, Gould GW 2000 Sex hormones induce insulin resistance in 3T3–L1 adipocytes by reducing cellular content of IRS proteins. Diabetologia 43:1374–1380
Kirwan JP, Haugel-de Mouzon S, Lepercq J, Challier J-C, Huston-Presley L, Friedman JE, Kalhan SC, Catalano PM 2002 TNF- is a predictor of insulin resistance in human pregnancy. Diabetes 51:2207–2213
Brussaard HE, Gevers-Leuven JA, Fllich M, Kluft C, Krans HMJ 1997 Short-term oestrogen replacement therapy improves insulin resistance, lipids and fibrinolysis in postmenopausal women with NIDDM. Diabetologia 40:843–849
Hernandez-Ono A, Monter-Carreola G, Zamora-Gonzalez J, Cardoso-Saldana G, Posadas-Sanchez R, Torres-Tamayo M, Posadas-Romero C 2002 Association of visceral fat with coronary risk factors in a population-based sample of postmenopausal women. Int J Obes Relat Metab Disord 26:33–39
Brochu M, Starling RD, Tchernof A, Matthews DE, Garcia-Rubi E, Poehlman ET 2000 Visceral adipose tissue is an independent correlate of glucose disposal in older obese postmenopausal women. J Clin Endocrinol Metab 85:2378–2384
Inoue G, Cheatham B, Emkey R, Kahn CR 1998 Dynamics of insulin signaling in 3T3–L1 adipocytes: differential compartmentalization and trafficking of insulin receptor substrate (IRS)-1 and IRS-2. J Biol Chem 273:11548–11555
Czech MP, Corvera S 1999 Signaling mechanisms that regulate glucose transport. J Biol Chem 274:1865–1868
Saltiel AR, Kahn CR 2001 Insulin signalling and the regulation of glucose and lipid metabolism. Nature 414:799–806
Virkamki A, Ueki K, Kahn CR 1999 Protein-protein interaction in insulin signaling and the molecular mechanisms of insulin resistance. J Clin Invest 103:931–943
Alessi DR, Andjelkovic M, Caudwell B, Cron P, Morrice N, Cohen P, Hemmings BA 1996 Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15:6541–6551
Green S, Walter P, Kumar V, Krust A, Bornert JM, Argos P, Chambon P 1986 Human oestrogen receptor cDNA: sequence, expression and homology to v-erb-A. Nature 320:134–139
Kuiper GGJM, Enmark E, Pelto-Huikko M, Nilsson S, Gustafsson J-A 1996 Cloning of a novel estrogen receptor expressed in rat prostate and ovary. Proc Natl Acad Sci USA 93:5925–5930
Enmark E, Gustafsson J-A 1999 Oestrogen receptors–an overview. J Intern Med 246:133–138
Mendelsohn ME 2002 Protective effects of estrogen on the cardiovascular system. Am J Cardiol 89(Suppl):12E–18E
Hager GL, Lim CS, Elbi C, Baumann CT 2000 Trafficking of nuclear receptors in living cells. J Steroid Biochem Mol Biol 74:249–254
Moss RL, Gu Q, Wong M 1997 Estrogen nontranscriptional signaling pathway. Rec Prog Horm Res 52:33–70
Pappas TC, Gametchu B, Watson CS 1995 Membrane estrogen receptors identified by multiple antibody labeling and impeded-ligand binding. FASEB J 9:404–410
Kitamura T, Ogawa W, Sakaue H, Hino Y, Kuroda S, Tanaka M, Matsumoto M, Maeda T, Konishi H, Kikkawa U, Kasuga M 1998 Requirement for activation of the serine-threonine kinase Akt (protein kinase B) in insulin stimulation of protein synthesis but not of glucose transport. Mol Cell Biol 18:3708–3717
Ueki K, Yamamoto-Honda R, Kaburagi Y, Yamauchi T, Tobe K, Burgering BMT, Coffer PJ, Komuro I, Akanuma Y, Yazaki Y, Kadowaki T 1998 Potential role of protein kinase B in insulin-induced glucose transport, glycogen synthesis, and protein synthesis. J Biol Chem 273:5315–5322
Takano A, Usui I, Haruta T, Kawahara J, Uno T, Iwata M, Kobayashi M 2001 Mammalian target of rapamycin pathway regulates insulin signaling via subcellular redistribution of insulin receptor substrate 1 and integrates nutritional signals and metabolic signals of insulin. Mol Cell Biol 21:5050–5062
Ishihara H, Sasaoka T, Hori H, Wada T, Hirai H, Haruta T, Langlois WJ, Kobayashi M 1999 Molecular cloning of rat SH2-containing inositol phosphatase 2 (SHIP2) and its role in the regulation of insulin signaling. Biochem Biophys Res Commun 260:265–272
Wada T, Sasaoka T, Funaki M, Hori H, Murakami S, Ishiki M, Haruta T, Asano T, Ogawa W, Ishihara H, Kobayashi M 2001 Overexpression of SH2-containing inositol phosphatase 2 results in negative regulation of insulin-induced metabolic actions in 3T3–L1 adipocytes via its 5'-phosphatase catalytic activity. Mol Cell Biol 21:1633–1646
Burgering BM, Coffer PJ 1995 Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature 376:599–602
Cong L-N, Chen H, Li Y, Zhou L, McGibbon MA, Taylor SI, Quon MJ 1997 Physiological role of Akt in insulin-stimulated translocation of GLUT4 in transfected rat adipose cells. Mol Endocrinol 11:1881–1890
Chen HL, Yang YP, Hu XL, Yelavarthi KK, Fishback JL, Hunt JS 1991 Tumor necrosis factor mRNA and protein are present in human placental and uterine cells at early and late stages of gestation. Am J Pathol 139:327–335
Innes KE, Wimsatt JH, McDuffie R 2001 Relative glucose tolerance and subsequent development of hypertension in pregnancy. Obstet Gynecol 97:905–910
Aguirre V, Werner ED, Giraud J, Lee YH, Shoelson SE, White MF 2002 Phosphorylation of Ser307 in insulin receptor substrate-1 blocks interactions with the insulin receptor and inhibits insulin action. J Biol Chem 277:1531–1537
Aguirre V, Uchida T, Yenush L, Davis R, White MF 2000 The c-Jun NH2-terminal kinase promotes insulin resistance during association with insulin receptor substrate-1 and phosphorylation of Ser307. J Biol Chem 275:9047–9054
Corbould AM, Judd SJ, Rodgers RJ 1998 Expression of types 1, 2, and 3 17-hydroxysteroid dehydrogenase in subcutaneous abdominal and intra-abdominal adipose tissue of women. J Clin Endocrinol Metab 83:187–194
Shao J, Catalano PM, Yamashita H, Ruyter I, Smith S, Youngren J, Friedman JE 2000 Decreased insulin receptor tyrosine kinase activity and plasma cell membrane glycoprotein-1 overexpression in skeletal muscle from obese women with gestational diabetes mellitus (GDM): evidence for increased serine/threonine phosphorylation in pregnancy and GDM. Diabetes 49:603–610
Rui L, Aguirre V, Kim JK, Shulman GI, Lee A, Corbould A, Dunaif A, White MF 2001 Insulin/IGF-1 and TNF- stimulate phosphorylation of IRS-1 at inhibitory Ser307 via distinct pathways. J Clin Invest 107:181–189
Gual P, Gremeaux T, Gonzalez T, Le Marchand-Brustel Y, Tanti J-F 2003 MAP kinases and mTOR mediate insulin-induced phosphorylation of insulin receptor substrate-1 on serine residues 307, 612, and 632. Diabetologia 46:1532–1542
Hirosumi J, Tuncman G, Chang L, Grgün CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS 2002 A central role for JNK in obesity and insulin resistance. Nature 420:333–336
Peraldi P, Hotamisligil GS, Buurman WA, White MF, Spiegelman BM 1996 Tumor necrosis factor (TNF)- inhibits insulin signaling through stimulation of the p55 TNF receptor and activation of sphingomyelinase. J Biol Chem 271:13018–13022
Hotamisligil GS, Peraldi P, Budavari A, Ellis R, White MF, Spiegelman BM 1996 IRS-1-mediated inhibition of insulin receptor tyrosine kinase activity in TNF- and obesity-induced insulin resistance. Science 271:665–668
Wakeling AE, Dukes M, Bowler J 1991 A potent specific pure antiestrogen with clinical potential. Cancer Res 51:3867–3873
Pietras RJ, Nemere I, Szego CM 2001 Steroid hormone receptors in target cell membranes. Endocrine 14:417–427
Ho KJ, Liao JK 2002 Nonnuclear actions of estrogen. Arterioscler Thromb Vasc Biol 22:1952–1961
Razandi M, Pedram A, Greene GL, Levin ER 1999 Cell membrane and nuclear estrogen receptors (ERs) originate from a single transcript: studies of ER and ER expressed in Chinese hamster ovary cells. Mol Endocrinol 13:307–319
Simoncini T, Hafezi-Moghadam A, Brazil DP, Ley K, Chin WW, Liao JK 2000 Interaction of oestrogen receptor with the regulatory subunit of phosphatidylinositol-3-OH kinase. Nature 407:538–541
Simoncini T, Rabkin E, Liao JK 2003 Molecular basis of cell membrane estrogen receptor interaction with phosphatidylinositol 3-kinase in endothelial cells. Arterioscler Thromb Vasc Biol 23:198–203
Rodriguez-Cuenca S, Monjo M, Proenza AM, Roca P 2005 Depot differences in steroid receptor expression in adipose tissue: possible role of the local steroid milieu. Am J Physiol 288:E200–E207(Kiyofumi Nagira, Toshiyasu Sasaoka, Tsut)