help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boileau, P.
Right arrow Articles by Hauguel-de Mouzon, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boileau, P.
Right arrow Articles by Hauguel-de Mouzon, S.
Endocrinology Vol. 142, No. 9 3974-3979
Copyright © 2001 by The Endocrine Society

ARTICLES

Dissociation between Insulin-Mediated Signaling Pathways and Biological Effects in Placental Cells: Role of Protein Kinase B and MAPK Phosphorylation

Pascal Boileau, Michèle Caüzac, Marie Ange Pereira, Jean Girard and Sylvie Hauguel-de Mouzon

Centre National de la Recherche Scientifique-Unité Propre de Recherche 1524, 92190 Meudon, France

Address all correspondence and requests for reprints to: Sylvie Hauguel-de Mouzon, Centre National de la Recherche Scientifique-Unité Propre de Recherche 1524, 9 rue Jules Hetzel, 92190 Meudon, France. E-mail: shm{at}infobiogen.fr


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Beyond the presence of insulin receptors, little is known of the mechanisms underlying the biological effects of insulin in the placenta. We show that phosphorylation of MAPK and protein kinase B were enhanced 286 ± 23% and 393 ± 17% upon insulin stimulation of JAr placental cells. MAPK activation was prevented by pretreatment with PD98059 but was unaffected by wortmannin. Insulin stimulation of protein kinase B phosphorylation was abolished by pretreatment with wortmannin, suggesting that it is dependent on phosphatidylinositol 3- kinase activation. Despite protein kinase B phosphorylation, GLUT4 translocation, glucose uptake, and glycogen synthesis were not stimulated by insulin. By contrast, glycogen synthesis was stimulated 20-fold in cells incubated with 11 mM glucose. Mitogenesis assessed by incorporation of [3H]thymidine into DNA was enhanced 1.9-fold in response to insulin. Stimulation of DNA synthesis was inhibited by pretreatment with PD98059 but was insensitive to wortmannin. These results indicate that stimulation of mitogenesis is one major biological effect of insulin in placenta cells that implicates the MAPK signaling pathway. Phosphatidylinositol 3-kinase- dependent protein kinase B activation is not sufficient to stimulate glucose transport and glycogen synthesis, highlighting the placenta as a nonclassic target of insulin for the regulation of glucose metabolism.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE METABOLIC AND growth-promoting effects of insulin are mediated by activation of insulin receptors (IRs) through a network of protein-protein interactions (1, 2). Activation of intracellular substrates such as IR substrates 1 to 4 and other proteins such as p85, the regulatory subunit of phosphatidylinositol 3-kinase (PI 3-kinase), or the more distal mediator MAPK, results in the stimulation of biological signals in target cells (3). The MAPK or ERK cascades are involved in the transduction of signals leading to cell growth and proliferation (4). By contrast, the activation of the MAPK pathway is not required for most of the metabolic effects of insulin in cultured adipocytes and muscle cells (5, 6). A role for PI 3-kinase activity in insulin-mediated glucose transport (7), GLUT4 translocation, and glycogen synthesis (8, 9) has been documented in vitro (10) and in vivo (11). Protein kinase B (PKB), a serine-threonine kinase acting as a downstream target of PI 3-kinase (12), has provided an attractive link between IR activation and the stimulation of metabolic functions in insulin-sensitive tissues. PKB has been implicated in several insulin-induced metabolic processes, including glucose transport, glycogen synthesis, protein synthesis, and antiapoptotic signaling (13, 14).

Placental IRs undergo autophosphorylation upon insulin stimulation in vitro (15). However, tyrosine kinase of placental IRs exhibits functional and kinetic properties that differ from those of liver and adipocyte receptors (16). Whether or not such differences have a biological significance is not known. Moreover, effects of insulin, such as enhancement of glucose uptake and stimulation of glycogen synthesis, have not been documented in the human placenta (17, 18); thus, the placenta is not considered an insulin target organ. For example, in insulinopenic hyperglycemic streptozotecin diabetic rats, glycogen content is decreased in liver and skeletal muscle but is markedly increased in the placenta (19). This "placental glycogen paradox" suggests that pathways located downstream of placental IRs could be differentially stimulated in the placenta compared with other insulin-sensitive tissues. The recent findings that the insulin-regulatable glucose transporter GLUT4 colocalizes with IRs in the human placenta suggests that placental glucose metabolism may be stimulatable by insulin (20).

This study was aimed at characterizing the signaling pathways leading to the main biological actions of insulin in placental cells. We show that PKB and MAPK are phosphorylated upon insulin action. PI 3-kinase-dependent phosphorylation of PKB does not lead to stimulation of glucose transport or glycogen synthesis. By contrast, activation of the MAPK pathway is required for stimulation of DNA synthesis.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
The choriocarcinoma cell line JAr was obtained from the American Type Culture Collection (Manassas, VA). RPMI 1640 medium was purchased from Life Technologies, Inc. (Gaithersburg, MD), and FCS was obtained from J. Boy (Reims, France). Other medium components were from Life Technologies, Inc. Wortmannin was purchased from Sigma (St. Louis, MO), and MEK1 inhibitor (PD98059) was purchased from New England Biolabs, Inc. (Beverly, MA). Human insulin was from Novo Nordisk (Copenhagen, Denmark). Antiphosphotyrosine antibody was from Transduction Laboratories, Inc. (Lexington, KY). Antiphospho-active MAPK was from Promega Corp. (Madison, WI), and antiphospho-Ser473 Akt (PKB) and anti-ERK1 antibodies were obtained from New England Biolabs, Inc. Anti-GLUT1 and anti-GLUT3 antibodies were from Biogenesis (Poole, UK), and monoclonal anti-GLUT4 antibody was a kind gift from Dr. Y. LeMarchand-Brustel (Institut National de la Santé et de la Recherche Médicale, Nice, France). For enhanced chemiluminescence detection, horseradish peroxidase-conjugated antirabbit IgG, horseradish peroxidase-conjugated antimouse IgG, and other reagents were purchased from Pierce Chemical Co. (Rockford, IL). Standard molecular weight markers were obtained from Promega Corp. 2-Deoxy-[1-3H]glucose ([3H]2DOG) (specific activity, 16 Ci/mmol), D-[6-14C]glucose (specific activity, 51.8 mCi/mmol), and [methyl-3H]thymidine (specific activity, 25 Ci/mmol) were from Amersham International (Les Ulis, France). Other reagents were from Sigma and were of the highest quality available.

Cell culture and treatments
JAr cells from passages 10 to 20 were cultured in RPMI 1640 medium supplemented with 10% FCS, penicillin G (100 U/ml), streptomycin (100 µg/ml), and Amphotericin (0.25 µg/ml) at 37 C in 5% CO2-95% air. Cells were cultured to 60 to 70% confluence and were incubated for 24 h in serum-free medium before stimulation. Cells were stimulated with insulin (100 nM) for the times indicated with or without prior treatment for 15 min with wortmannin (100 nM) or for 1 h with PD98059 (50 µM), and then they were immediately frozen in liquid nitrogen. Frozen cells were lysed with a Dounce homogenizer in 1 ml of 25 mM HEPES, 250 mM sucrose, 2 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1% aprotinin (10 mg/ml), 50 mM sodium fluoride, 5 mM sodium pyrophosphate, and 1 mM sodium orthovanadate (pH 7.4). Cell lysates were centrifuged at 500 x g for 5 min to preclear insoluble material, and the supernatant was centrifuged at 100,000 x g for 1 h. The resulting supernatant was stored at -80 C until use. Protein concentrations were determined by the Bio-Rad Laboratories, Inc. (Munich, Germany) protein assay using BSA as a standard.

DNA synthesis
Cells were cultured to 50% confluence in six-well cluster trays and were serum deprived for 24 h. In some experiments, the cells were pretreated with PD98059 or wortmannin and were incubated in the absence or presence of insulin (100 nM) for 16 h. [3H]Thymidine was added to the culture medium to a final concentration of 1 µCi/ml for 1 h at 37 C. After three PBS washes, 1 ml of 10% (wt/vol) trichloroacetic acid was added. Cells were then placed on ice for 30 min and were lysed in 0.5 ml of 1 M NaOH. After neutralization with 50 µl of 10 N HCl, 4 ml of scintillation fluid (Optiphase Highsafe 3, Fisher Scientific, Loughborough, UK) was added and [3H]thymidine incorporation was measured in triplicate using a scintillation counter (Betamatic 2, Kontron Instruments Ltd., St. Quentin en Yvelines, France).

Glycogen synthesis
Glycogen synthesis was quantitated by measuring the incorporation of [6-14C]glucose into glycogen. Serum- and glucose-deprived cells were incubated with 1, 5, or 11 mM glucose for 1 h, and D-[6-14C]glucose (1 µCi/ml) was added to the medium for 3 h at 37 C. The reaction was stopped by placing the plates on ice and washing twice with ice-cold PBS. Cells were solubilized in 1 ml of 30% (wt/vol) KOH, and the plates were placed on ice for 15 min. Cells were harvested and heated for 30 min at 100 C, and then 50 µl of 5% glycogen and 1 ml of ethanol were added. After precipitation overnight at -20 C, samples were centrifuged at 1500 x g for 15 min at 4 C. The resulting pellet containing glycogen was resuspended in 1 ml of water, and 4 ml of scintillation fluid was added. Radiolabeled glucose incorporation into glycogen was determined using a scintillation counter and was expressed in cpm/106 cells.

In some experiments, glycogen synthesis was also quantitated by determining the increment in cold glycogen concentration above basal levels (21). Cells were harvested and sonicated in 0.2 M sodium acetate buffer and incubated with amyloglucosidase (0.5 mg/ml) at 55 C for 60 min. Glucose concentrations were measured with glucose oxidase on aliquots of 10,000 x g supernatant.

2-Deoxy-glucose uptake
Serum-deprived cells cultured in six-well cluster trays were washed twice with PBS, and glucose transport assay was performed in 1 ml of RPMI 1640 medium containing 5 mM glucose. The cells were incubated in the absence (basal) or presence of insulin (100 nM) for 10, 20, 30, or 60 min. The assay was initiated by the addition of 1 µCi of [3H]2DOG. Uptake was performed for 10 min and was stopped by aspirating the medium. Plates were washed three times with ice-cold PBS and lysed by immersion in liquid nitrogen. The radioactivity incorporated into the cells was counted on 400-µl duplicate aliquots with a liquid scintillation counter. Each value was corrected for protein content and expressed in nmol/mg protein/min.

GLUT4 translocation
Serum-deprived cells were treated for 1 h with insulin, and the amount of GLUT4 at the plasma membrane was assessed by Western blotting using a monoclonal anti-GLUT4 antibody. Plasma and microsomal membrane fractions were prepared by subcellular fractionation. Briefly, cells were harvested and homogenized in 20 mM Tris, 250 mM sucrose buffer, pH 7.4, containing protease inhibitors. The homogenate was centrifuged for 5 min at 500 x g, and the supernatant was centrifuged for 30 min at 40,000 x g. The resulting supernatant was centrifuged for 60 min at 175,000 x g to obtain the low-density microsome fraction. The 40,000 x g pellet was resuspended in homogenization buffer and laid on top of a sucrose gradient (35%, vol/wt) and then centrifuged for 45 min at 75,000 x g to obtain the high-density microsome and plasma membrane fractions.

Immunoblotting
Equal amounts of proteins (80 µg) were heated at 100 C for 5 min before solubilizing in Laemmli buffer (22) containing 200 mM dithiothreitol and electrophoresed on 7.5% SDS-PAGE. Proteins were electrotransferred to nitrocellulose membranes, and GLUT1, GLUT3, and GLUT4 were detected as described previously (20, 23). For detection of phospho-MAPK, phosphorylated insulin receptor, MAPK/ERK1, and phospho-PKB, membranes were blocked overnight in Tris-buffered saline containing 0.1% Triton (0.1% TBST) with 5% BSA. Blots were incubated in 0.1% TBST containing 5% BSA with antiphospho-MAPK antibody (1:2000), antiphosphotyrosine antibody (1:1000), anti-MAPK antibody (1:6000), or antiphospho-Ser473 PKB (1:1500) for 3 h at room temperature. Blots were subjected to three 20-min washes in 0.1% TBST and incubated for 1 h with antimouse or antirabbit IgG conjugated to horseradish peroxidase diluted 1:5000 in 0.1% TBST with 5% BSA. After three 20-min washes in 0.1% TBST, immunoreactive signals were visualized by enhanced chemiluminescence detection and quantified by densitometry (Hoefer Scientific, San Francisco, CA). Apparent molecular weights were determined by comparison with standard molecular weight markers (Promega Corp.).

Statistical analysis
Results are expressed as means ± SEM. Statistical analysis was performed by t test for unpaired data (Statwork Software, Calabasas, CA).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Insulin stimulates IR, PKB, and MAPK phosphorylation in JAr placental cells
We first analyzed the characteristics of IR tyrosine phosphorylation in placental cells. Cells were treated with various concentrations of insulin for 10 min, and IR tyrosine phosphorylation was visualized after immunoblotting with a monoclonal antiphosphotyrosine antibody. As shown in Fig. 1Go, the IR ß subunit migrating at 97 kDa was not phosphorylated under basal conditions. Insulin stimulation caused a progressive phosphorylation of the ß subunit, which was maximal at 1 µM. The higher band (120 kDa) likely represents pp120, the phosphorylation of which has been shown to be stimulatable by insulin (24).



View larger version (17K):
[in this window]
[in a new window]
  		Figure 1. Insulin-induced tyrosine phosphorylation of IRs in placental cells. Cells were grown to 60 to 70% confluence, serum starved for 24 h, and treated with increasing concentrations of insulin for 10 min. Crude membrane fractions (50 µg of protein) were resolved on 7.5% SDS-PAGE and immunoblotted with antiphosphotyrosine antibody.

 
The activation of PKB and MAPK was visualized after Western blotting of cytosolic fractions from JAr cells using phosphospecific antibodies raised to the phospho-Ser473 of PKB and the tyrosine-phosphorylated activated MAPK (ERK1/ERK2 doublet). PKB phosphorylation was detected 5 min after the addition of insulin and became maximal at 30 min. By contrast, MAPK phosphorylation was maximal at 10 min after insulin stimulation and gradually decreased thereafter (Fig. 2Go). Densitometric analysis of the immunoblotting data show that maximal stimulation of MAPK and PKB phosphorylation were 286 ± 23% and 393 ± 17%, respectively (Fig. 2Go). Insulin-induced PKB activation was prevented by pretreatment of the cells with wortmannin, a potent inhibitor of PI 3-kinase, but was unaffected by PD98059, the specific inhibitor of the MAPK cascade (Fig. 3Go). The activation of phospho-MAPK by insulin was inhibited by pretreatment of the cells with PD98059, as shown by the disappearance of the internal 42- to 44-kDa doublet (Fig. 3Go).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 2. PKB and MAPK are phosphorylated in response to insulin in placental cells. Serum-deprived cells were stimulated with 100 nM insulin for the times indicated. Cytosolic fractions (80 µg of protein) were separated on 7.5% SDS-PAGE, and phosphoproteins were visualized after immunoblotting with antibodies raised to the phospho-Ser473 residue of PKB (phospho-PKB) and against the dually tyrosine-phosphorylated form ERK1/ERK2 (phospho-MAPK). Autoradiographs of immunoblots were scanned by densitometry. Top, Time course of insulin stimulation of PKB; bottom, time course of insulin stimulation of MAPK. Results are means ± SE of data from three separate experiments. Asterisks indicate statistical significance with P < 0.001 between insulin-stimulated and basal conditions.

 


View larger version (25K):
[in this window]
[in a new window]
  		Figure 3. Inhibition of insulin-induced phosphorylation of PKB and MAPK. Serum-deprived JAr placental cells were stimulated with 100 nM insulin for the times indicated. Cytosolic fractions (80 µg of protein) were separated on 7.5% SDS-PAGE and immunoblotted. Before stimulation with insulin (Ins), cells were treated with wortmannin (Wt) for 15 min or PD98059 (PD) for 1 h. Phosphorylation of PKB (phospho-PKB) and MAPK (phospho-MAPK) was visualized after immunoblotting using antibodies raised against phosphorylated forms of the proteins. Anti-MAPK antibody that does not distinguish between the phosphorylated and unphosphorylated MAPK proteins was used to control for the amount of protein in each lane. Each immunoblot is representative of three separate experiments.

 
Effects of insulin on glucose transport and glycogen synthesis in placental cell glucose transport
The effects of insulin on glucose transport were studied by measuring [3H]2DOG uptake without (basal) or with 100 nM insulin (Fig. 4Go). Basal glucose uptake measured during 10 min did not vary with 10.0 ± 0.5, 8.6 ± 0.5, 9.7 ± 0.6, and 8.5 ± 0.3 nmol/mg protein/min at 10, 20, 30, and 60 min, respectively. It was not modified in the presence of 100 nM insulin (9.8 ± 1.2, 9.3 ± 0.3, 10.0 ± 0.4, and 10.0 ± 0.6 nmol/mg protein/min). Glucose fluxes can be regulated not only by the total amount of glucose transporters expressed at the cell surface but also by the distribution of transporters between the plasma membrane and intracellular compartments. Therefore, we analyzed the subcellular distribution of the three major placental glucose transporter isoforms (GLUT1, GLUT3, and GLUT4) by Western blotting. Equal amounts of proteins from plasma and microsomal membranes were separated on SDS-PAGE, and glucose transporters were visualized in each fraction with specific antibodies (Fig. 5AGo). GLUT1 and GLUT3 were localized exclusively at the plasma membrane and in high-density microsomes. By contrast, GLUT4 was found in all fractions, but GLUT4 concentration was higher in intracellular microsomal fractions than in plasma membranes (Fig. 5AGo). We thus studied whether insulin induced a translocation of GLUT4 to the cell surface. No significant differences were observed in GLUT4 protein levels at the cell surface or in the intracellular compartment in the absence or presence of 100 nM insulin (Fig. 5BGo). The subcellular distribution of GLUT1 and GLUT3 was not modified in the absence or presence of insulin (data not shown).



View larger version (15K):
[in this window]
[in a new window]
  		Figure 4. Insulin does not stimulate 2-deoxy-glucose uptake in placental cells. Serum-deprived cells were incubated in the absence (open circles) or presence (closed circles) of 100 nM insulin for the indicated times. 2-Deoxy-glucose uptake was measured for 10 min with 0.1 mmol/liter [3H[2DOG. Each point represents the mean ± SE of duplicate determinations from three separate experiments.

 


View larger version (26K):
[in this window]
[in a new window]
  		Figure 5. Subcellular distribution of glucose transporters in placental cells. A, Subcellular fractions were separated on 10% SDS-PAGE and immunoblotted with anti-GLUT1-, anti-GLUT3-, and anti-GLUT4-specific antibodies. B, Serum-deprived cells were incubated in the absence (basal) or presence of 100 nM insulin (I) for 1 h, and GLUT4 was visualized after immunoblotting of cell fractions (50 µg of protein per lane). In some experiments, cells were pretreated with wortmannin before insulin stimulation (I+W). HDM, High-density microsomes; LDM, low-density microsomes; PM, plasma membrane.

 
Glycogen synthesis
Glycogen synthesis was assessed by measuring [14C]glucose incorporation into glycogen as a function of glucose concentration in the culture medium (Fig. 6Go). [14C]Glucose incorporation was enhanced 12- and 20-fold in cells incubated with 5 and 11 mM glucose, respectively (19.5 ± 2.0 and 28.8 ± 1.8 pmol/106 cells/h) compared with cells incubated with 1 mM glucose (1.54 ± 0.06 pmol/106 cells/h; P < 0.01). Glycogen synthesis estimated from the tracer-derived glycogen rates was not modified by the presence of insulin regardless of glucose concentration (Fig. 6Go). Glycogen synthesis was also quantitated by measuring total glycogen concentration under basal (absence of insulin) and insulin-stimulated conditions. No difference in total glycogen content was observed in the absence or presence of insulin (90.6 ± 5.2 vs. 84.0 ± 10.8 µg glycogen/mg protein).



View larger version (34K):
[in this window]
[in a new window]
  		Figure 6. Insulin does not stimulate glycogen synthesis in placental cells. Cells were treated with (solid bars) or without (hatched bars) 100 nM insulin for 1 h. Glycogen synthesis was assessed by measuring the incorporation of [6-14C]glucose into glycogen during 3 h. The amount of glycogen synthesized was determined by scintillation counting of precipitated glycogen. Results are means ± SE of three separate experiments in which each point was assayed in triplicate.

 
Mitogenic effect of insulin on placental cells
The effect of insulin on DNA synthesis was assessed by measuring the rate of [3H]thymidine incorporation into DNA. It was maximum between 10 and 1000 nM insulin (Fig. 7AGo); therefore, the following experiments were performed with 100 nM. DNA synthesis was enhanced 1.9-fold in response to insulin (6,599 ± 433 vs. 3,484 ± 212 cpm/106 cells/h) and 3.9-fold in response to FCS (11,323 ± 409 vs. 3,484 ± 212 cpm/106 cells/h) (Fig. 7BGo). The insulin-induced stimulation of DNA synthesis was inhibited by pretreatment with PD98059 (6,599 ± 433 vs. 4,509 ± 390 cpm/106 cells/h; P < 0.01). By contrast, the insulin-induced effect was unaffected by pretreatment of the cells with wortmannin (6,599 ± 433 vs. 6,751 ± 733 cpm/106 cells/h) (Fig. 7BGo).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 7. Effect of insulin on [3H]thymidine incorporation into DNA. Cells were serum starved for 24 h and incubated in the absence (basal) or presence of various concentrations of insulin or 10% FCS for 16 h. [3H]Thymidine was then added to the culture medium for 1 h. [3H]Thymidine incorporation into DNA was determined by scintillation counting. Results are means ± SE of three to five separate experiments with each condition assayed in triplicate. *P < 0.01 and **P < 0.001 compared with basal. A, Dose-dependent stimulation of insulin action; B, effect of PD98059 (PD) and wortmannin (W) on insulin (Ins)-induced DNA synthesis. Inhibitors were added before stimulation with 100 nM insulin.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
This study represents the first steps toward understanding the insulin-induced signaling pathways activated in placental cells. We show that PKB and MAPK phosphorylation are stimulated by insulin in JAr placental cells. The rapid induction of MAPK phosphorylation upon insulin stimulation is similar to that observed in other cells in which peak activity occurs within 5 to 10 min of exposure to insulin (25). Full activation of PKB by insulin requires phosphorylation of both Thr308 and Ser473 (26). However, only the phosphorylation of Ser473 is sensitive to wortmannin and thus appears to be under the control of PI 3-kinase (12, 26, 27). Insulin-induced PI 3-kinase-dependent stimulation of glucose transport has been demonstrated in a variety of insulin-responsive cell lines (9, 10), and constitutive activation of PKB is sufficient to stimulate glucose uptake and GLUT4 translocation (6, 28). We show here that neither glucose uptake nor GLUT4 translocation was enhanced in spite of PI 3-kinase-dependent activation of PKB in placental cells. The inability of GLUT4 to translocate to the cell surface despite PI 3-kinase activation and PKB phosphorylation strongly suggests that additional pathways and/or molecules are required for GLUT4 translocation in placental cells. Compartmentalization is a critical process for kinase activity and full phosphorylation of PKB (29, 30) and has not been studied in the placenta. Alternatively, other downstream effectors of PI 3-kinase, such as the atypical PKC {zeta} and {alpha} (31), could also be necessary for the translocation of placental GLUT4. Overexpression of GLUT4 protein in the skeletal muscle-like cell line C2C12 was insufficient to confer insulin-stimulated glucose uptake, possibly because of the high levels of GLUT1 and GLUT3 expression at the cell surface (32). This last hypothesis is worthy of consideration in cells expressing high levels of transporter isoforms other than GLUT4, such as placental cells.

The second puzzling issue regarding the signaling pathway recruited by PKB in the placenta is the absence of stimulation of glycogen synthesis by insulin. This is in contrast to other insulin-sensitive tissues (14) and is regarded as the glycogen paradox. Hormonal regulation of glycogen synthesis partially occurs by insulin-mediated dephosphorylation of glycogen synthase. Glycogen synthase kinase (GSK-3) has been identified as a major target of PKB (12), and dephosphorylation of GSK-3 was implicated in the control of glycogen synthesis in 3T3-L1 adipocytes (33). Although GSK-3 is expressed in JAr placental cells, the present findings show that placental glycogen accumulation is more related to increases in glucose than in insulin concentration. Glycogen synthesis was enhanced 20-fold by increasing glucose concentration from 1 to 11 mM. This is consistent with previous observations showing that [3H]2DOG uptake is enhanced in placenta of hyperglycemic insulin-deficient diabetic rats (34) and that the regulation of placental glycogen synthase is primarily mediated by its allosteric activator, glucose 6-phosphate (35).

Biological actions induced in the placental cell through PI 3-kinase and PKB signaling remain a matter of debate. The involvement of PKB in pleiotropic functions such as the regulation of cell survival and the induction of transcription factors (27) suggests that PKB phosphorylation triggers pathways other than stimulation of placental glucose uptake and metabolism.

Glucose is the major fuel for growth and energy metabolism of placental and fetal tissues (36). Nevertheless, under the conditions used to assay the effect of insulin, high glucose concentrations in the culture medium (25 vs. 5 mM) do not modify [3H]thymidine incorporation into DNA (Boileau, P., and S. Hauguel-de Mouzon, unpublished results). This finding prompted us to speculate that factors other than hyperglycemia may be responsible for the placental macrosomia of streptozotecin diabetic rats. Because many IRs are located on the fetal side of the placenta, enhanced mitogenesis secondary to fetal hyperinsulinism could contribute to the development of placental macrosomia. It is currently believed that insulin, the most important growth factor for the fetus, exerts growth-promoting effects during implantation and placentation through metabolic processes. However, insulin growth-promoting effects could also occur through a direct action on mitogenesis or cell proliferation. It has been stated for more than 30 yr that diabetes during pregnancy induces feto-placental macrosomia through the growth-promoting effects of insulin (37). So far, direct proof of the mitogenic actions of insulin is lacking because none of the intracellular substrates that mediate insulin action have yet been identified in the placenta. Pretreatment of cells with PD98059, the specific MEK1 inhibitor, inhibited phosphorylation of the 42- to 44-kDa MAPK doublet and impaired stimulation of DNA synthesis, demonstrating that activation of the MAPK cascade is essential for the stimulatory action of insulin on mitogenesis in the placenta as in other cell types (38, 39). Mitogenesis was unaffected by pretreatment of the cells with wortmannin. This indicates that neither activation of the PI 3-kinase pathway nor phosphorylation of PKB was required for insulin-mediated proliferation in JAr cells, consistent with the findings in Swiss 3T3 cells that inhibition of PI 3-kinase activity does not modify MAPK phosphorylation (40). The parallel dose-dependent stimulation of insulin on both IR ß phosphorylation (Fig. 1Go) and mitogenesis (Fig. 7AGo) in JAr cells suggests that the mitogenic action of insulin is mediated through the IR.

In conclusion, we have identified MAPK and PKB as downstream targets of IR in placental insulin signaling. One unexpected and puzzling issue is the lack of an insulin-mediated effect to stimulate glucose uptake and glycogen synthesis despite PKB phosphorylation. By contrast, insulin stimulates DNA synthesis via a MAPK-dependent and PI 3-kinase-independent pathway, indicating that stimulation of mitogenesis is one major biological effect of insulin in placenta cells. These findings open the way to further studies in human placenta to fully establish the physiological relevance of these molecular pathways.


   Acknowledgments
 


   Footnotes
 
Abbreviations: GSK-3, Glycogen synthase kinase; IR, insulin receptor; PI 3-kinase, phosphatidylinositol 3-kinase; PKB, protein kinase B; 0.1% TBST, Tris-buffered saline containing 0.1% Triton.

Received January 23, 2001.

Accepted for publication May 28, 2001.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Cheatham B, Kahn CR 1995 Insulin action and the insulin signaling network. Endocr Rev 16:117–142[CrossRef][Medline]
  2. White MF 1998 The IRS-signalling system: a network of docking proteins that mediate insulin action. Mol Cell Biochem 182:3–11[CrossRef][Medline]
  3. Pessin JE, Saltiel AR 2000 Signaling pathways in insulin action: molecular targets of insulin resistance. J Clin Invest 106:165–169[Medline]
  4. Robinson MJ, Cobb MH 1997 Mitogen-activated protein kinase pathways. Curr Opin Cell Biol 9:180–186[CrossRef][Medline]
  5. Lazar DF, Wiese RJ, Brady MJ, et al. 1995 Mitogen-activated protein kinase kinase inhibition does not block the stimulation of glucose utilization by insulin. J Biol Chem 270:20801–20807[Abstract/Free Full Text]
  6. Tanti JF, Gremeaux T, Grillo S, et al. 1996 Overexpression of a constitutively active form of phosphatidylinositol 3-kinase is sufficient to promote Glut 4 translocation in adipocytes. J Biol Chem 271:25227–25232[Abstract/Free Full Text]
  7. Sakaue H, Ogawa W, Takata M, et al. 1997 Phosphoinositide 3-kinase is required for insulin-induced but not for growth hormone- or hyperosmolarity-induced glucose uptake in 3T3–L1 adipocytes. Mol Endocrinol 11:1552–1562[Abstract/Free Full Text]
  8. Shepherd PR, Nave BT, Siddle K 1995 Insulin stimulation of glycogen synthesis and glycogen synthase activity is blocked by wortmannin and rapamycin in 3T3–L1 adipocytes: evidence for the involvement of phosphoinositide 3-kinase and p70 ribosomal-S6 kinase. Biochem J 305:25–28
  9. Martin SS, Haruta T, Morris AJ, Klippel A, Williams LT, Olefsky JM 1996 Activated phosphatidylinositol 3-kinase is sufficient to mediate actin rearrangement and GLUT4 translocation in 3T3–L1 adipocytes. J Biol Chem 271:17605–17608[Abstract/Free Full Text]
  10. Shepherd PR, Withers DJ, Siddle K 1998 Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem J 333:471–490
  11. Terauchi Y, Tsuji Y, Satoh S, et al. 1999 Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase. Nat Genet 21:230–235[CrossRef][Medline]
  12. Cross DAE, Alessi DR, Cohen P, Andjelkovic M, Hemmings BA 1995 Inhibition of glycogen-synthase kinase-3 by insulin mediated by protein- kinase-B. Nature 378:785–789[CrossRef][Medline]
  13. Tanti JF, Grillo S, Gremeaux T, Coffer PJ, Van Obberghen E, Le Marchand-Brustel Y 1997 Potential role of protein kinase B in glucose transporter 4 translocation in adipocytes. Endocrinology 138:2005–2010[Abstract/Free Full Text]
  14. Coffer PJ, Jin J, Woodgett JR 1998 Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. Biochem J 335:1–13
  15. Kasuga M, Fujita-Yamaguchi Y, Blithe DL, Kahn CR 1983 Tyrosine-specific protein kinase activity is associated with the purified insulin receptor. Proc Natl Acad Sci USA 80:2137–2141[Abstract/Free Full Text]
  16. O’Hare T, Pilch PF 1989 Intrinsic kinase activity of the insulin receptor: the intact ({alpha}2ß2) insulin receptor from rat liver contains a kinase domain with greater intrinsic activity than the intact insulin receptor from human placenta. J Biol Chem 264:602–610[Abstract/Free Full Text]
  17. Challier JC, Hauguel-de Mouzon S, Desmaizières V 1986 Effect of insulin on glucose transport and metabolism in the human placenta. J Clin Endocrinol Metab 62:803–807[Abstract]
  18. Schmon B, Hartmann M, Jones CJ, Desoye G 1991 Insulin and glucose do not affect the glycogen content in isolated and cultured trophoblast cells of human term placenta. J Clin Endocrinol Metab 73:888–893[Abstract]
  19. Desoye G, Shafrir E 1994 Placental metabolism and its regulation in health and diabetes. In Baum H, ed. Molecular aspects of medicine. Kidlington, UK: Elsevier Science; vol 15:531–550
  20. Xing A, Caüzac M, Challier JC, Girard J, Hauguel-de Mouzon S 1998 Unexpected expression of GLUT4 glucose transporter in villous stromal cells of human placenta. J Clin Endocrinol Metab 83:4097–4101[Abstract/Free Full Text]
  21. Chan TM, Exton JH 1976 A rapid method for the determination of glycogen content and radioactivity in small quantities of tissue or isolated hepatocytes. Anal Biochem 71:96–105[CrossRef][Medline]
  22. Laemmli UK 1970 Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685[CrossRef][Medline]
  23. Boileau P, Mrejen C, Girard J, Hauguel-de Mouzon S 1995 Overexpression of GLUT3 placental glucose transporter in diabetic rats. J Clin Invest 96: 309–317
  24. Accili D, Perrotti N, Rees-Jones R, Taylor SI 1986 Tissue distribution and subcellular localization of an endogenous substrate (pp120) for the insulin receptor-associated tyrosine kinase. Endocrinology 119:1274–1280[Abstract]
  25. Denton RM, Tavare JM 1995 Does mitogen-activated-protein kinase have a role in insulin action? The cases for and against. Eur J Biochem 227:597–611[Medline]
  26. Alessi DR, Andjelkovic M, Caudwell B, et al. 1996 Mechanism of activation of protein kinase B by insulin and IGF-1. EMBO J 15:6541–6551[Medline]
  27. Franke TF, Kaplan DR, Cantley LC 1997 PI3K: downstream AKT ion blocks apoptosis. Cell 88:435–437[CrossRef][Medline]
  28. Kohn AD, Summers SA, Birnbaum MJ, Roth RA 1996 Expression of a constitutively active Akt Ser/Thr kinase in 3T3–L1 adipocytes stimulates glucose uptake and glucose transporter 4 translocation. J Biol Chem 271:31372–31378[Abstract/Free Full Text]
  29. Toker A, Newton AC 2000 Akt/protein kinase B is regulated by autophosphorylation at the hypothetical PDK-2 site. J Biol Chem 275:8271–8274[Abstract/Free Full Text]
  30. Sable CL, Filippa N, Filloux C, Hemmings BA, Van Obberghen E 1998 Involvement of the pleckstrin homology domain in the insulin-stimulated activation of protein kinase B. J Biol Chem 273:29600–29606[Abstract/Free Full Text]
  31. Kotani K, Ogawa W, Matsumoto M, et al. 1998 Requirement of atypical protein kinase C lambda for insulin stimulation of glucose uptake but not for Akt activation in 3T3–L1 adipocytes. Mol Cell Biol 18:6971–6982[Abstract/Free Full Text]
  32. Kotliar N, Pilch PF 1992 Expression of the glucose transporter isoform GLUT 4 is insufficient to confer insulin-regulatable hexose uptake to cultured muscle cells. Mol Endocrinol 6:337–345[Abstract]
  33. Orena SJ, Torchia AJ, Garofalo RS 2000 Inhibition of glycogen-synthase kinase 3 stimulates glycogen synthase and glucose transport by distinct mechanisms in 3T3–L1 adipocytes. J Biol Chem 275:15765–15772[Abstract/Free Full Text]
  34. Zhou J, Bondy C 1993 Placental glucose transporter gene expression and metabolism in the rat. J Clin Invest 91:845–852
  35. Barash V, Gutman A, Shafrir E 1983 Mechanism of placental glycogen deposition in diabetes in the rat. Diabetologia 24:63–68[Medline]
  36. Battaglia FC, Meschia G 1978 Principal substrates of fetal metabolism. Physiol Rev 58:499–527[Free Full Text]
  37. Pedersen J 1954 Weight and length at birth of infants of diabetic mothers. Acta Endocrinol 16:330–342
  38. Waters SB, Yamauchi K, Pessin JE 1993 Functional expression of insulin receptor substrate-1 is required for insulin stimulated mitogenic signaling. J Biol Chem 268:22231–22234[Abstract/Free Full Text]
  39. Sale EM, Atkinson PG, Sale GJ 1995 Requirement of MAP kinase for differentiation of fibroblasts to adipocytes, for insulin activation of p90 S6 kinase and for insulin or serum stimulation of DNA synthesis. EMBO J 14:674–684[Medline]
  40. Duckworth BC, Cantley LC 1997 Conditional inhibition of the mitogen-activated protein kinase cascade by wortmannin: dependence on signal strength. J Biol Chem 272:27665–27670.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Diabetes CareHome page
B. M. Shields, G. Spyer, A. S. Slingerland, B. A. Knight, S. Ellard, P. M. Clark, S. Hauguel-de Mouzon, and A. T. Hattersley
Mutations in the Glucokinase Gene of the Fetus Result in Reduced Placental Weight
Diabetes Care, April 1, 2008; 31(4): 753 - 757.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. D. Jayanthi, B. Annamalai, D. J. Samuvel, U. Gether, and S. Ramamoorthy
Phosphorylation of the Norepinephrine Transporter at Threonine 258 and Serine 259 Is Linked to Protein Kinase C-mediated Transporter Internalization
J. Biol. Chem., August 18, 2006; 281(33): 23326 - 23340.
[Abstract] [Full Text] [PDF]

Home page
 J. Clin. Endocrinol. Metab.Home page
R. B. Lathi, A. P. Hess, S. Tulac, N. R. Nayak, M. Conti, and L. C. Giudice
Dose-Dependent Insulin Regulation of Insulin-Like Growth Factor Binding Protein-1 in Human Endometrial Stromal Cells Is Mediated by Distinct Signaling Pathways
J. Clin. Endocrinol. Metab., March 1, 2005; 90(3): 1599 - 1606.
[Abstract] [Full Text] [PDF]

Home page
 Hum ReprodHome page
A. Ericsson, B. Hamark, T.L. Powell, and T. Jansson
Glucose transporter isoform 4 is expressed in the syncytiotrophoblast of first trimester human placenta
Hum. Reprod., February 1, 2005; 20(2): 521 - 530.
[Abstract] [Full Text] [PDF]

Home page
 ReproductionHome page
A. Navarrete Santos, S. Tonack, M. Kirstein, M. Pantaleon, P. Kaye, and B. Fischer
Insulin acts via mitogen-activated protein kinase phosphorylation in rabbit blastocysts
Reproduction, November 1, 2004; 128(5): 517 - 526.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
G. Bifulco, C. Di Carlo, M. Caruso, F. Oriente, A. Di Spiezio Sardo, P. Formisano, F. Beguinot, and C. Nappi
Glucose Regulates Insulin Mitogenic Effect by Modulating SHP-2 Activation and Localization in JAr Cells
J. Biol. Chem., June 28, 2002; 277(27): 24306 - 24314.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Purchase Article
Right arrow View Shopping Cart
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow Request Copyright Permission
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Boileau, P.
Right arrow Articles by Hauguel-de Mouzon, S.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Boileau, P.
Right arrow Articles by Hauguel-de Mouzon, S.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology Endocrine Reviews J. Clin. End. & Metab.
Molecular Endocrinology Recent Prog. Horm. Res. All Endocrine Journals