help button home button Endocrine Society Endocrinology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Endocrinology, doi:10.1210/en.2005-0902
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Google Scholar
Google Scholar
Right arrow Articles by Mounier, C.
Right arrow Articles by Posner, B. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mounier, C.
Right arrow Articles by Posner, B. I.
Endocrinology Vol. 147, No. 5 2383-2391
Copyright © 2006 by The Endocrine Society

Regulation of Hepatic Insulin-Like Growth Factor-Binding Protein-1 Gene Expression by Insulin: Central Role for Mammalian Target of Rapamycin Independent of Forkhead Box O Proteins

Catherine Mounier, Victor Dumas and Barry I. Posner

Polypeptide Hormone Laboratory (V.D., B.I.P.), Faculty of Medicine, McGill University, Montréal, Quebéc, Canada H3A 2B2; and Biomed Departement Sciences Biologiques (C.M.), Université du Québec à Montréal, Montréal, Québec, Canada H2X 3Y7

Address all correspondence and requests for reprints to: Dr. Barry I. Posner, Polypeptide Hormone Laboratory, Strathcona Anatomy Building, 3640 University Street, Montréal, Québec, Canada H3A 2B2. E-mail: barry.posner{at}staff.mcgill.ca.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The expression of IGF-binding protein-1 (IGFBP-1) is induced in rat liver by dexamethasone and glucagon and is completely inhibited by 100 nM insulin. Various studies have implicated phosphatidylinositol 3-kinase, protein kinase B (Akt), phosphorylation of the transcription factors forkhead in rhabdomyosarcoma 1 (Foxo1)/Foxo3, and the mammalian target of rapamycin (mTOR) in insulin’s effect. In this study we examined insulin regulation of IGFBP-1 in both subconfluent and confluent hepatocytes. In subconfluent hepatocytes, insulin inhibition of IGFBP-1 mRNA levels was blocked by inhibiting PI3 kinase activation, and there was a corresponding inhibition of Foxo1/Foxo3 phosphorylation. In these same cells, inhibition of the insulin effect by rapamycin occurred in the presence of insulin-induced Foxo1/Foxo3 phosphorylation. In confluent hepatocytes, insulin could not activate the phosphatidylinositol 3-kinase (PI3 kinase)-Akt-Foxo1/Foxo3 pathway, but still inhibited IGFBP-1 gene expression in an mTOR-dependent manner. In subconfluent hepatocytes, the serine/threonine phosphatase inhibitor okadaic acid (100 nM) partially inhibited IGFBP-1 gene expression by 40%, but did not produce phosphorylation of either Akt or Foxo proteins. In contrast, 1 nM insulin inhibited the IGFBP-1 mRNA level by 40% and correspondingly activated Akt and Foxo1/Foxo3 phosphorylation to a level comparable to that observed with 100 nM insulin. These results suggest a potential role for a serine/threonine phosphatase(s) in the regulation of IGFBP-1 gene transcription, which is not downstream of mTOR and is independent of Akt. In conclusion, we have found that in rat liver, insulin inhibition of IGFBP-1 mRNA levels can occur in the absence of the phosphorylation of Foxo1/Foxo3, whereas activation of the mTOR pathway is both necessary and sufficient.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE BINDING OF insulin to the insulin receptor (IR) tyrosine kinase is followed by IR tyrosine kinase autophosphorylation, activation, and the phosphorylation of specific substrates (viz. IRS1 and -2) on tyrosine residues (1). The phosphotyrosine motifs in the IRSs bind adaptor proteins, such as the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3 kinase). The consequent activation of PI3 kinase results in the phosphorylation and activation of downstream protein kinases, especially protein kinase B/Akt (2). In turn, Akt phosphorylates and alters the behavior of various substrates (3)

The mammalian target of rapamycin (mTOR), a large protein (289 kDa) with a catalytic domain homologous to that of PI3 kinase (4), has been implicated in insulin-induced protein synthesis (5) and mitogenesis (6). Its activation has been attributed to the activation of PI3 kinase (7, 8, 9). The serine/threonine (Ser/Thr) phosphatase 2A (PP2A) regulates various processes, including aspects of insulin signaling (10, 11, 12, 13). The effects of insulin can be mediated by the inhibition of PP2A and structurally related enzymes, leading to increased protein phosphorylation (11, 12, 13). In fact, several studies have implicated PP2A in the control of mTOR pathway (14). Thus, Peterson and co-workers (15) provided evidence that mTOR phosphorylates and inactivates PP2A, preventing it from dephosphorylating and inactivating downstream effectors, such as 4E-BP1 and S6 kinase. In addition, evidence has been provided (16) that Akt is a direct target of PP2A, preventing Akt from activating downstream effectors, such as mTOR.

Insulin regulates the expression of numerous genes (17), including IGF-binding protein-1 (IGFBP-1), which is induced by dexamethasone and glucagon and inhibited by insulin (18) in a PI3 kinase-dependent manner (19). Two potential pathways, downstream of PI3 kinase, have been proposed to regulate IGFBP-1 mRNA levels. One involves activation of Akt (20), which directly affects the transcription factors forkhead in rhabdomyosarcoma 1 (Foxo1) (21), Foxa2 (22), and Foxo3 (3), each of which has been shown to mediate the transcription of insulin-responsive genes. In particular, Akt-dependent phosphorylation of Foxo1 and Foxo3 leads to their nuclear exclusion (3, 23, 24) and the inhibition of their binding to the insulin response element in the promoter of the IGFBP-1 gene (3, 21, 25, 26), thus negating its contribution to the induction of IGFBP1. Alternatively, mTOR, activated downstream of PI3 kinase in rat hepatocytes (19) and H4IIE hepatoma cells (27), may affect IGFBP-1 mRNA levels via a mechanism independent of the modulation of Foxo1 (27). Furthermore, it has been observed in H4IIE hepatoma cells that the binding of Foxo3 to the PEPCK and IGFBP-1 insulin response elements did not correlate with the effect of insulin on gene transcription (26).

In previous work we compared insulin and epidermal growth factor action in confluent (C) and subconfluent (SC) cultured hepatocytes (6). In this study we extended this comparison to the mechanism by which insulin regulates IGFBP-1 mRNA downstream of PI3 kinase. The work demonstrates that in confluent hepatocytes, insulin suppresses the induction of IGFBP-1 mRNA by activating mTOR independently of the activation of PI3 kinase and Akt and the phosphorylation of Foxo1/Foxo3. We provide evidence for a possible role of a Ser/Thr phosphatase(s) in this process, which does not appear to operate downstream of mTOR and seems to be independent of Akt.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Porcine insulin was a gift from Eli Lilly Research Laboratories (Indianapolis, IN). Glucagon, dexamethasone, wortmannin, okadaic acid (OA), and calf intestinal alkaline phosphatase were purchased from Sigma-Aldrich Corp. (St. Louis, MO); rapamycin and Ly290042 were obtained from Calbiochem (La Jolla, CA). The Tyr(P) (PY99) antibody was obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Akt, antiphospho-Akt (Ser473), Foxo1, and antiphospho-Foxo1 (Thr24)/Foxo3 (Thr32) antibodies were purchased from Cell Signaling Technology, Inc. (Beverly, MA). The PI3 kinase p85 and PP2A antibodies as well as the Ser/Thr phosphatase assay kit 1 were purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). Protein A-Sepharose was obtained from Amersham Biosciences (Baie d’Urfe, Québec, Canada). Collagenase was obtained from Worthington Biochemical Corp. (Lakewood, NJ). Cell culture medium and antibiotics were purchased from Invitrogen Life Technologies Canada, Inc. (Burlington, Canada), and Vitrogen-100 was obtained from Collagen Biomaterials (Toronto, Canada). [{gamma}-32P]ATP was purchased from PerkinElmer (Wellesley, MA). [{alpha}-32P]Deoxy-CTP and [125I]antirabbit IgG were provided by ICN Biomedicals Canada Ltd. (Mississauga, Canada). Most other reagents and chemicals were obtained from Sigma-Aldrich Corp. and were of the highest grade available.

Cell culture and hormone treatments
Primary hepatocytes, isolated from 120- to 140-g male Sprague Dawley rats (Charles River Laboratories, St. Constant, Canada) by in situ liver perfusion with collagenase (protocol 4110, approved by McGill University), were plated on a collagen matrix (Vitrogen-100; Collagen Corp., Toronto, Ontario, Canada) . SC and C cultures were prepared by seeding 1 x 106 and 3 x 106 cells onto 9.6-cm2 six-well plates, respectively (Corning, Costar, Cambridge, MA) or 5 x 106 and 1.5 x 107 cells, respectively, onto 78-cm2 culture dishes (Starstedt Canada, St. Laurent, Canada). Cells were bathed for 24 h in seeding medium (DMEM/Ham’s F-12 containing 10% fetal bovine serum, 10 mM HEPES, 20 mM NaHCO3, 500 IU/ml penicillin, and 500 µg/ml streptomycin) and then for 48 h in serum-free medium that differed from the seeding medium in that it lacked fetal bovine serum and contained 1.25 µg/ml fungizone, 0.4 mM ornithine, 2.25 µg/ml L-lactic acid, 2.5 ± 10–8 M selenium, and 1 ± 10–8 M ethanolamine. Hormone and drug treatments, carried out in serum-free medium, were initiated 72 h after plating for the times and at the concentrations indicated in the figure legends. Cells overexpressing dominant-negative p85 were infected with stocks of recombinant (Ad{Delta}p85) adenovirus containing a cDNA encoding the p85 regulatory subunit, whose p110 binding region was deleted ({Delta}p85) (28). After viral exposure, {Delta}p85-infected cells were serum starved for 20 h in serum-free medium and treated with the hormones indicated in the figure legends.

Wild-type HTC rat hepatoma cells were transfected with expression plasmids containing the human IR cDNA (29). These HTC-IR cells were used to measure PP2A activity. Cells were grown to 75% confluence in DMEM with 10% FCS and were serum starved for 48 h before stimulation with hormones and drugs for the times and at the concentrations indicated in the figure legends.

RNA extraction and dot-blot hybridization analysis
Total RNA was extracted from primary rat hepatocytes using a previously described procedure (30). Previous work compared Northern blotting with dot blots and established that the latter were as quantitatively accurate in assessing IGFBP-1 mRNA levels (19, 31). As in previous work, dot-blot analyses of 20 µg total RNA were performed on Hybond-N nylon membranes (GE Healthcare Life Sciences, Baie d’Urfé, Québec, Canada) in a dot-blot manifold (Bio-Rad Laboratories, Inc., Hercules, CA), according to the manufacturer’s protocol. RNA was fixed to the membranes by UV cross-linking and hybridized sequentially, with intermittent stripping, with IGFBP-1 and 18S cDNA probes.

Western blot analysis
To prepare the Triton X-100-soluble (TS) fraction, rat primary hepatocytes were rinsed twice with ice-cold PBS (pH 7.4) and solubilized with lysis buffer [50 mM HEPES (pH 7.5), 150 mM NaCl, 10 mM sodium pyrophosphate, 100 mM sodium fluoride, 1.5 mM MgCl2, 1 mM EGTA, 200 µM sodium orthovanadate, 1 mM phenylmethylsulfonylfluoride, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 10% glycerol, and 1% Triton X-100]. Cell lysates were clarified by centrifugation at 10,000 x g for 10 min at 4 C, and protein concentrations in the resulting supernatants were determined using the Bio-Rad protein assay (32). To determine the presence and the phosphorylation state of the Foxo1/Foxo3 transcription factors in C cells, total lysates where prepared as previously described for the TS fraction, except that after solubilization in Laemmli buffer [2% sodium dodecyl sulfate, 2% ß-mercaptoethanol, 10% (vol/vol) glycerol, and 50 mg/liter bromophenol blue in 0.1 M Tris-HCl buffer (pH 6.8)], cell lysates were sonicated for 5 sec using the Vivracell sonicator (Sonics & Materials, Newton, CT) at an amplitude of 21%. To prepare the Triton-insoluble (TI) fraction, cells were rinsed twice with ice-cold PBS (pH 7.4) and directly solubilized in hot Laemmli buffer. To fragment DNA after lysis of the nuclei, the TI fraction of cell lysates was passed through a syringe before loading onto a gel. To evaluate the total phosphorylation state of Foxo proteins, the TS fraction prepared from SC cells (500 µg protein) was incubated for 5 min at 30 C with 500 U calf intestinal alkaline phosphatase, then boiled for 5 min in Laemmli buffer. After separation on SDS-PAGE, extracted proteins were transferred to Immobilon-P membranes (Millipore Ltd., Mississauga, Canada) and probed with the indicated first antibody for 90 min, followed by a 1-h incubation with [125I]antirabbit IgG. Immunoreactive proteins were detected by autoradiography, and quantification of densitometry signals was performed using a Bio-Rad densitometer (model GS-700).

PP2A activity
After treatment with the test agents for the times and concentrations indicated in the figure legends, HTC-IR cells were rinsed twice with 20 mM Tris (pH 7.4) and 150 mM NaCl and solubilized in PTPase lysis buffer [20 mM Tris (pH 7.4), 150 mM NaCl, 1% (vol/vol) Nonidet P-40, 2 µM phenylmethylsulfonylfluoride, 2 µM leupeptin, and 10 µU/ml aprotinin]. Cell lysates were clarified by centrifugation at 10,000 x g for 10 min at 4 C, and protein concentrations in the resulting supernatants were determined using the Bio-Rad protein assay (32). Cell lysates were then incubated for 2 h at 4 C with an anti-PP2A antibody in the presence of protein A-Sepharose. The beads were collected by centrifugation and washed three times in PTPase lysis buffer, and PP2A activity was measured using the Ser/Thr phosphatase assay kit 1 according to the manufacturer’s protocol, except that the reaction was incubated for 10 min and not 1 h as recommended in the standard protocol. The phosphatase activity was then quantified using an Emax precision microplate reader (Molecular Devices, Sunnyvale, CA).

PI3 kinase activity
TS cell lysates (500 µg protein) from insulin-treated (100 nM insulin for 2 min) or nontreated SC and C cells were immunoprecipitated in the presence of protein A-Sepharose using anti-PY antibody. Immunoprecipitates were extensively washed, and the protein A-Sepharose pellets were resuspended in 50 µl kinase assay buffer [20 mM Tris-HCl (pH 7.5), 100 mM NaCl, and 0.5 mM EGTA] containing 0.5 mg/ml L-{alpha}-phosphatidylinositol (Avanti Polar Lipids, Inc., Alabaster, AL) and assayed for PI3 kinase activity as described previously (33).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Inhibition of IGFBP-1 gene expression by insulin in SC and C hepatocyte cultures: roles of PI3 kinase and mTOR
In previous work we showed that in primary rat hepatocytes seeded at low density (SC), but not in cells seeded at high density (C), insulin induced [3H]thymidine incorporation into DNA by a mechanism involving insulin-induced activation of both PI3 kinase and mTOR (6). In the present work we explored this difference further by examining the effect of insulin on IGFBP-1 mRNA levels in both SC and C cells. As previously observed the incubation of serum-deprived rat hepatocytes for 6 h in the presence of dexamethasone and glucagon (Dex/Gluc) markedly increased IGFBP-1 mRNA levels (19, 31), and insulin completely prevented this stimulation in both SC and C hepatocytes (Fig. 1Go). Interestingly the PI3 kinase inhibitors, wortmannin and LY294002 (34, 35, 36), prevented insulin from suppressing IGFBP-1 mRNA expression in SC, but not in C, cells (Fig. 1Go).


Figure 1
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 1. Effects of wortmannin, Ly294002, and rapamycin on insulin-mediated inhibition of IGFBP-1 mRNA levels in SC and C hepatocytes. Serum-starved SC ({square}) or C ({blacksquare}) hepatocytes were incubated for 6 h in the absence of hormone or in 100 nM dexamethasone and 100 nM glucagon with and without 100 nM insulin as indicated. Before incubation with the hormones, cells were treated for 30 min with vehicle (–), 500 nM wortmannin, 50 µM Ly294002, or 200 nM rapamycin. The ratios of the densitometric reading of the dot blots of IGFBP-1 mRNA and 18S mRNA are expressed as a percentage of that in control cells, which were normalized to 100%. Results are expressed as the mean of three separate experiments; bars indicate the SD.

 
We evaluated the efficacy of overexpressing {Delta}p85, a dominant-negative construct of the p85 regulatory subunit of PI3 kinase (28), on insulin suppression of IGFBP-1 gene expression. The expression of {Delta}p85 in SC cells dramatically suppressed insulin-induced PI3 kinase activation (Fig. 2AGo) and correspondingly inhibited insulin-induced suppression of IGFBP-1 gene expression (Fig. 2BGo). However, in C cells the expression of {Delta}p85 failed to inhibit the suppressive effect of insulin on IGFBP-1 expression (Fig. 2BGo). Thus, insulin-induced suppression of IGFBP-1 gene expression requires the activation of PI3 kinase in SC, but not in C, cells. In contrast, rapamycin inhibited the effect of insulin in both SC and C hepatocytes (Fig. 1AGo), indicating that this effect is mediated by mTOR (33, 37) in both culture conditions. These studies indicate that independently of PI3 kinase activation, mTOR can mediate insulin-induced suppression of IGFBP-1 gene expression in rat hepatocytes.


Figure 2
View larger version (17K):
[in this window]
[in a new window]
  		FIG. 2. Effect of dominant-negative p85 construct on total PI3 kinase activity and insulin-mediated inhibition of IGFBP-1 mRNA level. A, Hepatocytes were infected with (+) or without (–) dominant-negative p85 ({Delta}p85) recombinant adenovirus for 3 h, serum deprived for 48 h, then treated with (+) or without (–) 100 nM insulin for 2 min. Cell lysates were immunoprecipitated with {alpha}PY antibody, and the immunoprecipitates were analyzed for PI3 kinase activity as described in Materials and Methods. The autoradiogram shows a representative experiment performed in triplicate. The arrow indicates the location of the reaction product, phosphatidylinositol-3-phosphate (PIP3). The bar chart represents a quantification of the autoradiography. Results are expressed as a percentage of the noninfected, nonstimulated value. B, Serum-starved SC ({square}) or C ({blacksquare}) hepatocytes were infected with a dominant-negative p85 ({Delta}p85) recombinant adenovirus for 3 h, serum deprived for 48 h, and incubated for 6 h in the absence of hormone or in 100 nM dexamethasone and 100 nM glucagons with or without 100 nM insulin as indicated. IGFBP-1 mRNA levels were quantified as described in Fig. 1Go. Results are expressed as the mean ± SD of three separate experiments.

 
Phosphorylation and activation of PI3 kinase, Akt, and Foxo1/Foxo3 in SC vs. C cells in response to insulin
Previous studies have implicated Akt and Foxo1/Foxo3, downstream of PI3 kinase, in the regulation by insulin of IGFBP-1 gene expression (3, 20, 21, 23, 26, 38). We, therefore, analyzed the expression and activation of key signaling molecules leading to Akt activation and Foxo1/Foxo3 phosphorylation in both SC and C hepatocyte cultures. In preliminary studies we found that in C hepatocytes there was a redistribution of cytosolic proteins, such as IRS-1, IRS-2, and p85, from the TS to the TI fraction. Therefore, in studying events in these cells, we examined both the TS and TI fractions prepared as described in Materials and Methods (Fig. 3Go). Western blot analysis of protein extracted from SC cells showed that p85 was almost exclusively present in the TS fraction, whereas in C hepatocytes, it was only detected in the TI fraction (Fig. 3AGo, top panels). In agreement with the Western blot analysis, PI3 kinase activity, measured in the TS fraction, was only detected in SC cells (Fig. 3AGo, bottom panels). Western blot analysis also showed that Akt is present in the TS fraction from SC and the TI fraction from C hepatocytes (Fig. 3BGo, left panels). Using antiphospho-Akt (Ser473) antibody, we demonstrated that insulin produced phosphorylation of Akt in the TS fraction of SC cells, but had no effect on Akt phosphorylation in the TI fraction from C hepatocytes (Fig. 3BGo, right panels).


Figure 3
View larger version (35K):
[in this window]
[in a new window]
  		FIG. 3. Analysis of PI3 kinase and Akt activation in SC and C hepatocytes. A, TS or TI fractions were prepared from SC or C hepatocytes treated (+) or not (–) with insulin for 2 min (100 nM). Isolated proteins where either resolved on 8% SDS-PAGE and immunoblotted with an anti-p85 (top panels) or immunoprecipitated with an {alpha}PY antibody to measure PI3 kinase activity (bottom panel). PIP3, The reaction product phosphatidylinositol-3-phosphate. B, TS and TI fractions were isolated from SC and C hepatocytes treated with (+) or without (–) 100 nM insulin for 15 min. Isolated proteins were resolved on 8% SDS-PAGE and immunoblotted with either an anti-Akt antibody (left panels) or an antiphospho-Akt (Ser473; right panels). The autoradiographs are representative of at least three different experiments.

 
We then analyzed the effect of insulin on Foxo1 and Foxo3 phosphorylation in SC rat hepatocytes. Using an antibody that specifically recognized Foxo1, we found that in nontreated cells, Foxo1 migrated at 77 kDa (Fig. 4AGo, top panel). It would thus appear that in SC cells the bulk of Foxo1 is not phosphorylated in the absence of exogenous stimulation by insulin. Upon insulin treatment of SC cells, the band migrating at 77 kDa disappeared, and a band migrating at approximately 80 kDa appeared on the Western blot. This band corresponds to the band observed on immunoblotting with antiphospho-Foxo1 (Thr24; Fig. 4AGo, lower panel). The antiphospho-Foxo1 (Thr24) also recognized phosphorylated Foxo3 (Thr32), whose phosphorylation paralleled that of Foxo1 (Fig. 4AGo, bottom panel). Total phosphorylation of the Foxo proteins in SC cells was reduced by phosphatase treatment, validating the observations made with the phosphospecific antibodies (Fig. 4BGo). The phosphorylation of both Foxo1 and Foxo3 was markedly reduced by treatment with the potent PI3 kinase inhibitor, Ly290042, but not by the mTOR inhibitor, rapamycin (Fig. 4AGo). Because Foxo1/Foxo3 phosphorylation persists in the presence of rapamycin, we conclude that such phosphorylation is independent of mTOR and is not sufficient for the insulin effect on IGFBP-1 gene expression (Fig. 1Go) (19).


Figure 4
View larger version (35K):
[in this window]
[in a new window]
  		FIG. 4. Analysis of the phosphorylation of Foxo1/Foxo3 in SC and C hepatocytes. SC hepatocytes (A and B) and C hepatocytes (C) were treated for 15 min with insulin (100 nM) after 30 min of preincubation with vehicle (–),100 nM Ly294002 (Ly), or 200 nM rapamycin (Rap). Cells were lysed, and extracted proteins were resolved on 8% SDS-PAGE and immunoblotted with either an anti-Foxo1 antibody (top panels) or an antibody that recognized the phosphorylated isoforms of Foxo1 and Foxo3 [antiphospho-Foxo1 (Thr24)/Foxo3 (Thr32); bottom panels]. B, Cell lysates were incubated for 5 min at 30 C in the presence or absence of phosphatase before SDS-PAGE. C, Protein lysates (provided by Cell Signaling Technology) isolated from serum-treated NIH-3T3 (C+) were used as a positive control for the phosphorylated Foxo isoforms. The molecular weights are indicated at the left margin. The autoradiographs are representative of three different experiments.

 
TI and TS fractions were also prepared to analyze insulin’s effect on Foxo1 and Foxo3 in C hepatocytes. Surprisingly, we were unable to detect the presence of Foxo1 or Foxo3 in the two fractions, perhaps indicating a sequestration of these factors in a particular cellular compartment (data not shown). The cells were extracted using a more stringent method involving sonication, as described in Materials and Methods. In such fractions we observed Foxo1. However, insulin did not appear to induce phosphorylation of Foxo1, as reflected by the absence of shift on the Western blot depicted in Fig. 4CGo (top panel) and our failure to identify phosphorylated forms of either Foxo1 or Foxo3 using specific antibodies to the phosphorylated forms of Foxo1/Foxo3 (Fig. 4CGo, bottom panel). The failure of insulin to effect Foxo1 and Foxo3 phosphorylation in C hepatocytes is compatible with its inability to activate PI3 kinase and Akt in these cells (Fig. 3Go). These observations indicate that insulin-induced Foxo1/Foxo3 phosphorylation is not essential for insulin action on IGFBP-1 gene expression.

Effect of insulin and OA on Ser/Thr phosphatase activity, IGFBP-1 gene expression, and upstream signaling molecules
Various studies have implicated Ser/Thr phosphatases in insulin action (10, 39, 40, 41, 42). Furthermore, the activation of mTOR has been linked to the inhibition of Ser/Thr phosphatases, such as PP2A, PP4, and PP6 (43, 44, 45, 46). It was also demonstrated that Akt is a direct target of PP2A, which could induce dephosphorylation and down-regulation of Akt activity (16, 47, 48). OA is a potent tumor promoter (49) that binds to and inhibits PP1c and PP2Ac with dissociation constants (Ki) of 147 and 0.032 nM, respectively (50). We assessed the effects of both insulin and OA on hepatic phosphatase activities in HTC-IR cells, a hepatoma cell line overexpressing the IR that was previously used in our laboratory to study insulin signaling (33). This cell line was employed because phosphatase activity could be reproducibly measured in immunoprecipitates from lysates of these cells as described in Materials and Methods. As shown in Table 1Go, there was significant basal PP2A activity detected in HTC-IR cells. In agreement with previous reports (11, 12, 13), insulin (100 nM) alone inhibited PP2A activity by about 20%, whereas 100 nM OA reduced basal PP2A activity by approximately 85%.


View this table:
[in this window]
[in a new window]
  		TABLE 1. PP2A activity in HTC-IR cells

 
When incubating serum-deprived rat SC hepatocytes for 6 h in the presence of Dex/Gluc and 100 nM OA, we found a suppression of Dex/Gluc-induced IGFBP-1 mRNA expression by about 40%. A similar level of inhibition was observed in the presence of 1 nM insulin (Fig. 5AGo). As expected, increasing insulin concentrations increased the inhibition of IGFBP-1 mRNA levels, with a maximal inhibition of 93% observed in the presence of 100 nM insulin. Despite considerable differences in the extent of suppression of IGFBP1 mRNA by insulin at low vs. high doses, there was a substantial degree of Akt phosphorylation on Ser473 at 1 and 3 nM insulin compared with that at 100 nM insulin (Fig. 6Go). Interestingly, 100 nM OA, which inhibited the expression of IGFBP-1 mRNA to a similar level as that in the presence of 1 nM insulin (Fig. 5AGo), did not induce Akt phosphorylation at Ser473 (Fig. 6Go). Similar results were obtained for the phosphorylation of Akt at Thr 308 (data not shown). Parallel with our observations for Akt, 100 nM OA did not effect phosphorylation of Foxo1, as indicated by the lack of shift in Foxo1 migration, whereas 1 nM insulin did effect a shift of Foxo1 mobility not markedly different from that observed with 100 nM insulin (Fig. 6Go). The same observation was made using the phosphospecific antibody that recognizes phosphorylated forms of Foxo1 and Foxo3 (Fig. 6Go, bottom panel). Incubating the cells with a combination of OA and rapamycin revealed that the OA effect on Dex/Glu-induced IGFBP-1 mRNA level was lost in the presence of the mTOR inhibitor (Fig. 5BGo). Taken together, these data indicate that OA, a Ser/Thr phosphatase inhibitor, can induce a significant decrease in the level of expression of the IGFBP-1 mRNA independently of the activation of Akt and phosphorylation of Foxo1/Foxo3. Thus, in SC cells, the phosphorylation of Foxo1/Foxo3 is not required to observe the suppression of IGFBP-1 gene transcription.


Figure 5
View larger version (14K):
[in this window]
[in a new window]
  		FIG. 5. Comparison between the effects of insulin and OA on IGFBP-1 mRNA levels. A, SC hepatocytes (serum starved for 48 h) were incubated for 6 h in the absence of hormone ({square}), in 100 nM dexamethasone and 100 nM glucagon ({blacksquare}) with or without 100 nM OA, or in 100 nM dexamethasone, 100 nM glucagon, and insulin (Figure 5) at the indicated doses (*, P = 0.006, OA- vs. DG-treated cells). B, SC hepatocytes (serum starved for 48 h) were incubated for 6 h in the absence of hormone with or without 100 nM OA ({square}) or in 100 nM dexamethasone and 100 nM glucagon with 100 nM OA in the presence and absence of 200 nM rapamycin ({blacksquare}). IGFBP-1 mRNA levels were quantified as described in Fig. 1Go. Results are expressed as the mean of three separate measurements; bars indicate the SD.

 

Figure 6
View larger version (63K):
[in this window]
[in a new window]
  		FIG. 6. Comparison between the effects of insulin and OA on Akt and Foxo1/Foxo3 phosphorylation. Total cell lysates were obtained from SC hepatocytes after treatment with vehicle, the noted doses of insulin, or 100 nM OA for 15 min. Proteins were then resolved on 8% SDS-PAGE and immunoblotted with an antiphospho-Akt (Ser473) antibody (top panels), an anti-Akt antibody (second panels), an anti-Foxo1 antibody (third panels), or an antibody that recognized the phosphorylated isoforms of Foxo1 and Foxo3 [antiphospho-Foxo1 (Thr24)/Foxo3 (Thr32); bottom panels]. The autoradiographs are representative of two different experiments.

 
In addition, the results demonstrate that the phosphatase does not operate downstream of mTOR, because its inhibitory effect is prevented by coincubation with rapamycin. However, though 100 nM OA inhibited PP2A by 85% (Table 1Go), it only induced 40% inhibition of the level of IGFBP-1 mRNA (Fig. 5AGo). Therefore, the role of inhibiting PP2A in the regulation of IGFBP-1 mRNA levels must be regarded as limited at best and functioning in concert with other processes yet to be fully defined. In conclusion, the present study emphasizes the importance of an mTOR-dependent pathway that can be activated in a PI3 kinase-independent manner and can regulate IGFBP-1 mRNA levels in the absence of Foxo1/Foxo3 phosphorylation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
We previously found that in primary rat hepatocytes, insulin inhibited the induction of IGFBP-1 mRNA levels by Dex/Gluc via the activation of both PI3 kinase- and mTOR-dependent pathways (19). Various studies of the regulation of IGFBP-1 mRNA expression have identified a role for Akt and Foxo1/Foxo3 downstream of PI3 kinase (21, 25, 26). The involvement of mTOR in the regulation of this process has also been noted in the hepatoma cell line H4EII (27).

Most studies of the roles of Foxo1 and Foxo3 in the regulation of IGFBP-1 transcription have used systems overexpressing Foxo proteins (3, 21, 26, 27, 38, 51) and have not sought to establish the relative roles of these specific isoforms in the regulation of IGFBP-1 gene expression. In the present study we tried to evaluate the roles of the endogenous factors in cultured primary hepatocytes as a possibly more accurate reflection of physiological processes.

In SC hepatocytes, the inhibition of PI3 kinase activity by both specific inhibitors (wortmannin and LY294002) and the transfection of a dominant-negative construct of p85 ({Delta}p85) abrogated insulin suppression of IGFBP-1 gene expression and Foxo1/Foxo3 phosphorylation. Rapamycin also inhibited the effect of insulin without affecting Foxo1/Foxo3 phosphorylation. This latter observation of a dissociation between phosphorylation of Foxo1/Foxo3 and insulin action on gene transcription has been clearly established in other studies. Thus, phorbol esters induced activation of Akt and phosphorylation of Foxo1 without suppressing IGFBP-1 gene expression (52), and H2O2 attenuated the insulin effect on IGFBP-1 expression without affecting insulin-induced phosphorylation of Akt and Foxo1 (37). From these data, it seems clear that the phosphorylation of Foxo1/Foxo3 is not sufficient to mediate the effect of insulin on IGFBP-1 gene transcription.

Surprisingly, we found that insulin failed to activate PI3 kinase and Akt or phosphorylate Foxo1/Foxo3 in C hepatocytes. Nevertheless, insulin inhibited Dex/Gluc-induced IGFBP-1 mRNA production. This latter effect of insulin in C hepatocytes was fully reversed by rapamycin, indicating that mTOR mediated insulin’s effect on IGFBP-1 gene transcription independently of PI3 kinase activation. The existence of a pathway bypassing PI3 kinase-Akt-Foxo1/Foxo3 was also demonstrable in SC hepatocytes, because we could demonstrate that the Ser/Thr phosphatase inhibitor OA significantly decreased the IGFBP-1 mRNA level by 40% without affecting the activation and phosphorylation state of Akt and Foxo1/Foxo3. Taken together, these observations indicate that the phosphorylation of Foxo1/Foxo3 is neither necessary nor sufficient for the insulin effect on IGFBP-1 gene transcription, but may play a role in augmenting and consolidating the effect of insulin in SC hepatocytes. In the latter, the activation of PI3 kinase by insulin appears necessary to activate mTOR. However, in C hepatocytes, where there are important differences in the cellular distribution of molecules, mTOR activation appears to have become independent of the requirement for PI3 kinase activation. Perhaps this occurs as a consequence of a change in cellular localization of mTOR, which has been suggested as one possible mechanism by which its activation is accomplished (53, 54, 55). The implication of mTOR in insulin-regulated gene expression seems to be specific for IGFBP-1, because the regulation by insulin of the transcription of genes for glucose-6-phosphatase and phosphoenolpyruvate carboxykinase was not antagonized by rapamycin (56, 57).

Patel et al. (37) used the expression of active S6 kinase to demonstrate that this enzyme was not the key element downstream of mTOR leading to the insulin-dependent suppression of IGFBP-1 gene expression. A role for Ser/Thr phosphatases in insulin action has been previously established (10, 11, 12, 13). In this study we found that the Ser/Thr phosphatase inhibitor OA partially mimicked insulin by inhibiting the induction of IGFBP-1 gene expression by Dex/Gluc. Furthermore, this occurred in the absence of activation of the Akt-Foxo1/Foxo3 pathway. Other work has implicated phosphatase inhibition in the mTOR pathway (14). Thus, genetic screening has identified several phosphatases and a phosphatase-associated protein as part of a rapamycin-sensitive signaling pathway in Saccharomyces cerevisiae (44, 58). However, several studies implicating PP2A, PP4, and PP6 in mTOR signaling have located these enzymes downstream of mTOR itself (43, 44, 45, 46, 59, 60). In the present study, it appears that the phosphatase targeted by OA does not operate downstream of mTOR, because the effect of OA on IGFBP-1 mRNA levels was reversed by rapamycin. Both the identity of the putative phosphatase as well as its precise target in response to insulin remain to be clarified.

C hepatocytes represent a cellular model in which insulin suppressed the induction of IGFBP-1 mRNA levels by a mechanism involving mTOR in a manner completely independent of activation of the PI3 kinase-Akt-Foxo1/Foxo3 signaling pathway. Indeed, in contrast to SC cells, we found that insulin did not activate the PI3 kinase pathway at all in C cells. Although most work involving primary rat hepatocytes has been performed in SC cells, there are studies that have raised the possibility that SC hepatocytes may not accurately reflect the actual cellular environment within whole liver. For example, it is known that the expression of several liver-specific functions is inversely related to the degree of cell proliferation (61, 62), a feature readily observed in SC, but not C, primary rat hepatocytes (6). Furthermore, evaluation of urea, albumin, and glucose synthesis as well as the expression of specific hepatic genes demonstrated a lower level of liver-specific function in SC than in C hepatocytes (63). Indeed, C and not SC cells were noted to retain the adult phenotype, thus reflecting the in vivo state of fully differentiated hepatocytes (64). Therefore, our observations made in C primary hepatocytes may be relevant to in vivo physiology. It is possible that there are different cell populations in intact liver, some of which behave like C hepatocytes and others, perhaps located differently in the liver lobule, which behave like SC hepatocytes.

In summary, the present work compared the regulation of IGFBP-1 gene expression in SC and C hepatocytes. In the latter, we showed that insulin suppressed Dex/Gluc-induced IGFBP-1 mRNA levels by an mTOR-dependent mechanism and in the absence of activating the PI3 kinase-Akt-Foxo1/Foxo3 pathway. The phosphorylation of Foxo1/Foxo3 appears to be neither necessary nor sufficient for this insulin effect.


   Footnotes
 
This work was supported by the Canadian Institutes for Health Research, and through the continuing generosity of the Cleghorn Fund at McGill University and the Maurice Pollack Foundation of Montréal.

The authors have no affiliations that relate to the topic of this manuscript.

First Published Online February 2, 2006

Abbreviations: C, Confluent; Dex, dexamethasone; Foxo1, forkhead in rhabdomyosarcoma formerly referred to as FKHR; G6Pase, glucose-6-phosphatase; Gluc, glucagon; IGFBP-1, IGF-binding protein-1; IR, insulin receptor; IRS, insulin receptor substrate; mTOR, mammalian target of rapamycin; OA, okadaic acid; PEPCK, phosphoenolpyruvate carboxykinase; PI3 kinase, phosphatidylinositol 3-kinase; PP1, protein phosphatase 1; SC, subconfluent; Ser/Thr, serine/threonine; TI, Triton X-100 insoluble; TS, Triton X-100 soluble.

Received July 19, 2005.

Accepted for publication January 23, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Yenush L, Zanella C, Uchida T, Bernal D, White MF 1998 The pleckstrin homology and phosphotyrosine binding domains of insulin receptor substrate 1 mediate inhibition of apoptosis by insulin. Mol Cell Biol 18:6784–6794[Abstract/Free Full Text]
  2. Alessi DR, Downes CP 1998 The role of PI 3-kinase in insulin action. Biochim Biophys Acta 1436:151–164[Medline]
  3. Brunet A, Bonni A, Zigmond MJ, Lin MZ, Juo P, Hu LS, Anderson MJ, Arden KC, Blenis J, Greenberg ME 1999 Akt promotes cell survival by phosphorylating and inhibiting a Forkhead transcription factor. Cell 96:857–868[CrossRef][Medline]
  4. Abraham RT, Wiederrecht GJ 1996 Immunopharmacology of rapamycin. Annu Rev Immunol 14:483–510[CrossRef][Medline]
  5. Shah OJ, Anthony JC, Kimball SR, Jefferson LS 2000 4E-BP1 and S6K1: translational integration sites for nutritional and hormonal information in muscle. Am J Physiol 279:E715–E729
  6. Band CJ, Mounier C, Posner BI 1999 Epidermal growth factor and insulin-induced deoxyribonucleic acid synthesis in primary rat hepatocytes is phosphatidylinositol 3-kinase dependent and dissociated from protooncogene induction. Endocrinology 140:5626–5634[Abstract/Free Full Text]
  7. Lin TA, Lawrence Jr JC 1996 Control of the translational regulators PHAS-I and PHAS-II by insulin and cAMP in 3T3–L1 adipocytes. J Biol Chem 271:30199–30204[Abstract/Free Full Text]
  8. Petritsch C, Woscholski R, Edelmann HM, Parker PJ, Ballou LM 1995 Selective inhibition of p70 S6 kinase activation by phosphatidylinositol 3-kinase inhibitors. Eur J Biochem 230:431–438[Medline]
  9. Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J 1994 PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature 370:71–75[CrossRef][Medline]
  10. Brady MJ, Saltiel AR 2001 The role of protein phosphatase-1 in insulin action. Recent Prog Horm Res 56:157–173[Abstract]
  11. Begum N, Ragolia L 1996 cAMP counter-regulates insulin-mediated protein phosphatase-2A inactivation in rat skeletal muscle cells. J Biol Chem 271:31166–31171[Abstract/Free Full Text]
  12. Chen J, Martin BL, Brautigan DL 1992 Regulation of protein serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science 257:1261–1264[Abstract/Free Full Text]
  13. Srinivasan M, Begum N 1994 Regulation of protein phosphatase 1 and 2A activities by insulin during myogenesis in rat skeletal muscle cells in culture. J Biol Chem 269:12514–12520[Abstract/Free Full Text]
  14. Hay N, Sonenberg N 2004 Upstream and downstream of mTOR. Genes Dev 18:1926–1945[Abstract/Free Full Text]
  15. Peterson RT, Desai BN, Hardwick JS, Schreiber SL 1999 Protein phosphatase 2A interacts with the 70-kDa S6 kinase and is activated by inhibition of FKBP12-rapamycinassociated protein. Proc Natl Acad Sci USA 96:4438–4442[Abstract/Free Full Text]
  16. Andjelkovic M, Jakubowicz T, Cron P, Ming XF, Han JW, Hemmings BA 1996 Activation and phosphorylation of a pleckstrin homology domain containing protein kinase (RAC-PK/PKB) promoted by serum and protein phosphatase inhibitors. Proc Natl Acad Sci USA 93:5699–5704[Abstract/Free Full Text]
  17. Pilkis SJ, Granner DK 1992 Molecular physiology of the regulation of hepatic gluconeogenesis and glycolysis. Annu Rev Physiol 54:885–909[CrossRef][Medline]
  18. O’Brien RM, Granner DK 1996 Regulation of gene expression by insulin. Physiol Rev 76:1109–1161[Abstract/Free Full Text]
  19. Band CJ, Posner BI 1997 Phosphatidylinositol 3'-kinase and p70s6k are required for insulin but not bisperoxovanadium 1,10-phenanthroline (bpV(phen)) inhibition of insulin-like growth factor binding protein gene expression. Evidence for MEK-independent activation of mitogen-activated protein kinase by bpV(phen). J Biol Chem 272:138–145[Abstract/Free Full Text]
  20. Cichy SB, Uddin S, Danilkovich A, Guo S, Klippel A, Unterman TG 1998 Protein kinase B/Akt mediates effects of insulin on hepatic insulin-like growth factor-binding protein-1 gene expression through a conserved insulin response sequence. J Biol Chem 273:6482–6487[Abstract/Free Full Text]
  21. Guo S, Rena G, Cichy S, He X, Cohen P, Unterman T 1999 Phosphorylation of serine 256 by protein kinase B disrupts transactivation by FKHR and mediates effects of insulin on insulin-like growth factor-binding protein-1 promoter activity through a conserved insulin response sequence. J Biol Chem 274:17184–17192[Abstract/Free Full Text]
  22. Wolfrum C, Asilmaz E, Luca E, Friedman JM, Stoffel M 2004 Foxa2 regulates lipid metabolism and ketogenesis in the liver during fasting and in diabetes. Nature 432:1027–1032[CrossRef][Medline]
  23. Rena G, Prescott AR, Guo S, Cohen P, Unterman TG 2001 Roles of the forkhead in rhabdomyosarcoma (FKHR) phosphorylation sites in regulating 14–3-3 binding, transactivation and nuclear targetting. Biochem J 354:605–612[CrossRef][Medline]
  24. Brownawell AM, Kops GJ, Macara IG, Burgering BM 2001 Inhibition of nuclear import by protein kinase B (Akt) regulates the subcellular distribution and activity of the forkhead transcription factor AFX. Mol Cell Biol 21:3534–3546[Abstract/Free Full Text]
  25. Durham SK, Suwanichkul A, Scheimann AO, Yee D, Jackson JG, Barr FG, Powell DR 1999 FKHR binds the insulin response element in the insulin-like growth factor binding protein-1 promoter. Endocrinology 140:3140–3146[Abstract/Free Full Text]
  26. Hall RK, Yamasaki T, Kucera T, Waltner-Law M, O’Brien R, Granner DK 2000 Regulation of phosphoenolpyruvate carboxykinase and insulin-like growth factor-binding protein-1 gene expression by insulin. The role of winged helix/forkhead proteins. J Biol Chem 275:30169–30175[Abstract/Free Full Text]
  27. Patel S, Lochhead PA, Rena G, Fumagalli S, Pende M, Kozma SC, Thomas G, Sutherland C 2002 Insulin regulation of insulin-like growth factor-binding protein-1 gene expression is dependent on the mammalian target of rapamycin, but independent of ribosomal S6 kinase activity. J Biol Chem 277:9889–9895[Abstract/Free Full Text]
  28. Kong M, Mounier, C, Wu, J, Posner BI 2000 Epidermal growth factor-induced phosphatidylinositol 3-kinase activation and DNA synthesis. Identification of Grb2-associated binder 2 as the major mediator in rat hepatocytes. J Biol Chem 275:36035–36042[Abstract/Free Full Text]
  29. Sbraccia P, Wong KY, Brunetti A, Rafaeloff R, Trischitta V, Hawley DM, Goldfine ID 1990 Insulin down-regulates insulin receptor number and up-regulates insulin receptor affinity in cells expressing a tyrosine kinase-defective insulin receptor. J Biol Chem 265:4902–4907[Abstract/Free Full Text]
  30. Chomczynski P, Sacchi N 1987 Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 162:156–159[Medline]
  31. Kachra Z, Yang CR, Murphy LJ, Posner BI 1994 The regulation of insulin-like growth factor-binding protein 1 messenger ribonucleic acid in cultured rat hepatocytes: the roles of glucagon and growth hormone. Endocrinology 135:1722–1728[Abstract]
  32. Khan MN, Baquiran, G, Brule, C, Burgess, J, Foster, B, Bergeron, JJ, Posner BI 1989 Internalization and activation of the rat liver insulin receptor kinase in vivo. J Biol Chem 264:12931–12940[Abstract/Free Full Text]
  33. Band CJ, Posner BI, Dumas V, Contreres JO 1997 Early signaling events triggered by peroxovanadium [bpV(phen)] are insulin receptor kinase (IRK)-dependent: specificity of inhibition of IRK-associated protein tyrosine phosphatase(s) by bpV(phen). Mol Endocrinol 11:1899–1910[Abstract/Free Full Text]
  34. Ui M, Okada T, Hazeki K, Hazeki O 1995 Wortmannin as a unique probe for an intracellular signalling protein, phosphoinositide 3-kinase. Trends Biochem Sci 20:303–307[CrossRef][Medline]
  35. Wymann MP, Bulgarelli-Leva G, Zvelebil MJ, Pirola L, Vanhaesebroeck B, Waterfield MD, Panayotou G 1996 Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol 16:1722–1733[Abstract]
  36. Vlahos CJ, Matter WF, Hui KY, Brown RF 1994 A specific inhibitor of phosphatidylinositol 3-kinase, 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one (LY294002). J Biol Chem 269:5241–5248[Abstract/Free Full Text]
  37. Patel S, Van Der Kaay J, Sutherland C 2002 Insulin regulation of hepatic insulin-like growth factor-binding protein-1 (IGFBP-1) gene expression and mammalian target of rapamycin (mTOR) signalling is impaired by the presence of hydrogen peroxide. Biochem J 365:537–545[CrossRef][Medline]
  38. Nakae J, Park BC, Accili D 1999 Insulin stimulates phosphorylation of the forkhead transcription factor FKHR on serine 253 through a wortmannin-sensitive pathway. J Biol Chem 274:15982–15985[Abstract/Free Full Text]
  39. Nouspikel T, Iynedjian PB 1992 Insulin signalling and regulation of glucokinase gene expression in cultured hepatocytes. Eur J Biochem 210:365–373[Medline]
  40. Hartley D, Cooper GM 2002 Role of mTOR in the degradation of IRS-1: regulation of PP2A activity. J Cell Biochem 85:304–314[CrossRef][Medline]
  41. De Luca JP, Garnache AK, Rulfs J, Miller Jr TB 1999 Wortmannin inhibits insulin-stimulated activation of protein phosphatase 1 in rat cardiomyocytes. Am J Physiol 276:H1520–H1526
  42. Cazzolli R, Carpenter L, Biden TJ, Schmitz-Peiffer C 2001 A role for protein phosphatase 2A-like activity, but not atypical protein kinase C{zeta}, in the inhibition of protein kinase B/Akt and glycogen synthesis by palmitate. Diabetes 50:2210–2218[Abstract/Free Full Text]
  43. Gingras AC, Raught B, Sonenberg N 2001 Regulation of translation initiation by FRAP/mTOR. Genes Dev 15:807–826[Free Full Text]
  44. Di Como CJ, Arndt KT 1996 Nutrients, via the Tor proteins, stimulate the association of Tap42 with type 2A phosphatases. Genes Dev 10:1904–1916[Abstract/Free Full Text]
  45. Chen J, Peterson RT, Schreiber SL 1998 {alpha}4 associates with protein phosphatases 2A, 4, and 6. Biochem Biophys Res Commun 247:827–832[CrossRef][Medline]
  46. Nanahoshi M, Tsujishita Y, Tokunaga C, Inui S, Sakaguchi N, Hara K, Yonezawa K 1999 {alpha}4 protein as a common regulator of type 2A-related serine/threonine protein phosphatases. FEBS Lett 446:108–112[CrossRef][Medline]
  47. Stratford S, Hoehn KL, Liu F, Summers SA 2004 Regulation of insulin action by ceramide: dual mechanisms linking ceramide accumulation to the inhibition of Akt/protein kinase B. J Biol Chem 279:36608–36615[Abstract/Free Full Text]
  48. Kyoung Pyo H, Lovati E, Pasinetti GM, Ksiezak-Reding H 2004 Phosphorylation of {tau} at THR212 and SER214 in human neuronal and glial cultures: the role of AKT. Neuroscience 127:649–658[CrossRef][Medline]
  49. Fujiki H, Suganuma M 1993 Tumor promotion by inhibitors of protein phosphatases 1 and 2A: the okadaic acid class of compounds. Adv Cancer Res 61:143–194[Medline]
  50. Takai A, Sasaki K, Nagai H, Mieskes G, Isobe M, Isono K, Yasumoto T 1995 Inhibition of specific binding of okadaic acid to protein phosphatase 2A by microcystin-LR, calyculin-A and tautomycin: method of analysis of interactions of tight-binding ligands with target protein. Biochem J 306:657–665[Medline]
  51. Nasrin N, Ogg S, Cahill CM, Biggs W, Nui S, Dore J, Calvo D, Shi Y, Ruvkun G, Alexander-Bridges MC 2000 DAF-16 recruits the CREB-binding protein coactivator complex to the insulin-like growth factor binding protein 1 promoter in HepG2 cells. Proc Natl Acad Sci USA 97:10412–10417[Abstract/Free Full Text]
  52. Patel S, Lochhead PA, Rena G, Sutherland C 2001 Antagonistic effects of phorbol esters on insulin regulation of insulin-like growth factor-binding protein-1 (IGFBP-1) but not glucose-6-phosphatase gene expression. Biochem J 359:611–619[CrossRef][Medline]
  53. Withers DJ, Ouwens DM, Nave BT, van der Zon GC, Alarcon CM, Cardenas ME, Heitman J, Maassen JA, Shepherd PR 1997 Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells. Biochem Biophys Res Commun 241:704–709[CrossRef][Medline]
  54. Tang SJ, Reis G, Kang H, Gingras AC, Sonenberg N, Schuman EM 2002 A rapamycin-sensitive signaling pathway contributes to long-term synaptic plasticity in the hippocampus. Proc Natl Acad Sci USA 99:467–472[Abstract/Free Full Text]
  55. Schmelzle T, Hall MN 2000 TOR, a central controller of cell growth. Cell 103:253–262[CrossRef][Medline]
  56. Dickens M, Svitek CA, Culbert AA, O’Brien RM, Tavare JM 1998 Central role for phosphatidylinositide 3-kinase in the repression of glucose-6-phosphatase gene transcription by insulin. J Biol Chem 273:20144–20149[Abstract/Free Full Text]
  57. Sutherland C, O’Brien RM, Granner DK 1995 Phosphatidylinositol 3-kinase, but not p70/p85 ribosomal S6 protein kinase, is required for the regulation of phosphoenolpyruvate carboxykinase (PEPCK) gene expression by insulin. Dissociation of signaling pathways for insulin and phorbol ester regulation of PEPCK gene expression. J Biol Chem 270:15501–15506[Abstract/Free Full Text]
  58. Jiang Y, Broach JR 1999 Tor proteins and protein phosphatase 2A reciprocally regulate Tap42 in controlling cell growth in yeast. EMBO J 18:2782–2792[CrossRef][Medline]
  59. Murata K, Wu J, Brautigan DL 1997 B cell receptor-associated protein {alpha}4 displays rapamycin-sensitive binding directly to the catalytic subunit of protein phosphatase 2A. Proc Natl Acad Sci USA 94:10624–10629[Abstract/Free Full Text]
  60. Inui S, Sanjo H, Maeda K, Yamamoto H, Miyamoto E, Sakaguchi N 1998 Ig receptor binding protein 1 ({alpha}4) is associated with a rapamycin-sensitive signal transduction in lymphocytes through direct binding to the catalytic subunit of protein phosphatase 2A. Blood 92:539–546[Abstract/Free Full Text]
  61. Nakamura T, Yoshimoto K, Nakayama Y, Tomita Y, Ichihara A 1983 Reciprocal modulation of growth and differentiated functions of mature rat hepatocytes in primary culture by cell-cell contact and cell membranes. Proc Natl Acad Sci USA 80:7229–7233[Abstract/Free Full Text]
  62. Tygstrup N, Jensen SA, Krog B, Pietrangelo A, Shafritz DA 1996 Expression of messenger RNA for liver functions following 70% and 90% hepatectomy. J Hepatol 25:72–78[CrossRef][Medline]
  63. Grunnet N, Peng X, Tygstrup N 1999 Growth factors and gene expression in cultured rat hepatocytes. J Hepatol 31:117–122[CrossRef][Medline]
  64. Greuet J, Pichard L, Ourlin JC, Bonfils C, Domergue J, Le Treut P, Maurel P 1997 Effect of cell density and epidermal growth factor on the inducible expression of CYP3A and CYP1A genes in human hepatocytes in primary culture. Hepatology 25:1166–1175[CrossRef][Medline]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
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 Google Scholar
Google Scholar
Right arrow Articles by Mounier, C.
Right arrow Articles by Posner, B. I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Mounier, C.
Right arrow Articles by Posner, B. I.

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