From the Department of Biochemistry, School of Medical Sciences,
University of Bristol, BS8 1TD, United Kingdom and
Department of Molecular Physiology and Biophysics,
Vanderbilt University Medical School, Nashville, Tennessee 37232
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ABSTRACT |
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Transcription of the gene encoding the catalytic subunit of glucose-6-phosphatase (G6Pase) is stimulated by glucocorticoids and strongly repressed by insulin. We have explored the signaling pathways by which insulin mediates the repression of G6Pase transcription in H4IIE cells. Wortmannin, a phosphatidylinositide 3-kinase (PtdIns 3-kinase) inhibitor blocked the repression of G6Pase mRNA expression by insulin. However, both rapamycin, which inhibits p70S6 kinase activation, and PD98059, an inhibitor of mitogen-activated protein kinase activation, were without effect. Insulin inhibited dexamethasone-induced luciferase expression from a transiently transfected plasmid that places the luciferase gene under the control of the G6Pase promoter. This effect of insulin was mimicked by the overexpression of a constitutively active PtdIns 3-kinase but not by a constitutively active protein kinase B. Taken together, these data demonstrate that PtdIns 3-kinase activation is both necessary and at least partly sufficient for the repression of G6Pase expression by insulin, but neither mitogen-activated protein kinase nor p70S6 kinase are involved. In addition, activation of protein kinase B alone is not sufficient for repression of the G6Pase gene. These results imply the existence of a novel signaling pathway downstream of PtdIns 3 kinase that is involved in the regulation of G6Pase expression by insulin.
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INTRODUCTION |
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The hydrolysis of glucose-6-phosphate to glucose by glucose-6-phosphatase (G6Pase)1 represents the final step of both gluconeogenesis and glycogen breakdown in the liver. G6Pase is therefore a key enzyme in the regulation of blood glucose. Expression of the gene encoding the catalytic subunit of G6Pase is increased in starved and diabetic animals and repressed by re-feeding or insulin treatment, respectively (1-3). In H4IIE rat hepatoma cells, insulin inhibits both basal and glucocorticoid-stimulated G6Pase gene transcription (4, 5).
A multi-component insulin response sequence (IRS) has recently been identified in the G6Pase promoter that mediates a strong repression of mouse G6Pase gene transcription by insulin (5). This IRS is composed of two promoter regions, one of which contains three copies of the sequence T(G/A)TTT(T/G)(G/T) (5). The IRSs identified in the phosphoenolpyruvate carboxykinase, tyrosine aminotransferase, and apolipoprotein CIII promoters all contain this same motif, whereas the IRS in the insulin-like growth factor-binding protein-1 promoter has two copies of this motif arranged as an inverted palindrome (6-11). Like G6Pase, insulin also inhibits the transcription of these other genes. This raises the possibility that the expression of these genes may be regulated in a coordinated fashion by insulin, both at the level of the transcription factors involved as well as in the signaling pathways utilized.
One of the central enzymes involved in insulin signaling is
phosphatidylinositide 3-kinase (PtdIns 3-kinase (12)). This enzyme is
activated when the SH2 domain of its p85 subunit binds to
tyrosine-phosphorylated IRS-1 (13, 14). This leads to the production of
3-phosphorylated phosphatidylinositides, which serve to activate the
downstream protein kinase, protein kinase B (PKB). Hence wortmannin, an
inhibitor of PtdIns 3-kinase, is able to block activation of PKB by
insulin (15-18). PtdIns 3-kinase and PKB appear to be upstream
activators of p70S6 kinase; the stimulation of this enzyme being
inhibited by wortmannin as well as rapamycin, the latter acting via
FRAP/TOR (15, 19-22). PKB is also thought to be the enzyme responsible
for the insulin-dependent phosphorylation and inactivation
of glycogen synthase kinase-3 (GSK3) (23). Insulin is also able to
activate the small GTP-binding protein Ras, which then initiates the
Raf1 MAP kinase kinase
MAP kinase cascade (reviewed in Refs. 24
and 25).
Although the general definition of insulin signaling pathways has progressed dramatically, the elucidation of a complete signaling pathway from insulin receptor to transcription factor involved in the regulation of a specific gene remains to be established. In fact, the available data suggests that multiple divergent insulin signaling pathways regulate the expression of distinct genes (25-27). Thus, the activation of the Erk1 and Erk2 isoforms of MAP kinase appear to be important in the regulation of the AP-1 complex by insulin (28) but not of the phosphoenolpyruvate carboxykinase or hexokinase II genes (29, 30). By contrast, although the induction of hexokinase II and repression of phosphoenolpyruvate carboxykinase expression are blocked by wortmannin, only the former effect is blocked by rapamycin (29, 30).
The signaling pathway(s) involved in the repression of G6Pase gene transcription by insulin have not been explored. In the current study, we have combined the use of membrane-permeant kinase inhibitors and the transient overexpression of constitutively active PtdIns 3-kinase and protein kinase B to examine the mechanism of insulin action on G6Pase gene transcription.
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EXPERIMENTAL PROCEDURES |
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Materials-- General laboratory reagents were from BDH (Poole, UK). Dexamethasone and bovine insulin were from Sigma. Wortmannin and rapamycin were from Calbiochem. The S6 peptide, KEAKEKRQEQIAKKRRLSSLRASTSKSESSQK (32-mer) and GSK3 phosphopeptide, RRAAEELDSRAGS(P)PQL, were synthesized by Dr. Graham Bloomberg, Department of Biochemistry, University of Bristol. Rabbit polyclonal antipeptide antibody to human p70S6 kinase (residues 502-525, Ref. 17) and rabbit polyclonal anti-PKB antibody were kindly provided by Dr. Emily Foulstone, Department of Biochemistry, University of Bristol. Anti-active MAP kinase antibodies were a kind gift of Dr. E. Schaefer (Promega Corp., Madison).
Tissue Culture-- H4IIE rat hepatoma cells were routinely cultured in Dulbecco's modified Eagle's medium (Life Technologies, Inc.) containing 10% fetal calf serum in an humidified atmosphere of 95% O2 , 5% CO2 in 75 cm2 tissue culture flasks (Corning Inc., Corning, NY). Subconfluent cells were trypsinized and plated into 6-well plates (35-mm wells; Costar) for mRNA analysis, 12-well plates (22 mm wells; Costar) for transfection experiments and luciferase reporter assays, or 35-mm dishes (Falcon) for p70S6 kinase assays.
Constructs and Transfections--
A G6Pase firefly luciferase
reporter plasmid designated pGL3-G6P was generated by subcloning a
mouse G6Pase promoter fragment corresponding to bases 751 to +66
relative to the transcription start site into the polylinker of the
plasmid pGL3-Basic (Promega). Transfection efficiencies were monitored
by co-transfection with a pRL-SV40 plasmid (Promega) that expresses
Renilla reniformis luciferase under the control of a
constitutively active SV40 promoter. Expression plasmids containing the
p110 subunit of PtdIns 3-kinase have been described previously and were
kind gifts from Dr. Julian Downward (I.C.R.F., London). These include
pSG5-p110-K227E (expresses a constitutively active PtdIns 3-kinase
catalytic subunit (31)) and pSG5-p110-CAAX and
pSG5-p110-CAAX-R916P (express membrane-targeted forms of the
catalytic subunit), which are constitutively active and catalytically
inactive, respectively. Constructs expressing HA epitope-tagged,
wild-type, and constitutively active PKB (pCMV5-HA-PKB and
pCMV5-HA-PKB-T308D/S473D, respectively) were provided by Dr. D. R. Alessi (18). HA epitope-tagged GSK3
(pcDNA3-GSK3) was provided
by Dr. Jim Woodget (Ontario Cancer Institute, Toronto, Canada).
Measurement of G6Pase mRNA Levels by RT-PCR or Northern
Blotting--
Total RNA was isolated from 35-mm dishes of H4IIE cells
after treatment with ligands. Cells were washed with 2 ml of ice-cold phosphate-buffered saline and extracted in 1 ml of Tri-Reagent (Sigma).
Total RNA was prepared from the aqueous phase by isopropanol precipitation according to the manufacturer's instructions. cDNA was prepared by reverse transcription of 1 µg of total RNA using oligo(dT)15 primers and a reverse transcription kit
(Promega). G6Pase and -actin cDNA were amplified by 35 cycles of
PCR using Taq polymerase (Boehringer, Germany) and the
following primers: G6Pase, sense
5'-TAA GTG GAT TCT TTT TGG ACA-3' and antisense 5'-GAA GAG GCT GGC AAA GGG TGT-3', which gives a product of 562 base pairs;
-actin, sense 5'-GGT TCC GCT GCC CTG AGG CAC-3'
and antisense 5'-CAC TGT GTT GGC GTA GAG GTC-3', which gives a
133-bp product. PCR products were separated by electrophoresis on 2% agarose gels. Ethidium bromide-stained bands were visualized by UV
illumination and quantitated using ImageQuant software (Molecular Dynamics).
Luciferase Assays--
Lysates were prepared from transfected
H4IIE cells 18 h after ligand addition. Cells were washed in 2 ml
of ice-cold phosphate-buffered saline, and cells were extracted by
scraping into 80 µl of passive lysis buffer (Promega) or 200 µl of
ice-cold immunoprecipitation buffer (IPB; 20 mM Hepes, pH
7.5, 137 mM NaCl, 25 mM -glycerolphosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 10%
glycerol, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml
each pepstatin, antipain, and leupeptin, 0.5 mM
dithiothreitol, 1 mM Na3VO4.
Lysates were clarified by centrifugation for 15 min at 14,000 rpm.
Firefly and Renilla luciferases were assayed sequentially in
10 µl of lysate using the dual luciferase assay kit (Promega). The
activity of the G6Pase promoter was expressed as the ratio of the
firefly/Renilla luciferase activities and is thus corrected
for differences in transfection efficiency and cell viability.
Protein Kinase Assays--
p70S6 kinase was precipitated from
150 µl of cell extract by incubation with 5 µl of a rabbit
polyclonal antibody raised to rat p70S6 kinase and 5 mg of
pre-equilibrated protein A-Sepharose in a final volume of 500 µl of
IPB. Precipitates were tumbled at 4 °C for 3 h and then washed
with 3 × 1 ml IPB followed by 1 ml of kinase assay buffer (25 mM Hepes, pH 7.5, 25 mM -glycerolphosphate, 25 mM MgCl2, 0.2 mM
Na3VO4, 2 mM EGTA, 0.2 mM dithiothreitol). Immune complex kinase assays were
performed in 50 µl of kinase assay buffer containing 50 µM [
-32P]ATP (5 µCi/assay) and 500 µg/ml S6 peptide. Incubations were continued for 30 min at 30 °C
and terminated by spotting 40-µl aliquots onto P81 phosphocellulose
paper squares and washing extensively in 150 mM
orthophosphoric acid. Papers were dried, and 32P
incorporation into S6 peptide was determined by Cerenkov counting.
Western Blotting-- Transfected, HA epitope-tagged PKB was precipitated from H4IIE cell lysates using a rabbit anti-PKB polyclonal antibody as described for p70S6 kinase above. Immunoprecipitates were subjected to SDS-PAGE on 8% polyacrylamide gels. Proteins were transferred to Immobilon-P, and HA-tagged PKB was detected by Western blotting with anti-HA antibody (Boehringer, Germany) and enhanced chemiluminescence (Amersham Pharmacia Biotech). Active MAP kinase, dually phosphorylated on threonine and tyrosine, was detected in H4IIE cell lysates by Western blotting using anti-active MAP kinase antibody (25 ng/ml) as described by the manufacturer (Promega).
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RESULTS AND DISCUSSION |
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Repression of G6Pase Transcription by Insulin Is Blocked by
Wortmannin--
PtdIns 3-kinase is potently activated by insulin in
many cell types and has been shown to be important in the actions of
insulin on a number of cellular processes (25). To determine whether PtdIns 3-kinase activation is also required for insulin to suppress G6Pase gene expression, H4IIE cells were incubated in the absence or
presence of various combinations of 100 nM wortmannin and
200 nM insulin. Total mRNA was then isolated, and the
levels of G6Pase and -actin mRNA were determined by quantitative
RT-PCR. As shown in Fig. 1, there was a
significant repression of G6Pase mRNA expression by insulin at both
2 and 4. Wortmannin completely blocked the repression by insulin at
both time points and caused an additional elevation of G6Pase mRNA
levels in both the absence and presence of insulin as compared with
nontreated cells. Neither insulin nor wortmannin had any effect on
-actin mRNA expression. These data indicate that PtdIns 3-kinase
activity is necessary for the suppression of G6Pase gene expression by
insulin. In addition, the data also suggest that there is a tonic level
of PtdIns 3-kinase activity in the basal state which partially
represses G6Pase gene expression but that is also inhibited by
wortmannin.
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Constitutively Active PtdIns 3-Kinase Mimics the Effect of Insulin
on G6Pase Gene Transcription--
A transient transfection strategy
was used to determine whether PtdIns 3-kinase activation was sufficient
to repress G6Pase gene expression. H4IIE cells were co-transfected with
a reporter plasmid (pGL3-G6P) containing the mouse G6Pase promoter
(nucleotides 751 to +66) upstream of the firefly luciferase reporter
gene and either of two different plasmids that express constitutively active forms of PtdIns 3-kinase. One plasmid encodes a PtdIns 3-kinase
catalytic subunit (p110-CAAX) made constitutively active by
virtue of a carboxyl-terminal CAAX motif that directs
isoprenylation and membrane targeting. The other encodes a p110 subunit
with a single point mutation (K227E) in the Ras interaction domain, which appears to increase its basal activity (31).
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Repression of G6Pase Transcription by Insulin Is Not Blocked by the
MAP Kinase Kinase Inhibitor, PD98059--
Since the data suggest that
PtdIns 3-kinase is both necessary and substantially sufficient for the
ability of insulin to repress the expression of G6Pase, the nature of
the downstream target for the action of this lipid kinase was
investigated. It has been reported in some cell types that wortmannin
can block the activation of MAP kinase by insulin (33, 34). Thus, to
examine a role for MAP kinase in the repression of G6Pase gene
transcription by insulin, we used the MAP kinase kinase inhibitor
PD98059 (35). We found by Western blotting using anti-active MAP kinase
antibodies that MAP kinase was stimulated by insulin in H4IIE cells and
that this effect was completely reversed by PD98059 (Fig.
3A). Despite this, PD98059 had
no effect on the ability of insulin to repress G6Pase mRNA levels
but did cause an approximate 2-fold increase in the effect of
dexamethasone (Fig. 3B; as observed with wortmannin in Fig.
1). This excludes a role for MAP kinase in the repression of G6Pase
expression by insulin, but we speculate that the activity of the
glucocorticoid receptor may be regulated by phosphorylation by MAP
kinase. Such a phenomenon has been previously reported for both the
oestrogen receptor (36) and peroxisome proliferator-activated receptor
(37). However, this type of regulation does not form the basis for
the effect of insulin on G6Pase promoter activity.
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Rapamycin Does Not Block the Suppression of G6Pase Gene Transcription by Insulin-- It is well established that PtdIns 3-kinase is responsible for the activation of the p70S6 kinase (22). To determine whether p70S6 kinase activation is required for the repression of G6Pase gene transcription by insulin, H4IIE cells were transiently transfected with the G6P-luciferase fusion gene and treated with various combinations of dexamethasone, insulin, and rapamycin, a well characterized inhibitor of p70S6 kinase activation (38). Cells were extracted at either 2 or 18 h, and the resulting lysates were assayed for both luciferase and p70S6 kinase activity.
At both time points rapamycin completely blocked the activation of p70S6 kinase by insulin but had no effect on the basal kinase activity (Fig. 4A). Interestingly, treatment of cells for 18 h with dexamethasone caused a small but significant stimulation of p70S6 kinase activity, but this induction was also suppressed by rapamycin.
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Constitutively Active Protein Kinase B Does Not Mimic the Action of Insulin on G6Pase Gene Transcription-- Another, perhaps more direct, target for PtdIns 3-kinase is PKB (15), which is activated by insulin in H4IIE cells.2 To assess the potential role of PKB in mediating the action of insulin on G6Pase gene transcription, the effect of co-transfecting a plasmid encoding a constitutively active derivative of this enzyme on G6Pase-luciferase fusion gene expression was investigated. In this PKB derivative, the phosphorylation sites responsible for activation of the enzyme have been substituted with acidic residues (T308D/S473D; Ref. 16). Overexpression of PKB-T308D/S473D in 3T3 L1 adipocytes has a potent insulin-like effect on the translocation of GLUT4 to the plasma membrane,3 which is similar to the effect of overexpression of a constitutively active membrane-targeted GagPKB (39, 40).
To confirm that the transfected PKB-T308D/S473D was catalytically active when expressed in H4IIE cells, its ability to inhibit GSK3 as reported (23) was measured. As the transfection efficiency of H4IIE cells is very low (<2%), PKB-T308D/S473D was co-expressed with HA-tagged GSK3
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General Conclusions-- The data presented in this study support a model for the inhibitory action of insulin on G6Pase gene transcription in which PtdIns 3-kinase activation, but not MAP kinase, PKB, or p70S6 kinase activation, plays a central role. Future work must be directed at identifying the target for PtdIns 3-kinase that regulates the signaling pathway leading to the G6Pase promoter and how activation of this pathway affects the transcriptional machinery. As yet such a target has not been identified, but the likely protein may possess a plextrin-homology domain, which appears to be a recognition motif for 3-phosphorylated lipids (41). Calcium-insensitive isoforms of protein kinase C are activated by 3-phosphorylated inositol lipids (42, 43), and the possibility that these isoforms of protein kinase C may mediate the effect of insulin on the G6Pase and other promoters warrants further investigation.
In view of the homology between the G6Pase IRS and those in several other hepatic genes (see the Introduction), it will be of considerable interest to determine whether the transcription of these other genes can be similarly repressed by constitutively active PtdIns 3-kinase and whether PKB plays a role. Thus, although it was previously reported that PtdIns 3-kinase but not p70S6 kinase was required for the effect of insulin on phosphoenolpyruvate carboxykinase gene expression (29), it remains to be shown whether the activation of PtdIns 3-kinase is sufficient. However, it appears likely that the expression of these two crucial gluconeogenic enzymes is regulated by insulin and glucocorticoids in a co-ordinated fashion by very similar signaling pathways and transcriptional apparatus. ![]() |
ACKNOWLEDGEMENTS |
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We are very grateful to Dr. R. Taub (University of Pennsylvania) for the G6Pase promoter, Dr. J. Downward (I.C.R.F., London) for the PtdIns 3-kinase plasmids, and Drs. B. Hemmings, M. Andjelkovic (Friedrich Miescher-Institut), and D. Alessi (University of Dundee) for the protein kinase B expression plasmids. We also thank Dr. J. Woodget (Ontario Cancer Institute, Toronto, Canada) for the GSK3 construct and Dr. E. Schaefer (Promega Corp.) for the anti-active MAP kinase antibody.
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FOOTNOTES |
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* This work was supported by grants from the Medical Research Council and British Diabetic Association (to J. M. T.) and the American Diabetes Association, Juvenile Diabetes Foundation, and the Mark Collie Foundation (to R. O'B.). This work was also supported by a NATO travel grant (to J. M. T., and R. O'B).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ A British Diabetic Association senior research fellow. To whom correspondence should be addressed: Dept. of Biochemistry, School of Medical Sciences, University of Bristol, BS8 1TD, UK. Tel.: 44-0-117-928-8273; Fax: 44-0-117-928-8274. E-mail: j.tavare{at}bristol.ac.uk.
The abbreviations used are: G6Pase, glucose-6-phosphatase; IRS, insulin response sequence; PtdIns 3-kinase, phosphatidylinositide 3-kinase; PKB, protein kinase B; CMV, cytomegalovirus; HA, hemagglutinin; DMEM, Dulbecco's modified Eagle's medium; RT-PCR, reverse transcription-polymerase chain reaction; IPB, immunoprecipitation buffer; GSK3, glycogen synthase kinase-3.
2 M. Dickens and J. M. Tavaré, unpublished data.
3 P. Oatey and J. M. Tavaré, unpublished data.
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REFERENCES |
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