From the Department of Cellular and Molecular Physiology, The
Pennsylvania State University, College of Medicine,
Hershey, Pennsylvania 17033
Transcription of the phosphoenolpyruvate
carboxykinase (PEPCK) gene is induced by glucagon, acting through cAMP
and protein kinase A, and this induction is inhibited by insulin.
Conflicting reports have suggested that insulin inhibits induction by
cAMP by activating the Ras/mitogen-activated protein kinase (MAPK) pathway or by activating the phosphatidylinositol 3-kinase
(PI3-kinase), but not MAPK, pathway. Insulin activated PI3-kinase
phosphorylates lipids that activate protein kinase B (PKB) and
Ca2+/diacylglycerol-insensitive forms of protein
kinase C (PKC). We have assessed the roles of these pathways in insulin
inhibition of cAMP/PKA-induced transcription of PEPCK by using dominant
negative and dominant active forms of regulatory enzymes in the
Ras/MAPK and PKB pathways and chemical inhibitors of PKC isoforms.
Three independently acting inhibitory enzymes of the Ras/MAPK pathway, blocking SOS, Ras, and MAPK, had no effect upon insulin inhibition. However, dominant active Ras prevented induction of PEPCK and also
stimulated transcription mediated by Elk, a MAPK target. Insulin did
not stimulate Elk-mediated transcription, indicating that insulin did
not functionally activate the Ras/MAPK pathway. Inhibitors of
PI3-kinase, LY294002 and wortmannin, abolished insulin inhibition of
PEPCK gene transcription. However, inhibitors of PKC and mutated forms
of PKB, both of which are known downstream targets of PI3-kinase, had
no effect upon insulin inhibition. Dominant negative forms of PKB did
not interfere with insulin inhibition and a dominant active form of PKB
did not prevent induction by PKA. Phorbol ester-mediated inhibition of
PEPCK transcription was blocked by bisindole maleimide and by
staurosporine, but insulin-mediated inhibition was unaffected. Thus,
insulin inhibition of PKA-induced PEPCK expression does not require
MAPK activation but does require activation of PI3-kinase, although
this signal is not transmitted through the PKB or PKC pathways.
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INTRODUCTION |
Insulin stimulates a variety of changes in growth and metabolism
in different cell types, ranging from the stimulation of replication, translation, and protein synthesis to the covalent modification of enzymes of intermediary metabolism (1). In addition,
insulin regulates the transcription of specific genes, whose products
catalyze committed reactions in hepatic glucose metabolism (2). In
particular, the amounts of the enzymes that catalyze committed steps at
either end of the glucose utilization pathway, glucokinase and
phosphoenolpyruvate carboxykinase
(PEPCK)1 are regulated solely
through modulation of gene transcription in an opposite fashion by
insulin and glucagon, which acts through cAMP and PKA (3).
Transcription of the gene encoding glucokinase, which is required for
glycolysis, is induced by insulin and inhibited by cAMP. In contrast,
transcription of the gene encoding PEPCK, which is required for
gluconeogenesis, is induced by cAMP/PKA and inhibited by insulin (4).
The mechanism of insulin action has remained elusive.
The binding of insulin to its cell surface receptors activates their
intrinsic tyrosine kinase activity, leading to receptor autophosphorylation and phosphorylation of cytosolic proteins, known as
IRSs, which serve as adapters in intracellular signaling (1). IRS-1,
the predominant and most thoroughly characterized IRS, binds a variety
of signaling molecules when specific tyrosines are phosphorylated,
including the regulatory subunit of phosphatidylinositol 3-kinase
(PI3-kinase), Shc-1, and Grb 2 (1, 5, 6). Interaction among these
IRS-associated molecules initiates signaling cascades leading to the
activation of a variety of protein kinases, including MAPK, protein
kinase B, protein kinase C, glycogen synthase kinase-3, pp90rsk II, and
p70S6 kinase (7-14). All of these kinases have been implicated in one
or more of the growth or metabolic effects attributed to insulin. In
some cases, activation of a single pathway may suffice for regulation,
whereas in others more than one of these pathways may need to be
activated for regulation by insulin. For example, activation of both
PI3-kinase and MAPK is required for stimulation of general protein
synthesis by insulin, whereas only the PI3-kinase pathway needs to be
activated for stimulation of growth-related protein synthesis by
insulin (14). The lipid products of PI3-kinase, which is essential for
mediating many of the metabolic effects of insulin, activate PKB
(also known as Rac and Akt) (15-17) and novel isoforms of PKC not
regulated by calcium and diacylglycerol (12, 18).
We previously showed that multiple binding sites for CREB-GAL4 ligated
to a minimal PEPCK promoter (5XGT) could mediate induction by cAMP/PKA
and that this induction was inhibited, at least in part, by insulin in
H4IIe hepatoma cells (19). We proposed that insulin targeted the
CREB·CBP·RNA polymerase II complex to inhibit PEPCK gene
transcription. However, we recently reexamined this question with a
more sensitive luciferase reporter gene and found that insulin
inhibition of 5XGT was cAMP-independent, as it was indistinguishable
from insulin inhibition of basal PEPCK gene transcription (20). In
addition to the CRE, induction of the PEPCK gene by cAMP requires
heterologous binding sites located in the (AC) region (
271/
225)
(20-23). Factors binding to the AC region and CREB, which is targeted
by cAMP-activated PKA, form a cAMP response unit (CRU) that mediates
both induction by PKA and inhibition by insulin in the presence of the
minimal PEPCK promoter (20).
Blenis and Montminy and colleagues (11) provided evidence that CBP is
targeted by insulin to inhibit PEPCK transcription. Their data
indicated that activation of the Ras/MAPK pathway by insulin in H4IIe
rat hepatoma cells resulted in activation of pp90rskII by MAPK, leading
to its binding to CBP and inhibition of cAMP-induced transcription. On
the other hand, Gabbay et al. (24) demonstrated that
PD98059, a potent and specific MEK inhibitor, had no effect upon
insulin inhibition of PEPCK gene transcription in H4IIe cells. In
addition, Sutherland et al. (9) provided evidence that
inhibition of PI3-kinase activation abrogated insulin regulation of the
PEPCK gene. Thus, there is directly conflicting evidence regarding the
insulin-stimulated pathway(s) required for inhibition of PEPCK gene
transcription.
The present study was undertaken to examine the role of potential
insulin signaling pathways in the inhibition of PKA-induced PEPCK gene
transcription. We expressed dominant negative and dominant active forms
of regulatory signaling enzymes in the Ras/MAPK and PKB/Akt pathways
and utilized chemical inhibitors of PI3-kinase and protein kinase C
isoforms to assess the contribution of these different signaling
pathways to insulin inhibition of PEPCK expression.
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EXPERIMENTAL PROCEDURES |
Plasmids--
The PEPCK-Luc plasmid is derived from
PEPCK-chloramphenicol acetyltransferase, containing
600/+69 of the
PEPCK promoter (25). The G4-PEPCK-Luc plasmid contains the entire
promoter region (
600/+69) of the PEPCK gene, in which the CRE is
replaced by a GAL4 site (26). The CREB-GAL4 fusion protein expression
vector contains the activation domain of CREB (amino acids 1-277)
fused to amino acids 4-147 of the GAL4 DNA binding domain and has been
described previously (27, 28). The PKA expression vector, RSV-C
,
contains the cDNA for the catalytic subunit of protein kinase A
under control of the RSV promoter (28). The pRL-SV plasmid is the
expression vector for Renilla luciferase, which is used for
normalization of the transfection efficiency as assayed by firefly
luciferase. The expression vector for dominant-negative Ras,
pM2N-RasN17 and for dominant-active Ras, pRSV-Leu61 were provided by S. Cook of Onyx Pharmaceuticals (29). The expression vector for
dominant-negative Raf, RSV-RAF-C4, was from U. Rapp of New York
University Medical Center (30). The expression vector for
dominant-active PAC-1 was provided by K. Kelly, NIH (31). The G4-Elk-1
plasmid contains the GAL4 DNA binding domain (amino acids 1-147) fused
to the Elk-1 carboxyl-terminal activation domain (307-428) in
pcDNA3a with the Neo gene removed and was provided by R. Maurer of
Oregon Health Sciences University (32). The HA-Akt, HA-Akt-R25C, and
HA-Akt(pleckstrin homology (PH)) plasmids were obtained from T. Franke,
Montreal Neurological Institute (33). HA-Akt is the expression vector for protein kinase B (PKB/Akt). HA-Akt-R25C is the expression vector
for the dimerization-defective PKB. HA-Akt(PH) is the expression vector
for a mutant PKB which contains only the pleckstrin homology domain.
The pSG5-PKB (wild type), pSG5-PKB, K
A, and pSG5-gagPKB plasmids
were obtained from P. Coffer (University Hospital, Utrecht) (34).
pSG5-PKB is the expression vector for wild type PKB. pSG5-PKB, K
A
is the expression for kinase-defective PKB with a residue change at
amino acid 179. The pSG5-gagPKB plasmid is the expression vector for
constitutively active PKB.
Transfections--
H4IIe cells were cultured and transfected as
described previously (19, 26). Briefly, 1 ml of calcium phosphate
precipitate contained 20 µg of luciferase reporter vector + 2 µg
pRL-SV and 2 µg RSV-C
and/or 2-10 µg expression vector, as
indicated in the figure legends. An equal volume of cells was added to
the calcium precipitate and incubated for 15 min at room temperature. The cells were plated in replicate dishes, incubated for 4 h at 37 °C, 5% CO2, and treated with 20% Me2SO
for 3 min. Where indicated the following were added at the
concentration indicated for the last 20 h of the experiment:
insulin (Lilly), 10 nM; bisindolyl maleimide I, HCl (BIM)
(Calbiochem), 10 µM; and phorbol 12-myristate 13-acetate, (PMA) (Sigma), 1 µM. Following incubation for
20 h, cells were harvested with trypsin treatment and lysed with
1× passive lysis buffer (Promega) and stored at
80 °C for at
least 15 min. Lysates (100 µl) were obtained by spinning down the
lysed cells, and 10 µl of lysate was used for the luciferase assay. A
dual injector Monolight 3010 Luminometer was utilized to measure luciferase activity. 50 µl of each Promega reagent were added to the
lysate for the assay. Values were normalized for transfection efficiency and the mean was computed for several experiments. Data
shown in figures were obtained from independent transfection experiments performed with different preparations of the various plasmids.
RNA Isolation and Primer Extension--
H4IIe cells were
incubated overnight at 37 °C in serum-free medium containing 0.1%
bovine serum albumin, after which they were pretreated with the
PI3-kinase inhibitors, wortmannin, or LY294002, for 15 min, prior to
adding hormones and incubating with hormone + inhibitor for 3 h.
Wortmannin (Sigma) was added to a final concentration of 0.1 mM. LY294002 (Biomole) was added to a final concentration
of 10 µM. Insulin (Lilly) was added to a final
concentration of 10 nM. 8-(4-Chlorophenylthio)-cAMP (Sigma) was added to a final concentration of 0.1 mM. Cells were
harvested and total RNA was isolated as described by Chomczynski and
Sacchi (35). At the end of the procedure, the RNA was dissolved and reextracted with phenol:chloroform, ethanol-precipitated and dissolved in water. The amount of PEPCK mRNA in 50 µg of total RNA was
quantitated by primer extension analysis, as described previously
(36).
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RESULTS |
Alternative Signaling Pathways--
The molecular pathways
proposed to mediate glucagon-stimulated and insulin-inhibited PEPCK
gene expression are illustrated in Fig.
1, as are the targets of relevant
inhibitors. There is general agreement that glucagon stimulates
adenylate cyclase and cAMP activates PKA, a portion of which is
translocated to the nucleus where it phosphorylates CREB, leading to
CBP binding and activation of gene transcription (37-40). A region
containing putative binding sites for transcription factors of the AP-1
and C/EBP families, as well as the CREB binding site (CRE), are
required to form a functional CRU in the PEPCK promoter (20-23). This
CRU is necessary and sufficient for both induction by PKA and
inhibition by insulin of a minimal promoter, whereas CREB alone is not
(20). Thus, more specificity is required for insulin to inhibit
PKA-induced PEPCK gene transcription than is provided by the
P-CREB/CBP/RNA polymerase II complex alone (20). However, it has been
suggested that Ras/MAPK-mediated activation of pp90rskII and its
binding to CBP is the mechanism utilized by insulin to inhibit PEPCK
gene transcription (11).

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Fig. 1.
Potential pathways of glucagon and
insulin signaling to the PEPCK gene. Alternate signaling pathways
are shown together with the inhibitors (black ovals) that
block specific enzymes. Circled Ps represent
phosphorylations of proteins believed to be important to regulation.
C, catalytic subunit; R, regulatory subunit;
GSK-3, glycogen synthase kinase 3; PD, MEK
inhibitor PD98059; LY, PI3-kinase inhibitor LY294002;
Wort, PI3-kinase inhibitor wortmannin; Staur,
staurosporine.
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Role of Activation of the Ras/MAPK Pathway in Insulin
Inhibition--
To examine the role of the Ras/MAPK pathway in insulin
signaling relevant to inhibition of PEPCK transcription, we determined the effects of expression of dominant negative (dn) and dominant active
(da) forms of enzymes involved in transmission and regulation of
signals through the Ras/MAPK pathway. In general, dominant negative
forms of enzymes interact with the signaling component immediately
upstream and prevent further transmission of the signal, whereas
dominant active forms of signaling enzymes stimulate all pathway
members downstream of that particular enzyme without any requirement
for input from receptors or other enzymes upstream. For the experiments
shown in Fig. 2, H4IIe cells were
cotransfected with PEPCK-Luc and expression vectors for dn-Ras, dn-Raf,
da-PAC-1, and da-Ras. Dominant negative Ras interferes with the
exchange activity of SOS, preventing exchange of GDP for GTP on
endogenous Ras and interfering with downstream signaling (29, 41).
Dominant negative Raf binds to GTP-activated Ras but cannot be
activated itself and thus prevents downstream signaling (30). Dominant active PAC-1 dephosphorylates and inactivates MAPK, preventing it from
activating downstream targets (31). As shown in Fig. 2, expression of
dn-Ras, dn-Raf, or da-PAC-1 had no affect upon insulin inhibition of
PKA-induced PEPCK-Luc. However, all three Ras-pathway inhibitors
relieved constitutive restraint of PKA induction through the Ras
pathway and enhanced induction. Dominant active Ras (da-Ras) activates
downstream components independently of signaling input (42).
Cotransfection of H4IIe cells with PEPCK-Luc and da-Ras blocked
induction by PKA at least as effectively as treatment with insulin.
Thus, activation of the Ras/MAPK pathway at the level of Ras is
sufficient to inhibit PKA-induced PEPCK expression but not necessary
for insulin inhibition.

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Fig. 2.
Effects of dominant negative and dominant
active regulatory enzymes in the Ras/MAPK pathway on hormonal
regulation of PEPCK-Luc. H4IIe cells were cotransfected with 10 µg of PEPCK-Luc and 1 µg of pRLSV, plus 1 µg of pRSV-PKAc and/or
5 µg of Ras pathway mutants, as indicated. The mutants are: dn-Ras,
dominant negative Ras (RasN17); dn-Raf, dominant negative Raf-1;
da-PAC-1, dominant active PAC-1; da-Ras, dominant active Ras (RasL61).
PEPCK-Luc expression was normalized for expression of pRL-SV and the
value for the untreated control set to 1. The data illustrated
represent the results of four to eight independent experiments: four
(dn-Ras, da-Ras), seven (dn-Raf), or eight (da-PAC-1).
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Assessment of Insulin Activation of the Ras/MAPK Pathway in
H4IIe Cells--
To determine whether insulin functionally activated
the Ras/MAPK pathway affecting gene expression, we employed G4-Elk, a MAPK-activated transcription factor. H4IIe cells were cotransfected with G4-PEPCK-Luc and G4-Elk and tested for induction by insulin and
inhibition of this induction by the Ras pathway mutant enzymes (Fig.
3). In G4-Elk, the activation domain of
the MAPK-activated transcription factor Elk is fused to the GAL4 DNA
binding domain. The G4-PEPCK promoter contains a GAL4 site in place of
the CRE. Thus, if insulin activated the Ras/MAPK pathway in H4IIe
cells, G4-PEPCK-Luc expression should be induced by insulin in the
presence of G4-Elk, and this induction should be inhibited by the
Ras/MAPK signaling pathway mutants. As illustrated in Fig. 3, insulin
did not activate G4-PEPCK-Luc through the G4-Elk factor. However, da-Ras did potently activate transcription mediated by G4-Elk, and this
induction was unaffected by insulin. These results demonstrate that
insulin stimulation of H4IIe cells did not significantly activate the
Ras/MAPK pathway. Together with the results above, these data strongly
argue against any role for activation of the Ras/MAPK pathway in
insulin-mediated inhibition of PKA-induced PEPCK gene expression.

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Fig. 3.
Effects of dominant negative and dominant
active regulatory enzymes on G4-Elk-mediated transcription of
G4-PEPCK-Luc. H4IIe cells were cotransfected with 10 µg of
PEPCK-Luc and 1 µg of pRLSV, plus 1 µg of pRSV-PKAc, 1 µg of
G4-Elk, and/or 5 µg of Ras pathway mutants, as indicated. The mutants
are dn-Ras, dominant negative Ras (RasN17); dn-Raf, dominant negative
Raf-1; da-PAC-1, dominant active PAC-1; da-Ras, dominant active Ras
(RasL61). G4-PEPCK contains a GAL4 site in place of the CRE. In G4-Elk,
the activation domain of the MAPK-regulated Elk factor is fused to the
GAL4 DNA binding domain. PEPCK-Luc expression was normalized for
expression of pRL-SV and the value for the untreated control set to 1. The data illustrated represent the results of three independent
experiments.
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Activation of PI3-Kinase Is Required for Insulin
Inhibition--
Next, we examined the effects of two different
chemical inhibitors of PI3-kinase, wortmannin and LY294002, on
endogenous PEPCK mRNA expression (Fig.
4). Total mRNA was prepared from
H4IIe cells after 3 h of hormone treatment, a time at which
changes in PEPCK mRNA are near maximal (4). PEPCK mRNA was
quantitated by primer extension analysis. H4IIe cells were treated with
nothing, cAMP alone or cAMP + insulin, in the absence or presence of
Me2SO vehicle, 0.1 mM or 0.5 mM
wortmannin, as indicated in Fig. 4A. Wortmannin completely
blocked insulin inhibition of PKA-induced PEPCK mRNA accumulation.
We also tested another chemical inhibitor of PI3-kinase, LY294002 (10 µM), in the same manner. As shown in Fig. 4B,
LY294002 also completely blocked insulin inhibition of PKA-induced
accumulation of PEPCK mRNA. These results confirm those of Granner
and colleagues (9) and indicate that activation of PI3-kinase by
insulin is obligatory for insulin inhibition of PKA-induced PEPCK gene
expression. Based on other studies of insulin signaling, the most
probable downstream targets of PI3-kinase are 1) protein kinase B, also known as Akt (13, 43) and 2) nonclassical forms of PKC (12, 14,
18).

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Fig. 4.
Effects of the PI3-kinase inhibitors,
wortmannin, and LY294002, on insulin inhibition of cAMP-induced PEPCK
expression. H4IIe cells were treated with inhibitor or vehicle for
15 min prior to treatment with 0.1 mM
8-(4-chlorophenylthio)-cAMP and/or 10 nM
insulin for 3 h, as indicated. Total RNA was prepared from the
cells and quantitated by primer extension analysis. The data were
normalized to the cAMP-induced sample to allow more direct comparison
of the effects of wortmannin (A) and LY294002
(B) on insulin inhibition. The data illustrated represent
the results of six independent experiments each for A and
B.
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Role of Activation of PKB/Akt in Insulin Inhibition--
We used
recently developed mutants of PKB/Akt to determine whether this enzyme
plays an obligatory role in the insulin signal that causes inhibition
of PKA-induced PEPCK expression. PKB/Akt is activated by the lipid
products of PI3-kinase (33, 44) and this requires dimerization through
its PH domain (16). A mutant of PKB/Akt that is defective for
dimerization, Akt-R25C (dim-def in Fig.
5), cannot be activated in
vitro (33). Likewise a mutant protein containing only the PH
domain, PH-Akt (dim only in Fig. 5) dimerizes with full-length PKB and
prevents its activation in a dominant negative manner (33). As can be
seen in Fig. 5, cotransfection of H4IIe cells with PEPCK-Luc and wild
type PKB/Akt, the dimerization defective mutant, Akt-R25C, or the
dominant negative form, PH-Akt, had no significant effect on insulin
inhibition of PKA-induced PEPCK expression.

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Fig. 5.
Effects of pleckstrin homology domain mutants
of PKB on hormonal regulation of PEPCK-Luc. H4IIe cells were
cotransfected with 10 µg of PEPCK-Luc and 1 µg of pRLSV, plus 1 µg of pRSV-PKAc and/or 5 µg of PKB mutants, as indicated. The
mutants are: Akt-R25C; dimerization defective PKB; PH-Akt, pleckstrin
homology dimerization domain only of PKB. PEPCK-Luc expression was
normalized for expression of pRL-SV and the value for the untreated
control set to 1. The data illustrated represent the results of five
independent experiments.
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To further examine the potential role of PKB in insulin signaling, we
tested a kinase defective form of the enzyme, PKB, K
A, as well as
a constitutively active, viral form of the enzyme, gagPKB (34). If PKB
were obligatory for insulin signaling, the kinase defective form of the
enzyme would be expected to interfere with the activation of native PKB
and prevent insulin inhibition. Likewise, the constitutively active
form would be expected to inhibit induction by PKA if insulin
activation of PKB was responsible for inhibition. H4IIe cells were
cotransfected with PEPCK-Luc and these mutated PKB/Akt enzymes (Fig.
6). Expression of the kinase defective
isoform, PKB, K
A, resulted in augmentation of induction by PKA,
but had no effect upon insulin inhibition. Expression of the
constitutively active isoform, gagPKB, drastically reduced both basal
and PKA-induced expression to similar extents, but had no effect upon
insulin inhibition. These data indicate that PKB is not an obligatory
enzyme in the pathway utilized by insulin to signal inhibition of
PKA-induced PEPCK expression.

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Fig. 6.
Effects of kinase-defective and
constitutively active enzymes in the PKB pathway on hormonal regulation
of PEPCK-Luc. H4IIe cells were cotransfected with 10 µg of
PEPCK-Luc and 1 µg of pRLSV, plus 1 µg of pRSV-PKAc and/or 5 µg
of PKB mutants, as indicated. The mutants are: PKB, K A, kinase
defective PKB; gagPKB, constitutively active PKB. PEPCK-Luc expression
was normalized for expression of pRL-SV and the value for the untreated
control set to 1. The data illustrated represent the results of three
independent experiments.
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Role of Activation of PKC in Insulin Inhibition--
Classical
isoforms of PKC are regulated by Ca2+ and phospholipids.
This is mimicked by the phorbol ester PMA and is inhibited by the
phorbol ester analogue BIM (45). Recent work has suggested a role for
unique isoforms of PKC, which are insensitive to phorbol ester
regulation, in insulin action (12, 14, 18). Within this class, the
activity of PKC
has been demonstrated to be regulated by
insulin; in particular, by the lipid products of PI3-kinase (12, 14).
Both classical and unique isoforms of PKC are sensitive to inhibition
by the less selective inhibitor staurosporine (46). We tested both BIM
and staurosporine for their ability to block inhibition by either PMA
or insulin of PKA-induced PEPCK-Luc expression. As illustrated in Fig.
7A, BIM blocked inhibition of
PKA-induced gene transcription by the phorbol ester, PMA, but had no
effect upon insulin inhibition. There was a modest reduction in
PKA-induced activity in the presence of BIM, consistent with its
overlapping specificity for PKA (45). However, the inhibitory effects
of PMA were entirely blocked by BIM. These data indicate that classical
isoforms of PKC are not involved in the signaling pathway utilized by
insulin, which is consistent with a previous report employing
down-regulation of PKC with PMA (47). In contrast, staurosporine, which
is proposed to nonselectively inhibit all isoforms of PKC, had
paradoxical effects on PEPCK-Luc expression (Fig. 7B).
Treatment with staurosporine alone caused marked induction of
PEPCK-Luc. Both insulin and PMA effectively inhibited induction by
staurosporine alone and insulin inhibited induction by PKA in the
presence of staurosporine. PMA inhibition was partially blocked by
staurosporine. A higher concentration of the inhibitor
(10
6 M) was toxic to H4IIe cells and a lower
concentration (10
8 M) resulted in more potent
induction of PEPCK-Luc. Thus, although staurosporine apparently
inhibits kinases other than PKC, leading to the complicated pattern
observed, the effective inhibition of PKA induction by insulin in the
presence of staurosporine argues against any role for
PKC
or other novel isoforms of PKC in insulin-mediated
inhibition of the PEPCK gene.

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Fig. 7.
Effects of inhibition of PKC isoforms on
hormonal regulation of PEPCK-Luc. H4IIe cells were cotransfected
with 10 µg of PEPCK-Luc, 1 µg of pRLSV, and/or 1 µg of pRSV-PKAc,
as indicated. A, cells were treated with or without BIM (10 µM) and with either insulin (10 nM) or PMA (1 µM) during the final 20 h of the experiment.
B, cells were treated with or without staurosporine
(Staur, 10 7 M) and with either
insulin (10 nM) or PMA (1 µM) during the
final 20 h of the experiment. PEPCK-Luc expression was normalized
for expression of pRL-SV and the value for the untreated control set to
1. The data illustrated represent the results of six independent
experiments in A and nine independent experiments in
B.
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 |
DISCUSSION |
The results presented here demonstrate that activation of the
insulin, Ras/MAPK, or PKC signaling pathways inhibited PKA-induced PEPCK transcription. However, inhibition of either the Ras/MAPK or PKC
pathways had no effect upon insulin inhibition. Although dominant
active Ras mimicked insulin inhibition of PKA-induced PEPCK
transcription, three independently acting dominant negative inhibitors
of the Ras/MAPK pathway had no effect upon insulin inhibition. In
addition, we showed that insulin does not functionally activate the
Ras/MAPK pathway in H4IIe cells. In contrast, inhibition of
PI3-kinase activity with either wortmannin or LY294002 abolished inhibition by insulin. Inhibition of PKB/Akt and PKC
,
known downstream targets of PI3-kinase, or of PMA-activated PKC had no
effect upon insulin inhibition of PKA-induced PEPCK-Luc activity. Thus,
several potential insulin signaling pathways converge on some common
transcription factor or complex, but most of them are dispensable for
insulin inhibition of PKA-induced PEPCK gene transcription. Overall,
our data suggest that an as yet uncharacterized target of PI3-kinase
mediates insulin inhibition of cAMP-induced PEPCK gene transcription or
that alternate pathways may be utilized. Our data are summarized in
Fig. 8, which also specifies the elements determined to be necessary for opposing regulation of PEPCK gene expression by cAMP and insulin (20).

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Fig. 8.
Role of alternate signaling pathways to
insulin inhibition of PKA-induced transcription of the PEPCK gene.
Alternate signaling pathways are shown together with the inhibitors
(black ovals) that block specific enzymes. A line is drawn through the
name of enzymes whose blockade had no effect upon insulin inhibition of
PKA-induced PEPCK transcription. Based on this information, insulin
likely activates PI3-kinase which signals through an as yet
unidentified mediator to modify the function of the CRU-associated
factors (CREB, activator protein-1, C/EBP) or cofactors, that mediate
induction by PKA. This summary is based on the results of the present
study, except for the PD result, which is based on Gabbay et
al. (24), and the Ly294002/wortmannin (LY/Wort) result,
which is also based on Sutherland et al. (9). The
abbreviations are the same as in Fig. 1.
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Lack of Involvement of the Ras/MAPK Pathway in Insulin
Inhibition--
We found that three different inhibitors of the
Ras/MAPK pathway, dn-Ras, dn-Raf, and da-PAC-1, had no effect upon
insulin inhibition. The fact that these three inhibitors target three distinct steps in the Ras/MAPK pathway strengthens the argument that
the Ras/MAPK pathway plays no obligatory role in insulin signaling. In
addition, we showed that insulin does not functionally stimulate the
Ras/MAPK pathway in H4IIe cells, as indicated by its failure to
stimulate G4-PEPCK-Luc expression in the presence of G4-Elk, a
classical MAPK target (32). In contrast, da-Ras both inhibited
PEPCK-Luc and stimulated G4-PEPCK-Luc expression by 20-fold in the
presence of G4-Elk. Thus, if the Ras/MAPK pathway had been activated by
insulin, it would have prevented induction by PKA.
Interestingly, all of the Ras/MAPK pathway inhibitors enhanced
induction by PKA, suggesting that the Ras/MAPK pathway may exert a
constitutive restraining influence on PEPCK gene transcription. This
restraint was relieved when the pathway was inhibited. This relief of
restraint was consistently seen in all experiments and with all enzyme
mutants that interfere with Ras/MAPK signaling. It was most pronounced
with lower amounts of expression vector (data not shown), arguing
against it being an artifact of the expression vector. Other examples
of constitutive restraint are seen with the kinase-defective PKB, K
A mutant and with phosphorylation of Ser-142 in CREB, the mutation of
which potentiates CREB-mediated transcription induction (20,
48).
Recently, a model was proposed for insulin inhibition of PKA-induced
PEPCK gene transcription by Nakajima et al. (11). They used
an H4IIe cell line stably transfected with a truncated PEPCK promoter
(
134/+69) and Ras pathway mutants to argue that insulin activates the
Ras/MAPK pathway, culminating in activation of pp90rsk. The binding of
pp90rsk to CBP was proposed to disrupt the P-CREB·CBP·RNA polymerase II complex and terminate activation by cAMP.
However, recent findings are not consistent with this model. First of
all, the single CREB binding site in the PEPCK promoter is insufficient
for activation of transcription by PKA; the
134/+69 PEPCK promoter
can not mediate induction by PKA in H4IIe cells (20) or in HepG2 cells
(23). Both Roesler and colleagues (21-23, 49) and we (20) have shown
that additional elements from the upstream PEPCK promoter that bind
factors other than CREB are absolutely required for induction of PEPCK
transcription by PKA and inhibition of PKA-induced transcription (20).
When induction is due to CREB alone, as in CREB-GAL4 + PKA-stimulated
transcription of 5XGT-Luc (5 GAL4 sites fused to the minimal PEPCK
promoter), it is inhibited to no greater extent than basal
transcription by insulin (20). Thus, on these grounds alone, more must
be involved than simply the P-CREB·CBP·RNA polymerase II complex, or insulin would have effectively inhibited CREB-dependent
induction by PKA in 5XGT, as it does with the complete PEPCK
promoter.
Second, we show here that the Ras/MAPK pathway is not activated in
response to insulin and that inhibition of the Ras/MAPK at three
different and independent steps has no effect upon insulin inhibition
of PKA-induced PEPCK gene transcription. Furthermore, insulin did not
functionally activate the Ras/MAPK pathway, as evidenced by the lack of
induction of G4-PEPCK with G4-Elk. Finally, using an independent
approach, Granner and colleagues (24) showed that a MEK inhibitor had
no effect upon insulin inhibition of PEPCK transcription and recently
showed that the effects of dn-Ras at very high concentrations involve
inhibition of PI3-kinase activity (50). Thus, the combined evidence
from these studies, utilizing both chemical inhibitors and dominant
negative enzymes, argues against a mechanism for insulin inhibition of
PEPCK gene transcription action based on activation of the Ras/MAPK
pathway.
Signaling through PI3-Kinase Is Required for Insulin
Inhibition--
PI3-kinase activation by insulin is mediated by
phosphorylation of specific tyrosines in IRSs in response to insulin
activation of its receptor tyrosine kinase and is independent of
activation of the Ras/MAPK pathway by insulin (5, 51, 52). Many of the
cellular effects of insulin, including stimulation of protein synthesis, mitogenesis, translocation of GLUT4 transporters, and the
regulation of gene expression require activation of PI3-kinase (1, 9,
10, 14, 53). Treatment of H4IIe cells with LY294002 or wortmannin,
chemical inhibitors of PI3-kinase, directly blocked kinase activity as
well as the ability of insulin to inhibit PEPCK gene transcription
induced by PKA (Fig. 4) or PKA + Dex (9). Thus, activation of
PI3-kinase appears to be an obligatory part of the insulin signaling
pathway.
PI3-kinase is activated by binding to tyrosine phosphorylated IRS (52)
and phosphorylates phosphatidylinositol-4-phosphate and
phosphatidylinositol-4,5-bisphosphate to produce the physiologically significant regulators, phosphatidylinositol-3,4-diphosphate and phosphatidylinositol-3,4,5-trisphosphate (54). These lipid products activate both PKB and PKC. Phosphatidylinositol-3,4-diphosphate and
phosphatidylinositol-3,4,5-trisphosphate directly and indirectly activate protein kinase B (15, 17, 44, 55). Binding of phosphatidylinositol-3,4-diphosphate to the pleckstrin homology domain
of PKB directly activates the enzyme in vitro (16, 33). In
addition, the products of phosphatidylinositol-3-kinase contribute to a
concerted mechanism for activation of PKB (56, 57). The protein kinase,
PDK-1, binds phosphatidylinositol-3,4,5-trisphosphate and
phosphorylates PKB bound to phosphatidylinositol-3,4-diphosphate, resulting in full activation in vivo (58). In addition,
binding of phosphatidylinositol-3,4-diphosphate and/or
phosphatidylinositol-3,4,5-trisphosphate to novel isoforms of PKC (not
regulated by Ca2+ and phospholipids) directly activates
these kinases (12, 18).
Lack of Involvement of the PKB/Akt Pathway in Insulin
Inhibition--
Protein kinase B was first identified as the viral
oncogene, Akt, of which a constitutively active form has
been isolated, gagPKB (17, 59). Insulin-activated PKB phosphorylates
and inactivates glycogen synthase kinase-3, mediating insulin
stimulation of glycogen synthesis (60). Insulin also stimulates GLUT4
glucose transporter translocation through activation of PI3-kinase and PKB (53, 61). In addition, inhibition of either PI3-kinase or of PKB
prevents activation of a survival factor and results in increased
apoptosis (34, 62, 63). PKB dimerization through its pleckstrin
homology domain is required for activation of the purified enzyme
in vitro (33). Mutants of PKB that are defective for
dimerization or that contain only the dimerization domain inhibited
activation of PKB in vitro (33), as well as relieving repression of apoptosis in vivo (34, 62, 63). In addition, a
phosphorylation defective mutant, PKB, K
A, enhanced apoptosis (63)
and blocked insulin-stimulated translocation of the GLUT4 transporter
(61). On the other hand, expression of the constitutively active form
of PKB inhibited apoptosis (34) and overexpression of wild type PKB
enhanced GLUT 4 translocation (61).
In contrast, using these same reagents, we found that overexpression of
PKB mutants with defective dimerization or kinase domains had no effect
upon insulin inhibition of PKA-induced PEPCK-Luc transcription.
Although constitutively active gagPKB depressed transcription, it had
no effect upon inhibition by insulin. Thus, our results suggest that
PKB is not required as a downstream mediator of PI3-kinase in the
insulin signaling pathway utilized for inhibition of PEPCK gene
transcription.
Activation of PKC Is Not Required for Insulin
Inhibition--
Previous work demonstrated that either insulin or
PMA-activated PKC inhibits PEPCK gene transcription (64). The mechanism for inhibition by insulin is independent of that used by PKC because insulin can still suppress hormone-induced PEPCK transcription following down-regulation of PKC by PMA (64). The BIM inhibition data
presented here demonstrate in a new way that the mechanisms utilized by
PMA and insulin are initially independent, although they likely
converge at some mediator (which also can be targeted by da-Ras) that
is required for modification of a crucial transcription factor
interaction. PKC
and other diacylglycerol-independent isoforms of PKC are activated by the lipid products of PI3-kinase (12,
18). Based on inhibition by staurosporine but not phorbol ester
analogs, PKC
was shown to be involved in the stimulation of protein synthesis by insulin (14). The lack of effect of staurosporine on insulin inhibition seen here suggests that
PKC
is not involved in insulin signaling to the PEPCK
gene. Even though our data are complicated by the unexpected
observation of staurosporine induction of PEPCK, this effect was
inhibited by insulin, as was induction by PKA. Thus, neither
PMA-activated PKC isoforms nor novel PI3-kinase-activated PKC isoforms
appear to play an obligatory role in insulin inhibition of PKA-induced
PEPCK gene transcription.
Several Pathways Converge on a Common Target or Complex That
Mediates Insulin Inhibition--
Our current results show that
activation of the Ras/MAPK pathway by da-Ras can inhibit induction of
PEPCK-Luc by PKA, as does insulin. Stimulation of the EGF receptor also
activates MAPK and results in inhibition of hormone-induced PEPCK gene
transcription (65), as does activation of PKC by phorbol esters (Fig.
7) (64). Activation of reactivating kinase (or p38) by oxidant stress
also mimicked the effect of insulin inhibition of PEPCK expression, but
that insulin inhibition was unaffected by an inhibitor of reactivating
kinase (66). Thus, activation of any of several signaling pathways can
inhibit gluconeogenic hormone-induced PEPCK gene expression in a manner
indistinguishable from insulin. This strongly suggests that all of
these pathways converge on a common transcription factor or complex
that is targeted by insulin. Our results indicate that activation of
the Ras/MAPK, PKB, or PKC pathways either does not occur (Ras/MAPK,
PKC) and/or can not account for insulin inhibition of PEPCK
transcription (PKB). On the other hand, inhibition of PI3-kinase
activity abolished insulin inhibition. It should be stressed that the
possibility that insulin works through alternate, parallel paths,
either in vivo or in the H4IIe cell model, can not be
excluded with the reagents available.
We suggest that factors bound to the CRU of the PEPCK promoter interact
with CBP and/or other integrator complexes in some unique way that
permits discrimination of this gene by insulin signals generated
through activation of PI3-kinase. The observation that G4-Elk mediated
induction by da-Ras, while PKA induction of PEPCK-Luc was inhibited by
da-Ras, illustrates how switching a single factor (CREB
Elk) in a
complex regulatory array can alter the response to kinase signals,
i.e. from inhibition to activation of transcription in this
case. In addition, the lack of effect of insulin on Elk-mediated
induction is further evidence that insulin specifically targets a
factor in the CRU rather than acting through a more general mechanism.
The identity of the specific transcription factors within the CRU of
the PEPCK gene that are modified by an insulin-generated signal to
inhibit transcription induction remain to be identified, as does the
mediator activated by PI3-kinase that transmits the signal for
modification of these factors. One or more of the CRU factors and/or
their coactivators that cooperate in a unique way to confer induction
by PKA must be targeted for inhibition by insulin.
We thank Justin Cho for technical assistance.
We thank Drs. R. Maurer, S. Cook, F. McCormick, U. Rapp, P. Coffer, and
T. Frank for their generous gifts of plasmids, and David Spector for
critical reading of the manuscript.