Assessment of the Roles of Mitogen-activated Protein Kinase, Phosphatidylinositol 3-Kinase, Protein Kinase B, and Protein Kinase C in Insulin Inhibition of cAMP-induced Phosphoenolpyruvate Carboxykinase Gene Transcription*

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.


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 Ca 2؉ /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.
Insulin stimulates a variety of changes in growth and me-tabolism 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)(8)(9)(10)(11)(12)(13)(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)(16)(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 CREB⅐CBP⅐RNA 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 (Ϫ271/Ϫ225) (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 PKAinduced 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.

EXPERIMENTAL PROCEDURES
Plasmids-The PEPCK-Luc plasmid is derived from PEPCK-chloramphenicol acetyltransferase, containing Ϫ600/ϩ69 of the PEPCK promoter (25). The G4-PEPCK-Luc plasmid contains the entire promoter region (Ϫ600/ϩ69) 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 dominantactive 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 3 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 3 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 g of luciferase reporter vector ϩ 2 g pRL-SV and 2 g RSV-C␣ and/or 2-10 g 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% CO 2 , and treated with 20% Me 2 SO 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 M; and phorbol 12-myristate 13-acetate, (PMA) (Sigma), 1 M. Following incubation for 20 h, cells were harvested with trypsin treatment and lysed with 1ϫ passive lysis buffer (Promega) and stored at Ϫ80°C for at least 15 min. Lysates (100 l) were obtained by spinning down the lysed cells, and 10 l of lysate was used for the luciferase assay. A dual injector Monolight 3010 Luminometer was utilized to measure luciferase activity. 50 l 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 ϩ 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 M. 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 g of total RNA was quantitated by primer extension analysis, as described previously (36).

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)(38)(39)(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 mecha-nism utilized by insulin to inhibit PEPCK gene transcription (11).
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.

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.
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 ϩ insulin, in the absence or presence of Me 2 SO 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 M), 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).
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.
To further examine the potential role of PKB in insulin signaling, we tested a kinase defective form of the enzyme, PKB, K 3 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 3 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.
Role of Activation of PKC in Insulin Inhibition-Classical isoforms of PKC are regulated by Ca 2ϩ 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 PI3kinase (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 PKAinduced 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 Ϫ6 M) was toxic to H4IIe cells and a lower concentration (10 Ϫ8 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. 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 insu-lin 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 PI3kinase 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).
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 20fold 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 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. 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 3 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 (Ϫ134/ϩ69) 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-CREB⅐CBP⅐RNA 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 Ϫ134/ϩ69 PEPCK promoter can not mediate induction by PKA in H4IIe cells (20) or in HepG2 cells (23). Both Roesler and colleagues (21)(22)(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 ϩ 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-CREB⅐CBP⅐RNA 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 PI3kinase, directly blocked kinase activity as well as the ability of insulin to inhibit PEPCK gene transcription induced by PKA (Fig. 4) or PKA ϩ Dex (9). Thus, activation of PI3-kinase appears to be an obligatory part of the insulin signaling pathway.
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 3 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 PKAinduced 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 stauro-sporine 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 PMAactivated 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 3 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 insulingenerated 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.