Dominant Negative Forms of Akt (Protein Kinase B) and Atypical Protein Kinase Cλ Do Not Prevent Insulin Inhibition of Phosphoenolpyruvate Carboxykinase Gene Transcription

Ko Kotani,Wataru Ogawa,Yasuhisa Hino,Tadahiro Kitamura,Hikaru Ueno,Wataru Sano,Calum Sutherland,Daryl K Granner,Masato Kasuga

doi:10.1074/jbc.274.30.21305

Abstract

Transcriptional regulation of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting enzyme in hepatic gluconeogenesis, by insulin was investigated with the use of adenovirus vectors encoding various mutant signaling proteins. Insulin inhibited transcription induced by dexamethasone and cAMP of a chloramphenicol acetyltransferase (CAT) reporter gene fused with the PEPCK promoter sequence in HL1C cells stably transfected with this construct. A dominant negative mutant of phosphoinositide (PI) 3-kinase blocked insulin inhibition of transcription of the PEPCK-CAT fusion gene, whereas a constitutively active mutant of PI 3-kinase mimicked the effect of insulin. Although a constitutively active mutant of Akt (protein kinase B) inhibited PEPCK-CAT gene transcription induced by dexamethasone and cAMP, a mutant Akt (Akt-AA) in which the phosphorylation sites targeted by insulin are replaced by alanine did not affect the ability of insulin to inhibit transcription of the fusion gene. Akt-AA almost completely inhibited insulin-induced activation of both endogenous and recombinant Akt in HL1C cells. Furthermore, neither a kinase-defective mutant protein kinase Clambda (PKClambda), which blocked insulin-induced activation of endogenous PKClambda, nor a dominant negative mutant of the small GTPase Rac prevented inhibition of PEPCK-CAT gene transcription by insulin. These data suggest that phosphoinositide 3-kinase is important for insulin-induced inhibition of PEPCK gene transcription and that a downstream effector of phosphoinositide 3-kinase distinct from Akt, PKClambda, and Rac may exist for mediating the effect of insulin.

Highlights

The primary role of insulin is to control the plasma glucose concentration by stimulating glucose transport into muscle and adipose cells as well as by reducing glucose output from the liver [1]
The combination of dexamethasone and 8-chlorophenylthio-cAMP induced a 30 –50-fold increase of Phosphoenolpyruvate carboxykinase (PEPCK) promoter in HL1C cells (Fig. 1B), and incubation of these cells with insulin resulted in inhibition of dexamethasone-cAMP-stimulated transcription from the PEPCK-chloramphenicol acetyltransferase (CAT) reporter gene by ϳ80 –90% (Fig. 1B), which is consistent with the results of previous studies [10, 14, 16]
The stimulation of CAT activity by dexamethasone-cAMP in cells infected with AxCA⌬p85 was similar to that noted in noninfected cells (Fig. 1B), indicating that ⌬p85 did not affect the induction of PEPCK gene transcription by dexamethasone-cAMP

Summary

The primary role of insulin is to control the plasma glucose

Concentration by stimulating glucose transport into muscle and adipose cells as well as by reducing glucose output from the liver [1] These actions of insulin are mediated by activation of effectors, such as glucose transporters and glycogen synthase, or by regulation of the amount of specific protein participants in metabolic pathways [1,2,3,4]. A constitutively active mutant of Ras was shown to inhibit transcription of the PEPCK gene induced by cAMP-dependent protein kinase [9]. We have further explored the role of PI 3-kinase in the inhibition of PEPCK gene transcription by insulin, by using dominant negative and constitutively active mutants of the enzyme. We have investigated the roles of several downstream targets of PI 3-kinase, including Akt, atypical protein kinase C (PKC), and the small GTPase Rac, in this action of insulin

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION