Analysis of the role of protein kinase B (cAKT) in insulin-dependent induction of glucokinase and sterol regulatory element-binding protein 1 (SREBP1) mRNAs in hepatocytes

• Published in: Biochemical journal Vol. 376; no. Pt 3; pp. 697 - 705
• Main Authors: Ribaux, Pascale G, Iynedjian, Patrick B
• Format: Journal Article
• Language: English
• Published: England 15.12.2003
• Subjects: Animals; CCAAT-Enhancer-Binding Proteins - biosynthesis; CCAAT-Enhancer-Binding Proteins - genetics; Cells, Cultured; Ceramides - pharmacology; DNA-Binding Proteins - biosynthesis; DNA-Binding Proteins - genetics; Enzyme Induction - drug effects; Gene Expression Regulation; Glucokinase - biosynthesis; Glucokinase - genetics; Hepatocytes - drug effects; Hepatocytes - enzymology; Hepatocytes - metabolism; Insulin - pharmacology; Insulin Antagonists - pharmacology; Male; Mutation; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - physiology; Proto-Oncogene Proteins c-akt; Rats; Rats, Wistar; RNA, Messenger - biosynthesis; Signal Transduction; Sterol Regulatory Element Binding Protein 1; Transcription Factors
• ISSN: 0264-6021; 1470-8728
• DOI: 10.1042/bj20031287

Previous work showed that acute stimulation of a conditionally active protein kinase B (PKB or cAKT) was sufficient to elicit insulin-like induction of GCK (glucokinase) and SREBP1 (sterol regulatory element-binding protein 1) in hepatocytes [Iynedjian, Roth, Fleischmann and Gjinovci (2000) Biochem. J. 351, 621-627; Fleischmann and Iynedjian (2000) Biochem. J. 349, 13-17]. The objective of the present study was to determine whether activation of PKB during insulin stimulation of hepatocytes was a necessary condition for the induction of the two genes. Activation of PKB by insulin was inhibited by pretreatment of the hepatocytes with C2 ceramide. This resulted in the inhibition of insulin-dependent increases in GCK and SREBP1 mRNAs. A triple mutant of PKB failed to interfere with insulin activation of PKB in hepatocytes even at high overexpression levels achieved after adenovirus transduction. A PKB-CaaX fusion protein, which can act as a dominant-negative inhibitor of PKB activation in other cells, was shown to be constitutively activated in hepatocytes and to trigger insulin-like induction of GCK and SREBP1. In addition, constitutive PKB-CaaX activity caused refractoriness of the hepatocytes to insulin signalling at an upstream step resulting in the inhibition of both extracellular-signal-regulated kinase 1/2 and endogenous PKB activation. The stimulation of gene expression by constitutively active PKB-CaaX and inhibition of the insulin effect by ceramide are compatible with a role for PKB in the insulin-dependent induction of GCK and SREBP1.