Use of RNA Interference-mediated Gene Silencing and Adenoviral Overexpression to Elucidate the Roles of AKT/Protein Kinase B Isoforms in Insulin Actions*

Insulin plays a central role in the regulation of glucose homeostasis in part by stimulating glucose uptake and glycogen synthesis. The serine/threonine protein kinase Akt has been proposed to mediate insulin signaling in several processes. However, it is unclear whether Akt is involved in insulin-stimulated glucose uptake and which isoforms of Akt are responsible for each insulin action. We confirmed that expression of a constitutively active Akt, using an adenoviral expression vector, promoted translocation of glucose transporter 4 (GLUT4) to plasma membrane, 2-deoxyglucose (2-DG) uptake, and glycogen synthesis in both Chinese hamster ovary cells and 3T3-L1 adipocytes. Inhibition of Akt either by adenoviral expression of a dominant negative Akt or by the introduction of synthetic 21-mer short interference RNA against Akt markedly reduced insulin-stimulated GLUT4 translocation, 2-DG uptake, and glycogen synthesis. Experiments with isoform-specific short interference RNA revealed that Akt2, and Akt1 to a lesser extent, has an essential role in insulin-stimulated GLUT4 translocation and 2-DG uptake in both cell lines, whereas Akt1 and Akt2 contribute equally to insulin-stimulated glycogen synthesis. These data suggest a prerequisite role of Akt in insulin-stimulated glucose uptake and distinct functions among Akt isoforms.

[1]  S. Staal.  Molecular cloning of the akt oncogene and its human homologues AKT1 and AKT2: amplification of AKT1 in a primary human gastric adenocarcinoma. , 1987, Proceedings of the National Academy of Sciences of the United States of America.

[2]  J. Hartley,et al.  Thymic lymphoma induction by the AKT8 murine retrovirus , 1988, The Journal of experimental medicine.

[3]  J. Woodgett,et al.  Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. , 1991, European journal of biochemistry.

[4]  J. Testa,et al.  A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region. , 1991, Science.

[5]  Y. Ebina,et al.  Direct demonstration of insulin-induced GLUT4 translocation to the surface of intact cells by insertion of a c-myc epitope into an exofacial GLUT4 domain. , 1993, The Journal of biological chemistry.

[6]  Y. Ebina,et al.  Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase. , 1993, Biochemical and biophysical research communications.

[7]  P. Cohen,et al.  Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B , 1995, Nature.

[8]  M. Birnbaum,et al.  Expression of a Constitutively Active Akt Ser/Thr Kinase in 3T3-L1 Adipocytes Stimulates Glucose Uptake and Glucose Transporter 4 Translocation* , 1996, The Journal of Biological Chemistry.

[9]  F. McCormick,et al.  Dual role of phosphatidylinositol-3,4,5-trisphosphate in the activation of protein kinase B. , 1997, Science.

[10]  P. Cohen,et al.  Characterization of a 3-phosphoinositide-dependent protein kinase which phosphorylates and activates protein kinase Bα , 1997, Current Biology.

[11]  David R. Kaplan,et al.  Direct Regulation of the Akt Proto-Oncogene Product by Phosphatidylinositol-3,4-bisphosphate , 1997, Science.

[12]  M. Birnbaum,et al.  Insulin Increases the Association of Akt-2 with Glut4-containing Vesicles* , 1998, The Journal of Biological Chemistry.

[13]  M. Kasuga,et al.  Requirement for Activation of the Serine-Threonine Kinase Akt (Protein Kinase B) in Insulin Stimulation of Protein Synthesis but Not of Glucose Transport , 1998, Molecular and Cellular Biology.

[14]  J. Woodgett,et al.  Protein kinase B (c-Akt): a multifunctional mediator of phosphatidylinositol 3-kinase activation. , 1998, The Biochemical journal.

[15]  Y. Nakaya,et al.  Bradykinin directly triggers GLUT4 translocation via an insulin-independent pathway. , 1998, Diabetes.

[16]  M. Kasuga,et al.  Requirement of Atypical Protein Kinase Cλ for Insulin Stimulation of Glucose Uptake but Not for Akt Activation in 3T3-L1 Adipocytes , 1998, Molecular and Cellular Biology.

[17]  F. McCormick,et al.  Protein kinase B kinases that mediate phosphatidylinositol 3,4,5-trisphosphate-dependent activation of protein kinase B. , 1998, Science.

[18]  A. Klippel,et al.  Protein Kinase B/Akt Mediates Effects of Insulin on Hepatic Insulin-like Growth Factor-binding Protein-1 Gene Expression through a Conserved Insulin Response Sequence* , 1998, The Journal of Biological Chemistry.

[19]  H. Maegawa,et al.  High glucose-induced abnormal epidermal growth factor signaling. , 1998, Journal of biochemistry.

[20]  P. Cohen,et al.  Phosphorylation of Serine 256 by Protein Kinase B Disrupts Transactivation by FKHR and Mediates Effects of Insulin on Insulin-like Growth Factor-binding Protein-1 Promoter Activity through a Conserved Insulin Response Sequence* , 1999, The Journal of Biological Chemistry.

[21]  D. James,et al.  A Role for Protein Kinase Bβ/Akt2 in Insulin-Stimulated GLUT4 Translocation in Adipocytes , 1999, Molecular and Cellular Biology.

[22]  A. Fire,et al.  RNA-triggered gene silencing. , 1999, Trends in genetics : TIG.

[23]  J. Woodgett,et al.  Protein Kinase B/Akt Participates in GLUT4 Translocation by Insulin in L6 Myoblasts , 1999, Molecular and Cellular Biology.

[24]  K. Kandror,et al.  Akt-2 Binds to Glut4-containing Vesicles and Phosphorylates Their Component Proteins in Response to Insulin* , 1999, The Journal of Biological Chemistry.

[25]  R. Lehmann,et al.  Targeted mRNA degradation by double-stranded RNA in vitro. , 1999, Genes & development.

[26]  P. Sharp,et al.  RNAi and double-strand RNA. , 1999, Genes & development.

[27]  M. Greenberg,et al.  Akt Promotes Cell Survival by Phosphorylating and Inhibiting a Forkhead Transcription Factor , 1999, Cell.

[28]  J. Laine,et al.  The protooncogene TCL1 is an Akt kinase coactivator. , 2000, Molecular cell.

[29]  Toshiyuki Obata,et al.  Peptide and Protein Library Screening Defines Optimal Substrate Motifs for AKT/PKB* , 2000, The Journal of Biological Chemistry.

[30]  K. Kishi,et al.  Overexpression of wild-type Akt1 promoted insulin-stimulated p70S6 kinase (p70S6K) activity and affected GSK3 beta regulation, but did not promote insulin-stimulated GLUT4 translocation or glucose transport in L6 myotubes. , 2000, The journal of medical investigation : JMI.

[31]  A. Newton,et al.  Akt/Protein Kinase B Is Regulated by Autophosphorylation at the Hypothetical PDK-2 Site* , 2000, The Journal of Biological Chemistry.

[32]  Dan Liu,et al.  Identification of CISK, a new member of the SGK kinase family that promotes IL-3-dependent survival , 2000, Current Biology.

[33]  P. Hawkins,et al.  Gi-mediated translocation of GLUT4 is independent of p85/p110α and p110γ phosphoinositide 3-kinases but might involve the activation of Akt kinase , 2000 .

[34]  B. Hemmings,et al.  Ten years of protein kinase B signalling: a hard Akt to follow. , 2001, Trends in biochemical sciences.

[35]  T. Tuschl,et al.  RNA interference is mediated by 21- and 22-nucleotide RNAs. , 2001, Genes & development.

[36]  K. Kaestner,et al.  Insulin Resistance and a Diabetes Mellitus-Like Syndrome in Mice Lacking the Protein Kinase Akt2 (PKBβ) , 2001 .

[37]  K. Nishikura,et al.  A Short Primer on RNAi RNA-Directed RNA Polymerase Acts as a Key Catalyst , 2001, Cell.

[38]  I. Roninson,et al.  Growth retardation and increased apoptosis in mice with homozygous disruption of the Akt1 gene. , 2001, Genes & development.

[39]  N. Masuyama,et al.  Akt Inhibits the Orphan Nuclear Receptor Nur77 and T-cell Apoptosis* , 2001, The Journal of Biological Chemistry.

[40]  T. Tuschl,et al.  Functional anatomy of siRNAs for mediating efficient RNAi in Drosophila melanogaster embryo lysate , 2001, The EMBO journal.

[41]  M. Birnbaum,et al.  Akt1/PKBα Is Required for Normal Growth but Dispensable for Maintenance of Glucose Homeostasis in Mice* , 2001, The Journal of Biological Chemistry.

[42]  T. Tuschl,et al.  Duplexes of 21-nucleotide RNAs mediate RNA interference in cultured mammalian cells , 2001, Nature.

[43]  K. Morino,et al.  Membrane Localization of 3-Phosphoinositide-dependent Protein Kinase-1 Stimulates Activities of Akt and Atypical Protein Kinase C but Does Not Stimulate Glucose Transport and Glycogen Synthesis in 3T3-L1 Adipocytes* , 2002, The Journal of Biological Chemistry.

[44]  N. LaRusso,et al.  Channel-mediated water movement across enclosed or perfused mouse intrahepatic bile duct units. , 2002, American journal of physiology. Cell physiology.

[45]  M. Birnbaum,et al.  Akt1 Regulates a JNK Scaffold during Excitotoxic Apoptosis , 2002, Neuron.

[46]  J. Laine,et al.  Differential Regulation of Akt Kinase Isoforms by the Members of the TCL1 Oncogene Family* , 2002, The Journal of Biological Chemistry.

[47]  M. Stern,et al.  Identification of Akt Association and Oligomerization Domains of the Akt Kinase Coactivator TCL1 , 2002, Molecular and Cellular Biology.

[48]  P. Kang,et al.  Akt/Protein Kinase B Promotes Organ Growth in Transgenic Mice , 2002, Molecular and Cellular Biology.

[49]  M. Birnbaum,et al.  Role of Akt/protein kinase B in metabolism , 2002, Trends in Endocrinology & Metabolism.

[50]  D. Carling,et al.  Regulation of glycogen synthase by glucose and glycogen: a possible role for AMP-activated protein kinase. , 2003, Diabetes.