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

• Published in: The Journal of biological chemistry 2003-07, Vol.278 (30), p.28312-28323
• Main Authors: Katome, Takashi, Obata, Toshiyuki, Matsushima, Rie, Masuyama, Norihisa, Cantley, Lewis C., Gotoh, Yukiko, Kishi, Kazuhiro, Shiota, Hiroshi, Ebina, Yousuke
• Format: Article
• Language: English
• Subjects: 3T3 Cells; Adenoviridae - genetics; Animals; Base Sequence; Brain - metabolism; CHO Cells; COS Cells; Cricetinae; Deoxyglucose - pharmacokinetics; Dose-Response Relationship, Drug; Gene Library; Gene Silencing; Genetic Techniques; Glucose Transporter Type 4; Glycogen - metabolism; Immunoblotting; Insulin - metabolism; Luciferases - metabolism; Mice; Molecular Sequence Data; Monosaccharide Transport Proteins - metabolism; Muscle Proteins; Plasmids - metabolism; Precipitin Tests; Protein Isoforms; Protein Transport; Protein-Serine-Threonine Kinases; Proto-Oncogene Proteins - chemistry; Proto-Oncogene Proteins c-akt; Rats; RNA Interference; Time Factors
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M302094200

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.