Use of RNA Interference-mediated Gene Silencing and Adenoviral Overexpression to Elucidate the Roles of AKT/Protein Kinase B Isoforms in Insulin Actions
| Autor: | Yukiko Gotoh, Kazuhiro Kishi, Lewis C. Cantley, Norihisa Masuyama, Hiroshi Shiota, Yousuke Ebina, Rie Matsushima, Takashi Katome, Toshiyuki Obata |
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| Rok vydání: | 2003 |
| Předmět: |
Time Factors
Monosaccharide Transport Proteins Immunoblotting Molecular Sequence Data Muscle Proteins AKT1 AKT2 CHO Cells Deoxyglucose Protein Serine-Threonine Kinases Biochemistry AKT3 Adenoviridae Mice Cricetinae Proto-Oncogene Proteins Animals Insulin Protein Isoforms Glucose homeostasis Gene Silencing Luciferases Glycogen synthase Molecular Biology Protein kinase B PI3K/AKT/mTOR pathway Gene Library Glucose Transporter Type 4 Base Sequence Dose-Response Relationship Drug biology Brain 3T3 Cells Cell Biology Precipitin Tests Molecular biology Rats Protein Transport Insulin receptor Genetic Techniques COS Cells biology.protein RNA Interference Proto-Oncogene Proteins c-akt Glycogen Plasmids |
| Zdroj: | Journal of Biological Chemistry. 278:28312-28323 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.m302094200 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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