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.
Division of Molecular Genetics, Institutes for Enzyme Research and Department of Ophthalmology, Graduate School of Medicine, University of Tokushima, 3-18-15 Kuramoto, Tokushima City, Tokushima 770-8503, Japan.
Takashi
T
Katome
Toshiyuki
T
Obata
Rie
R
Matsushima
Norihisa
N
Masuyama
Lewis C
LC
Cantley
Yukiko
Y
Gotoh
Kazuhiro
K
Kishi
Hiroshi
H
Shiota
Yousuke
Y
Ebina
D016475:3T3 Cells; D000256:Adenoviridae / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D001921:Brain / Q000378:metabolism; D016466:CHO Cells; D019556:COS Cells; D006224:Cricetinae; D003847:Deoxyglucose / Q000493:pharmacokinetics; D004305:Dose-Response Relationship, Drug; D015723:Gene Library; D020868:Gene Silencing; D005821:Genetic Techniques; D051275:Glucose Transporter Type 4; D006003:Glycogen / Q000378:metabolism; D015151:Immunoblotting; D007328:Insulin / Q000378:metabolism*; D008156:Luciferases / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009004:Monosaccharide Transport Proteins / Q000378:metabolism; D009124:Muscle Proteins; D010957:Plasmids / Q000378:metabolism; D011233:Precipitin Tests; D020033:Protein Isoforms; D017346:Protein Serine-Threonine Kinases; D021381:Protein Transport; D011518:Proto-Oncogene Proteins / Q000737:chemistry*; D051057:Proto-Oncogene Proteins c-akt; D034622:RNA Interference; D051381:Rats; D013997:Time Factors
D051275:Glucose Transporter Type 4; D007328:Insulin; D009004:Monosaccharide Transport Proteins; D009124:Muscle Proteins; D020033:Protein Isoforms; D011518:Proto-Oncogene Proteins; C495693:Slc2a4 protein, mouse; C495694:Slc2a4 protein, rat; D006003:Glycogen; D003847:Deoxyglucose; D008156:Luciferases; C494920:Akt1 protein, rat; C494922:Akt2 protein, mouse; C494923:Akt2 protein, rat; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt