Physiological Role of Akt in Insulin-Stimulated Translocation of GLUT4 in Transfected Rat Adipose Cells

Abstract

Stimulation of glucose transport is among the most important metabolic actions of insulin. Studies in adipose cells have demonstrated that insulin stimulates its receptor to phosphorylate tyrosine residues in IRS-1, leading to activation of phosphatidylinositol 3-kinase, which plays a necessary role in mediating the translocation of the insulin-responsive glucose transporter GLUT4 to the cell surface. Akt is a serine-threonine kinase recently identified as a direct downstream target of phosphatidylinositol 3-kinase. A previous study in 3T3-L1 cells showed that overexpression of a constitutively active mutant of Akt is sufficient to recruit GLUT4 to the cell surface. Since effects of overexpression of signaling molecules in tissue culture models do not always reflect physiological function, we have overexpressed a dominant inhibitory mutant of Akt in rat adipose cells to investigate the effects of inhibiting endogenous Akt in a physiologically relevant insulin target cell. Cells were transfected with either wild type (Akt-WT), constitutively active (Akt-myr), or dominant inhibitory (Akt-K179A) forms of Akt, and effects of overexpression of these constructs on insulin-stimulated translocation of a cotransfected epitope-tagged GLUT4 were studied. Overexpression of Akt-WT resulted in significant translocation of GLUT4 to the cell surface even in the absence of insulin. Interestingly, overexpression of Akt-myr resulted in an even larger effect that was independent of insulin. More importantly, overexpression of Akt-K179A (kinase-inactive mutant) significantly inhibited insulin-stimulated translocation of GLUT4. Taken together, our data suggest that Akt is not only capable of stimulating the translocation of GLUT4 but that endogenous Akt is likely to play a significant physiological role in insulin-stimulated glucose uptake in insulin targets such as muscle and adipose tissue.

Inactivation of glycogen synthase kinase-3β contributes to brain-derived neutrophic factor/TrkB-induced resistance to chemotherapy in neuroblastoma cells

“…The GSK-3h (S9A9) plasmid (21) was used in this study, and a constitutively active Akt and empty vector (EV) were provided by Dr. M.J. Quon (Hypertension-Endocrine Branch, National Heart, Lung, and Blood Institute, National Institutes of Health, Bethesda, MD) (22).…”

Cyclic Nucleotide Phosphodiesterase 3B Is a Downstream Target of Protein Kinase B and May Be Involved in Regulation of Effects of Protein Kinase B on Thymidine Incorporation in FDCP2 Cells

“…For example, PKB has been implicated in insulin-stimulated translocation of GLUT4 transporter and glucose transport in rat adipocytes (4,5), insulin stimulation of glycogen synthesis (6,7), meiotic maturation of frog oocytes (8), and antiapoptotic actions of IGF-1 (9) and IL-3 (10,11).…”

“…The following plasmid vector constructs (4,27) were transformed into Escherichia coli, amplified, and purified.…”

“…These agents did not block IGF-1-induced activation of PKB (assessed by phosphorylation of Crosstide peptide by immunoprecipitated PKB) (data not shown). 4 were immunoprecipitated with anti-PDE3B-NT Ab and subjected to SDS-PAGE, and PDE3B bands were visualized by scanning the dried gel on a PhosphorImager (Molecular Dynamics). Lysates (ϳ2.5 mg protein) of cells incubated without 32 PO 4 were immunoprecipitated and immunoblotted with anti-PDE3B Ab (B), or immunoprecipitated with anti-PKB-CT Ab (D) for assay of PKB activity by phosphorylation of Crosstide (mean cpm Ϯ SEM, n ϭ 3).…”

Effect of Akt inhibition on scatter factor-regulated gene expression in DU-145 human prostate cancer cells

“…Vectors encoding wt-Akt, an active (myr) mutant Akt and a DN kinase inactive (K179A) mutant Akt (DN-Akt) (in the pCIS2 vector) were provided by Dr Michael Quon (National Heart, Lung and Blood Institute, Bethesda, MD, USA) (Cong et al, 1997). An expression vector encoding wt-Pak1 (Adam et al, 2000) was provided by Dr R Kumar (University of Texas MD Anderson Cancer Center, Houston, TX, USA).…”