Zhang Wei1,2,, Ville Hietakangas3, Sheena Wee1, Rachel Lim1, Jayantha Gunaratne1 and Stephen M. Cohen1,2.
1- Institute of Molecular and Cell Biology
61 Biopolis Drive
2- Department of Biological Sciences
National University of Singapore
3- Institute of Biotechnology and Department of Biosciences
00014 University of Helsinki
Published in Genes & Development on 15 February 2013.
Endoplasmic reticulum (ER) stress is emerging as a potential contributor to the onset of type 2 diabetes, by making cells insulin resistant. However, our understanding of the mechanisms by which ER stress affects insulin response remains fragmentary. Here we present evidence that the ER stress pathway acts via a conserved signaling mechanism
involving the protein kinase PERK to modulate cellular insulin responsiveness. Insulin signaling via AKT reduces activity of FOXO transcription factors. In some cells, PERK can promote insulin responsiveness. However, we find that PERK also acts oppositely via phosphorylation of FOXO, to promote FOXO activity. Inhibition of PERK improves cellular insulin responsiveness at the level of FOXO activity. We suggest that the protein kinase PERK may be a promising pharmacological target for ameliorating insulin resistance.
Figure Legend: Depletion of human PERK limits FOXO activity
(A) Luciferase reporter assays for endogenous FOXO3 activity in H1299 cells. H1299 cells were transfected to express a FOXO3 responsive luciferase reporter containing 4 synthetic FOXO3 binding sites (4FRE). Cells were serum starved to reduce insulin signaling and treated with the PI3K inhibitor LY294002 to further reduce AKT activity. Data: average ± SD for 6 independent experiments. *** p<0.001.
(B) FOXO3 Luciferase assays as in (A), except that cells were treated with siRNA to deplete endogenous PERK or left untreated. Data: average ± SD for 3 independent experiments. ** p<0.01.
(C) Normalized mRNA levels of FOXO1 targets measure by qPCR. HEPG2 cells were serum starved to reduce insulin signaling and treated with the PI3K inhibitor LY294002 to further reduce AKT activity. 4 of 14 FOXO targets tested showed a consistent increase in mRNA levels (gray bars) compared to control cells in normal mediaum (open bars). Black bars: PERK siRNA treatment. * p<0.05 with/without PERK siRNA treatment. Efficacy of PERK siRNA treatment is shown at the far right.
(D) Immunoblot of HepG2 cells treated with insulin and with siRNA to deplete endogenous PERK. Upper panel probed with antibody to S473-phosphorylated human AKT. Lower panel probed with antibody to total AKT protein.
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