Fig. 5

mTOR/p70S6K is the key for metabolic-disorder-induced endoplasmic reticulum stress. A Phosphoproteomic analysis of insulin and/or PA-stimulated Ishikawa cells. After treatment with vehicle, 100 nM insulin, 0.5 mM PA or 0.5 mM PA plus 100 nM insulin for 3 h, the expression levels of phosphorylation sites were assessed by phospho-kinase array. Signals of relevant kinases in response to insulin and/or PA stimulation are indicated by numbers. B The relative intensities of p-p70 S6K (T389), Lck (Y394) and STAT2 (Y689) sites. C Immunoblot of p-p70 S6K (Thr389) and its upstream molecular mTOR after insulin and/or PA treatment. D Expression level of mTOR and GRP78 under insulin and/or PA treatment with mTOR inhibition. Non-specific siRNA or mTOR siRNA was transfected into Ishikawa cells for 48 h first. Insulin and/or PA was then added into the treated cells. E Immunoblot of GRP78 and CHOP under insulin and PA combined treatment with or without pre-treatment of ATF6α inhibitor Ceapin-A7 (6 μM), PERK inhibitor ISRIB (0.2 μM) and IRE1 inhibitor 4μ8C (50 μM). F Schematic model of insulin enhancing PA-induced ER stress in EC cells