Fig 2: Acute ER stress does not inhibit insulin-stimulated phosphorylation of IRS1 at four specific tyrosine phosphorylation sites in C2C12 myotubes. Tyrosine phosphorylation at (A) Y608, (B) Y628, (C) Y891, and (D) Y935 was analyzed by Western blotting. C2C12 myotubes were serum-starved for 18 h before exposure to 1 µM thapsigargin for 10, 20, or 30 min (left side of figure panels) or to 0.1, 1.0, or 10 µg/ml tunicamycin for 30 min (right side of figure panels), followed by stimulation with the indicated concentrations of insulin for 5 min in the continued presence of thapsigargin or tunicamycin. Bars represent SEs (n = 3). p values for comparison of effects of thapsigargin or tunicamycin within one insulin concentration and for comparison of effects of different insulin concentrations were calculated by ordinary two-way ANOVA with Tukey’s multiple comparisons test (Tukey, 1949a). Abbreviation: a-Tub, a-tubulin.

Fig 3: ER stress does not elicit IRS1 S307/S312 phosphorylation. (A, B) Serum-starved 3T3-F442A cells, (C, D) C2C12 myotubes, and (E, F) Hep G2 cells were treated with 1 µM thapsigargin or 5 µg/ml anisomycin (ANI) for the indicated times. (A, C, E) Cell lysates were analyzed by Western blotting. (B, D, F) Quantification of the Western blots shown in panels A, C, and E. Bars represent SEs (n = 3). p values for comparison of every sample to every other sample were calculated by ordinary one-way ANOVA with Tukey’s multiple comparisons test. Anisomycin-treated samples are positive controls in the phospho-S307/S312-IRS1 Western blots.

Fig 4: Elenbecestat treatment restored decreased InsR levels and insulin-pathway defects in the liver from HFD mice. (A) Western blot images showing pro-InsR, InsRa and InsRβ levels in WT-NCD and WT-HFD+Veh/Ele mice. (B) Statistical analysis of hepatic Pro-InsR, INSR-α, InsRβ in WT-NCD and WT-HFD+Veh/Ele mice (n = 5, 5, 5). (C) Representative immunostaining images of InsRβ and the statistical analysis of hepatic InsRβ in WT-NCD and WT-HFD+Veh/Ele mice (n = 8, 5, 8). Scale bar, 50 μm. (D) Representative western blots and statistical analysis of hepatic p-IRS1/IRS1, p-PI3K/PI3K, p-AKT/AKT and p-GSK3β/GSK3β ratio in WT-NCD and WT-HFD+Veh/Ele mice (n = 5–6). Data are represented as mean ± SEM. (B–D) One-way ANOVA followed by Tukey’s multiple comparisons test. * p ˂ 0.05, ** p ˂ 0.01, *** p ˂ 0.001, ns means no significant differences.