Fig 2: DYRK1A is increased in insulin-resistant cell models. A, establishment of the insulin-resistant model in SH-SY5Y cells. Vehicle indicates absence of insulin and High ins indicates a chronic high insulin condition (1 μm for 24 h). After pretreatment, the media were removed and the cells were washed three times and then rechallenged with 10 nm insulin for 15 min. Levels of pAKT Ser-473, AKT, pGSK3β Ser-9, GSK3β, IRS-1, and DYRK1A were detected by Western blot, β-actin was used as loading control. B–E, quantification of A. F, establishment of the insulin-resistant model in primary neurons. Vehicle indicates the absence of insulin and High ins indicates a chronic high insulin condition (3 μm for 24 h). After pretreatment, the media were removed and the cells were washed three times and then rechallenged with 10 nm insulin for 15 min. Levels of pAKT Ser-473, AKT, pGSK3β Ser-9, GSK3β, IRS-1, and DYRK1A were detected by Western blot, β-actin was used as loading control. G–J, quantification of F. All quantified results were obtained from three independent experiments. Data are presented as mean ± S.D., *, p < 0.05, p values were calculated by Student's t test (for pAKT Ser-473/AKT and pGSK3β Ser-9/GSK3β changes after 10 nm insulin stimulation) or two-way ANOVA followed by Tukey's multiple comparisons test (for IRS-1 and DYRK1A changes at different conditions).

Fig 3: Comparison of simulated graphs of both un-treated and treated IGF-1R associated HPN models.The simulation graph represents the relative expression level (x-axis) of key entities with respect to time (y-axis). The black sigmoid curves represent the un-treated behaviours and green curves represent the treated behaviours of entities involved in IGF-1R associated signaling. Fig 10A–10H represents the relative change in dynamical behaviours of key proteins (ER-α, IGF-1R, p53, BRCA1, Mdm2, Akt, PI3k and IRS-1) before and after treated with anti-estrogen to be occurred.

Fig 4: C-terminal CX3CL1 fragment (CX3CL1-ct) overexpression in transgenic mice induces insulin/IGF-1 signaling pathways.A, neuron-specific overexpression of CX3CL1-ct in mice was achieved by breeding CX3CL1-ct mice with CaMKIIa-Tet mice. The transgene was turned on by doxycycline withdrawal at P45. Hippocampal and cortical protein lysates from the indicated genotypes of mice were examined with indicated antibodies by Western blotting. CX3CL1-ct/tTa mice had significantly increased expression of insulin receptor (anti-IGFβ) and insulin growth factor-1 (IGF-1) receptor (IGF-1R; detected by anti-IGF-1Rβ). Downstream molecules insulin substrate 1 (IRS1) and IRS2, Akt, PDK1, and Foxo3 were more obviously activated. B, bar graphs show comparative levels normalized to the loading control. N = 3 independent experiments (∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001, one-way ANOVA). IGF, insulin growth factor; P45, postnatal day 45.

Fig 5: DYRK1A ameliorates chronic high insulin-induced insulin resistance in SH-SY5Y cells. A, SH-SY5Y cells were transfected with DYRK1A or control plasmid and then treated in vehicle or high ins. conditions for 24 h to induce insulin resistance, and then rechallenged with 10 nm insulin for 15 min. Levels of pAKT Ser-473, AKT, pGSK3β Ser-9, GSK3β, IRS-1, DYRK1A, and β-actin were detected by Western blot. B–D, quantification of A. All quantified results were obtained from three independent experiments. Data are presented as mean ± S.D., *, p < 0.05, p values were calculated by Student's t test (for C and D) or two-way ANOVA followed by Tukey's multiple comparisons test (for B). E, SH-SY5Y cells were treated with DMSO or 1 μm harmine in vehicle or high ins. conditions for 24 h, and then rechallenged with 10 nm insulin for 15 min. Levels of pAKT Ser-473, AKT, pGSK3β Ser-9, GSK3β, IRS-1, DYRK1A, and β-actin were detected by Western blot. F–H, the quantification of C. All quantified results were obtained from three independent experiments. Data are presented as mean ± S.D., *, p < 0.05, p values were calculated by Student's t test (for G and H) or two-way ANOVA followed by Tukey's multiple comparisons test (for F).