Fig. 1

Induction of neuronal insulin resistance (IR) in 21DIV primary neuronal cultures. a The schematic illustrates the protocol to induce IR in primary cortical and hippocampal neurons. Representative immunoblots (b) probed with antibodies detecting pAKT/pan-AKT, pInsR/pan-InsR, pIRS1/pan-IRS1; and quantitation of pInsR/pan-InsR ratio (c), pAKT S473/AKT ratio (d) and pIRS1 S612/IRS1 ratio (e) as well as total IRS1 (f) and InsR (g) protein levels in cortical primary neurons upon indicated treatments are shown. β-Actin was used as internal standard for protein loading. c Induction of IR results in a significantly reduced InsR phosphorylation in response to 15-min stimulation with insulin. d Scatter dot-bar plots showing significant reduction in pAKT level responsiveness of neurons to 15-min stimulation with insulin upon induction of IR (n = 10). Treatment of cortical neurons with insulin/TNFα for 24 h resulted in enhanced levels of pIRS1 S612 (e, n = 11 for groups 1 and 2; n = 7 for groups 3 and 4) and reduced levels of total IRS1 (f, n = 11 for groups 1 and 2; n = 9 for groups 3 and 4). h, i Immunoblot analysis of synaptosomes (Syn) and the respective crude membrane fraction (P2) after induction of IR (n = 5). i Induction of IR significantly reduced responsiveness to insulin as measured by pAKT/AKT ratio in synaptosomal fraction. j IR reduced levels of pAKT at synapses labelled with Shank3. Confocal images depict dendrites of hippocampal neurons immunostained with antibodies directed against pAKT S473 and Shank3. Original pixel intensities from 0 to 255 are presented as a gradient lookup table. Scale bar, 10 μm. k Scatter dot plot depicting synaptic pAKT immunofluorescence intensity quantified within a Shank3 mask normalized to control. n = 45 for groups 1 and 4; n = 46 for group 2; n = 43 for group 3; n numbers refer to the number of dendrite segments from different neurons acquired from at least three independent coverslips. l Schematic representation of PI3Kγ activity assay where orange circles represent PI(3,4)P₂ and green circles represent PI(3,4)P₃. m Quantification of the substrate PI(4,5)P₂ through colorimetric reaction in PI(3,4,5)P₃-coated detection plate for competitive binding. Cell extracts treated with the PI3Kγ selective inhibitor AS-605240 during the enzymatic reaction were used as a negative control. n = 8 for groups 1 and 2; n = 6 for group 3. ***P < 0.001, **P < 0.01, *P < 0.05 versus control, by two-way ANOVA followed by Bonferroni’s post-hoc test. Two-tailed Student’s t-test was used in m. n.s. = non-significant. Data are presented as the mean ± SEM