Fig 2: Ex vivo cholesterol replenishment in hippocampal slices of old mice restores insulin signaling. (a) Addition of cyclodextrin‐cholesterol (referred as MβCD‐Ch or Cholesterol) solution to old mice hippocampal slices induces a 20% increase in membrane cholesterol. (b, c) Cholesterol addition (as in a) reduces hyperactivated Akt and its downstream target p70S6K. (b) Detection of Akt Phospho‐Serine 473; (c) detection of p70S6K Phospho‐Threonine 389. (d) Cholesterol addition to old hippocampal slices reduces Phospho‐Serine 9 GSK3β inhibitory residue. (e) Addition of the cholesterol solution to hippocampal slices of old mice rescues the impairment of insulin‐LTD. Graphic showing insulin‐LTD induction in old mice slices (n = 7) and old mice slices incubated with MβCD‐Ch (n = 9). Black box indicates time of insulin application. Gray box indicates the time of MβCD‐Ch mix application. Representative analogue traces below were collected at the indicated time points. (f, g) Cholesterol replenishment decreases insulin resistance marks on IRS‐1 protein. (f) IRS‐1, Phospho‐Serine 632; (g) Phospho‐Serine 307. The value inside the bars indicates the number of independent experiments. Data are represented as mean ± SEM. t test for (a, b, d, g), Wilcoxon test for (c, f), one‐way ANOVA with post hoc Bonferroni's test for (e). The asterisks indicate the p values (*p < 0.05; **p < 0.01)

Fig 3: IκBα and p65 siRNA Rescued Insulin-stimulated Glucose Uptake and GLUT4 Membrane Translocation on Olanzapine-induced Adipocyte IR. (A) Effect of transfection reagents on insulin-stimulated glucose uptake (black bar) in olanzapine-treated adipocytes (olanzapine (lane 3), IκBα siRNA (lane 5), co-incubation of olanzapine and p65 siRNA (lane 6)). (B) Effect of transfection reagents (olanzapine (3# both left and right), IκBα siRNA (5#), co-incubation of olanzapine and p65 siRNA (6#) on insulin-stimulated GLUT4 translocation in olanzapine-treated adipocytes. (C) Effect of transfection reagents (olanzapine (3# both upper and lower panels), IκBα siRNA (5#), co-incubation of olanzapine and p65 siRNA (6#)) on p-IRS1 protein expression in olanzapine-treated adipocytes. (D) Effect of transfection reagents (olanzapine (3# both upper and lower panels), IκBα siRNA (5#), co-incubation of olanzapine and p65 siRNA (6#)) on p-AKT protein expression in olanzapine-treated adipocytes. lane 1, Control without Control siRNA and olanzapine; lane 2, Control siRNA transfected; lane 3, olanzapine treatment; lane 4, Control siRNA transfected and olanzapine treatment; lane 5, IκBα siRNA transfected; lane 6, p65 siRNA transefected and olanzapine treatment. One-way ANOVA statistical analyses, post hoc t-test, were performed for multiple comparisons of levels of plasma GLUT4, p-AKT, or p-IRS-1 among different treatments. Data in bar graphs are expressed as mean ± SD. *p < 0.05 compared with insulin group; #p < 0.05 compared with olanzapine and insulin treatment group.

Fig 4: ADP induced hepatic gluconeogenesis in mice. (A) Expression of gluconeogenic genes. The mRNA levels of gluconeogenic genes (G6pase, Fbp1, Pck1) were determined with qRT-PCR in the liver tissue of mice after injection of ADP (125 mg/kg) at 1h (n=6). (B) Pyruvate tolerance test (PTT). The mice were injected i.p. with sodium pyruvate (2 g/kg) and ADP (125 mg/kg) after 16h fasting (n=9). (C) Liver glycogen. Glycogen abundance was determined in the liver tissue of mice after ADP (125 mg/kg) injection at 1h (n=6). *p < 0.05; **p < 0.01; ***p < 0.001 vs. the sodium pyruvate group. Phosphorylation level of IRS1 (D), Akt (E), CREB (F) , AMPK (G) in the liver tissue at 1h after ADP injection (n=3). *p < 0.05 vs normal saline control group. The data in this figure represents mean ± SEM.

Fig 5: Effect of high-sucrose diet over the insulin-signaling pathway in peripancreatic and gonadal adipose tissue. Representative blots of IRS1, AKT, p-AKT(S473), P70S6K1, p-P70S6K1(T389), and β-actin as load control, from pooled peripancreatic adipose tissue (pWAT) (A) and gonadal adipose tissue (gWAT) (C) from control and HSD groups. Normalized ratio of phosphorylated AKT and P70S6K1 and total forms in pWAT (B) and gWAT (D). Mean density ± s.e.m. from three independent experiments. Two-way ANOVA was performed for p-AKT (Diet F(1,8) = 22.4, P = 0.0014) *P < 0.01 compared to control; p-P70S6K1 (Diet F(1,8) = 7.89, P = 0.022) with post hoc Tukey’s test with interaction between sex and diet. *P < 0.05 compared to control in pWAT. For p-AKT in gWAT (Sex F(1,8) = 7.51, P = 0.02). One-way ANOVA with post hoc Tukey’s test *P < 0.008 compared to control.