Figure 8. The role of S1P-derived sPRR on insulin signaling in differentiated 3T3 cells.

The cells were pretreated for 24 hours with vehicle, sPRR-His, PF, or PF + sPRR-His and treated for 20 minutes with vehicle or insulin, followed by measurement of glucose uptake and examination of p-AKT/AKT and Glut4 protein abundances. (A) Glucose uptake (n = 6, repeated for 3 times). (B) Immunoblotting analysis of p-AKT, AKT, and Glut4 in the whole cell lysates of the cells in A exposed to vehicle or sPRR-His for 24 hours (n = 3, repeated for 3 times). The blot was stripped and reprobed with anti-AKT antibody. The same protein samples were run on a separate gel for detecting GAPDH. (C and D) Immunoblotting analysis of the effect of sPRR-His on insulin-induced activation of p-AKT and Glut4 (n = 5, repeated for 2 times). The blot was stripped and reprobed with anti-AKT antibody or anti-Calnexin2 antibody. The cells were pretreated for 24 hours with vehicle or sPRR-His and then treated for 20 minutes with insulin, followed by immunoblotting analysis of p-AKT, AKT, and Glut4. (E) Effect of short-term sPRR-His treatment on basal and insulin-induced glucose uptake (n = 10). (F) ELISA measurement of medium sPRR in cells treated with vehicle or insulin for 20 minutes (n = 10). (G) Effect of PRR-neutralizing antibody on insulin-induced glucose uptake (n = 10). The cells were pretreated for 1 hour with vehicle or antibody and then treated with insulin for 20 minutes, followed by measurement of glucose uptake. *P < 0.05 vs. vehicle/CTR group in A or vehicle group in B–D, ^#P < 0.05 vs. vehicle/insulin group in A or insulin group in C and D. Statistical significance was determined by using ANOVA with the Bonferroni test for multiple comparisons. Data are shown as mean ± SEM.