Figure 6

(a) Adipocytes were insulin-stimulated (0.1 nM) for 30 min, followed by western blot analysis against IRS-1 (total and pY612), Akt (total and pS473), AS160 (total and pT642), using adipocytes from c57BL6/J mice fed chow (4 weeks), HFD (4 weeks) or HFD 2 weeks followed by chow 2 weeks (reversed group). n = 4–6 biologic replicates/condition. HSP90 used as a loading control. (b) Epididymal (left graph) and Inguinal (right graph) adipocytes isolated after chow, HFD or reversed feeding were subjected to tracer glucose uptake assay, either non-stimulated (basal), or insulin-stimulated (0.1 nM). n = 3 independent experiments, each sample measured in triplicates. (c) GLUT4 protein expression in adipocyte lysates from chow, HFD and reverse. n = 3 biologic replicates/condition, HSP90 used as a loading control. (d) Epididymal adipocytes (chow) were pre-treated with either DMSO (control), Latrunculin B (LatB) or Jasplakinolide (JASP) for 20 min, and thereafter subjected to tracer glucose uptake assay, either non-stimulated (basal), or insulin-stimulated (0.01 nM or 0.1 nM) for 20 min. n = 3 independent experiments, each sample measured in triplicates. Data expressed as fold of control (DMSO in basal state). Far right graph: quantification (western blot) of Akt and AS160 phosphorylation (pS473 and pT642, respectively) in cells treated with either DMSO, LatB or JASP, with or without 0.1 nM insulin. (e) Western blot analysis to detect protein expression of total and phosphorylated IRS-1 (pS632/635) in adipocyte lysates from the three feeding groups. n = 4–6 biologic replicates/condition, HSP90 used as a loading control. Data presented as mean ± SD, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, and ****p ≤ 0.0001.