Figure 3.

mTORC2 phosphorylation is necessary for PClP-mediated glucose uptake in brown adipocytes. (A) Representative immunoblot with the relative quantification of p-mTOR (S2481) in brown adipocytes treated with (5 μM) PClP or isoprenaline (1 μM) for 15 min (n = 6). (B) The effect of 5 μM of KU, an mTORC inhibitor, on PClP (5 μM) and isoprenaline (1 μM)-stimulated glucose uptake in brown adipocytes normalized to basal. Cells were pretreated with KU for 30 min prior to stimulation with either PClP or isoprenaline for 5 h (n = 4). The effect of KU pretreatment on p-mTOR (S2481) in brown adipocytes is shown as a representative immunoblot. (C) Representative epifluorescence and TIRF images and their relative quantification showing total GLUT1 and GLUT1 at the plasma membrane, respectively, for brown adipocytes stimulated with PClP (5 μM) or isoprenaline (1 μM) for 5 h in the presence or absence of 2 ug/ml of actinomycin D (n = 4), scale bars represent 50 μm and 10 μm for epifluorescence and TIRF images, respectively. Each value represents mean ± SEM. Statistics for (B) and (C) were calculated using two-way ANOVA followed by Fischer's LSD post-test. Statistics for the remaining data presented were calculated using a paired t-test. ∗P < 0.05, ∗∗P < 0.005, and ∗∗∗P < 0.0005 for differences compared to controls. ## represents statistical differences for P < 0.005 within groups.