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. 2016 Jun 20;65(9):2591–2605. doi: 10.2337/db16-0243

Figure 5.

Figure 5

Bnip3 KD impairs insulin-stimulated glucose uptake in 3T3-L1 adipocytes. A: Basal and insulin-mediated 2-DG uptake in 3T3-L1 adipocytes transduced with either shCtrl, shB3_1 or shB3_2 and differentiated for 8 days. B: Relative mRNA levels of adipogenic markers. C: Phospho-Akt (p-Akt)/PKB, phospo-ERK1/2 (pERK), and phospho-rpS6 (prpS6) protein levels in 3T3-L1 adipocytes starved for 2 h and treated with or without insulin (10 nmol/L) for 15 min. D: Flow cytometric analysis of 7XMyc-GLUT4 plasma membrane occupancy in 3T3-L1 adipocytes transduced with shCtrl or shB3_2, together with 7XMyc-GLUT4-GFP, starved for 5 h, and incubated with insulin (0–100 nmol/L) for 15 min. E: Basal and insulin-stimulated 3-MG transport. A.U., arbitrary units. F: Analysis of steady-state levels of cellular ATP, glucose (Glc), and glucose-6-phosphate (G6P). G: Insulin-stimulated 2-DG uptake in the presence of oligomycin (200 nmol/L) for 2 h. Nontreated (N.T.) values are expressed as 100% for both shCtrl and shB3_2 to address the relative sensitivity to oligomycin. H and I: Bnip3 and Nix protein levels (H) and basal and insulin-stimulated 2-DG transport (I) in shCtrl or in shB3_1 adipocytes stably transduced with inducible constructs expressing the indicated Bnip3 deletion mutants. Values are expressed as mean ± SEM (n = 3 or 4 in A–I, except for n = 6 in D and F). *P < 0.05; **P < 0.01.