An improved in vitro 3T3-L1 adipocyte model of inflammation and insulin resistance

Figure 1.. Tnf-α and hypoxia downregulated insulin sensitivity gene expression, upregulated inflammatory gene expression, and inhibited insulin-stimulated glucose uptake: mature differentiated 3T3-L1 adipocytes were treated with tnf-α and/or hypoxia for 24 h. Insulin sensitive gene markers adipoq (a) and Slc2a4 (b), and inflammation gene markers Ccl2 (c) and IL6 (d) and were assessed using rt-qPCR. gene expression was normalized using Eef2 as reference gene, and the δδct method was used to assess fold changes (n = 3 independent experiments). (e) mature differentiated 3T3-L1 adipocytes were treated with tnf-α and hypoxia for 24 h. Glucose uptake was assessed using a glucose analog (2-NBDG). Cells were stimulated with or without 100 nM insulin for 20 min. Cytochalasin B (cytB) treated cells were used to assess non-specific glucose uptake. The non-specific transport values obtained from the cytB group were subtracted from the values obtained from the cells stimulated with or without insulin (n = 3 independent experiments). The data is presented as the percent of the non-stimulated control. Data was analysed using one-way ANOVA with Tukey post-hoc test (A-E). Bar = mean ± SE. Means that do not…

Figure 2.. Modulating the treatment time with tnf-α in the presence of hypoxia increased the viability of cells in adipocyte insulin resistance model yet maintained the inflammatory and insulin resistant phenotype. Mature differentiated 3T3-L1 adipocytes were treated with tnf-α and hypoxia for 24 h or with tnf-α and hypoxia for 12 h and 24 h, respectively. (a) Quantitation of live/dead staining shown as the percentage of live cells. (b) Flow chart to test the effect of modulating the treatment time of tnf-α in the presence of hypoxia on cell death and makers of inflammation and insulin resistance. (c) Representative fluorescence images of live (green) and dead (red) cells in the control, tnf-α/Hypoxia treated for 12 h/24 h (TNF/HYP, 12 h/24 h) and the tnf-α/Hypoxia treated for 24 h/24 h (TNF/HYP, 24 h/24 h) groups. Images were taken at 20X magnification. (d) Quantitation of live/dead staining shown as the percentage of live cells (n = 3 independent experiments). (E-H) rt-qPCR to assess insulin sensitive and inflammation gene markers. Insulin sensitive gene markers adipoq (E) and Slc2a4 (F), and inflammation gene markers IL6 (G) and Ccl2 (H) are shown. Gene expression was normalized using…

Figure 3.. Tnf-α (12 h) and hypoxia (24 h) induce long-term insulin resistance in an in vitro adipocyte insulin resistance model. (a) Mature differentiated 3T3-L1 adipocytes were treated with tnf-α for 12 h and hypoxia for 24 h to create the insulin resistance group and the untreated cells served as a control. After 24 h, 4.5 g/L glucose and 10% FBS were added to both the treated and control adipocytes and monitored at 48 and 72 h post tnf-α/Hypoxia treatment. (b) Representative fluorescence images of live (green) and dead (red) cells in the control group versus the tnf-α/Hypoxia treated for 12 h/24 h at different time points. Images were taken at 10X magnification. (c) Cell viability was assessed by live/dead staining at 24 h, 48 h, and 72 h (n = 3 independent experiments). Insulin sensitive markers adipoq (d), Slc2a4 (e) and inflammatory marker Ccl2 (f) were assessed using rt-qPCR at different time points. Gene expression was normalized using Eef2 as reference genes, and the δδct method was used to assess fold changes (n = 3 independent experiments). (g-h) glucose uptake was assessed using a glucose analog (2-NBDG) in control and 48 h treated cells (g) and control and 72 h treated cells…