Figure 1.
Effect of leptin on HCT116 cells respiration.
HCT116 cells were treated with DMEM (control), vs. leptin (100 ng/ml), for 24 hours. (A) Basal OCR, (B), Basal ECAR, (C), FCCP-induced maximal OCR (0.4 µM) were measured using the XF24 Analyzer (n=5). *, P< 0.01 vs. control (Student’s t-test). **, P< 0.05 vs. control (Student’s t-test). The results were normalized to cell number and expressed as percentage of control.
Figure 2.
Effect of leptin on HCT116 glycolysis.
HCT116 cells were treated with DMEM (control) vs. leptin (100 ng/ml), for 24 hours. (A) Gene expression levels were detected using quantitative real-time PCR (n=4). (B) Total cell lysates were analyzed by Western blot. (C) Densitometric analysis of the Western blot data was made. * P> 0.05, vs. respective control of each protein (Student’s t-test).
Figure 3.
Effect of leptin on HCT116 cells mitochondria.
HCT116 cells were treated with DMEM (control) vs. leptin (100 ng/ml), for 24 hours. (A, B) Gene expression levels were detected using quantitative real-time PCR (n=4). *, P< 0.05, **, P< 0.01 vs. respective control of each gene (Student’s t-test). (C) Cell lysates were analyzed for cytochrome C (CytC, top panel) and β-actin (bottom panel) by Western blot, and densitometric analysis of the data was made. * P> 0.01, vs. control (Student’s t-test).
Figure 4.
Effects of the obese CM on HCT116 cells respiration.
HCT116 cells were treated for 24 hours with DMEM (control), CM collected from visceral AT of non-obese subjects (n=4) or obese subjects (n=10) and analyzed for their OCR (A) and ECAR (B) levels by using the XF24 Analyzer. *, P< 0.05 vs. non-obese or control (Student’s t-test). (C) Maximal OCR levels following FCCP (0.4 µM) were measured using the XF24 Analyzer. Control (n=9), non-obese (n=4), obese (n=10). *, P< 0.05 vs. non-obese sample (Mann Whitney test). Results were normalized to protein concentration and expressed as percentage of control.
Figure 5.
Effects of the obese CM on HCT116 glycolysis.
HCT 116 cells were treated for 24 hours with CM collected from visceral AT of non-obese subjects (n=4) or obese subjects (n=9). (A) Bax gene expression levels were detected using quantitative real-time PCR. *, P< 0.05 vs. respective non-obese sample of each gene (Mann Whitney test). (B) Cell lysates were analyzed by Western blot. HM-7 and Caco2 were used as controls (see Results) (C), Densitometric analysis of the Western blot data. *, P> 0.01, **, P<0.05 (Two Way ANOVA, Bonferroni test).
Figure 6.
Effects of the obese CM on HCT116 cells mitochondria.
HCT116 cells were treated for 24 hours with DMEM (control), CM collected from visceral AT of non-obese subjects. (A, B) Gene expression levels were detected using quantitative real-time PCR. Obese (n=8), non-obese (n=4). *, P< 0.05, **, P< 0.01 vs. respective non-obese sample of each gene (Mann Whitney test). (C) Gene expression levels were detected using quantitative real-time PCR. Control (n=3), non-obese (n=4), obese (n=9). *, P< 0.05, vs. control (Mann Whitney), ** P< 0.05, vs. non-obese (Mann Whitney). (D) Cell lysates were analyzed for Bax (top panel) or β-actin (bottom panel) antibodies, by Western blot and densitometric analysis was made. Vertical white lines denote image splicing to present only relevant bands, for clarity (shown is a single blot). Control (n=3), non-obese (n=3), obese (n=6). *, P> 0.01 vs. Control (One Way ANOVA, Tukey test). **, P< 0.001, vs. the non-obese samples (One Way ANOVA, Tukey test).
Figure 7.
Leptin involvement in mediating obesity-reduced OCR.
HCT116 cells were treated for 24 hours with CM collected from visceral AT of non-obese or obese subjects, with or without leptin antagonist (1 ng/ml). OCR levels were measured using the XF24 Analyzer. Non-obese (n=4), obese (n=7), non-obese antagonist (LepA non-obese, n=3), obese antagonist (LepA Obese, n=6). *, P< 0.05 vs. samples from obese (Student’s t-test). Results were normalized to protein concentration and expressed as percentage of control.