Fig. 1. Critical role of IL-1β signaling in leptin-induced cell cycle arrest in rat hepatocytes. (A) Hepatocytes were isolated from rats as indicated in the methods and treated with leptin (250 ng/mL) for the indicated time periods. The amount of mature IL-1β secreted into the media were determined by western blot analysis using TCA-precipitated protein as described in methods. Loading of the equal amounts of proteins was confirmed by Ponceau red staining. (B, C) Rat hepatocytes were pretreated with IL-1Ra (100 ng/mL) for 1 h (B), or transfected with siRNA (100 nM) targeting IL-1R1 or scrambled control siRNA for 24 h (C), followed by treatment with leptin (250 ng/mL) for additional 24 h. Cells were stained with propidium iodide (PI) and cell cycle analysis was carried out by flow cytometry. Representative images from three independent experiments are presented. The percentage of the cells distributed in each cell cycle phase from three experiments is presented in the right panel. (D) Cells were treated with leptin (250 ng/mL) for the indicated time points. Messenger RNA level of p16 was determined by qRT-PCR using GAPDH as an internal control. (E, F) Hepatocytes were treated with leptin (250 ng/mL) for the indicated time durations (E) or treated with different doses of leptin for 8 h (F). Protein expression level of p16 was determined by western blot analysis. (G) Cells were transfected with p16 siRNA (100 nM) for 24 h followed by treatment with leptin (250 ng/mL) for additional 24 h. Cell cycle analysis was performed by flow cytometry. Representative images from three independent experiments are presented. The percentage of the cells in each cell cycle phase from three experiments is displayed in the right panel. Gene silencing efficiency was monitored by western blot analysis after 24 h of transfection and presented in the upper panel. (H) Hepatocytes were treated with leptin (250 ng/mL) for 6 h in the absence or presence of IL-1Ra (100 ng/mL). p16 mRNA level was determined by qRT-PCR. (I, J) Hepatocytes were pretreated with IL-1Ra (100 ng/mL) (I), or MCC950 (1 µM) or Ac-YVAD-cmk (1 µM) (J) for 1 h followed by incubation with leptin (250 ng/mL) for 8 h. Protein expression levels of p16 were measured by western blot analysis. (K) Hepatocytes were treated with conditioned media obtained from hepatocytes culture treated with leptin in the absence or presence of Ac-YVAD-cmk for 8 h. Expression level of p16 was measured by western blot analysis. β-actin was used as an internal control. In the western blot analyses, the band intensities were analyzed by densitometric analysis and are presented in the lower panel of the images. Values are presented as mean ± SEM, n=3. *p<0.05 compared with control cells. #p<0.05 in comparison to the cells treated with leptin.
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