Table 1.
Primers for semiquantitative PCR.
Figure 1.
Effect of inflammatory stress on hepatic CD36 protein and mRNA expression.
HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. The protein level of CD36 in the cells (A) and livers (B) was examined by western blotting, and β-actin served as the internal reference. The mRNA expression of CD36 in the cells (C) and livers (D) was determined by real-time PCR, and β-actin served as the housekeeping gene. The results are depicted as the mean ± SD from at least three separate experiments. *P<0.05 versus the control, #P<0.01 versus the control.
Figure 2.
Effect of inflammatory cytokines on hepatic CD36 protein stability.
HepG2 cells were pre-incubated in serum-free medium for 24 hours; 14 mg/L CHX in the presence or absence of 25 ng/mL TNF-α or 20 ng/mL IL-6 was then added to the medium, and the cells were incubated for the indicated time. The CD36 protein levels were analysed by western blotting and were normalised to β-actin. The results are depicted as the mean ± SD from three separate experiments. The statistical significance was set at P<0.05.
Figure 3.
Effect of inflammatory stress on CD36 translational efficiency and phosphorylation of the mTOR signalling pathway.
HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 20 ng/mL IL-6 (IL-6). C57BL/6J mice were fed a normal chow diet (Control) or a normal chow diet plus casein injection (Casein) for 14 weeks. A polysomal analysis was performed to detect the CD36 translational efficiency in the cells (A) and livers (B). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (C) and livers (D). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. The results are depicted as the mean ± SD from three separate experiments. *P<0.05 versus the control, #P<0.01 versus the control.
Figure 4.
Effect of rapamycin on the phosphorylation of the mTOR signalling pathway and CD36 translational efficiency under inflammatory stress.
HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet and received a casein injection (Casein) or casein and rapamycin injection (Casein+Rapa) for 14 weeks. Western blotting analyses were performed for p-mTOR (phospho S2448), total mTOR, p-p70S6K (Thr 421/Ser 424), total p70S6K, p-4E-BP1 (Ser 65/Thr 70), total 4E-BP1, p-eIF4E (Ser 209), total eIF4E, and β-actin in the cells (A) and livers (B). The relative band intensities of the phosphorylated protein were normalised to that of the total protein. A polysomal analysis was performed to detect CD36 translational efficiency in the cells (C) and livers (D). The absorbance at 254 nm was given for those conditions, and the positions of CD36, 28S rRNA, 18S rRNA, and β-actin were detected by semiquantitative PCR. The results are depicted as the mean ± SD from three separate experiments. * denotes a significant difference at P<0.05, and # denotes a significant difference at P<0.01.
Figure 5.
Effect of rapamycin on hepatic CD36 mRNA and protein expression under inflammatory stress.
HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet and received a casein injection (Casein) or casein and rapamycin injection (Casein+Rapa) for 14 weeks. The mRNA expression of CD36 in the cells (A) and livers (B) was determined by real-time PCR, and β-actin served as the housekeeping gene. The protein level of CD36 in the cells (C) and livers (D) was examined by western blotting, and β-actin served as the internal reference. The results are depicted as the mean ± SD from at least three separate experiments. * denotes a significant difference at P<0.05, and # denotes a significant difference at P<0.01.
Figure 6.
Effect of CD36 siRNA and rapamycin on hepatic FFA uptake under inflammatory stress.
HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). (A) The effect of CD36 siRNA on FITC-labeled hexadecanoic acid uptake by HepG2 cells under inflammatory stress was determined using fluorescence microscopy. (B) The effect of rapamycin on FITC-labeled hexadecanoic acid uptake by HepG2 cells under inflammatory stress was detected using flow cytometry. The results are depicted as the mean ± SD from three separate experiments. * denotes a significant difference at P<0.05, and # denotes a significant difference at P<0.01.
Figure 7.
Effect of rapamycin on hepatic lipid accumulation under inflammatory stress.
HepG2 cells were pre-incubated for 24 hours in serum-free medium and then incubated for another 24 hours in serum-free medium (Control) or medium containing 25 ng/mL TNF-α (TNF-α) or 25 ng/mL TNF-α plus 10 ng/mL rapamycin (TNF-α+Rapa) or 20 ng/mL IL-6 (IL-6) or 20 ng/mL IL-6 plus 10 ng/mL rapamycin (IL-6+ Rapa). C57BL/6J mice were fed a normal chow diet (Control), a normal chow diet plus casein injection (Casein), or a normal chow diet plus casein and rapamycin injection (Casein+Rapa) for 14 weeks. The lipid accumulation in the cells (A) and livers (B) was observed by Oil Red O staining (original magnification ×400). The concentrations of FFA and TG in the cells (C) and livers (D) were measured as described in the Materials and Methods. The results are depicted as the mean ± SD from at least three separate experiments. * denotes a significant difference at P<0.05, and # denotes a significant difference at P<0.01.