Table 1.
Primers for quantitative real-time PCR.
Table 2.
Clinical features of subjects with liver biopsy evaluated in the study.
Figure 1.
HFD determines mild steatosis and hepatic iron accumulation with increased ferritin.
Rats were fed for 12 weeks with standard chow (n = 6), or high fat diet (HFD, n = 6), or HFD plus s.c. iron administration at week 8 (250 mg iron sulfate, n = 6). A) Representative images of hematoxylin-eosin staining of liver section with magnification of 10X as indicated. B) Concentrations of serum cholesterol, triglyceride and glucose. C) Serum iron levels. D) Dietary iron intake and hepatic iron concentration (HIC) measured by atomic absorption spectrometry. E) Ferritin H mRNA levels in HFD and HFD+iron rats compared to controls. Gene expression was evaluated by qRT-PCR. F) Ferritin H protein levels evaluated by Western Blotting. G) Densitometric analysis of ferritin H protein levels; β-actin is shown as the loading control. The figure is representative of results obtained in 6 animals per group, replicated in two independent experiments. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs controls.
Figure 2.
Effect of HFD on hepatic iron metabolism genes.
A) Hepatic hepcidin and Fp-1 mRNA levels. Gene expression was evaluated by qt-PCR. B) Serum hepcidin levels were evaluated by an EIA kit. C) Hepatic Fp-1 protein levels evaluated by Western Blotting. D) Densitometric analysis of ferroportin protein levels; β-actin is shown as the loading control. The figure is representative of results obtained in 6 animals per group in two independent experiments. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs controls.
Figure 3.
Effect of HFD on TfR-1 levels.
A) Hepatic TfR-1 mRNA levels in HFD and HFD+iron rats compared to controls. Gene expression was evaluated by qRT-PCR. B) Hepatic TfR-1 protein levels evaluated by Western Blotting. C) Densitometric analysis of TfR-1 protein levels; β-actin is shown as the loading control. The figure is representative of results obtained in 6 animals per group in two independent experiments. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs. controls.
Figure 4.
A) Total IRP1 activity was measured by RNA band shift assay. B) Densitometric analysis of IRP1 activity. C) Hepatic IRP1 protein levels evaluated by Western Blotting. C) Densitometric analysis of IRP1 protein levels; β-actin is shown as the loading control. D). Effect of HFD on IRP1 and IRP2 mRNA levels. Gene expression was evaluated by qRT-PCR. The figure is representative of results obtained in 6 animals per group in two independent experiments. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs controls.
Figure 5.
Effect of FFA treatment on iron metabolism in HepG2 hepatocytes.
HepG2 hepatocytes were incubated with PA (0.66 mM) + OA (1 mM) or with FAC (150 μM) alone or in combination with FFA for 24 hours. A) Cells after Red Oil O staining. Intensity of red color is proportional to lipid accumulation. B) Absorbance of Red Oil O (540 nm). C) Intracellular iron concentration was measured by atomic absorption spectrometry. D) Ferritin H protein levels were evaluated by Western Blotting (lower part). Densitometric analysis of ferritin H protein levels is shown in the upper part of the figure; β-actin is shown as the loading control. E) TfR-1 mRNA levels evaluated by qRT-PCR. F) TfR-1 protein levels were evaluated by Western Blotting (lower part). Densitometric analysis of TfR-1 protein levels is shown in the upper part of the figure; β-actin is shown as the loading control. Results are mean values of three independent experiments, each experimental condition was evaluated in triplicate. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs. controls.
Figure 6.
Effect of fatty acids on IRP1 expression in HepG2 hepatocytes.
A) IRP1 protein levels were evaluated by Western Blotting. B) Densitometric analysis of IRP1 protein levels: β-actin is shown as the loading control. C) IRP1 mRNA levels evaluated by qRT-PCR. Results are mean values of three independent experiments, each experimental condition was evaluated in triplicate. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs. controls.
Figure 7.
IRP1 gene silencing in HepG2 hepatocytes.
A) IRP1 siRNA treatment of HepG2 cells reduced IRP1 mRNA levels compared to control cells (about 60%). B) TfR-1 mRNA levels evaluated by qRT-PCR in HepG2 treated with IRP1 siRNA or Negative silencer siRNA. C) Intracellular iron concentration was measured by atomic absorption spectrometry in HepG2 cells treated with IRP1 siRNA or Negative silencer siRNA. Results are mean values of three independent experiments, each experimental condition was evaluated in triplicate. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs. untreated cells plus transfection reagent (lipofectamine).
Figure 8.
Association between steatosis and expression of iron genes in patients with fatty liver and healthy controls.
A) Expression of hepatic hepcidin, Fp-1, TfR-1 mRNA levels according to liver damage (normal liver and simple steatosis, vs. nonalcoholic steatohepatitis: NASH), evaluated by qRT-PCR. B) Relative expression of TfR-1, Fp-1, and hepcidin according to the presence of siderosis. Values are expressed as means±SD. AU, arbitrary units. *p<0.05 vs. controls.