Table 1.
Body weight gain, serum metabolite parameters and hepatic lipid content.
Fig 1.
Hepatic mitochondrial lipid utilization.
Fatty acid utilization capacity was tested measuring both CPT system activity (A) and β-oxidation rate (B). Data are reported as means±ES of 8 different rats for each group. Significant of differences is shown: ** p<0.01 vs. N; *** p<0.001 vs. N; ## p<0.01 vs. D; ### p<0.001 vs. D.
Fig 2.
Sections of liver stained with Hematoxylin and Eosin. A) Control rats (N group). Lipid accumulation in HFD condition (black arrows, panel C, D group). Perivasal cellular vacuolization in presence of DDE (dashed arrows, panels B (N+DDE) and D (D+DDE). Eosinophilic cells in HFD (panel C) and in DDE-treated animals (panel B, N+DDE & panel D, D+DDE) were evidenced (arrowheads). Magnification used: 10X; Scale bar 50 μm.
Fig 3.
Hepatic Kupffer cells detection.
Sections of liver were immunostained with anti-CD68 antibody to detect Kupffer cells in the liver. Panel A, N group; panel B, N+DDE group; panel C, D group; panel D, D+DDE group. Positive cells were evidenced with black arrows. Magnification used: 10X; scale bar applied: 50 μm.
Fig 4.
Analysis of the hepatic lipid peroxidation measured as MDA content in rat liver homogenate. Significance of differences is shown: *p<0.05 vs. N; ** p<0.01 vs. N; #p<0.05 vs. D.
Fig 5.
The level of GSSG was evaluated on total liver homogenate. Significant of differences is shown: ***p<0.001 vs. N; ## p<0,01 vs. D; ### p<0.001 vs. D.
Fig 6.
Western blotting and enzymatic activity of antioxidant enzymes.
(A1) Hepatic cytosolic SOD1 content; (A2) Total SOD activity; (B1) Hepatic mitochondrial SOD2 content; (B2) SOD2 activity; (C1) Hepatic GPx1 content; (C2) Total GPx Activity. WB intensity of the bands were normalized to that of β-actin (in A1 and C1) or COX4 (in B1). Lines legend: 1, N; 2, D; 3, D+DDE; 4, N+DDE. Significance of differences is shown. *p<0.05 vs. N; **p<0.01 vs. N; ***p<0.001 vs. N; #p<0.05 vs. D; ##p<0.01 vs. D; ###p<0.001 vs. D; •p<0.05 vs. N+DDE.
Fig 7.
UCP2 gene expression in rat liver.
UCP2 mRNA levels were determined by using real-time PCR analysis. The amount of UCP2 transcripts was normalized to that of β-actin mRNA and converted in fold change, compared with rats fed with a standard diet (N group). Significance of differences is shown. **p<0.01 vs. N; ***p<0.001 vs. N; ##p<0.01 vs. D; ###p<0.001 vs. D; ••p<0.01 vs. N+DDE.
Fig 8.
UCP2 mRNA localization in rat liver.
Section of liver from rats fed with a standard diet (N, panel A): transcripts are localized only in Kupffer cells (black arrow), hepatocytes are unlabeled (arrowhead). In all the other groups (N+DDE, D, D+DDE, panels B, C and D respectively) transcripts are localized in both Kupffer cells and hepatocytes. Magnification used: 20X; Scale bar: 20 μm.
Fig 9.
(A) Western blotting for the mitochondrial protein UCP2. The amount of UCP2 protein content was normalized to COX4 and converted in fold change, compared with rats fed with a standard diet (N group). (B) Western blotting for UCP1; (C) Western blotting for UCP3. Significance of differences for UCP2 protein levels are shown: *p<0.05 vs. N; **p<0.01 vs. N; #p<0.05 vs. D; ##p<0.01 vs. D; •p<0.05 vs. N+DDE. Lines legend: 1, N; 2, D; 3, D+DDE; 4, N+DDE.
Fig 10.
(A) Under normal diet condition (N group), only phagocytic cells were marked with primary antibody (black arrows), whilst no positivity was detected in the hepatocytes (arrowhead). (C) Immunoreactive hepatocytes (black arrows) were evident following HFD condition and in DDE-treated groups (panel B, D group; panel D, D+DDE group). Notice mitochondria labeled by anti-UCP2 antibody, thus confirming the reliability of the immunostaining. Magnification used: 100X; Scale bar 5μm.
Fig 11.
CD68 and UCP2 localization in N rats.
Immunostaining for CD68 (A) and UCP2 (B) reelevates colocalization of the two antigens. Different arrows were used to compare the same cells positives to the both antibodies used (big arrow, thin arrow, dashed arrow). Magnification used: 40X; Scale bar 50μm.
Fig 12.
Western blotting analysis of CYP450 2B. Data were obtained as media ± standard deviation and graphically represented as fold change vs. N. Significant of difference between groups is shown: ***p<0.001 vs. N; ##p<0.01 vs. D.