Table 1.
Mouse primers used in the experiment.
Figure 1.
Effects of the nor-NOHA on diet-induced obesity and liver weight.
(A) body weight, (B) body weight gain, (C) epididymal, perirenal, retroperitoneal, and mesenteric fat weight, (D) liver weight, (E) epididymal fat morphology, and (F) liver morphology in mice fed with normal or high-fat diets (ND and HFD, respectively). C57BL/6 mice were fed an ND or HFD for 12 weeks. In HFD-fed mice, after 7 weeks of obesity induction, the animals were placed on either only HFD or HFD orally gavaged with 40 mg kg−1 nor-NOHA (arginase inhibitor) dissolved in 0.9% NaCl solution for 5 weeks. Body weight was measured weekly. Food intake per mouse was recorded every other day. Representative liver and epididymal fat paraffin sections were stained with hematoxylin and eosin from 3 groups: (i) ND, (ii) HFD, and (iii) HFD with nor-NOHA. Images were captured at 400× magnification. Scale bar, 200 µm. The results are expressed as means ± SEs of mice, and groups were compared by analysis of variance (ANOVA) with Duncan’s multiple range test. The same letter indicates no significant difference between two groups (p<0.05).
Table 2.
Effect of nor-NOHA on body weight, food intake, lipid profile and NO in plasma and the liver among the experimental groups.
Figure 2.
Effects of the nor-NOHA, on the mRNA levels of several genes in mouse livers.
Total RNA was extracted from each liver and subjected to real-time PCR analyses using primers specific for PPAR-γ2 (A), SREBP-1c (B), ADRP (C), SCD-1 (D), ACC (E), FAS (F), PPAR-α (G), and CPT-1α (H). The results are expressed as mean ± SE of mice, and differences between groups were tested by analysis of variance (ANOVA) with Duncan’s multiple range test. The same letter indicates no significant difference between two groups (p<0.05).
Figure 3.
Effects of the nor-NOHA, on the mRNA levels of several genes in the liver.
Total RNA was extracted from each liver and subjected to real-time PCR analyses using primers specific for PGC-1α (A), and PGC-1β (B). The results are expressed as mean ± SE of mice, and differences between groups were tested by analysis of variance (ANOVA) with Duncan’s multiple range test. The same letter indicates no significant difference between two groups (p<0.05).
Figure 4.
Effect of the nor-NOHA on levels of phosphorylated AMPKα at Thr172 (p-AMPKα) in the liver.
Densitometric analysis of p-AMPKα protein levels, using an enzyme-linked immunosorbent assay (ELISA) kit (upper) and protein levels of p-AMPKα and AMPK levels, using a representative western blot image (lower). The results are expressed as mean ± SE of mice, and differences between groups were tested by analysis of variance (ANOVA) with Duncan’s multiple range test. The same letter indicates no significant difference between two groups (p<0.05).
Figure 5.
Effect of the nor-NOHA on lipid accumulation in OLA-induced hepatic steatosis in HepG2 cells.
(A) Cell viability, (B and C) triglyceride contents, accumulation of intracellular lipid, (D and E) nitric oxide (NO, nitrate+nitrite), arginase activity, and (F and G) protein levels of p-AMPKα, arginase 1, and eNOS. The HepG2 cells were treated with different concentrations of oleic acid (0.2–2.0 mM) or ethanol as a control (CTL) (A and B) or with 1.5 mM oleic acid with nor-NOHA (CTL, distilled water, or 1–100 µM) for 30 min (E) or for 24 h (C, D, F, and G). Cell viability was analyzed using an MTT assay after treatment with different concentrations of oleic acid or ethanol for 24 h. TG content was measured with the AdipoRed assay and intracellular lipids were stained with Oil Red O (400× magnification) and then quantified. The data represent the mean percentage levels compared with ethanol-treated cells. Protein levels of p-AMPKα and AMPK levels, showing a representative western blot. The results are expressed as mean ± SE of at least three independent experimental results, and differences between groups were tested by analysis of variance (ANOVA) with Duncan’s multiple range test. The same letter indicates no significant difference between two groups (p<0.05).