Punica granatum L.-derived omega-5 nanoemulsion improves hepatic steatosis in mice fed a high fat diet by increasing fatty acid utilization in hepatocytes

Abstract

Pomegranate seed oil (PSO) is mainly composed of punicic acid (PA), a polyunsaturated fatty acid also known as omega-5 (ω-5), a potent antioxidant associated with a variety of metabolic and cellular beneficial effects. However, the potential benefits of a nanoemulsified version of ω-5 (PSOn) have not been evaluated in a pathological liver condition. Here, we examined whether PSOn had beneficial effects on C57BL/6N mice fed a high-fat diet (HFD), specifically on hepatic steatosis. We observed that PSOn supplementation decreased body weight and body fat mass in control mice, whereas glucose intolerance, insulin resistance, energy expenditure, and hepatic steatosis were improved in both control mice and in mice fed a HFD. Interestingly, PSOn increased fatty acid oxidation in primary hepatocytes and antioxidant gene expression. Altogether, our data indicate that PSOn effectively reduces some of the HFD-derived metabolic syndrome indicators by means of an increase in fatty acid oxidation within hepatocytes.

Conflict of interest statement

KZL received a scholarship and CPM has been funded by Distribuidora BioLife, S.A. de C.V. The rest of the authors declare no potential conflicts of interest.

Figures

Figure 1

Overall effect of the HFD-fed mice supplemented with PSOn. (A) The whole body weight of the different mouse groups is shown during the diet time-course administration; lines represent the mean ± standard error of the mean (SEM) from each group (***p < 0.0001 between the indicated groups). The total calorie (B) and food amount (C) intake are plotted for each of the dietary groups, where the bars indicate the mean (SEM) from each group (***p < 0.01). (D) Representative photographs of mice and their corresponding liver organs for each dietary group at the end of the treatment.

Figure 2

PSOn supplementation does not have an impact on whole body composition. (A) The tissue/body weight ratio is shown as a percentage of each of the indicated organs (WAT, white adipose tissue; BAT, brown adipose tissue; MS, soleus muscle; GM, gastrocnemius muscle); bars represent the mean ± SEM for each mouse group. (B) Body composition analysis by magnetic resonance imaging (MRI) indicates the difference between fat and lean mass in mice before (B) and after (A) the diet and supplementation time (*p < 0.05 & ***p < 0.01).

Figure 3

PSOn treatment reduces glucose intolerance and insulin resistance in HFD-fed mice. The time-course blood glucose levels are plotted for each group after the ipGTT (A) or ipITT (B) as described in the “Materials and methods”. The area under the curve (AUC) was determined and is shown on the right panel for each test. The data are shown as the mean ± SEM. *p < 0.05 by Student’s t test vs. HFD; **p < 0.01 by Student’s t test C vs. C-P; ANOVA **p < 0.01 C and C-P vs. H, H-P1 and H-P7; ANOVA ***p < 0.001 C and C-P vs. H, H-P1 and H-P7.

Figure 4

PSOn increased energy expenditure without modification of substrate. Mice from each diet group were subjected to the indirect calorimetric assay in fasting (grey areas in A & B) and feeding (yellow areas in A & B) conditions. Oxygen (VO2) consumption (A) and the respiratory exchange ratio (RER) (B) were measured at the indicated time points. The quantitation for both parameters is shown after the feeding condition on the right panels. Data represent the mean ± SEM for each group; *p < 0.05; ***p < 0.001.

Figure 5

PSOn supplementation reverses HFD-induced hepatic steatosis. Representative micrographs of liver histological sections stained with haematoxylin–eosin (H&E, left panel) and oil red O (ORO, right panel) from the indicated mouse groups. The objective magnification is indicated at the top of each panel. Scale bars in ×20 and ×40 are 100 μM and 50 μM, respectively.

Figure 6

PSOn increases fatty acid oxidation in hepatocytes. Assessment of the mitochondrial function based on the oxygen consumption rate (OCR) in a time-course experiment is indicated for control (A) or stimulated (B) conditions. The endogenous (BSA alone) (C) and exogenous (Palmitate:BSA) (D) fatty acid oxidation was evaluated in basal or maximal conditions for control or PSOn-supplemented cells in the presence ( +) or absence (−) of etomoxir (eto) and plotted as the OCR. Bars indicate the mean ± SEM for each group (***p < 0.01; FCCP, carbonyl cyanide-p-trifluoromethoxyphenylhydrazone).

Figure 7

PSOn increases the antioxidant- and lipid metabolism-related gene expression. Determination of transcripts was made by total RNA extraction from liver tissue and quantitated by qPCR. Representative data are shown. The bars represent the mean ± SEM of the relative expression for each gene and the indicated mouse groups (Aox1, aldehyde oxidase; Gsta4, glutathione S-transferase 4; Nqo1, NADPH-quinone dehydrogenase 1; Nfe2, nuclear factor erythroid 2; Prdx1, peroxiredoxin 1). *p < 0.05; **p < 0.001 vs. HFD group.