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

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

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

PSOn oral gavage administration has no detrimental effects on mice

As a first approach, we decided to perform toxicological experiments on mice to test whether PSOn has any harmful health risk on global homeostasis. In previous reports, other researchers have administered PSO or other organic oils by direct addition to chow diets; however, this strategy has some disadvantages due to the uncertainty of the total and most reliable amount of food consumption per mice. To overcome this problem, we decided to take advantage of the water solubility of PSOn and feed the mice with an oral gavage strategy. First, we tested the administration of three different single doses (1, 2 or 4 mg/g) of PSOn in 8-week-old C57BL/6N mice. Fifteen days later (acute protocol, see Supplementary Materials), peripheral blood and internal organs (liver, kidneys, among others) were obtained to perform histological and biochemical analyses. Mice did not show any signs of abnormal behaviour after oral administration or at the time of death. We did not find any gross disturbance in body weight gain or organ/body weight ratio (Supplementary Fig. 1A,B). In addition, liver and kidney histology did not reveal tissue damage at the cellular level (Supplementary Fig. 1C) or in the biochemical parameters (alanine-amino-transferase (ALT), aspartate-amino-transferase (AST), triglycerides, cholesterol, albumin, and glucose) (Supplementary Fig. 1D). In an independent experiment, we also evaluated the long-term effect of PSOn in an every-other-day administration basis (chronic protocol, Supplementary Materials). Behaviour, food consumption, and body weight were monitored and, as before, blood and tissues were collected. The same parameters were evaluated; however, we did not observe any histological or biochemical differences (Supplementary Fig. 2A–D). Thus, we concluded that PSOn had no detrimental effects on mouse homeostasis.

PSOn supplementation does not impact whole body weight in mice fed a high-fat diet

In previous reports, PSO supplementation has been related to prevention of diet-induced obesity and other beneficial effects. As high-fat diet-mediated fatty liver disease is tightly associated with obesity, we hypothesized that PSOn could prevent or reverse obesity and triglyceride hepatic deposition in our mice. Interestingly, as shown in Fig. 1A, we found that control mice supplemented with PSOn (C-P) gained significantly less body weight compared to chow-fed mice (3.48 g vs. 8.04 g, respectively, p < 0.001); on the contrary, and as expected, the high-fat diet (HFD) significantly increased mouse body weight compared to the chow or chow-PSOn group (p < 0.001); unexpectedly, our HFD-fed mice supplemented with PSOn at week 1 (H-P1) or at week 7 (H-P7) did not lose any body weight compared to the HFD-fed group (Fig. 1A). Interestingly, the higher body weight gain observed in the HFD group was not due to differences in food consumption, since all of the groups showed similar food intake behaviour, but to an increase in energy intake (Fig. 1B,C). In addition, and as expected, the HFD-fed mice had an obese body phenotype and their livers showed signs of steatosis when compared to control mice (Fig. 1D). With regard to the lipid profile, we did not observe any favourable improvement. However, AST and ALT enzymes were slightly lower after PSOn supplementation in both the H-P1 and H-P7 mouse groups compared to the HFD group; albumin was also significantly augmented in the H-P7 group, indicating that PSOn might improve hepatic damage induced by HFD (Table 1).

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.

Table 1 Biochemical characteristics.

PSOn administration does not impact body composition

We then decided to assess the relationship between whole mouse body weight and organ weight in order to determine whether mice suffer from any gross organ size disturbance after PSOn treatment. As observed in Fig. 2A, mice under HFD had a significantly lower liver/body weight ratio, which can be explained by the higher body mass when compared to controls, whereas white adipose tissue mass showed the opposite ratio (p < 0.001), surely due to the fat mass gain at the expense of muscle mass loss, compared to the control mouse groups (Fig. 2A). To further corroborate these data, we performed magnetic resonance imaging to evaluate whether those mice were different with respect to body fat or lean mass. Here, we confirmed that HFD-fed mice gained significantly more fat mass on average, 21.4 g (p < 0.001), compared to their basal levels and the control groups, while the muscle mass was proportionally lower (Fig. 2B), and PSOn treatment did not modify the loss of lean mass. These results indicate that PSOn has no effect on body composition in mice fed a HFD.

Figure 2
figure2

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).

PSOn supplementation reduces glucose intolerance and insulin resistance in HFD-fed mice

As PSO has been reported to play a role in insulin and glucose regulation, we sought to investigate whether the supplementation of PSOn modifies the glucose intolerance induced by the HFD. Thus, we performed intraperitoneal glucose and insulin tolerance tests. As expected, control mice efficiently regulate their glucose levels along the time interval, reaching basal levels at the end of the experiment (Fig. 3A). In contrast, HFD-fed mice showed glucose intolerance (p < 0.0001 vs. C or C-P groups) and remained hyperglycaemic after 120 min (Fig. 3A, left panel). The area under the curve plots showed that PSOn supplementation in the H-P1 group did not improve the glucose intolerance induced by HFD; however, when administered at week 7 (H-P7), PSOn significantly (p < 0.05) diminished blood glucose levels (Fig. 3A, right panel). Interestingly, the insulin tolerance test showed a significant drop in glucose levels in the C-P mouse group when compared with the control group (Fig. 3B, p < 0.01); we observed that HP1 has no effect on insulin sensitivity, whereas the glucose levels showed a striking decline in the H-P7 group, although they did not reach a significant difference when compared to the HFD group (p < 0.17) (Fig. 3B, right panel). Based on these observations, we conclude that PSOn is able to improve glucose and insulin metabolism in control and HFD-fed mice.

Figure 3
figure3

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.

PSOn supplementation increases energy expenditure without modifying substrate utilization

To evaluate whether PSOn modifies energy expenditure or substrate utilization in HFD-fed mice, we conducted an indirect calorimetric assay. Notably, we observed that PSOn significantly increased oxygen consumption by 1.2-fold in mice fed the control diet (p < 0.0001). As expected, HFD-fed mice significantly reduced their oxygen consumption rate by a 0.9-fold change (p < 0.001) compared to control mice (Fig. 4A, right plot). Interestingly, when HFD-fed mice were supplemented with PSOn since the beginning of the diet regime, oxygen consumption did not improve and, instead, it remained the same as for mice from the high-fat diet group (3,465.5 vs. 3,494.5, ml/kg/h, p < 0.99) (Fig. 4A). However, HFD-fed mice supplemented in week 7 (H-P7) successfully and significantly increased their oxygen consumption rate almost to the level of the chow control mouse group (3,681 vs. 3,748, ml/kg/h, p < 0.99) (Fig. 4A). With regard to the respiratory exchange ratio (RER), as can be observed in Fig. 4B, all of the groups had a RER of approximately 0.75 under fasted conditions, indicating that fatty acids are the main substrate being oxidized. As expected, the control groups increased their RER to 1 in re-fed conditions, indicating the switch to glucose as the substrate being oxidized. However, the HFD-fed mice continued to have a RER of 0.75, indicating the presence of metabolic inflexibility. PSOn supplementation, either from the beginning (H-P1) or in week 7 (H-P7), did not improve this metabolic inflexibility (Fig. 4B). Altogether, these results indicate that PSO increases energy expenditure without modifying substrate utilization.

Figure 4
figure4

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.

PSOn supplementation reverses high-fat diet-induced hepatic steatosis

Given that hepatic steatosis, glucose intolerance and insulin resistance are common complications during obesity and that our data indicate that PSOn treatment effectively improved glucose and insulin levels in HFD-fed mice, we decided to evaluate if the hepatic steatosis could also be improved. To this end, we performed histological oil red O (ORO) staining on mouse liver tissue sections. As observed in Fig. 5, HFD-fed mice clearly showed lipid droplet accumulation in the liver parenchyma compared to control diet-fed mice. This pattern was also observed in the H-P1 group, although to a lesser extent. In contrast, mice treated with PSOn at week 7 (H-P7) clearly displayed a reversion of this phenotype at the end of the experiment (Fig. 5). Taken together, these data suggest that PSOn does not cause tissue damage and reverses but does not prevent liver lipid droplet accumulation.

Figure 5
figure5

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.

PSOn increases fatty acid oxidation in hepatocytes

As PSOn treatment reduced hepatic steatosis, we evaluated mitochondrial function and fatty acid oxidation in primary hepatocytes to determine whether PSOn exerts a direct effect on these cells. First, we tested the safety of PSOn exposure using different cell lines in vitro. We measured cell viability and whole integrity by means of dose–response and time-course experiments in myoblasts (C2C12 cells), fibroblasts (3T3-L1, cells) and mouse brain primary astrocytes. Interestingly, increasing concentrations of PSOn did not modify cell viability or whole cell morphology in any of the cells tested (Supplementary Fig. 3A,B). Therefore, we incubated mouse primary hepatocytes with 1 mg/ml PSOn for 3 or 18 h and performed a mitochondrial stress test in order to determine the effect of PSOn on mitochondrial function using an extracellular flux analyser (SeahorseXFe96 analyser, Agilent Technologies). Unexpectedly, the basal oxygen consumption rate (OCR) and key parameters of mitochondrial function such as ATP production, proton leak, maximal respiration, or spare respiratory capacity were not different between hepatocytes incubated with vehicle or PSOn at the indicated times (Supplementary Fig. 4), indicating that PSOn does not modify mitochondrial function. Thus, we then evaluated the oxidation of exogenous and endogenous fatty acids to determine whether the decrease in hepatic lipid droplets was specifically due to an increase in fatty acid oxidation. To test this hypothesis, we performed a mitochondrial stress test in primary hepatocytes incubated in the presence or absence of PSOn (1 mg/ml) for 3 h in a medium containing BSA to measure endogenous FA oxidation or with palmitate-BSA to measure exogenous FA oxidation, with or without the carnitine-palmitoyltransferase-1A (CPT1A) inhibitor, etomoxir. Interestingly, PSOn increased basal respiration and maximal respiration when glucose was limited (Fig. 6A,B). Furthermore, PSOn increased the endogenous FA oxidation capacity of hepatocytes in both basal and maximal respiration conditions (p < 0.01) (Fig. 6C,D). Altogether, these data indicate that PSOn significantly increases fatty acid oxidation capacity in hepatocytes and thus helps to reverse the hepatic steatosis induced by a HFD.

Figure 6
figure6

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).

PSOn increases the antioxidant- and lipid metabolism-related gene expression

As PSO has been reported to have antioxidant properties11, we decided to measure the expression of several antioxidant transcripts in the liver of the different mouse groups to evaluate whether PSOn preserved the antioxidant properties. As we expected, the PCR experiments showed that PSOn supplementation increased the expression of several antioxidant genes (aldehyde oxidase 1 (Aox1), glutathione S-transferase A4 (Gst4), NAD(P)H quinone dehydrogenase 1 (Nqo1), nuclear factor erythroid 2-like 2 (Nrf2), and peroxiredoxin 1 (Prdx1), among others), when administered since the beginning of the diet supplementation (H-P1); however, the increase was larger in the livers of mice supplemented at week 7 (H-P7), consistent with the rest of our metabolic findings (Fig. 7A). In addition, we also observed that lipid metabolism-associated transcripts such as peroxisome proliferator activated receptor-alpha (Pparα), Pparβ or Pparγ, as well as fatty acid synthase (Fasn), and sterol regulatory element binding transcription factor (Srbp1), were significantly up regulated in the liver of high-fat diet-fed mice (Fig. 7B). These results indicate that PSOn increased the expression of antioxidant- and lipid metabolism-related genes in the livers of mice fed a high-fat diet.

Figure 7
figure7

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.

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