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

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.


S1. Animal care and PSOn supplementation maintenance
The Animal Care and Ethics Committee of the INCMNSZ approved all of the procedures related to animal handling and experimentation. Eight-week-old male C57BL/6N mice were housed in environmentally enriched cages. Food (a standard rodent chow diet) and water were provided ad libitum. The circadian rhythm was controlled with a 12/12-h light/dark cycle. For the acute protocol, mice were divided into a control group and 1 mg/kg, 2 mg/kg and 4 mg/g PSOn groups (n=5 each). The PSOn was administered in mice once by oral gavage, and mice were followed up to 15 days. We also sought to test the long-term effect of PSOn in an every other day administration basis (chronic protocol); here, mice were divided into a control or PSOn group (n=5 each) and the same oral gavage strategy was used to administer three different doses (8.7 mg/g, 26 mg/g and 35 mg/g of PSOn body weight); mice were followed up to 28 days. Body weight and food consumption were monitored every 3 days. At the end of both protocols, mice were euthanized by pentobarbital injection and decapitated; peripheral blood, liver and kidneys were collected for further analysis. Tissues from both protocols were processed for H&E staining.

S2. Biochemical and cellular peripheral blood measurements
Plasma was obtained from peripheral blood and used to determine the alanine aminotransferase (ALT), aspartate aminotransferase (AST), albumin, total cholesterol, glucose and urea levels in colourimetric assays using a COBASs c111 analyser (Roche Diagnostics, Mannheim. Switzerland). Blood cell counts and haematological values were recorded with a DxH 800 (Beckman Coulter) automatic cell counter.

S3. Cell culture and treatment
3T3-L1 preadipocytes and C2C12 myoblast cells were grown in supplemented (10% foetal bovine serum (FBS), 1% penicillin/streptomycin) DMEM (DMEM-S) maintained in a 95% O 2 , 5% CO 2 atmosphere at 37°C. The cells were seeded at a density of 40x10 4 cells/well in 24-well plates and allowed to reach confluence before treatment. The cells were exposed to 0.1, 0.25, 0.50, 0.80 or 1.0 mg/ml PSOn for 3, 6, 12, 24 or 48 h. Control cells were treated with PSOn solvent only. After treatment, the cells were evaluated for viability according to the quick protocol of the Vybrant MTT Cell Proliferation Assay (Thermo Fisher Scientific, USA). The absorbance of the control wells at 470 nm was considered to indicate 100% viability, and the viability of the treated cells was extrapolated accordingly.

S4. Primary astrocyte cell culture
Primary astrocytes were obtained from neonatal mice as previously described [1]. Briefly, postnatal P1-P4 pups were cleaned with ethanol and decapitated to obtain the brains. Under a stereoscopic microscope, the meninges were removed, and the cortices were separated and placed in cold Hank's balanced saline solution (HBSS). The cortices from 4 mice were collected and then triturated with scissors. The minced tissue was digested in 0.25% (final) trypsin solution for 30 min. The digested tissue was further mechanically homogenized using fire-polished Pasteur pipets. The cell suspensions were plated in 75 cm 2 culture flasks coated with poly-D-lysine (50 µg/ml). The cells were maintained in astrocyte culture medium (high-glucose DMEM (Gibco, Life Technologies, USA) supplemented with 10% heat-inactivated foetal bovine serum); after 7 days in culture, the microglia and oligodendrocyte precursors were detached by vigorous shaking.
After intensive PBS washing, the cells were trypsinized and collected. The astrocyte-enriched cell suspensions were used to seed astrocytes at a density of 4x10 4 cells/well in astrocyte culture medium, and the cells were treated as described above.