Fish Oil Supplementation Mitigates High-Fat Diet-Induced Obesity: Exploring Epigenetic Modulation and Genes Associated with Adipose Tissue Dysfunction in Mice

Abstract

This study investigated the effects of fish oil (FO) treatment, particularly enriched with eicosapentaenoic acid (EPA), on obesity induced by a high-fat diet (HFD) in mice. The investigation focused on elucidating the impact of FO on epigenetic modifications in white adipose tissue (WAT) and the involvement of adipose-derived stem cells (ASCs). C57BL/6j mice were divided into two groups: control diet and HFD for 16 weeks. In the last 8 weeks, the HFD group was subdivided into HFD and HFD + FO (treated with FO). WAT was removed for RNA and protein extraction, while ASCs were isolated, cultured, and treated with leptin. All samples were analyzed using functional genomics tools, including PCR-array, RT-PCR, and Western Blot assays. Mice receiving an HFD displayed increased body mass, fat accumulation, and altered gene expression associated with WAT inflammation and dysfunction. FO supplementation attenuated these effects, a potential protective role against HFD-induced obesity. Analysis of H3K27 revealed HFD-induced changes in histone, which were partially reversed by FO treatment. This study further explored leptin signaling in ASCs, suggesting a potential mechanism for ASC dysfunction in the obesity-rich leptin environment of WAT. Overall, FO supplementation demonstrated efficacy in mitigating HFD-induced obesity, influencing epigenetic and molecular pathways, and shedding light on the role of ASCs and leptin signaling in WAT dysfunction associated with obesity.

Keywords: H3K27; WAT; inflammation; leptin; n-3 PUFA; obesity.

Figure 1

Obesity Model Characterization. (A) Caloric (kcal/day/animal), Food and Fat (g/day/animal) intake, (B) Body mass evolution, (C) Fasting glucose, (D) Glucose tolerance test or GTT, and (E) Incremental area under the glycemic curve in control (CO) and obese animals induced by a high fat diet (HFD) for 12 weeks. In (A,B), the measurements were performed weekly throughout the experimental protocol. In (C–E), the glycemic curve or glucose concentration versus time was calculated after glucose administration (2 g/Kg b.w.). Data were analyzed using Student’s t-test, and show mean ± SEM (n = 12). * p < 0.05 or **** p < 0.0001 versus control.

Figure 2

Body mass evolution (A), depot mass of visceral epididymal (Epi) (B), retroperitoneal (Rp) (C), and subcutaneous inguinal (Ing) (D) adipose tissues in milligrams (mg), after 16 weeks of experimental diets and fish oil (FO) supplementation. In the initial 8 weeks, the animals were submitted to either a control (CO) or high-fat diet (HFD). During the last 8 weeks of the experimental protocol, the diets were continued, and the animals underwent gavage (CO and HFD groups received water, while the HFD + FO group received FO) three times a week. Data were analyzed using one-way Analysis of Variance (ANOVA) followed by Tukey’s post-test, and show mean ± SEM (n = 6). * p < 0.05 vs. CO, ^# p < 0.05 vs. CO and HFD + FO, **** p < 0.0001.

Figure 3

Heatmap of gene expression in Epi WAT from mice subjected to 16 weeks of experimental diets and fish oil supplementation. Control diet (CO), high-fat diet (HFD), and high-fat diet plus fish oil (HFD + FO). The gene expression level is indicated using a color scale, where red indicates higher expression and green indicates lower expression.

Figure 4

Gene expression of Acly (A) and genes encoding histone modifiers Ezh2 (B), Crebbp (C), Ep300 (D), Kdm6a (E), and Kdm6b (F), in the visceral Epi WAT from animals that received control diet (CO), high-fat diet (HFD), or HFD and fish oil (HFD + FO). Target genes were normalized by the constitutive Gapdh. Data were analyzed using one-way ANOVA followed by Tukey’s post-test, and show mean ± SEM (n = 4–6). * p < 0.05 or ** p < 0.01 or *** p < 0.001.

Figure 5

Graphical representation of the protein content of ACL (A), H3K27ac (B), and H3K27me3 (C), in visceral Epi WAT from animals that received a control diet (CO), a high-fat diet (HFD), or an HFD diet and fish oil (HFD + FO). Data were analyzed using one-way ANOVA followed by Tukey’s post-test. Values were expressed as mean ± SEM, in relation to the control and corrected by the expression of the constitutive beta-actin and total protein by Ponceau. A representative image of protein expression levels from 2 independent experiments is shown above each graph (n = 3 animals) quantified by ImageJ (Software v1.5.4e). * p < 0.05 or ** p < 0.01 or **** p < 0.0001.

Figure 6

Gene expression of Lepr1 (A) and Lepr2 (B) in ASC isolated from WAT of animals that received a control diet (CO) or a high-fat diet (HFD). Total content of LEP R1 (Ob-Rb) protein (C) and gene expression of Acly (D) in ASC isolated from WAT of HFD-induced obese animals, treated in vitro with 100 ng/mL leptin for 24 h. Data were analyzed using Student’s t-test, and show mean ± SEM (n = 4–6). In (A–C), target genes were normalized by the constitutive 36B4. In C, total content of protein was quantified by ImageJ and expressed in relation to the control and corrected by the expression of the constitutive beta-actin and total protein by Ponceau. A representative image of protein expression level is shown above the graphic. * p < 0.05.