Chia Oil Supplementation Attenuates Obesity‐Induced Hepatic Steatosis, PVAT Inflammation, and Endothelial Dysfunction in Mice

Figures

• 

  FIGURE 1. Dietary chia oil supplementation improves body composition and glucose homeostasis in obese mice. (A) Experimental design. (B) Body mass gain over time. (C) Energy intake curve. (D) Area under the curve (AUC) of energy intake. (E) Lean mass percentage. (F) Fat mass percentage as measured by NMR. (G) Intraperitoneal glucose tolerance test (ipGTT) and (H) its corresponding AUC. (I) Intraperitoneal insulin tolerance test (ipITT). (J) Corresponding AUC. (K) Fasting insulin levels. (L) Serum adiponectin. (M) Serum leptin concentrations. Data are presented as mean ± SEM. Statistical analyses were performed using two‐way RM ANOVA (B, C, G, and I) and one‐way ANOVA for endpoint measurements (D, E, F, H, J, K, L, and M), followed by appropriate post‐hoc tests as described in Experimental section. *p < 0.05, **p < 0.01, ***p <0,005, ****p < 0,001. n = 5–6 animals/group. C = Control; H = High‐fat diet; HC = High‐fat diet + chia oil.

• 

  FIGURE 2. Chia oil supplementation improves circulating lipid profile in obese mice. (A) Serum triglyceride levels, (B) total cholesterol, (C) low‐density lipoprotein cholesterol (LDL‐c), (D) high‐density lipoprotein cholesterol (HDL‐c), (E) very‐low‐density lipoprotein cholesterol (VLDL‐c), and (F) nonesterified fatty acids (NEFA). Data are presented as mean ± SEM. Statistical analyses were performed using one‐way ANOVA followed by appropriate post hoc tests as described in experimental section. *p < 0.05, **p < 0.01, ***p <0,005, ****p<0,001. n = 5–6 animals/group. C = Control; H = High‐fat diet; HC = High‐fat diet + chia oil.

• 

  FIGURE 3. Chia oil supplementation reduces hepatic steatosis and lipid accumulation in obese mice. (A) Serum levels of alanine aminotransferase (ALT) and (B) aspartate aminotransferase (AST). (C) Representative hematoxylin and eosin (H&E)‐stained liver sections from each group, showing hepatic architecture and steatosis (magnification: 20×). (D) Quantification of hepatic triglyceride content. Data are presented as mean ± SEM. Statistical analyses were performed using one‐way ANOVA followed by appropriate post hoc tests as described in experimental section. *p < 0.05, **p < 0.01, ***p <0,005, ****p<0,001. n = 6 animals/group. C = Control; H = High‐fat diet; HC = High‐fat diet + chia oil.

• 

  FIGURE 4. Chia oil supplementation modulates hepatic expression of genes and proteins involved in lipid metabolism and inflammation in obese mice. Relative mRNA expression of (A) acetyl‐CoA carboxylase (ACC), (B) adipose triglyceride lipase (ATGL), (C) fatty acid‐binding protein 4 (FABP4), (D) hormone‐sensitive lipase (HSL), and (E) carnitine palmitoyltransferase 1 alpha (CPT‐1α). Hepatic expression of inflammatory and macrophage polarization markers: (F) TNF‐α, (G) CD11b, (H) CD86, and (I) CD206. (J‐L) Protein expression of lipoprotein lipase (LPL) and PGC‐1α assessed by Western blotting. Gene expression levels were normalized to 18S rRNA and are expressed relative to the control group. Anti‐GAPDH antibody was used as the loading control in western blotting. Data are presented as mean ± SEM. Statistical analyses were performed using one‐way ANOVA followed by appropriate post‐hoc tests as described in experimental section. *p < 0.05, **p < 0.01. n = 3–7 animals/group. C = Control; H = High‐fat diet; HC = High‐fat diet + chia oil.

• 

  FIGURE 5. Chia oil supplementation promotes anti‐inflammatory remodeling of mesenteric perivascular adipose tissue (PVAT) in obese mice. (A) Mass of mesenteric PVAT. (B) Representative hematoxylin and eosin (H&E)‐stained sections of PVAT (magnification: 20×). (C) Quantification of adipocyte area. (D) Number of adipocytes. (E–H) Secretion levels of leptin, TNF‐α, MCP‐1 and IL‐1 measured by ELISA. (I) Gene expression of macrophage marker F4/80. (J‐M) mRNA expression of polarization markers CD80, CD86, CD163, and CD301. (N) mRNA expression of CD206, a marker of M2 macrophages. (O), (P) mRNA expression of inducible nitric oxide synthase (iNOS) and arginase‐1. Gene expression levels were normalized to 18S rRNA and expressed relative to the control group. Data are presented as mean ± S.E.M. Statistical analyses were performed using one‐way ANOVA followed by appropriate post‐hoc tests as described in experimental section. *p < 0.05, **p < 0.01, ***p<0.005, ****p<0.0001. n = 5–6 animals/group. C = Control; H = High‐fat diet; HC = High‐fat diet + chia oil.

• 

  FIGURE 6. Chia oil supplementation improves vascular function and enhances AMPK signaling in mesenteric PVAT of obese mice. (A) Endothelium‐dependent vasodilation in response to acetylcholine (Ach) and (B) vasoconstriction response to norepinephrine (NE) in isolated mesenteric vascular beds. (C‐E) Protein expression of total and phosphorylated AMPK (Thr172) and total and phosphorylated eNOS (Ser1177) in mesenteric perivascular adipose tissue (PVAT), analyzed by Western blotting. Anti‐β‐actin antibody was used as the loading control. Data are presented as mean ± S.E.M. Statistical analyses were performed using two‐way RM ANOVA (A and B) and one‐way ANOVA for endpoint measurements (D and E), followed by appropriate post‐hoc tests as described in experimental section. *p < 0.05, **p < 0.01; #p < 0.05 vs. H. n = 5–6 animals/group. C = Control; H = High‐fat diet; HC = High‐fat diet + chia oil.