Feeding fish oil to DIO mice increased rectal temperature and improved obesity
As shown in Fig. 2a, at 12 weeks of age, the DIO model mice (LD and FOD) weighed significantly more than the nonobese mice (33.44 ± 2.09 g vs. 27.16 ± 1.08 g, p < 0.001 by independent t test).
During the experimental period, the body weight of mice fed the LD increased in a time-dependent manner (Fig. 2a, b). The body weight of mice fed the CD remained unchanged after 12 weeks. Mice fed the LD had a higher caloric intake per day than the other two groups(Fig. 2c, d). Mice fed the FOD had a temporary decrease in caloric intake when switching from LD to FOD at 13 weeks, but thereafter, caloric intake was similar to that of mice fed CD. A temporary decrease in caloric intake following the change from LD to FOD has been reported in previous studies [13]. Rectal temperatures in the mice fed LD and FOD were similarly higher than those in mice fed the CD (Fig. 2e, f). Thus, mice fed the LD showed higher caloric intake and higher rectal temperatures compared to mice fed the CD, whereas mice fed the FOD showed similar caloric intake to mice fed the CD but higher rectal temperatures, suggesting the mechanism of bodyweight reduction by FOD.
Figure 2: Fish oil increases rectal temperature and improves obesity.
Feeding a FOD to DIO mice resulted in caloric intake similar to that of mice fed a CD, body temperature remained as high as that of mice fed the LD, and obesity improved. (a) Body weight; (b) body weight gain between 12 and 20 weeks of age; (c) food consumption; (d) average food consumption between 12 and 20 weeks of age; (e) body temperature; (f) average body temperature between 12 and 20 weeks of age. The data are presented as the mean ± standard error of the mean, n = 4–6 animals per group. * P < 0.05, *** P < 0.001 compared with CD group, ‡‡ P < 0.01, ‡‡‡ P < 0.001 compared with LD group using one-way ANOVA. FOD, fish oil-based high-fat diet; CD, control diet; LD, lard-based high-fat diet.
Feeding fish oil to DIO model mice reduced subcutaneous and visceral fat and improved fatty liver
Consistent with body weight, WAT weights of the mesentery (Fig. 3a), inguinal (Fig. 3b), and epididymis regions (Fig. 3c) were higher in mice fed the LD than in mice fed the CD, and mice fed FOD showed a significant reduction in weight in these tissues. Liver weight increased in both mice fed the LD and the FOD (Fig. 3d), but Oil Red O-stained images of liver tissue revealed that remarkable fat deposition was observed only in mice fed the LD (Fig. 3e), indicating that the increase in liver weight of mice fed the FOD does not reflect fatty liver. It has been reported that FO increases liver weight by inducing peroxisome proliferation [14].
Figure 3: Fish oil reduces subcutaneous and visceral fat.
Mice were dissected at 21 weeks, and liver and adipose tissue weights were compared. The weights of mesenteric fat, inguinal fat, and epididymal fat increased in LD-fed mice compared to CD-fed mice but were reduced in FOD-fed mice. Liver weights increased in both LD-fed and FOD-fed mice compared to CD-fed mice, but Oil Red O staining showed fat droplets only in LD-fed mice. (a) Mesenteric adipose tissue (Mes) mass; (b) inguinal adipose tissue (Ing) mass; (c) epididymal adipose tissue (Epi) mass; (d) liver mass; (e) representative Oil red o-stained liver histology. The data are presented as the mean ± standard error of the mean, n = 4–6 animals per group. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with CD group, ‡ P < 0.05, ‡‡‡ P < 0.001 compared with LD group using one-way ANOVA. LD, lard-based high-fat diet; CD, control diet; FOD, fish oil-based high-fat diet.
Feeding fish oil to DIO model mice improved glucose tolerance and insulin resistance
In the intraperitoneal ITT performed at 20 weeks, the area under the curve of blood glucose levels was larger in mice fed the LD than in mice fed the CD, and mice fed the FOD showed a significant reduction (Fig. 4a, b). In the intraperitoneal GTT, the area under the curve of blood glucose levels in mice fed the LD was larger than that in mice fed the CD, and the mice fed the FOD showed a significant reduction (Fig. 4c, d). Blood insulin levels were higher in mice fed the LD both before and after glucose administration (Fig. 4e, f). This indicates that insulin resistance was improved by administering FO to DIO mice.
Figure 4: Fish oil improves glucose tolerance and insulin resistance.
Mice fed the LD developed insulin resistance, which improved when mice were fed the with FOD. (a) Changes in blood glucose, as indicated by the ITT. (b) AUC of blood glucose levels during the ITT. (c) Changes in blood glucose, as indicated by the GTT. (d) AUC of blood glucose levels during the GTT. (e) Changes in plasma insulin, as indicated by the GTT. (f) AUC of plasma insulin levels during the GTT. The data are presented as the mean ± standard error of the mean, n = 4–8 animals per group. ** P < 0.01 compared with CD group, ‡ P < 0.05, ‡‡ P < 0.01 compared with LD group using one-way ANOVA. LD, lard-based high-fat diet; FOD, fish oil-based high-fat diet; ITT, insulin tolerance test; AUC, area under the curve; GTT, glucose tolerance test.
Feeding fish oil to DIO mice decreased FBG, insulin, T-Chol, FGF21, and Leptin.
Plasma total cholesterol (T-Chol) concentration was higher in mice fed the LD compared to mice fed the CD; furthermore, mice fed the FOD showed a significant reduction in T-Chol. Plasma triglyceride concentrations did not differ between groups. Blood free fatty acid concentrations tended to be lower in mice fed the FOD, but the difference was not significant. Blood FGF21 concentrations were significantly lower in mice fed the FOD than in mice fed the LD. Blood leptin levels were higher in mice fed the LD than in mice fed the CD, and mice fed the FOD showed a significant reduction, suggesting leptin resistance in mice fed the LD and an improvement when mice were fed the FOD (Table 2).
Table 2
Plasma concentrations of various parameters in 21-week-old male C57BL/6J mice.
| CD | LD | FOD |
FBG (mg/dL) | 71.2 ± 5.02 | 110.75 ± 5.27*** | 70.2 ± 3.95‡‡‡ |
Insulin (ng/mL) | 0.28 ± 0.01 | 0.58 ± 0.09* | 0.37 ± 0.02* |
T-Chol (mg/dL) | 45.1 ± 11.74 | 69.7 ± 6.54** | 52.2 ± 8.3‡ |
TG (mg/dL) | 69.67 ± 23.98 | 97 ± 17.59 | 90.67 ± 22.17 |
NEFAs (mEq/L) | 0.15 ± 0.03 | 0.20 ± 0.04 | 0.1 ± 0.04 |
FGF21 (pg/mL) | 1146.364 ± 375.21 | 1276.091 ± 188.37 | 282.72 ± 44.61‡ |
Leptin (ng/mL) | 14.43 ± 3.26 | 42.31 ± 3.74*** | 16.91 ± 3.67‡‡‡ |
* P < 0.05, ** P < 0.01, *** P < 0.001 compared with CD group, ‡ P < 0.05, ‡‡‡ P < 0.001 compared with LD group using one-way ANOVA. FBG, fasting blood glucose; T-Chol, total cholesterol; TG, triglyceride; NEFAs, nonesterified fatty acids; FGF21, fibroblast growth factor 21.
Fish oil-fed and LD-fed DIO mice had increased expression of heat-producing genes in brown adipose tissue but not in white fat
To confirm the mechanism by which rectal temperature increased in mice fed LD and FOD, the mRNA expression of genes involved in heat production was measured in adipose tissue obtained from mice of each group (Fig. 5, 6).
FOD increased β3-adrenergic receptor (β3AR) mRNA expression by 3.7-fold in BAT (Fig. 5a), PRDM16 by 3.3-fold (Fig. 5b), PGC1α by 4.3-fold (Fig. 5c), and UCP1 by 4.7-fold (Fig. 5f) compared to mice fed the CD and increased PPARα (Fig. 5d) and PPARγ (Fig. 5e). Mice fed the LD also showed increased expression of genes involved in heat production, but not to the extent as observed in mice fed the FOD. The expression of the free fatty acid receptor FFAR4 increased in mice fed the LD and in mice fed the FOD compared to mice fed the CD (Fig. 5i). FGF21 mRNA expression also increased in mice fed the LD and the FOD, showing a different trend from that of blood FGF21 levels (Fig. 5g). βklotho, which acts as a receptor for FGF21, increased in mice fed the FOD compared to mice fed the LD (Fig. 5h). Unlike the results related to BAT, no changes in gene expression involved in heat production were observed in inguinal WAT. (Fig. 6a-i)
Figure 5: Fish oil increased the expression of heat-producing genes in BAT.
The expression of genes involved in heat production increased in BAT of mice fed the LD and the FOD. Gene expression levels in BAT. (a) β3AR; (b) PRDM16; (c) PGC1α; (d) PPARα; (e) PPARγ; (f) UCP1; (g) FGF21; (h) βklotho; (i) FFAR4. The data are presented as the mean ± standard error of the mean, n = 6–7 animals per group. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with CD group, ‡ P < 0.05, ‡‡ P < 0.01, ‡‡‡ P < 0.001 compared with LD group using one-way ANOVA. BAT, brown adipose tissue; β3AR, β3-adrenergic receptor; PRDM16, PR domain containing 16; PGC1α, peroxisome proliferator-activated receptor-γ coactivator-1α; PPARα, peroxisome proliferator-activated receptor-α; PPARγ, peroxisome proliferator-activated receptor-γ; UCP1, uncoupling protein 1; FGF21, fibroblast growth factor 21; FFAR4, free fatty acid receptor 4.
Figure 6: Fish oil did not increase the expression of heat-producing genes in inguinal WAT.
No changes in gene expression were observed in inguinal WAT. (a) β3AR; (b) PRDM16; (c) PGC1α; (d) PPARα; (e) PPARγ; (f) UCP1; (g) FGF21; (h) βklotho; (i) FFAR4. The data are presented as the mean ± standard error of the mean, n = 6–7 animals per group. WAT, white adipose tissue; β3AR, β3-adrenergic receptor; PRDM16, PR domain containing 16; PGC1α, peroxisome proliferator-activated receptor-γ coactivator-1α; PPARα, peroxisome proliferator-activated receptor-α; PPARγ, peroxisome proliferator-activated receptor-γ; UCP1, uncoupling protein 1; FGF21, fibroblast growth factor 21; FFAR4, free fatty acid receptor 4.