Figure 1.
Schematic of diets and mouse groups.
(A) Schematic illustrates the dietary ω6/ω3 fatty acid ratio, and the mg/kg/day intake of total ω3 fatty acid, EPA, DHA and EPA+DHA in the HFH and HFL diets consumed by the mice. For comparison, the approximate mg/kg/day dose of EPA+DHA for an 80 kg human taking a standard prescription of Lovaza fish oil (four 1 g capsules, containing 840 mg EPA+DHA each, per day) is shown. (B) 15-week diet regimens for WT mice: normal chow diet (13% Kcal from fat), high fat diet (46% kcal from fat) with high dietary ω6/ω3 ratio (HFH), or high fat diet (46% kcal from fat) with low dietary ω6/ω3 (HFL). (C) 15-week diet regimens for WT and 12/15-LOKO mice: high fat diet (46% kcal from fat) with high dietary ω6/ω3 ratio (HFH). FA, fatty acids; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
Table 1.
Composition of diets.
Figure 2.
The effect of varying dietary ω6/ω3 ratio in WT (NC n = 4), open circles; HFH n = 7, black squares; and HFL n = 8, gray triangles) mice on (A) body weight over fist 10 weeks of diet regimens and (B) food intake averaged over weeks 5 to 10 on the respective diets. Values are mean ±SE. ***P<0.001 HFH vs. HFL. NC, normal control (chow); HFH, high dietary ω6/ω3 ratio; HFL, low dietary ω6/ω3 ratio.
Table 2.
Metabolic parameters in WT mice fed NC, HFH, and HFL diets.
Figure 3.
Assessment of hepatic inflammation and steatosis.
The effect of varying dietary ω6/ω3 ratio in high fat diet-fed WT mice on the hepatic mRNA expression of (A) cytokines driving a Th1 profile, (B) pro/anti-inflammatory cytokines, (C) neutrophil chemoattractants, and (D) regulators of lymphocyte homing response, all normalized to signal in WT NC mice (n = 7). (E) Haematoxylin-eosin and Oil red O staining of paraffin-embedded liver sections. Quantitative grading scale assessing the quantity of hepatic triglycerides, normalized to signal in WT NC mice (expressed as a fold increase in stained area relative to WT NC mice). (F) The effect of varying dietary ω6/ω3 ratio in high fat diet-fed WT mice on the hepatic mRNA expression of sterol regulatory element-binding protein 1 (Srebp1) normalized to signal in WT NC mice. WT HFH: black bars; WT HFL: gray bars. Values are mean ±SE. 5-7 animals per group. *P<0.05, **P<0.01, ***P<0.001 HFH vs. HFL. Scale bar, 50 µm. WT NC SEs for genes Il12a (p35), Il12b (p40), Il18, Ifng, Tnfa, Il10, Cxcl1 (KC), Cxcl2/3 (Mip2), Ccr7, Ccl19, Ccl21 and Srebp1 were 0.14, 0.09, 0.14, 0.08, 0.12, 0.17, 0.14, 0.13, 0.20, 0.21, 0.15, and 0.10, respectively. WT NC SE value for oil red O stain was 0.15. NC, normal control (chow); HFH, high dietary ω6/ω3 ratio; HFL, low dietary ω6/ω3 ratio.
Figure 4.
Plasma concentration of the major ω6 fatty acids and their corresponding pro-inflammatory metabolites.
The effect of varying dietary ω6/ω3 ratio in high fat diet-fed WT mice on plasma concentrations of oxidized metabolites of (A) arachidonic acid and (B) linoleic acid. (C) Plasma concentrations of arachidonic and linoleic acids. NC n = 3 (open bars), HFH n = 7 (black bars), HFL n = 8 (gray bars). T-test analysis was restricted to HF diet animals only. Values are mean ±SE. *P<0.05 HFH vs. HFL. NC, normal control (chow); HFH, high dietary ω6/ω3 ratio; HFL, low dietary ω6/ω3 ratio; HETE, hydroxyeicosatetraenoic acid; HODE, hydroxyoctadecadienoic acid; oxoODE, oxooctadecadienoic acid.
Figure 5.
Assessment of hepatic inflammation and steatosis in 12/15-LOKO mice on HFH diet.
The effect of 12/15-lipoxygenase deficiency on (A) terminal body weight, food intake and gonadal white adipose tissue (gWAT) weight expressed as percent total body weight, and hepatic expression of (B) regulators of lymphocyte homing response, (C) pro/anti-inflammatory cytokines and (D) neutrophil chemoattractants. (E) Haematoxylin-eosin and Oil red O staining of paraffin-embedded liver sections to show hepatic steatosis. (F) Quantitative grading scale assessing the quantity of hepatic triglycerides, normalized to signal in WT NC (n = 7) mice (expressed as a fold increase in stained area relative to WT NC mice). (G) Plasma concentrations of oxidized metabolites of arachidonic acid in KO HFH n = 4 (dark gray bars) compared to WT HFH n = 7 (black bars) mice. Values are mean ±SE. *P<0.05, **P<0.01 WT HFH vs. 12/15-LOKO HFH. Scale bar, 50 µm. WT NC SEs for genes Ccr7, Ccl19, Ccl21, Ifng, Tnfa, Il10, Cxcl1 (KC), Cxcl2/3 (MIP-2) were 0.20, 0.21, 0.15, 0.08, 0.12, 0.17, 0.14, 0.13, respectively. WT NC SE value for oil red O stain was 0.15. KO, 12/15 lipoxygenase knockout; HFH, high dietary ω6/ω3 ratio; gWAT, gonadal white adipose tissue; HETE, hydroxyeicosatetraenoic acid.