PPARα-independent actions of omega-3 PUFAs contribute to their beneficial effects on adiposity and glucose homeostasis

Abstract

Excess dietary lipid generally leads to fat deposition and impaired glucose homeostasis, but consumption of fish oil (FO) alleviates many of these detrimental effects. The beneficial effects of FO are thought to be mediated largely via activation of the nuclear receptor peroxisomal-proliferator-activated receptor α (PPARα) by omega-3 polyunsaturated fatty acids and the resulting upregulation of lipid catabolism. However, pharmacological and genetic PPARα manipulations have yielded variable results. We have compared the metabolic effects of FO supplementation and the synthetic PPARα agonist Wy-14,643 (WY) in mice fed a lard-based high-fat diet. In contrast to FO, WY treatment resulted in little protection against diet-induced obesity and glucose intolerance, despite upregulating many lipid metabolic pathways. These differences were likely due to differential effects on hepatic lipid synthesis, with FO decreasing and WY amplifying hepatic lipid accumulation. Our results highlight that the beneficial metabolic effects of FO are likely mediated through multiple independent pathways.

Figure 1. Glucose metabolism measures in chow, lard-HFD, fish oil-fed and WY-treated mice.

Basal plasma glucose (A) and insulin (B) levels. An intraperitoneal glucose tolerance test (C) was performed in mice that were fasted for 6 hours prior to receiving a glucose bolus (2 g/kg). (D) The incremental area under the curve (iAUC) indicates the animal's ability to clear the glucose load from the circulation. Shown are means ± SEMs, with n = 8–10 mice per group; a,b,c p < 0.05, 0.01, 0.001 vs chow; d,e p < 0.05, 0.01 vs. lard-HFD.

Figure 2. Food intake, Whole body energy metabolism and urinary energy output in chow, lard-HFD, fish oil-fed and WY-treated mice.

(A) Food intake was measured in every 2–3 days and the average cumulative intake per mouse over the course of the study is shown. Indirect calorimetry was performed in mice and the 24 hr average for energy expenditure (B) and respiratory exchange ratio (C) are presented. Energy output in the urine (D) was assessed by bomb calorimetry. Shown are means ± SEMs, with n = 7–8 per group; b,c p < 0.01, 0.001 vs chow; d,f p < 0.05, 0.001 vs. lard-HFD, *p < 0.05, † p < 0.01, ‡ p < 0.001.

Figure 3. Markers of lipid metabolism in liver.

Triacylglycerol content (A) was used as a measure of lipid accumulation. The activity of medium chain acyl coenzyme A dehydrogenase (MCAD, B), citrate synthase (CS, C) and acyl-CoA oxidase (AOX, D) were determined in liver homogenates as described previously. Protein levels of oxidative and peroxisomal markers in liver were measured by immunoblotting (E–H) as described in. Oxidation of [1-¹⁴C]-palmitic acid to CO₂ (I) and acid-soluble metabolites (ASM, J) was assessed in liver homogenates. Shown are means ± SEMs; with n = 8–10 mice per group. Representative immunoblots show n = 2, with densitometry calculated for n = 8–10 per group. a,b,c p < 0.05, 0.01, 0.001 vs chow; d, f p < 0.05, 0.001 vs lard-HFD; * p < 0.05, † p < 0.01, ‡ p < 0.001. Complex I, III and V represent sub-units of the complexes of the electron transport chain (ETC), PMP70 = peroxisomal membrane protein 70. AU – Arbitrary units.

Figure 4. Protein levels of fatty acid synthase (FAS), acetyl-CoA carboxylase (ACC) and stearoyl-CoA desaturase 1 (SCD1) in liver measured by immunoblotting.

Representative immunoblots show n = 2, with densitometry (means ± SEMs) calculated for n = 8 per group. a,b,c p < 0.05, 0.01, 0.001 vs chow; d, f p < 0.05, 0.001 vs lard-HFD; * p < 0.05, † p < 0.01, ‡ p < 0.001. AU – Arbitrary units.

Figure 5. Markers of lipid metabolism in skeletal muscle.

Triacylglycerol content (A) in quadriceps muscle was used as a measure of lipid accumulation. Beta-hydroxyacyl dehydrogenase (β-HAD; B), medium chain acyl coenzyme A dehydrogenase (MCAD, C) citrate synthase (CS, D) and acyl-CoA oxidase (AOX, E) activities were measured in muscle homogenates. Oxidation of [1-¹⁴C]-palmitic acid was assessed in muscle homogenates, with the ratio of CO2:acid soluble metabolites (ASM) shown (F). Shown are means ± SEMs; with n = 8–10 mice per group. a,b,c p < 0.05, 0.01, 0.001 vs chow; f, p < 00.001 vs lard; * p < 0.05, ‡ p < 0.001.