Polyunsaturated Fatty Acids Attenuate Diet Induced Obesity and Insulin Resistance, Modulating Mitochondrial Respiratory Uncoupling in Rat Skeletal Muscle

Abstract

Objectives: Omega (ω)-3 polyunsaturated fatty acids (PUFA) are dietary compounds able to attenuate insulin resistance. Anyway, the precise actions of ω-3PUFAs in skeletal muscle are overlooked. We hypothesized that PUFAs, modulating mitochondrial function and efficiency, would ameliorate pro-inflammatory and pro-oxidant signs of nutritionally induced obesity.

Study design: To this aim, rats were fed a control diet (CD) or isocaloric high fat diets containing either ω-3 PUFA (FD) or lard (LD) for 6 weeks.

Results: FD rats showed lower weight, lipid gain and energy efficiency compared to LD-fed animals, showing higher energy expenditure and O2 consumption/CO2 production. Serum lipid profile and pro-inflammatory parameters in FD-fed animals were reduced compared to LD. Accordingly, FD rats exhibited a higher glucose tolerance revealed by an improved glucose and insulin tolerance tests compared to LD, accompanied by a restoration of insulin signalling in skeletal muscle. PUFAs increased lipid oxidation and reduced energy efficiency in subsarcolemmal mitochondria, and increase AMPK activation, reducing both endoplasmic reticulum and oxidative stress. Increased mitochondrial respiration was related to an increased mitochondriogenesis in FD skeletal muscle, as shown by the increase in PGC1-α and -β.

Conclusions: our data strengthened the association of high dietary ω3-PUFA intake with reduced mitochondrial energy efficiency in the skeletal muscle.

Fig 1. Effect of ω-3 PUFA on glucose and lipid metabolism.

HOMA-IR index (A); Plasma insulin (B), and glucose (C) concentrations at different time intervals after glucose load and respective area under curve (AUC) (upper inserts) and insulin tolerance test (D) are shown. Representative western blots of insulin-induced Akt phosphorylation (Ser473) (E), and lipid content (F)in skeletal muscle are also reported. The graphic reported in panel 1E represent the densitometric analysis of protein band obtained in three separate experiments. Haematoxylin-eosin sections of glycogen (G upper panels), ADRP expression (G lower panels) are shown. PAS positive material was stained magenta at a magnification of 20x. Values are expressed as means±SEM from n = 8 animals/group. Different superscripted letters indicate statistically significant differences (P<0.05).

Fig 2. Effect of ω-3 PUFA on mitochondrial functions and energy efficiency.

IMF and SS mitochondrial respiration in the presence of succinate (A) or palmitoyl-carnitine (B) as substrates were determined. CPT activity (C); basal (D) or palmitate-induced (E) proton leakage in SS mitochondria and respiration rates at 160 mV (the highest membrane potential common to all the curves) (upper insert); aconitase activity (F); H₂O₂ yield (G), relative mRNA expression of FGF21 (H), PGC1α (I), PGC1β (L) are also shown. Values are expressed as means±SEM from n = 8 animals/group. Different superscripted letters indicate statistically significant differences (P<0.05).

Fig 3. Effect of ω-3 PUFA on oxidative- and ER-stress and AMPK activation.

Total thiols (A) and GSH/GSSG ratio (upper insert); protein carbonyl levels in skeletal muscle (B) and in serum (upper insert). Cytoplasmic GST and NQO1 activities and Nrf2 levels in nucleus (C). Representative immunoblots of TNFα (D) and BiP/GRP78 (E) p-eIF2-α (F), pAMPK (G) and pACC (H) are shown. Densitometric analysis of protein bands are reported: after normalisation the levels are expressed as the density ratio of target to control (tubulin, lamin or total protein). Values are expressed as means±SEM from n = 8 animals/group. Different superscripted letters indicate statistically significant differences (P<0.05).