Protective Role And Potential Mechanism Of Flaxseed Oil Against HFD-induced Insulin Resistance

Objective:1. To investigate the appropriate intake of flaxseed oil supplementation against chronic high-fat-diet (HFD) induced insulin resistance in mice.2. To explore the relationship between n-3 polyunsaturated fatty acids (n-3PUFA) status mediated by flaxseed oil administration and HFD-induced insulin resistance based on lipidomics.3. To investigate the role of hepatic endoplasmic reticulum (ER) stress and unfolded protein response (UPR), mitochondrial damage and autophagy dysfunction in HFD-induced insulin resistance and the potential mechanism of flaxseed oil supplementation.Methods:1. Eighty male C57BL/6J mice were divided randomly into five groups of sixteen animals each as follows:Low fat control group (LFD:1.9% lard+2.4% corn oil); High fat model group (HFD:31.9%lard+3.2% com oil); Low (L-FO:10% flaxseed oil+21.9% lard+3.2% corn oil), medium (M-FO:20% flaxseed oil+11.9% lard+3.2% corn oil) and high dose of flaxseed oil enriched high fat groups (H-FO:30% flaxseed oil+1.9% lard+3.2% corn oil). Mice were fed low fat diet (10% fat-derived calories) and/or high fat diet (60% fat-derived calories) with or without different dose of flaxseed oil supplementation for 16 weeks.2. Fasting serum glucose, insulin, inflammatory cytokines levels as well as hepatic triglyceride (TG) and total cholesterol (TC) contents were measured based on enzymatic kinetic method and ELISA, respectively. Liver tissue samples were fixed and tissue sections were stained with HE staining. Frozen hepatic sections were immediately incubated with DHE to detect the level of ROS.Measurements of malondialdehyde (MDA) and glutathione (GSH) in liver by enzymatic colorimetric methods were based on thiobarbituric acid and improved dithiodimorpholine nitrobenzoic acid, respectively. The serum and hepatic lipidomics were analyzed by solid phase microextraction and gas chromatography. The plasma membrane and mitochondria were obtained from fresh liver samples by a series of differential centrifugations, and the extracted lipids were analyzed by solid phase microextraction and gas chromatography. Transmission electron microscopy was performed on mice liver to examine mitochondrial morphology and mitophagy status. Mitochondrial membrane potential and respiratory control ratio were analyzed by using the fluorescent dye JC-1 and oxygen electrode methods following the isolation of mitochondria, respectively. Western blot was used to detect the expression of hepatic insulin signaling, ER stress and UPR, JNK signaling, mitophagy and mitochondrial biogenesis regulation related proteins.Results:1. The average caloric intakes among groups revealed no significant difference throughout the whole experimental period except the LFD group. Medium and high dose of flaxseed oil supplementation significantly alleviated chronic HFD-induced increases in body weight, liver and adipose ratios to body weight, and improved systematic insulin resistance in mice.2. Flaxseed oil treatment exerted significant inhibiting effects on HFD-induced increase in serum inflammatory cytokines, especially the most dramatic reduction of MCP-1.3. Chronic high fat feeding induced serious hepatic steatosis, accompanying remarkably increased TG concentration (+88%), and oxidative stress. Administration of medium and high dose of flaxseed oil significantly improved the aboved adverse changes.4. Flaxseed oil supplementation improved chronic HFD-induced decrease in protein expressions of p-Ser473Akt, p-Tyr632IRS-1 and p-Ser2448 mTOR under acute insulin stimulation, indicating improved the insulin-signaling pathway in mice liver.5. Flaxseed oil administration reversed HFD-induced increase in serum saturated fatty acid (SFA) and n-6 polyunsaturated fatty acids (n-6PUFA) contents with a dose-effect relationship being observed. Moreover, further decreased MUFA levels and significantly increased n-3 polyunsaturated fatty acids (n-3PUFA) in serum were observed in HFD-fed mice following flaxseed oil administration. The effect of flaxseed oil on hepatic fatty acid composition revealed similar changing trend to serum.6. Flaxseed oil dose-dependently attenuated HFD-induced increase in serum TG, cholesteryl ester (CE) and free fatty acid (FFA) contents. The levels of diglyceride (DG) and phospholipid (PL) in serum were not significantly different across the studied groups. Flaxseed oil mediated accumulation of n-3PUFAs was lipid classes specific. The increased ?-linolenic acid (ALA), eicoeapentaenoic acid (EPA), docosapentaenic acid (DPA) and docosahexaenoic acid (DHA) contents were specifically accumulated in TG, FFAs and CE but not DG and phospholipids pool. Notably, the increase in ALA and EPA levels was dose-dependent from L-FO to H-FO. However, the content of its downstream product DHA were decreased, indicating a feedback inhibition in a dose-dependent manner. Flaxseed oil-mediated changes in hepatic specific lipid species indicated similar changing trend similar to serum.7. Chronic high fat feeding induced remarked remodeling of plasma membrane fatty acid composition in mice liver, revealing increased n-3PUFA content and decreased MUFA and n-6/n-3 ratio. Flaxseed oil supplement decreased the contents of linoleic acid (LA) derived arachidonic acid and docosatetraenoic acid and further increased the accumulation of LA, ALA and ALA derived EPA and DPA in plasma membrane.8. The improved systemic insulin resistance following flaxseed oil supplementation may be based on the specific incorporation of ALA, EPA and DPA in serum lipid pool. The correlation map indicated that FFA/CE with ALA and EPA, TG containing ALA, EPA and DPA, and DG including ALA positively related to insulin sensitivity mediated by flaxseed oil administration.9. Flaxseed oil supplementation alleviated HFD-induced ER stress and unfolded protein response inactivation, and decreased the protein expression and phosphorylation levels of JNK, accompanied by improved insulin resistance.10. Flaxseed oil supplementation evidently alleviated the mitochondrial morphological abnormalities as a consequence of HFD treatment, increased the accumulation of ALA, DPA and DHA and decreased n-6/n-3 ratio in mitochondrial membrane, and improved mitochondrial membrane potential and respiratory control ratio. Flaxseed oil supplementation enhanced mitophagy and mitochondrial biosynthesis regulation, indicating an enhancing mitochondrial degradation by autophagy.Conclusion:1. Flaxseed oil supplementation significantly alleviated HFD-induced hepatic steatosis, oxidative stress and impairment of insulin-signaling pathway, accompanying by improved systematic inflammatory response and insulin resistance. In which, medium dose intake of flaxseed oil has revealed remarkably antagonistic effect.2. Medium and high dose of flaxseed oil mediated specific accumulation of ALA, EPA and DPA in serum and hepatic lipid pools and plasma membrane was positively related to improvement in systemic and hepatic insulin resistance in mice3. Flaxseed oil supplementation inhibited HFD-induced ER stress and unfolded protein response deactivation, mitochondrial dysfunction and defects in mitophagy, paralleling with improved hepatic insulin resistance.