The Study Of The Regulation Of N-3 Polyunsaturated Fatty Acids On Mitochondrial Energy Metabolism In Mouse Muscle

N-3 polyunsaturated fatty acids(n-3 PUFA)as important components of the plasma membrane and organelle membranes affect the metabolisms of cellular fatty acids and physiological activities by regulating gene expressions.A number of studies have proved that n-3PUFA improve muscle metabolism by promoting synthesis of myocutaneous proteins,enhancing the contractile function of muscle cells and inhibiting muscle atrophy.However,the effects of n-3 PUFA on generation and metabolism of ATP in muscle have not been clearly understood.In this study,transgenic mice that overexpressed Fad3b,a fatty acid desaturase gene from flax,were produced,the mice were used as a model to analyze the roles of n-3 PUFA in the regulation of ATP metabolism in muscle fibers.Analyses of the transgenic mice showed that the contents of PUFA in various tissues changed due to the expressions of the transgenic Fad3b.In the transgenic muscles,the n-3 PUFA were significantly increased.The level of DHA in the transgenic pups was 2.66times over that in the non-transgenic mice.These results suggested that the increased DHA be key for changes of n-3 PUFA in muscles.Transcriptomical data of the muscles in transgenic and nontransgenic mice showed that total of 1654 differentially expressed genes were identified,of which 909 genes were up–regulation,and 745genes were down-regulation in the Fad3b-overexpress group.Proteomic analysis showed that 407 different proteins were found in 1765 detected proteins,and 203 proteins were up-regulation,204 were down-regulation in Fad3b-overexpress group.The overexpression of Fad3b in muscles significantly promoted the metabolism of fatty acids,but inhibited the oxidative phosphorylation of mitochondria and the metabolism of glucoses.To further investigate the effect of n-3PUFA on muscle energy metabolism,the muscle satellite cells(MSCs)were isolated and cultured from the Fad3b-overexpressed and the wide-type pups.The established MSCs were respectively called F-MSCs and WT-MSCs.The morphology of the mitochondria in F-MSCs was thinner,shorter and scattered around the nucleus than those in the WT-MSCs.When the satellite cells were adipocyte-induced,the F-MSCs and DT-MSCs-derived adipocytes only had small triglyceride droplets,while the droplets induced from WT-MSCs were much large.The activities of triglyceride hydrolase in F-MSCs and DT-MSCs were significantly enhanced,and the glycerol was significantly accelerated,which indicated that n-3 PUFA could promote lipolysis.Meanwhile,the expressions of AMPK,PGC1?,PPAR?and ACAA1 that promoted fatty acid beta oxidation were significantly up-regulated in both F-MSCs and DT-MSCs,suggested that n-3 PUFA increase the level and rate of fatty acid beta oxidation in the muscle cells.In F-MSCs and DT-MSCs,the glucose levels and expressions of Glut4 and HK were significantly improved,but the pruvic acid levels and the expressions of PFK and PK were significantly lower than that in the WT-MSCs.These results revealed that the glycolysis process was significant inhibited.Furthermore,down-regulation of Akt2 suppressed the activity of GSK3,then activated glycogen synthesis,and subsequently increased glycogen levels in both F-MSCs and DT-MSCs.As key component in mitochondrial TCA and oxidative phosphorylation,high-level of acetyl coenzyme A was detected in the F-MSCs and DT-MSCs.The activity of CS and its products,citric acids,were significantly increased,while IDH decreased significantly.The expressions of TCA speed limiting enzymes such as OGDH,SDHA and MDH2 were significantly down-regulated.These results suggested that n-3 PUFA inhibit the TCA by decreasing the activities of the speed limiting enzymes,which resulted in abnormal accumulation of NAD~+in muscle cells and decreased the concentration of NADH.High-levels of n-3 PUFA also significantly decreased the activities of the key components of the electron respiratory chain,such as complex I,complex IV and ATP enzymes,which led to the decreased synthesis of ATPs.In summary,ovexpressions of Fad3b in mice increased the amount of n-3 polyunsaturated fatty acids,promoted the oxidative metabolism of fatty acids and the synthesis of glycogen,but suppressed the glycolysis and pentose phosphate cycle,and decreased the activities of the TCA and the respiratory chain complexes,and then inhibited the OXPHOS.