Mechanisms Of PPAR-? Regulated Energy Metabolism Of Muscle Satellite Cells From Fad3 Transgenic Mice

The N-3 polyunsaturated fatty acids(N-3 PUFA)have essential roles in the regulation of metabolisms of glucose,fatty acids and proteins,which are certainly beneficial for the reduction of insulin resistance,anti-inflammatory,and protection of cardiovascular system.In order to investigate the possible mechanisms of N-3 PUFA on metabolisms in both muscle tissue and cells,the fad3 transgene mice and satellite cells were used.First,the composition changes of PUFAs in the transgenic muscle,liver and adipose tissues were analyzed;Second,the expressions of key enzymes involved in the Kreb's cycle,and the expressions of PPARs genes either in transgenic satellite cells or DHA-supplemented satellite cells were detected;Third,the activator or inhibitor of PPAR-? were added to the culture of the satellite cells,the expressions of both PPARs and TCA defined enzymes were analyzed;Forth,target protein(s)of PPAR-? protein were identified by CO-IP technique.The results were as followings:(1)In the transgene mice,expressions of fad3 genes were tissue-specific,which significantly expressed in liver and muscles;conteNt of N-3 PUFA increased and n-6 PUFA decreased,and the transition of N-6 to N-3 PUFA improved;total qualitity of both short-and middle-chain fatty acids decreased.(2)the expression of PPAR-? protein significantly expressed in both fad3 satellite cells and DHA-supplemented satellite cells,while PPAR-? and PPAR-? were with changes compared to the control.N-3 PUFA natively regulated TCA cycle via inhibitions of the key enzymes involved in TCA including Idh2,Idh3 a,Ogdh and Mdh2.(3)protein spectroscopy data showed that the metabolic capacities of fatty acids in fad3 and control cells added with 100ng/ml DHA were enhanced,and the mitochondrial oxidative phosphorylation and TCA related protein expressions were significantly reduced;(4)quantitative PCR verified the expression of TCA related genes and showed that the increase of N-3 PUFA significantly reduced the expressions of Idh2,Ogdh,Sdha and Mdh2;(5)in the fad3 transgenic satellite cells,the PPAR-? mRNA significantly increased,the protein level especially in the nuclei greatly reduced;the expressions of PPAR-? and PPAR-? did not changed;(6)addition of MK886,an inhibitor of PPAR-?,significantly decreased the expressions of the key limiting enzymes in TCA including Idh2,Ogdh,Sdha and Mdh2,while addition of WY14643,an activator of PPAR-?,significantly increased the expressions of the TCA key enzymes;(7)Chip-seq results indicated that the increased cytoplasmic N-3 PUFA inhibited the binding ability of PAPR-? to the DNA sequence of the genes associated with mitochondrial energy metabolism.In conclusion,fad3 transgene significantly increased the N-3 PUFA in vivo by catalyzing the transformation of N-6 PUFA into N-3 PUFA,the high level cytoplasmic N-3 PUFA affected the mitochondrial energy metabolism of muscle satellite cells by inhibiting the binding ability of PAPR-? to the DNA sequence of the genes associated with mitochondrial energy metabolism.