Adiponectin And Its Receptors Mediated Adipogenic Gene Expression And Molecular Regulation Intramuscular,Visceral And Subcutaneous Adipocytes In Sheep

Fat trait,which plays an important role in life activities and physiological metabolism,is one of the most important economic traits in meat sheep industry.Adiponectin,which is the highest adipocyte cytokine and the only negatively correlated with obesity,is involved in the regulation of fat and glucose metabolism,the maintenance of energy balance,insulin resistance,anti-inflammatory response and other physiological processes in vivo.At present,fewer studies on the role of adiponectin and its receptor genes in adipose differentiation in sheep have been reported.In this study,the full-length cDNA sequence of adiponectin and its receptor genes in Lanzhou fat-tailed sheep were cloned at the first time using rapid amplification of cDNA end(RACE).The tissue specific expression pattern of three genes in 15 tissues in sheep was implemented by real-time fluorescence quantitative PCR(qPCR).The subcutaneous,visceral and intramuscular pre-adipocytes were isolated and cultured.The relative expression of adiponectin,adiponectin receptors,lipoprotein lipase(LPL),hormonesensitive lipase(HSL),fatty acid synthase(FAS)and peroxisome activator receptor ?/?(PPAR ?/?)in the differentiation and maturation from pre-adipocytes into adipocytes.The adipogenesis influence and adipogenic related gene expression of over expression and siRNA silence adiponectin receptor genes in intramuscular adipocytes were analyzed.Candidate proteins interacted with adiponectin receptors in fat metabolic pathway were screened by pull-down and mass spectrometry.The following results were obtained:1.Using homology cloning and RACE,the full-length cDNA of adiponectin(APN)with 2010 bp(GenBank accession number: KJ159213),adiponectin receptor 1(AdipoR1)with 2035 bp(KJ159212)and adiponectin receptor 2(AdipoR2)with 1809 bp(KF921623)were cloned for the first time in sheep.The relative expression pattern of three genes in 15 tissues including liver,kidney,heart,lung,spleen and subcutaneous fat,visceral fat,muscle,ovary,testis,large intestine,small intestine,brain,rumen and abomasum in sheep were investigated by qPCR.The expression of APN was the highest in muscle tissue,and following in heart and abomasum.AdipoR1 was the highest expression in visceral fat and subcutaneous fat,following in spleen,heart and small intestine.The expression of AdipoR2 was the highest in subcutaneous tissue and visceral tissue,but very low in other tissues.2.The vesceral and subcutaneous pre-adipocytes were isolated and cultured by type I collagenase digestion,inducing to differentiate into matured adipocytes by DMEM/F12+ insulin(5?g/mL)+ transferrin(50?g/mL)+ sodium selenite(5ng/mL)+ hydrocortisone(50ng/mL)for 11 days.The intramuscular pre-adipocytes of longissimus dorsi muscle were isolated and cultured by type II collagenase digestion and differential adhesion method,inducing to differentiate into matured adipocytes by DMEM/F12+IBMX(0.5mmol/L)+ DEX(1?mol/L)+ Insulin(10mg/L)for 10 days.During the differentiation of the three kinds of pre-adipocytes,the fat production increased gradually,and the growth curve was "S" type,and the exponential growth phase was observed from the third to the ninth day.The levels of LPL and PPAR? in visceral and subcutaneous pre-adipocytes on the 7th and 11 th day were significantly higher than those on the third day(p<0.05).The expression of FAS gene reached to the peak on the 7th day.There was no significant difference in the expression of adiponectin and its receptor genes in visceral and subcutaneous pre-adipocytes(p>0.05),but the expression of AdipoR2 was the highest(35.27)and APN was the lowest(0.60),while the AdipoR1 was in the middle(5.24).Similarly,the expression of AdipoR2 in intramuscular adipocytes was most significantly higher than that in AdipoR1 and APN(p<0.01),and the expression trend was similar to that in visceral adipose tissue and subcutaneous adipose tissue,but the expression level decreased.The expression patterns of LPL,PPAR? and FAS in intramuscular pre-adipocytes were consistent with those of visceral and subcutaneous pre-adipocytes,indicating that AdipoRs,LPL and PPAR? were early marker genes for adipocyte differentiation.3.AdipoR1 plays an important role in adipogenesis by regulating the expression of hormone-sensitive lipase(HSL)and peroxisome proliferator receptor ?(PPAR?)during the differentiation of sheep intramuscular pre-adipocytes.(1)On the 7th day after differentiation,the mRNA expression of HSL and AdipoR1 in intramuscular pre-adipocytes decreased,while the mRNA expression of other marker genes increased,but had no significant effect on the expression of AdipoR2.(2)Overexpression of AdipoR1 could significantly reduce the deposition of muscle pre-adipocytes,but overexpression of AdipoR2 had no significant effect on fat deposition.Overexpression of AdipoR1 increased the expression of HSL and decreased the expression of PPAR?,but did not affect the expression of FAS and PPAR?.The overexpression of AdipoR2 gene had no effect on the expression of HSL,FAS,PPAR? and PPAR?.(3)AdipoR1 gene silencing increased the volume and fat deposition content of intramuscular pre-adipocytes,significantly decreased the expression of HSL and increased the expression of PPAR?,but had no effect on the expression of FAS and PPAR?.After AdipoR2 gene silencing,it had no significant effect on the volume and fat deposition content of intramuscular pre-adipocytes,and had no effect on the expression of HSL,FAS,PPAR? and PPAR?.4.Pull-down and mass spectrometry analysis showed that L-lactate dehydrogenase(Llactate dehydrogenase)and 1-type long-chain acyl coenzyme A synthase(Acyl-CoA synthetase long chain family member-1)could interact with AdipoR1 in sheep visceral fat.Type 1-long chain acyl coenzyme A synthase(Acyl-CoA synthetase long chain family member-1)in the tail fat can interact with AdipoR2,which lays a foundation for further study of adiponectin receptor function.In conclusion,AdipoR1 can play an important role in adipogenesis by regulating the expression of HSL and PPAR? during the differentiation of sheep intramuscular preadipocytes.It can be used as an important genetic marker gene in meat sheep breeding industry.