Research On The Mechanism Of Acetate Regulating Adipocytes Metabolism In Rabbits

In animal production,lipid accumulation and distribution could affect the meat quality,and attracts more and more researcher's attention.Short-chain fatty acids?SCFAs?are the fermented production of gastrointestinal tract microorganism.In addition to providing energy,SCFAs are known to have regulator effects on rumen fermentation,insulin and glucose secretion,gastrointestinal motility,cell proliferation,apoptosis and differentiation.Acetate,as the main fermented production of gastrointestinal tract microorganism in the rabbit,plays an important role in lipid metabolism and cell proliferation,differentiation.In this study,based on the regulation of lipid metabolism by acetate,we established the stress model of rabbit lipid metabolism and explored the mechanism of acetate regulating lipid metabolism in rabbits.Identification and expression pattern analysis of rabbit GPR41 and GPR43 genes In this study,we coloned and sequenced the free fatty acid receptors GPR41 and GPR43 in rabbits,followed by the expression pattern analysis.Results showed that,the rabbit genome could express GPR41 and GPR43 mRNA.The rabbit GPR41 mRNA has high similarities with the human,bovine and Capra hircus?84%?,and GPR43 m RNA has high similarities with the rat and mouse?84%?.Real-time PCR results indicated that GPR41 and GPR43 mRNA were expressed throughout rabbit's whole development and were detected in all detected tissues,such as thymus,spleen,pancreas,lung,adipose tissue,duodenum,jejunum,ileum,colon and cecum.At 90^thday,GPR41 and GPR43 mRNA were most highly expressed in pancreas?P<0.05?and adipose tissue?P<0.05?,respectively.The expression level of GPR41m RNA was down-regulated in duodenum,cecum and pancreas?P<0.05?and up-regulated in jejunum,ileum,adipose tissue and spleen during growth.GPR43 mRNA was highly expressed in duodenum,jejunum,ileum,colon,cecum and lung at 15^thday?P<0.05?,whereas the expression level in pancreas and spleen increased later after birth,with the highest expression at 60^thh day?P<0.05?.The function of acetate on lipornetabolism in rabbits 80 New Zealand rabbits,after weaning and in good condition,were selected and divided into four groups randomly,received a subcutaneous injection of 0,0.5,1.0 or 2.0 g/kg/day body mass acetate,with same amount of saline for control.Our results showed that acetate induced a dose-dependent decrease in shoulder adipose?P<0.05?.Although 2.0 g/kg acetate injection did not alter the plasma leptin and glucose concentration?P>0.05?,it significantly decreased the plasma adiponectin,insulin and triglyceride concentrations?P<0.05?.The analysis of lipid metabolism related genes expression in adipose tissue showed that,2.0g/kg acetate injection significantly up-regulated the key transcriptional regulators of adipogenic differentiation gene expression,such as PPAR?,C/EBP?,ADD1 and aP2?P<0.05?,and increased the expression of lipolysis related genes,such as CPT1,CPT2 and HSL?P<0.05?.Acetate treatment had no effect on the expression of lipid synthesis genes,such as FAS and ACC1?P<0.05?,but the expression of adipocyte specific genes was stimulated,such as AdipoR1 and AdipoR2.In addition,the expression levels of GPR41 and GPR43 mRNA and protein phosphorylation levels of AMPK,P38 MAPK,ERK1/2 and JNK were increased by acetate treatment?P<0.05?.However,there was no significant effect on the expression of mTOR protein and its phosphorylated protein?P>0.05?.The above results indicated that acetate could promote rabbit adipose differentiation and lipid catabolism,inhibit lipid deposition,and the regulation effect of acetate on rabbit lipid metabolism maybe actualize by GPR41/43,AMPK and MAPK signal pathways.Effect of acetate on lipid metabolism of rabbit adipose-derived stem cells?ADSCs?1Analysis of adipogenic differentiation ability of rabbit ADSCs.Rabbit ADSCs were selected for adipogenic differentiation in vitro,and were induced to adipogenic differentiation for 15d.The results showed that the lipid droplets became visible on day 3 of differentiation in cells,and larger along with the prolongation of time.At the sametime,the morphology of the cells changed from long spindle to round.The expression levels of lipid metabolism related genes revealed that,expression of adipogenic differentiation genes,such as PPAR?,C/EBP?and ADD1,increased significantly?P<0.05?from day 3 with a peak on day 7,accompanied by the raise of FAS,ACC1 and CPT1 expression?P<0.05?as well as the raise of adipocyte-specific genes expression such as AdipoR1 and AdipoR2?P<0.05?.It indicated that rabbit ADSCs could be induced into mature adipocytes in vitro gradually,and the cells go into the active period of adipogenic differentiation on day 3.2 Effect of acetate on adipose metabolism of rabbit ADSCs.We selected the rabbit ADSCs on day 3 of adipogenic differentiation and different concentration acetate were used to treat the cells for different time.Results showed that,acetate treatment for 6h increased the expression of PPAR?,C/EBP?,ADD1,FAS,ACC1 and AMPK??P<0.05?,and activated the expression of phosphorylated AMPK and p38 MAPK?P<0.05?.Acetate treatment for 48h increased the expression of C/EBP?,ADD1 and ACC1,decreased the expression of AMPK?m RNA,and inhibited the expression of phosphorylated mTOR and ERK?P<0.05?.GPR41and GPR43 mRNA were not detected in rabbit ADSCs in vitro.It seems that the regulation effect of acetate on lipid metabolism of rabbit ADSCs is not achieved through GPR41 or GPR43 pathway.3 Effect of AMPK,p38 MAPK on acetate-induced adipose metabolism.Rabbit ADSCs were treated with acetate?6mM?for 6h,accompanied by the treatments of AMPK inhibitor?10?M Compound C?or p38 MAPK?10?M SB203580?.Results showed that,these inhibitors decreased the acetate-induced phosphorylated AMPK and p38 MAPK respectively?P<0.05?.And Compound C inhibited the up-regulation of acetate on PPAR?,C/EBP?,ADD1,ACC1and CPT1?P<0.05?,and activated reversely the down-regulation of acetate on HSL expression?P<0.05?,with the decrease of lipid deposition.But SB203580 increased the acetate-induced expression of lipid metabolism genes?P<0.05?and lipid deposition.Results showed that the positive regulation of acetate on rabbit fat deposition is achieved by AMPK.4 Effect of mTOR,ERK on acetate-induced adipose metabolism.Rabbit ADSCs were treated with acetate?9mM?for 48h,with ERK activator?10?M CeramideC6?or mTOR activator?10?M MHY1485?.Results showed that,the acetate-induced ADD1 and HSL were inhibited after the activation of ERK,along with the relieving of inhibited LPL?P<0.05?.The activation of acetate on ADD1,ACC1 and HSL was inhibited after the activation of mTOR.Results showed that acetate promoted the expression of ADD1 and HSL and decreased the expression of LPL via the inhibition of ERK pathway,and activated the expression of ADD1,ACC1 and HSL via the inhibition of mTOR pathway.The inhibition of mTOR and ERK by acetate resulted in the relative decrease of lipid synthesis and the increase of catabolism.In conclusion,acetate can promote the adipogenic differentiation ability of rabbit adipocytes,and the regulation effect on fat deposition is in time-dependent manner.In vivo,results showed that acetate may be through AMPK,MAPK,gpr41/43 and other signaling pathways to play the positive regulation on adipogenic differentiation ability,up-regulate fatty acid oxidation and lipolysis activity and promote lipid metabolism,reducing lipid deposition.In vitro,results showed that the short term?6h?acetate treatment promotes cell adipogenic differentiation through the activation of AMPK,which increased fatty acid biosynthesis,inhibited lipid catabolism,and enhanced lipid deposition.When acetate treatment time was further extended?48h?,it decreased the lipid deposition rate,accelerated the rate of lipid catabolism,and the lipid deposition amount began to decrease,through inhibiting the activity of mTOR and ERK.