Study On Irisin Regulate Common Carp Glucose Metabolism Through AMPK And PI3K/Akt Signaling Pathway

Skeletal muscle is not only the main exercise organ of body,but also can express,synthesize and secrete various biological active molecules,such as FGF-21,IL-15 and myostatin,etc.These muscle factors participate in the body`s metabolism regulation in paracrine or endocrine manner.Irisin is a new muscle factor discovered in 2012.It has the effects of improving insulin resistance,anti-inflammatory,and improve cardiovascular and endothelial functions.However,there are few reports on the research of irisin on glucose metabolism in fish.In this study,the TSA（Transcriptome Shotgun Assembly）database was used for splicing and comparison,and the FNDC5 sequence of the precursor gene of common carp（Cyprinus carpio L.）irisin was cloned by specific primers.The common carp FNDC5 gene sequence information was obtained through sequence alignment,phylogenetic tree analysis and gene collinearity analysis.Subsequently,the expression characteristics of common carp FNDC5 were analyzed.The recombinant irisin protein of common carp was obtained through prokaryotic expression system,and polyclonal antibody was prepared.Through intraperitoneal injection and primary hepatocyte incubation experiment,the effect of irisin on common carp glucose metabolism was detected.In addition,theRNAi experiment was used to knock down the expression of FNDC5 to detect the effect on common carp glucose metabolism.Finally,the signaling pathway of irisin on common carp glucose metabolism was clarified by incubating primary hepatocytes with irisin and AMPK or PI3K/Akt inhibitors in vitro.The main findings are as follows:1.In this study,the c DNA sequence of common carp FNDC5 was cloned from the hypothalamus of common carp for the first time.The c DNA of FNDC5 is 722 bp,of which the open reading frame is 666 bp,encoding 221 amino acids,and has a predicted molecular weight of 24.586 KDa.Signal P was used to predict that the first 25 amino acids of FNDC5 are signal peptides,and the subsequent 112 amino acid sequences are irisin mature peptides.Net Phos predicts that the common carp FNDC5 contains 24 phosphorylation sites,including 13 serine residues,10 threonine residues and 1 tyrosine residue.In addition,the prediction results showed that the common carp FNDC5 protein contains two N-glycosylation sites（NVS and NTT）.Phylogenetic tree analysis showed that common carp FNDC5 and fish FNDC5 clustered together.Combined with the results of gene collinearity analysis,we successfully obtained the common carp FNDC5.In order to analyze the expression pattern of FNDC5 in common carp tissues,19 tissues in the brain area and the periphery were detected by q PCR.The results showed that FNDC5 mRNA is highly expressed in the mesocerebrum,telencephalon,hypothalamus and gonads,and low expressed in the heart,liver and gills.2.In order to evaluate the effect of nutritional status on FNDC5 mRNA expression,starvation and refeeding,glucose tolerance（OGTT）,high-glucose and high-fat feeding experiments（8 weeks）were carried out.After 7 days of starvation,the expression level of FNDC5 in the liver and midgut was significantly upregulated,and returned to normal levels after refed.OGTT results showed that the expression of FNDC5 was significantly up-regulated in the brain,midgut and red muscle,and significantly down-regulated in the liver.High-sugar or high-fat feeding can up-regulate the expression of FNDC5 mRNA.Insulin injection（100 ng/g B.W.）inhibited the expression of FNDC5 in common carp liver and gut.On the contrary,glucagon（100 ng/g B.W.）injection promoted the expression of FNDC5 in liver.3.The recombinant common carp irisin protein prepared by Escherichia coli prokaryotic expression system,which provided a protein basis for future functional studies.After purification of common carp recombinant irisin protein,NIH negative mice were immunized to prepare polyclonal antiserum of common carp irisin.The titer of irisin antibody detected by polyclonal antibody titer was 1:90000.The antibody was used to analyze common carp serum samples by ELISA,and the content of serum irisin was detected to be1.26 ng/m L,and an experimental method for detecting irisin in fish was successfully established.The immunofluorescence experiment confirmed the endogenous irisin protein in common carp tissues.4.The functional study of common carp recombinant irisin protein was carried out by incubation of primary hepatocytes in vitro and intraperitoneal injection experiments.The results showed that irisin can significantly reduce the glucose content,and regulate the mRNA expression of glucose transporter sglt1 and glut2,promote the expression of glycogen synthesis related gene gs,and inhibit the expression of gsk3β.Enzyme activity results showed that irisin can significantly increase the activities of glycolysis-related enzymes,glucokinase（gk）,hexokinase（hk）,pyruvate kinase（pk）and phosphofructokinase（pfk）,reduce the activities of gluconeogenesis-related enzymes,fructose-1,6-bisphosphatase（fbpase）,phosphoenolpyruvate carboxykinase（pepck）and glucose-6-phosphatase（g6pase）,and promote the synthesis of liver glycogen.The above results indicate that irisin can inhibit the activity of gluconeogenic enzymes and promote glycolytic enzyme activity by regulating the expression of genes related to liver glucose transport and glycogen synthesis,thereby reducing serum glucose levels.5.Inject irisin-siRNA into the brain forRNAi experiment.We found that after siRNA injection,the mRNA expression of FNDC5 in the brain and liver was inhibited,and the inhibition rates reached 91% and97%,respectively,which proved thatRNAi can reduce the expression level of common carp FNDC5 effectively.After the injection of siRNA,the serum irisin content decreased significantly,and the serum glucose showed an increasing trend.The expression of glut2 in the brain decreased,but in the liver,the mRNA levels of glut2 and gs were significantly down-regulated,while pygl was significantly up-regulated.The gene expression of glycolysis hk and pfk was significantly down-regulated,and gluconeogenesis g6 pase and pepck were significantly up-regulated.It further proves that irisin plays an important role in common carp glucose metabolism.6.In order to clarify the mechanism of irisin,this study used irisin and AMPK or PI3K/Akt inhibitors to incubate common carp primary hepatocytes.The results showed that irisin activated AMPK signaling pathway and PI3K/Akt signaling pathway,and the phosphorylation reached the highest level at 10 min.Irisin regulated g6 pase through the AMPK/PI3K/Akt pathway,regulated the mRNA levels of fbpase,pepck and gk by activating the AMPK signaling pathway,regulated the mRNA levels of gs,pfk,hk and gsk3β by activating the PI3 K pathway.The results showed that irisin can act on different target genes through AMPK and PI3K/Akt signaling pathways to regulate common carp glucose metabolism.