The Regulation Of Berberine On Glycolipid Metabolism Of Blunt Snout Bream Megalobrama Amamblycephala And Investigation Of Related MiRNAs

Carbohydrates,as a low-cost energy substance in feed,plays a role in improving the utilization of protein and lipid in feed and reducing the emission of ammonia nitrogen in aquaculture.In recent years,due to the rising price of fishmeal and peoples attention to ecological protection,carbohydrates have been widely used in fish feed.However,fish are generally intolerant to carbohydrates.Excessive carbohydrate content in feed will cause metabolic disorders,which will affect the health of fish.More and more studies have shown that micro RNA（miRNA）plays an important role in the regulation of glycolipid metabolism in fish.In addition to,it is known that berberine（BBR）can effectively alleviate the adverse effects of high-fat diet on fish metabolism by regulating miRNA,but there are few research on the application of high-carbohydrate diet.In view of this,this study used blunt snout bream（Megalobrama amamblycephala）as the test object to explore the effect of BBR on glucose metabolism and its mechanism of action from the perspective of miRNA regulation,providing reference for the application of BBR in fish feed.1 The effect of berberine on the growth of blunt snout bream and the related genes of glycolipid metabolismThis trial was conducted to investigate the berberine added in high carbohydrate diet affects the growth,insulin signal transduction and glycolipid metabolism of blunt snout bream which were randomly assigned into three diets: normal-carbohydrate diet（NCD,30% carbohydrate,w/w）,high-carbohydrate diet（HCD,43% carbohydrate）,and HCB（HCD supplemented with 50 mg/kg BBR）.After 10 weeks’ feeding trial,the results showed that higher plasma glucose,triglyceride,total cholesterol were observed in HCD-fed fish than in NCD-fed fish,while HCB-feeding significantly ameliorated this effect.Moreover,HCBfeeding remarkably reversed HCD-induced hepatic glycogen and lipid contents.In insulin signaling,BBR inclusion restored HCD-induced suppression of insulin receptor substrate m RNA expression and elevation of forkhead transcription factor 1 m RNA expression.In glucose metabolism,up-regulated glucose transporter 2（glut2）and glycogen synthase（gs）m RNA expressions in HCD group were observed compared to NCD group.However,BBRadding reduced the m RNA expressions of gs,phosphoenolpyruvate carboxykinase,glucose-6-phosphatase and increased the transcriptional levels of glut2 and pyruvate kinase.In lipid metabolism,BBR supplementation could reverse down-regulated hepatic carnitine palmitoyl transferase I m RNA expression and up-regulated acetyl-Co A carboxylase and fatty acid synthetase m RNA expressions in HCD group.In conclusion,it demonstrates that BBR could improve glucose metabolism of this species via enhancing glycolysis and insulin signaling,while inhibiting glycogen synthesis and gluconeogenesis.Meanwhile,BBR could also reduce the metabolic burden of liver and enhance the absorption of abdominal fat to inhibit fat synthesis and promoting lipid decomposition.2 The mechanism of miRNA regulation on liver glycolipid metabolism of blunt snout bream under the effect of berberineAs a small RNA molecule,miRNA can participate in the regulation of gene expression by identifying and degrading target m RNAs,and play a role in glycolipid metabolism.In order to further investigate the role of miRNA in BBRs regulation of glucose metabo lism of blunt snout bream,16 fish were randomly selected from the control group（NCD,30% carbohydrate level）,the high-carbohydrate group（HCD,43% carbohydrate level）and the berberine group（HCB,HCD group supplemented with 50 mg/kg BBR）and intraperitoneal injection of glucose（1.67 g/kg body weight）,respectively.Then,the plasma and liver samples were collected at 0 h,1 h,2 h,6 h and 12 h.The results showed that plasma glucose and glycogen levels were significantly affected by sampling time,feed and their interaction.Different from the NCD and HCB groups,the plasma glucose in the HCD group did not decrease after 1 h,but remained high at 2 h,and the corresponding glycogen level was also opposite to the NCD group,showing a significant increase within 0-2 h.In order to investigate the causes of abnormal changes in plasma glucose and glycogen content in the HCD group at 2 h and the role of BBR,miRNA high-throughput sequencing was performed on the livers of the three groups of fish at 2 h.By comparing with the genomic information of blunt snout bream,20 and 12 miRNAs（DEMs）were differentially expressed in HCD vs NCD and HCB vs HCD,respectively.By predicting the target genes of DEMs and analyzing the function,we found that HCD may affect the liver metabolism of blunt snout bream under glucose loading through NF-κB pathway.However,miRNAs regulated by BBR mainly play roles in adipocyte liponysis,Niacin and Nicotinamide metabolism,and amino acid（arginine,ornithine,lysine）transmembrane transport.In addition,the newly discovered novel:Chr12₁8892 has become the main research object because it is regulated by BBR,and has a high expression level in liver and has rich target gene function.In the functional exploration of novel:Chr12₁8892,it was found that its target gene,adenylate cyclase 3（adcy3）,was widely involved in lipid decomposition,amino acid metabolism and other pathways,and a targeting relationship with novel:Chr12₁8892 was also confirmed by double luciferase assay.It may be the target of BBR in plasma glucose regulation of blunt snout bream.In conclusion,BBR can participate in glucose metabolism of blunt snout bream by affecting lipolysis,amino acid transport and nicotinamide metabolism of liver adipocytes.In addition,BBR may target novel:Chr12₁8892 to activate the expression of adcy3 and participate in metabolic regulation.