| Carbohydrate is the most readily available and the cheapest energy yielding macronutrient in the world,thus it is extensively incorporated in the feed of aquatic animals.The carbohydrate utilization capacity of the carnivorous fish is poor,especially obvious in largemouth bass Micropterus salmoides(LMB),which can tolerate dietary starch as low as 10%.Metformin has a strong glucose-lowering effect and can improve the glycolipid metabolism,thus it is currently the first-line drug of choice in the clinical treatment for human beings with type 2 diabetes.However,the potential role of metformin in the glycolipid metabolism of fish is still unclear.In this thesis,the effects of dietary starch and metformin levels on the growth,body composition,hepatic glycolipid metabolism and liver histology of LMB were investigated,and the effects of intraperitoneal injection of glucose and metformin on hepatic glycolipid metabolism were also evaluated.The main contents were as follows:1.Effects of dietary metformin and starch level on growth,body composition,glucose homeostasis and liver histology of LMB Micropterus salmoidesIn this study,an 8-week feeding trial was performed to evaluate the effects of dietary metformin inclusion on the growth performance,body composition,glucose homeostasis and liver histology of juvenile largemouth bass Micropterus salmoides(LMB)fed high starch diets.Using a 2 × 2 factorial design,four iso-proteinic(50%)and iso-lipidic(13%)diets were formulated with two starch levels(9% and 14%)with or without 0.25% metformin inclusion at each starch level.Each diet was assigned to triplicate tanks of LMB with an initial body weight of 10.8 g.The results showed that an increase of dietary starch from 9.25% to 14.3% retarded the growth performance(weight gain ratio and specific growth rate)of LMB through impairing feed utilization(feed efficiency ratio,protein efficiency ratio and protein productive value)(P<0.05).The glucose level of LMB was significantly increased with the dietary starch level increasing(P<0.05),moreover,with an increase of starch level,the ability of glucose tolerance was also significantly decreased after intraperitoneal injection glucose(P<0.05).At the molecular level,the relative expressions of glucokinase(gck),phosphofructokinase(pfkla)and glycogen synthase(gys1 and gys2)were significantly up-regulated by feeding high starch diet(P<0.05)while relative expressions of glucose-6-phosphatase(g6pca1)was down-regulated(P<0.05),suggested that LMB could cope with intaking high starch diet by regulating glycolysis,glycogen synthesis and gluconeogenesis.In addition,with the increase of dietary starch level,the expressions of acetyl-Co A carboxylase(acca),ATP citrate lyase(aclya and aclyb)and lipid synthetase(fas)were significantly up-regulated(P<0.05),while the expression levels of lipolysis such as carnitine palmitoyl transferase(cpt1a,cpt1a2 and cpt1b)were significantly down-regulated(P<0.05).Consistent with gene expression,the content of liver lipid,whole fish lipid,cholesterol and triglyceride in plasma were also significantly increased with increasing dietary starch level(P<0.05).The results of liver histology presented that hepatocyte vacuolation was increased in HC,and the phenomenon of nuclear deviation or even disappearance was aggravated.The activities of ALT and AST in plasma were also significantly increased in HC(P<0.05),indicating that high starch diets could induce liver injury of LMB.Though feed consumption increased,the growth performance of LMB did not benefit from dietary metformin administration as a result of the impaired feed utilization.Metformin significantly reduced glucose level of LMB,and the hypoglycemic effect was still present within 1-6 h after glucose load(P<0.05).At the molecular level,metformin supplementation significantly up-regulated the relative expressions of pfkla,fas and aclyb(P<0.05),while significantly down-regulated the relative expressions of gys1 and gys2(P<0.05).The results indicated that metformin could improve glucose homeostasis and glucose tolerance of LMB by promoting glycolysis and lipid synthesis.However,dietary metformin did not mitigate the liver injuries(the activities of alanine aminotransferase and aspartate aminotransferase in the plasma along with histological appearance)in LMB fed high starch diets.In conclusion,our results demonstrated that dietary metformin inclusion did not mitigate the growth retardation and liver injury of LMB fed high starch diets,but improved the glucose homeostasis and glucose tolerance through promoting the glycolytic and lipogenic pathways in the liver.2.Effects of intraperitoneal injection of glucose and metformin on hepatic glucolipid metabolism of LMB Micropterus salmoideTo explore the effects of injecting glucose and metformin on hepatic glycolipid metabolism of LMB at the molecular level,LMB with initial body weight of 65.0±2.0g were selected and divided into four treatment,they were PBS group(injected phosphoric acid buffer),glucose group(Glu,injected 1g/kg glucose),metformin group(Met,injected100mg/kg metformin)and the glucose plus metformin group(Glu+Met,injected 1g/kg glucose and 100mg/kg metformin),respectively.Then sampled at 3 h and 15 h after intraperitoneal injection.The results showed that at 3h,glucose levels were significantly affected by both glucose and metformin,with higher values in response to glucose and metformin(P<0.05).The relative expression levels of acetyl-Co A carboxylase(acca)and carnitine palmitoyl transferase(cpt1a2 and cpt2)were only affected by glucose,they were significantly up-regulated by glucose(P<0.05).The relative expression levels of phosphoenolpyruvate kinase(pck2),glucose-6-phosphatase(g6pca1,g6pca2)and lipid synthase(fas)were only affected by metformin,they were significantly up-regulated by metformin(P<0.05).The relative expression levels of accb,ATP citrate lyase(aclya and aclyb)and cpt1 b were affected by both glucose and metformin,and they were significantly up-regulated by glucose and metformin(P<0.05).Glucokinase(gck),glycogen phosphorylase(pygl)and pck1 were significantly affected by the interaction between glucose and metformin(P<0.05).The relative expression level of gck was up-regulated in Glu,and down-regulated in Met and Glu+Met;the relative expression of pygl had no change in Glu and Met,however,Glu+Met up-regulated pygl;as for pck1,in Glu and Met,there were no effect on it,however,Glu+Met down-regulated the relative expression level of pck1.At 15 h,glucose levels were significantly affected by both glucose and metformin,with higher values in response to glucose and metformin(P<0.05).The relative expression levels of acca and cpt2 were only affected by glucose,which were significantly up-regulated by glucose(P<0.05).The relative expression levels of phosphofructokinase(pfkla),gys1,accb and aclya were only affected by metformin,and the expression levels of pfkla,gys1 and accb were significantly down-regulated by metformin(P<0.05),the trend of aclya was opposite to those three.The relative expression levels of glucose transporter(glut2),pygl and pck1 were significantly affected by the interaction between glucose and metformin(P<0.05),the relative expression of glut2 was significantly up-regulated in Glu,injection of metformin alone did not affect the expression of glut2,however,in Glu+Met,the relative expression of glut2 was down-regulated;the relative expression level of pygl was not affected in Glu and Met,however,it was down-regulated in Glu+Met;pck1 was down-regulated in Glu and Met,while there was no change in Glu+Met.In conclusion,glucose activated glycolysis,lipogenic and lipolysis pathways in the liver of LMB,the lower ability of glucose tolerance in LMB may be due to the lag of glut2 and the fact that pfkla,g6pca1 and g6pca2 are not regulated by glucose,in this study,metformin increased glucose levels in LMB,the reason may be that metformin inhibited glucose transport,and glycolysis while promoted gluconogenesis pathways in the liver of LMB,in addition,we also confirmed that metformin promoted lipid synthesis in LMB. |
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