| Carbohydrate is the cheapest feed material.Increasing carbohydrate level in aquatic feed can save the cost of diets.However,fish are intolerant to high carbohydrate.The important factor is that the insulin secretion of fish lags behind the sugar absorption rate.Glucose uptake in mammalian muscle tissue is achieved by the non-insulin-dependent AMPKα-TBC1D1 pathway affecting intracellular GLUT4 translocation.But whether glucose uptake in fish occurs by regulating GLUT4 in this pathway is not very clear now.In addition,it is important to pay more attention on the nutritional regulation strategies to alleviate the damage of high starch diet on fish nutrition.The aim of the present study was to investigate the molecular mechanism and regulatory strategy of AMPKα pathway on mediating the response to high starch diet in Monopterus albus.Research contents are as follows:1.Cloning and expression analysis of ORF of AMPKα1 in rice field eelIn order to understand the function of AMPKα1 and further explore the mechanism of AMPKα involved in glucose metabolism of M.albus,the open reading frame(ORF)of AMPKα1 was obtained and identified by molecular cloning.The results showed that the ORF length of AMPKα1 of M.albus was 1722 bp,encoding 573 amino acids.The AMPKα1sequence of M.albus has high homology with other species,and 96.68% similarity with AMPKα1 of climbing perch,which is relatively conservative sequence in evolution.The expression level of AMPKα1 gene in brain,intestine and liver of M.albus was higher,followed by that in spleen and kidney,and the expression level in muscle was the lowest.2.Effects of different starch levels on AMPKα mediated glucose uptake and utilization metabolism in muscle of rice field eelGLUT4 mainly mediates glucose transport in muscles.AMPK,as the upstream regulator of GLUT4,may play an important role in glucose metabolism in fish muscles.Five isonitrogenous and isolipid diets with starch levels(15%,20%,25%,30% and 35%;S15,S20,S25,S30 and S35)were formulated to study the effects of different starch levels on gene expression of AMPKα-mediated glucose absorption and glycolysis pathway in the muscle of M.albus(initial body weight,30.00 ± 0.1g),and the experiment lasted for 8 weeks.The results showed that with the increase of dietary starch levels,the gene expressions of ampkα1,ampkα2,tbc1d1 and glut4 in the muscle of M.albus increased at first and then decreased,and the genes expression of S20 group were significantly higher than in the other groups(P < 0.05).Different starch levels did not significantly affect the content of muscle glycogen(P > 0.05).The activity of HK in S30 group was the highest and significantly higher than that in S25 group(P < 0.05)and had no significant difference with other groups(P > 0.05).The PFK activity increased first and then decreased,and the PFK activity in S20 group was the highest,which was significantly higher than that in S15 group(P < 0.05),and there was no significant difference between S25 group and S30 group(P > 0.05).PK activity in S25 group was the highest and significantly higher than that in other groups(P < 0.05),the PK activity in S30 group was significantly lower than that in other groups(P < 0.05).The m RNA expressions of hk,pfk,pk,g6 pase and fpbase were up-regulated and then downregulated with the increase of dietary starch level.The expressions of hk and pfk in S20 group were significantly higher than those in other groups(P < 0.05).The expressions of pk in S25 group was significantly higher than that in S30 group(P < 0.05),but had no significant difference with other groups(P > 0.05).The m RNA expression of g6 pd was first up-regulated,then down-regulated,and then up-regulated with the increase of glucose level.The m RNA expression level of g6 pd in S20 group was the highest and significantly higher than that in S30 group(P < 0.05).To sum up,the expression of glucose transport-related genes in muscle of M.albus could be increased by adding appropriate α starch levels(20%)in diet.Adding α starch levels 20% in the diet can enhance the glycolysis metabolism of muscle of M.albus.It was found that AMPKα-TBC1D1-GLUT4 pathway may mediate glucose absorption in muscle of M.albus.3.Effects of high starch diet supplemented with metformin on growth performance,tissue morphology and glucose metabolism mediated by AMPKα pathway of rice field eelFour isonitrogenous and isolipid diets with LC group(15% α starch levels),HC group(35% α starch levels),HCM0.25 group(35% α starch levels + 0.25% metformin)and HCM0.50 group(35% α starch levels + 0.50% metformin)were formulated to study the effects of metformin supplementation on the growth,tissue morphology and glucose metabolism of M.albus(initial body weight,17.14 ± 0.02 g),and the experiment lasted for8 weeks.The results showed that the growth performance of HC group was not significantly different from that of LC group(P > 0.05).There was no significant difference in growth performance between HCM0.25 group and LC group and HC group(P > 0.05).The final average weight,weight gain rate and specific growth rate in HCM0.50 group were significantly decreased,while the feed conversion rate was significantly increased(P < 0.05).Compared with LC group,hepatocytes in HC group showed swelling,increased vacuoles,smaller or disappeared nuclei,and cell membrane damage.The morphology of hepatocytes in HCM0.25 group was obviously normal and the number of vacuoles decreased.The glycogen content in HC group was significantly higher than that in LC group(P < 0.05).The glycogen content of HCM0.25 group was significantly reduced(P < 0.05).Compared with LC group,the activities of HK,PFK,FBPase and PEPCK in HC group were significantly increased(P < 0.05).Compared with HC group,PFK and PK activities in HCM0.25 and HCM0.50 groups were significantly increased,while FBPase activity was significantly decreased(P < 0.05).The m RNA expressions of pk,g6 pase and fbpase in liver of HC group were not significantly different from those of LC group(P > 0.05).Compared with LC group,the m RNA expression levels of hk,pfk and pepck in liver were significantly up-regulated(P< 0.05).Compared with HC group,the m RNA expression levels of pk in HCM0.25 and HCM0.50 groups was significantly up-regulated by 4.32 times and 4.59 times,and the m RNA expressions of g6 pase,fbpase and pepck in liver were significantly down-regulated(P < 0.05).Compared with LC group,the m RNA expressions levels of pfk,pk,g6 pase,fbpase and pepck in muscle of HC group had no significant changes(P > 0.05).Compared with LC group,the m RNA expressions level of hk in muscle of HC group was significantly down-regulated by 1.51 times,and the m RNA expressions level of g6 pd was significantly up-regulated by 1.46 times(P < 0.05).Compared with HC group,the m RNA expressions level of hk,pfk,g6 pase and pepck in HCM0.25 group were significantly up-regulated(P <0.05).The m RNA expressions level of ampkα1,ampkα2,tbc1d1 and glut4 in the muscle of HCM0.25 group were significantly up-regulated compared with those of HC group(P <0.05).These results indicated that 35% α starch diet did not affect the growth performance of M.albus,but could damage the liver morphology.Adding 0.25% metformin can relieve liver damage of M.albus when fed with high α starch.At the same time,metformin may increase muscle glucose absorption. |
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