Mechanism Of FoxO1 On Glucose And Lipid Metabolism In Micropterus Salmoides

High starch diets induced metabolic liver disease(MLD)in largemouth bass(Micropterus salmoides).However,low starch levels in the formula would negatively affect the processing of expanded floating feeds.Increasing starch levels in the formula can not only reduce the feed cost but also improve the feed quality.Therefore,it was the key issue for the sustainable development of the largemouth bass industry to improve starch utilization.In mammals,FoxO1 had been known as a key molecular site to regulate hepatic gluconeogenesis,enhance glucose tolerance and insulin sensitivity.Therefore,this study aims to explore the mechanism of FoxO1 on glycolipid metabolism of largemouth bass,which may help to investigate the reasons for glucose intolerance and improve the starch utilization capability.Experiment 1:Effects of high starch diets on nutritive metabolism in largemouth bass.Two isonitrogenous and isolipidic diets containing 9.06%(LS)and 13.56%(HS)starch levels were fed to largemouth bass(initial body weight 47.60 ± 0.20 g)for 8w to investigate the effects on the growth performance,glycolipid,and energy metabolism.The results indicated that HS diets did not affect SGR at 2w(P > 0.05),whereas significantly lower SGR was observed at 8w(P < 0.05).Plasma glucose levels at postprandial 24 h in the LS or HS group were significantly lower than that at postprandial 3h,while plasma insulin levels were not significantly different at 2w or 8w.At 2w,the hepatosomatic index,plasma AKP and TBA levels,and hepatic glycogen,TG,TC,TBA and NEFA contents were significantly increased in the HS group(P < 0.05).Moreover,HS diets significantly up-regulated(P < 0.05)the m RNA levels of hepatic glycolysis,gluconeogenesis,fatty acid synthesis,TG synthesis,TBA synthesis,energy metabolism,inflammatory,and apoptosis-related genes,while significantly down-regulated the m RNA levels of TG hydrolysis and β oxidation-related genes,and antioxidant capability.At 2w,HS induced liver nuclear dense and fibrosis.The phosphorylation of AKT-FoxO1 was significantly inhibited(P < 0.05)in the liver disease groups,while FoxO1 protein and transcriptional levels were significantly up-regulated(P < 0.05).Interestingly,all the metabolic parameters,FoxO1 protein and transcriptional levels were returned to homeostasis in the HS group at8 w,even the liver of largemouth bass was shown no obvious abnormality by histopathological analysis.Therefore,due to insufficient insulin secretion of largemouth bass,AKT-FoxO1 phosphorylation was significantly inhibited after feeding HS,which led to acute glucolipid metabolic disorders(up-regulated gluconeogenesis and TG accumulation)and induced MLD.However,in the long-term,largemouth bass owned the adaptability to HS by increasing bile acid synthesis and energy metabolism,which was associated with FoxO1 homeostasis.Experiment 2: Molecular cloning,tissue expression,and function analysis of largemouth bass FoxO1Sequence alignments analysis investigated that FoxO1 exhibited a high degree of conservation in forkhead domain,transactivation domain,and AKT phosphorylation sites.Phylogenetic analysis revealed that largemouth bass FoxO1 remained largely conserved compared with bony fish and was clustered closest to Perciformes.FoxO1 m RNA was high expressed in the brain and liver,but low in the muscle.Activation of FoxO1 phosphorylation at S267 or S329 increased the FoxO1 nuclear export and decreased FoxO1 protein levels.Experiment 3:Effects of glucose metabolism in largemouth bass after the glucose or insulin-glucose injection.Effects of AKT-FoxO1 pathway on glucose metabolism in the largemouth bass were explored by intraperitoneal injection of 10 ml 0.75% saline solution/kg BW,1 g glucose/kg BW,or a mixture of 1.35 U bovine insulin/kg BW and 1 g glucose/kg BW,respectively.Glucose loading resulted in persistent hyperglycemia and plasma insulin levels failed in responding positively(P > 0.05).After the insulin-glucose injection,insulin levels were significantly elevated(P < 0.05)and glucose levels recovered to the basal value after 6h,which indicated insufficient insulin secretion caused persistent hyperglycemia.Compared with the glucose injection group,transcriptional levels and enzyme activities of hepatic glycolysis-related genes(GK and PK)were significantly activated(P < 0.05),while gluconeogenesis-related genes(PCK and G6Pase)were significantly depressed(P < 0.05)at 3h after the insulin-glucose injection.Besides,the phosphorylation of AKT-FoxO1 pathway was significantly activated(P < 0.05).Therefore,insulin improved glucose metabolism by activating the phosphorylation AKT-FoxO1 pathway to decrease hyperglycemia stress after the meal.Experiment 4:Mechanism of FoxO1 on glucose and lipid metabolism in largemouth bassPrimary hepatocytes were treated with 5.5 m M glucose(control group),25 m M glucose(HG group),and 25 m M glucose +1 μM AS1842856(FoxO1 inhibitor),and it was shown that HG induced the glucose and lipid metabolic disorders in hepatocytes,mainly along with abnormal gluconeogenesis and TG accumulation symptoms.After treatment with AS1842856,the metabolic disorder induced by HG was effectively mitigated.Dual-luciferase reporter assay demonstrated that FoxO1 positively regulated the activity of chrebp and pck promoter,while negatively regulated srebp1.In conclusion,HS diets mediated gluconeogenesis and lipid metabolism through FoxO1-PCK and FoxO1-Chrebp/Srebp1 pathways,respectively,to induced glucolipid metabolism disorder and MLD,which was the main reason for poor starch utilization capability of largemouth bass.