Effects Of Supplementing Taurine To A High Carbohydrate Dietary On IGF-1R SUMOylation And Glucose Metabolism In Hybrid Grouper(♀ Epinephelus Fuscoguttatus × ♂ E.lanceolatus),Nile Tilapia(Oreochromis Niloticus) And Grass Carp(Ctenopharyngodon Idella)

Fishes are an innate"diabetic constitution"in vertebrates.Fish cannot accurately perform the insulin cascade reaction due to decreased insulin sensitivity and defective insulin receptor function,which prevents PI₃K/AKT from activating by phosphorylation and leading to ineffective glucose metabolism regulation and persistent hyperglycemia after ingesting high-carbohydrate dietary.And in fish,the insulin growth factor-1 signaling pathway is no less important than the insulin signaling pathway in terms of glucose metabolism.In addition to transducing insulin signals through phosphorylated receptors,the insulin-like growth factor-1 signaling pathway can also play a role through small ubiquitin-like modification（SUMOyaltion）.SUMOylation is a post-translational modification for Tyrosine kinase receptors and signaling pathway proteins including IR,IGF-1R,PDK-1,AKT,GSK3Β,FOXO1 and HIF-1,linking small ubiquitin-like protiens（SUMOs）to the target protein through multi-stage enzymatic reaction,which play important roles in the regulation of phosphorylation of target proteins,cell signal transduction and transcriptional activity of key protein elements,thereby regulating the process of glucose metabolism.Taurine can directly or indirectly interact with the insulin receptor due to an insulin-like structure,and then activate the PI₃K/AKT signaling pathway to promote glucose and lipid metabolism,and maintain blood glucose homeostasis.In present study,we took carnivorous hybrid grouper,omnivorous Nile tilapia and herbivorous grass carp as the research objects.Firstly,the gene of hybrid grouper’s SUMO1,SUMO2 and UBC9 were cloned by RACR-PCR technology,and their gene sequence characteristics and evolutionary relationships were analyzed soon.Then,we test the hypothesis that taurine can improve fish growth and glucose metabolism through Insulin/PI₃K/AKT signaling pathway and its SUMOylation.Therefore,we test the effects of supplementing taurine to high carbohydrate dietary on growth,glucose metabolism,IGF-1R signaling pathway and SUMOylation related gene expression of three fish species:hybrid grouper（♀Epinephelus fuscoguttatus×♂E.lanceolatus）,Nile tilapia（Oreochromis niloticus）and grass carp（Ctenopharyngodon idella）to explore the mechanism of taurine and IGF-1R SUMOylation.The content of studies and results are as followings:1.Molecular cloning and sequence analysis of SUMO1,SUMO2 and UBC9 of hybrid grouperThe full-length cDNA sequences of the hybrid grouper SUMO1,SUMO2 and UBC9 genes were cloned and identified by RACE-PCR technology.The full-length cDNAs of SUMO1,SUMO2,and UBC9 are 770,807,and 1048 bp,the open reading frame（ORF）is divided into 306,291 and 477 bp,encoding 101,96 and 158 amino acids,respectively.Homology analysis shows that the similarity of hybrid grouper with Nile tilapia and grass carp SUMO1 is 98%and 91%,and SUMO2 is 96%and 93%,UBC9 similarity is all 94%.And the functional domain analysis showed that the C-terminals of SUMO1 and SUMO2 of hybrid grouper have conserved double-glycine repeats and conserved domains of the ubiquitin-like folding superfamily.In addition,theψKXE/D motif was also found in the SUMO2 sequence can form a polyubiquitin chain.UBC9 has the conserved Cys93 and ubiquitin-like ligase E2 catalytic superfamily conserved domains.The tissue expression profiles of SUMO1,SUMO2 and UBC9 show that the pearl gentian grouper SUMOylation is highly expressed in brain and heart tissues,and is the lowest in muscle.2.The effect of supplementing taurine to the high carbohydrate dietary on the growth performance,IGF-1R SUMOylation,glucose metabolism and intestinal microflora in hybrid grouperIn this study,totally 6 kinds of isoproteic and isolipidic experimental dietary were designed,including the medium-carbohydrate positive control group（20%,PC）,the high-carbohydrate control group（30%,NC）,and the high-carbohydrate negative control group with 0.4%,0.8%,1.2%and 1.6%taurine experimental group（T1～T4）.540 fish（initial body weight 12.10±0.30 g）were randomly distributed into 6treatments with triplicate groups of 30 fish in each treatment.After 8 week feeding trial,the results showed that:compared with the PC group,the weight gain rate,specific growth rate,intestinal trypsin andα-amylase activities of the NC group were significantly decreased,the serum insulin and IGF-1 levels were significantly decreased,and the liver gene expression of SUMO1,UBC9,IGF-1,IR and PDK-1 decreased significantly,and the liver IGF-1 receptor,GK and G6Pase activities decreased significantly.After supplementing taurine in NC dietary,the weight gain rate and specific growth rate of the taurine group increased first and then decreased with the increase of taurine level（P<0.05）.Intestinal trypsin,lipase andα-The activity of amylase increased significantly,and the levels of serum insulin and IGF-1 increased significantly.The content of serum triglycerides and total cholesterol showed a trend of first increasing and then decreasing with the increase of taurine level（P<0.05）,liver SUMO1,SUMO2,UBC9,IGF-1,IGF-1R,IR,IRS-1,PI₃K,PDK-1,AKT,GSK3β,FOXO1 and HIF-1 gene expression increased first and then decreased with the increase of taurine level（P<0.05）,the level of liver IGF-1 receptor,the activities of HK,GK,and FAS showed a trend of first increase and then decrease with the increase of taurine level（P<0.05）.The activity of liver PEPCK and G6Pase first decreased and then increased with the increase of taurine level.At the same time,the dietary carbohydrate and taurine level did not significantly change the diversity and abundance of the intestinal flora of hybrid grouper（P>0.05）,but at the phylum level,NC group significantly increased the abundance of the Proteobacteria.And taurine significantly reduced the abundance of Planctomycetes;at the genus level,the carbohydrate level in the feed had no significant effect on the abundance of Ochrobactrum（P>0.05）,but the taurine level in the feed was significantly Increased the abundance of Ochrobactrum.The above results indicate that taurine may increase the SUMOylation modification and gene expression of IR,IGF-1R,PDK-1,AKT,GSK3βand HIF-1 to promote the glycolysis and fat synthesis pathways of hybrid grouper,and promote the carbohydrate utilization capacity and growth performance of hybrid grouper;In the meantime,high carbohydrate dietary increases the abundance of potential pathogenic bacteria at the phylum level,while taurine reduces the abundance of pathogenic bacteria at the genus level,improves the structure of the intestinal flora;finally,the optimal taurine level is1.31%at 30%carbohydrate level dietary of hybrid grouper according to the curve model of hybrid grouper weight gain rate and dietary taurine level.3.The effect of supplementing taurine to the high carbohydrate dietary on the growth performance,IGF-1R SUMOylation,glucose metabolism and intestinal microflora in Nile tilapiaA total of 6 kinds of equal isoproteic and isolipidic experimental dietary were designed,namely the medium-carbohydrate positive control group（30%,PC）,the high-carbohydrate negative control group（44%,NC）and the high-carbohydrate negative control group with 0.3%,0.6%,0.9%and 1.2%taurine experimental groups（T1～T4）.540 Nile tilapia（14.55±0.31 g）were randomly distributed into 6 treatments with triplicate groups of 30 fish in each treatment.After 8 week feeding trial,the results showed that:high-carbohydrate level dietary has no significant effect on the weight gain rate and specific growth rate of Nile tilapia（P>0.05）,but it significantly promotes the activity of intestinalα-amylase,improves protein efficiency,and reduces feed coefficient.In addition,compared with the PC group,the NC group Nile tilapia serum insulin,IGF-1 and liver IGF-1 receptor levels significantly decreased,liver IRS-1,PI₃K gene expression significantly decreased,liver UBC9,IGF-1R,AKT,GSK3βand FOXO1 gene expression increased significantly,whole fish crude fat content and serum triglyceride content increased significantly,liver HK and GK activity decreased significantly.After taurine supplementing,there was no significant difference in weight gain rate and specific growth rate between the taurine group and the high carbohydrate group（P>0.05）,the activities of intestinal trypsin,lipase andα-amylase were significantly increased,and serum insulin and IGF-1 levels increased significantly,serum triglyceride levels decreased significantly with the increase in dietary taurine levels,liver SUMO1,SUMO2,IGF-1,IGF-1R,IRS-1,GSK3βand FOXO1 gene expression decreased significantly,The expression of liver UBC9,AKT and HIF-1genes increased with the increase of feed taurine level,and significant differences began to appear in the T3 and T4 groups（P<0.05）.The expression of liver IR gene showed a trend of increasing first and then decreasing（P>0.05）with the increase of taurine level.The activities of HK,GK and PK in the liver were significantly increased,and the activities of PEPCK and G6Pase in the liver were significantly decreased.At the same time,the carbohydrate level and taurine level of dietary had no significant effect on the diversity and abundance of the intestinal flora of Nile tilapia（P>0.05）,but at the phylum level,the high carbohydrates dietary significantly increased the abundance of Chloroflexi,The abundance of Fusobacterium decreased first and then increased with the increase of dietary taurine level（P<0.05）;at the genus level,the carbohydrate level in dietary significantly reduced the abundance of Plesiomonas,the abundance of Cetobacterium showed a trend of first decline and then rise with the increase of the taurine level in the feed（P<0.05）.The above results indicate that omnivorous Nile tilapia may increase the expression of UBC9 gene in the liver,increase the SUMOylation modification of IGF-1R,GSK3βand FOXO1,and promote Nile tilapia to convert carbohydrates into glycogen and fat,and improve the utilization of carbohydrate.And on one hand,taurine may reduce the SUMOylation modification and gene expression levels of IGF-1R,PDK-1,GSK3βand FOXO1 by inhibiting the expression of SUMO1 and SUMO2,and reduce the synthesis of fat,on the other hand,taurine can promote the gene expression of UBC9 to increase the SUMOylation modification and gene expression levels of IR,AKT and HIF-1,promote glycolysis via improving the activities of HK,GK and PK,thereby regulating the glucose and lipid metabolism of Nile tilapia;in the meantime,dietary carbohydrate and taurine levels can reduce the abundance of potential pathogenic bacteria at the genus level improve the flora structure;finally,the optimal taurine level is 0.86%at 44%carbohydrate level dietary of Nile tilapia according to the curve model of Nile tilapia weight gain rate and dietary taurine level.4.The effect of supplementing taurine in high-carbohydrate dietary on the growth performance,IGF-1R SUMOylation,glucose metabolism and intestinal microflora of grass carpA total of 6 kinds of equal isoproteic and isolipidic experimental dietary were designed,namely the medium-carbohydrate positive control group（31.49%,PC）,the high-carbohydrate negative control group（38.61%,NC）and the high-carbohydrate negative control group with 0.05%,0.10%,0.15%and 0.20%taurine experimental groups（T1～T4）.540 grass carp（100.00±0.33 g）were casually distributed into 6treatments with triplicate groups of 30 fish in each treatment.After 8 week feeding trial,the results showed that:compared with the PC group,the weight gain rate and specific growth rate of grass carp in the NC group were not significantly different P>0.05),the activities of intestinal trypsin,lipase andα-amylase were significantly decreased,and the whole fish had crude lipid content was significantly increased,the crude protein content of whole fish was significantly decreased,the serum insulin and IGF-1 levels were significantly decreased,the liver SUMO1,IR,AKT,FOXO1 and HIF-1 gene expression decreased,and the liver IGF-1R and IRS-1 gene expression significantly increased,the blood carbohydrate level significantly decreased,the liver GK,PK and FAS enzyme activities significantly decreased,and the liver PEPCK enzyme activity significantly increased.After supplementing taurine,the weight gain rate and specific growth rate of grass carp increased first and then decreased with the increase of the taurine level in the feed（P<0.05）,intestinal trypsin,lipase andα-amylase activity increased first and then decreased with the increase of feed taurine level（P<0.05）,liver SUMO1,SUMO2,UBC9,IGF-1,IGF-1R,IR,IRS-1,HIF-1 and FOXO1 genes expression increased first and then decreased with the increase of feed taurine level（P<0.05）.Serum insulin,IGF-1 and triglyceride levels increased significantly,and liver IGF-1R,HK,GK,PK,FAS and G6Pase enzyme activities increased significantly,and liver PEPCK enzyme activity decreased significantly.At the same time,the diversity and abundance of grass carp intestinal flora were not significantly affected by the carbohydrate level（P>0.05）.The dietary taurine level significantly increases the diversity of the intestinal flora of grass carp,but has no significant effect on the abundance of the intestinal flora（P>0.05）;at the genus level,high-carbohydrate dietary significantly reduces the abundance of Erysipelatoclostridium.Taurine significantly increased the abundance of Erysipelatoclostridium in the T1 and T3 groups.The above results indicate that the high-carbohydrate diet may down-regulate the transcription of GK,PK and FAS by reducing the SUMO1 modification of grass carp liver AKT,FOXO1 and HIF-1,Leading to a decrease in its enzymatic activity;and taurine may increase the transcription and enzymatic activity of liver HK,GK,PK,FAS and G6Pase by promoting the SUMOylation modification of IGF-1R,FOXO1 and HIF-1,thereby increasing grass carp to carbohydrate utilization of carbohydrate;in the meantime,taurine can reduce the inhibitory effect of high-carbohydrate dietary on the abundance of Erysipelatoclostridium,and improving grass carp intestinal absorption of glucose and lipids via the help of Erysipelatoclostridium;finally,the optimal taurine level is0.08%at 38.61%carbohydrate level dietary of grass carp according to the curve model of grass carp weight gain rate and dietary taurine level.