Comparison Study On Clone And Expression Of Glucose Transporter 4(GLUT4) In Different Fish Species

In this experiment, three fishes of different food habits: omnivorous tilapia(Niloticus), moderate carnivorous golden pompano(Trachinotus ovatus) and ferocious carnivorous cobia(Rachycentron canadum), were taken as the research objects. the GLUT4 sequence of pompanos and cobia were cloned by RACE-PCR cloning technology, and analyze the difference in the protein structure and evolution.Compared the difference of GLUT4 gene expression in different trophic fish with nutrition status of three feed carbohydrate levels(high, middle and low).we studied the effect of different dietary carbohydrate levels on growth and glucose metabolism in Nile tilapia, and the influence of Glucose Tolerance Test(GTT)on serum biochemical indexes of blood glucose, insulin, IGF-1, triglyceride and liver/muscle glycogen, the GLUT4 gene expression, The aim of this article was to exploring the tolerance mechanism and molecular mechanism of fishes with weak ability of glucose utilization.The content of studies and results are as followings:1.Cloning and sequence analysis of Trachinotus ovatus and Rachycentron canadum GLUT4.The full-length cDNA of GLUT4 were obtained using 5′-RACE and 3′-RACE.Pompano GLUT4(GB: KP693688.1 containing 1937 bp, ORF of 1521 bp amino acid sequence of 506 aa. Cobia GLUT4(GB: KP684143.1) containing 2241 bp, ORF of1524 bp amino acid sequence of 507aa。All results indicate that the cloned GLUT4 gene were high homology with human GLUT4, and multiple sequences alignment showed that the mutation of key function motifs(FQQI,TELEY) may affect the traffic mode 、 function and efficiency of GLUT4, and may affect the glucose utilization ability of fish.2.Effects of three dietary carbohydrate levels on expression of GLUT4 in fishes.In the detection of pompano、cobia and tilapia, GLUT4 was highly expressed in skeletal muscle and heart, similar to the study in mammals.With three nutrition conditions of dietary glucose levels. In cobia muscle, GLUT4 expressed highest in the CM group(P<0.05). In Cobia heart in the highest expression of GLUT4 was in CM group, significantly higher than the other(P<0.05). GLUT4 expression in pompanomuscle showed the biggest in CM group, and in heart, the expression of GLUT4 was highest in CL group(P<0.05). GLUT4 expression in Tilapia muscle, CM group was significantly higher than the other two groups(P<0.05),and in heart, the highest was CL group(P<0.05). Dietary glucose levels can induce the expression of GLUT4 in tissues, but there was difference in heart and muscle. Effects of different dietary carbohydrate levels on GLUT4 expression wree also differ in fishes with different food habits.3.Effects of different dietary carbohydrate levels on grow performance、glucose metabolism and GLUT4 expression in Nile tilapia.Tilapia showed the best grows performance in CM group. Plasma glucose levels was proportional to the dietary glucose leveles, CH group was significantly higher than other two groups. CL group was significantly higher than the other groups of plasma insulin levels.The content of IGF-1 was similar to plasma glucose. Liver glycogen was highest in CH group. Activity of GK and PK enzyme showed the lowest in CL group(P<0.05). PEPCK showed the highest enzyme activity in the CM group(P<0.05), GLUT4 in muscle expressed supreme in the CM group(P<0.05). The expression of IGF-1 gene in liver was highest in CM group(P<0.05). Suitable level of dietary carbohydrate can promote the growth of Nile tilapia, stimulate glycolytic enzyme activity, increased the concentration of IGF-1 and expression of GLUT4,improve the utilize of dietary carbohydrate. But high level would suppresse growth,make excessive accumulation of hepatic glycogen, decrease GLUT4 expression.4.Under three level of dietary carbohydrate, the effect of glucose injection on tilapia glucose metabolism and GLUT4 expressionThe results showed that plasma glucose of three group raised to spike in 1h quietly then declined, at 6h CH and CM group return to the begining of the injection,CL group is significantly higher than 0 hours(P<0.05) at 12 h. Plasma insulin, CH group of plasma insulin levels declined at 1h(P<0.05) then rise to the 0h level. There was no significant change in CM group, CL shoued a decline trend. Plasma IGF-1levels was positive correlated with diets carbohydrate level, after injection CM and CH groups reached the maximum at 1h, and CL group at 3h(P<0.05), then decreased.The expression of IGF-1 in liver, CM group was significant higher than others, and the trends were the mirror image of the plasma IGF-1. In three levels of carbohydrate, tilapia liver glycogen of CM and CH groups were roughly smooth after rising. Muscle glycogen in CH group decreased at 1h(P<0.05) then rising. Nosignificant trend was found in CM group, CL group showed a trend of decline at 1h,then wen rising. There was a direct relationship between three carbohydrates levels and the activity of liver PK and GK enzyme activities, all above the activities raised first and then went down. All groups of tilapia muscle GLUT4 gene expression changed slightly in 0-3h and then increased sharply during 6-12 h. CH group reach the maximum at 6h, CM group was at 6h and CL group was at 6h. IGF-1 expression in liver fall after a rise in the trend of CH and CM group after injection in 1h peak.Results of this study indicated that a period of moderate level of carbohydrate dietary can improve tilapia glucose tolerance. Compaired with insulin, IGF-1 would response to blood glucose changes more quickly, what’s more, the delay response of GLUT4 expression may be the reason why fish with a weak glucose tolerance.