Effects Of Glucose Injection On Glucose Metabolism And Glucose Transporter Genes Expression In Cobia(Rachycentron Canadum)

Carbohydrate is the cheapest source of energy. The amount of utilization of the carbohydrate in fish feed was limited because of the limited ability to utilize carbohydrate of fish, particularly carnivorous species. How to improve the utilization of carbohydrates has become a hot topic in fish nutrition research areas. It is very necessary to clarify the mechanism of glucose metabolism in fish. The cobia(Rachycentron canadum)was selected as the model species for this paper to study the effects of glucose injection on plasma biochemical indexes, liver/muscle glycogen, glycolytic enzyme and expression of glucose transporters.In this experiment, cobia( weight 85±3g) were allotted to two groups and were intraperitoneally injected with 0. 68% saline and 60mg/100 g weight of glucose solution respectively. The fish were sampled at 0、1、2、4、8、12、24 and 48 h after injection. The result showed that the levels of plasma glucose, triglyceride and cholesterol significantly changed compared to control groups(p<0. 05), reaching the top at 1h、8h and 48 h respectively, whereas the plasma total protein did not change significantly(p>0. 05). After the injection of glucose, hepatic glycogen levels of cobia increased at first and went down latter, reaching the highest level at 8h( p<0. 05), whilst no significant change were observed in muscle glycogen(p>0. 05). Plasma insulin content reached the highest level at2 h and then decreased significantly(p<0. 05). The activity of pyruvate kinase(PK)reach the highest level at 4h after injection and then went a down trend(p<0. 05). However, the activity of hexokinase heterophosphatase(HK)and Phosphofructokinase(PFK)did not change significantly in current study(p>0. 05).GLUT4, one of the GLUT family member, had been reported. In this study, 5members of GLUT family, including GLUT1, GLUT2, GLUT3, GLUT5 and GLUT9 were cloned. RT-PCR was performed to detect the expression of 5 members of GLUT family.The m RNA of GLUT1 was mostly expressed in foregut, while the m RNA of GLUT5 was mostly expressed in gonad. The m RNA of GLUT2, GLUT3 and GLUT9 were expressed mostly in pylorus without exception. Analysis of the effects of glucose injection on m RNA expression of 6 GLUTs members in the liver and muscle of cobia revealed that theexpression of GLUT2 in liver significantly change, and reached the top at 24 h after glucose injection(p<0. 05). The expression of GLUT3 significantly increased within 24 h compared to the control group( p<0. 05). The level of GLUT5 increased first and decreased at last, reaching the peak at 12 h, and increased by 4 times compared to the control group(p<0. 05). However, there has not any significant change in the expression of GLUT1, GLUT4 and GLUT9 compared to the control(p>0. 05). In the muscle, glucose injection effect the expression of GLUT1 and GLUT3 significantly( p<0. 05). After glucose injection, the level of GLUT1 and GLUT3 in muscle increased first and then went a down trend. The GLUT1 m RNA level reached the top at 2h, and was 2. 1 times higher than the control(p<0. 05). The GLUT3 m RNA level reached the highest level at 4h(p<0.05).In order to lucubrate the function of GLUTs involved in the glycometabolism of cobia,RACE-PCR was performed to gain the full length of GLUT1 and GLUT2, and the bioinformatics was analysed. The result showed that c DNA sequence of GLUT1 in cobia was 2531 bp, and open reading frame(ORF)was 1440 bp, which encoding 479 amino acid(Gen Bank accession No. KU955598). The c DNA sequence of GLUT2 was 1380 bp, and ORF was 1170 bp, which encoding 389 amino acid(Gen Bank accession No. KU955599).Phylogenetic analysis supported that amino acid sequence of GLUT1 and GLUT2 had a high similarity and nearest relative to the homologous protein of other fish.In conclusion,the glucose injection had an significant effect on the plasma glucose,plasma triglyceride, plasma cholesterol, plasma insulin, hepatic glycogen, the activity of PK and the expression of GLUT in liver and muscle, which might helpful to clarify the mechanism of fish glucose metabolism and provide reference to the research of the reason for fish’s low utilization of carbohydrate.