| Carbohydrate is an essential ingredient in fish feed.Its advantages are easy to get and cheap.Adding sugar in the aquatic feed can save protein.But most fish are less tolerant of sugar and show a symptom similar to diabetes.How to regulate the metabolism of fish glycolipids through nutrition,and improve the utilization of carbohydrates by fish is particularly important.Orexin is a neuropeptide secreted by the hypothalamus.It participates in the body's feeding and energy homeostasis.Its function and mechanism in mammals are very thorough.However,in aquatic animals,the role of orexin is mainly concerned with the regulation of food intake,and its research on energy metabolism is less.Therefore,in this study,the full-length cDNA sequence of orexin-A gene was obtained by rapid amplification of cDNA ends(RACE),and the orexin-A protein was chemically synthesized for in vivo injection and in vitro cell biology experiments.In the in vivo injection experiment,a total of 450 healthy and well-prepared Cyprinus carpio were selected(average initial body weight was 22.02±0.17 g),and they were randomly divided into5 groups of 3 replicates each with 30 replicates.After fasting for 24 hours,the experimental group received orexin-A(0,10,100,1000 ng/g body weight)intraperitoneally with oral glucose at a dose of 1.67 mg/g body weight.The control group was fed with PBS solution.At the end of the experiment,blood glucose levels,glucose transporter-related gene expression,glucose transport activity,and glycolytic and gluconeogenesis-related genes were detected.In vitro cell biology experiments were carried out by isolation and culture of primary hepatocytes,and cells were incubated with glucose and orexin-A,respectively,and the expression of genes involved in glucose metabolism was detected after the experiment.In addition,in order to test the effect of orexin-a on feeding neuropeptides in the hypothalamus of carp,we also detected the expression of hypothalamic feeding-related neuropeptide genes after the end of the in vivo experiment.The experimental results are as follows:1.Cloning and phylogenetic analysis of prepro-orexin.The sequence of carp orexin cDNA was cloned by RT-PCR and rapid amplification of cDNA ends.The orexin precursor gene has a partial length of 513 bp and an ORF of 471 bp,encoding a 156amino acid orexin protein,including 49 amino acids of orexin-A and 28 amino acids of orexin-B.There are4 Cys in the Orexin-A sequence,and orexin has an insertion sequence located at positions 84-104(DGAHLPLLLPGGTVSAPLRLG).The homologous alignment revealed that carp orexin-B is more conserved than orexin-A.The orexin precursor gene distribution revealed that the orexin precursor gene was highly expressed in the brain,especially in the hypothalamus,and the expression of orexin in the liver,intestine and muscle was higher in peripheral tissues.2.The regulation of orexin-A on blood glucose and its effect on the expression of glucose transporter and glycolytic gluconeogenesisThe injection of orexin-A significantly reduced the hyperglycemia levels caused by oral glucose tolerance test(OGTT).After 1 h of experiment,the injection of orexin-a group was significantly lower than that of the non-injection group(p<0.05).The expressions of glucose transporter 2(glut2)and Na~+/glucose co-transporter 1(sglt1)in the intestine were significantly upregulated after OGTT administration,but the expressions of glut2 and sglt1 were significantly lower than those in the non-injected group after 2 hours of orexin-A injection(p<0.05).The expression levels of glut2 and sglt1 genes of the liver and glucose transporter 4(glut4)of the muscle were significantly higher in the injection group than in the non-injection group(p<0.05).After administration,intestinal glucose transport activity was also significantly increased(p<0.05),but the orexin-A injection group was significantly lower than the non-injected group(p<0.05).3.Effects of orexin-A on glucose metabolism in primary hepatocytes of carp.The effect of glucose concentration on glucose metabolism in primary hepatocytes showed that treatment with glucose(5 mM)had no effect on the expression of glucose metabolism-related genes in primary hepatocytes,while 15 mM glucose treatment significantly increased glut2 mRNA expression.It promoted the expression of glycolysis and gluconeogenesis mRNA.Orexin-A(100,1000 nM)stimulated glut2 mRNA expression in hepatocytes(p<0.05)in a dose-dependent manner;orexin-A(100,1000 nM)significantly increased hepatocyte glycogen synthase(gys)gene expression(p<0.05),also in a dose-dependent manner;orexin-A(100,1000 nM)stimulates hepatocyte glycolytic hexokinase(hk)and glucokinase(gk)gene expression,inhibits the expression of the gluconeogenes fructose 1,6-bisphosphatase(fbpase)gene.4.The effect of orexin-A on the expression of ingestion-related neuropeptides of the hypothalamus.Real-time fluorescent quantitative PCR(qRT-PCR)technology was used to detect the expression levels of neuropeptide Y(neuropeptide Y,npy)(promoting ingestion),cocaine-amphetamines regulating transcription product(cart)and the Proopiomelanocortin(pomc)(inhibition of feeding)in carp after glucose tolerance test.After injection of orexin-A,the expression of npy gene in the injection group was significantly lower than that in the uninjected group,and the cart mRNA expression was significantly higher in the injection group than in the uninjected group.In summary,the results of in vivo experiments show that orexin-A can significantly reduce the blood sugar level after infusion of glucose,and its blood sugar lowering ability may be achieved by inhibiting the absorption of intestinal glucose and promoting the utilization of liver and muscle glucose;Somatic experiments also confirmed that orexin-A can promote the absorption and utilization of glucose by the liver.At the same time,orexin-A can significantly inhibit the gene expression of the fasting peptide npy and promote the gene expression of the inhibitory peptides cart and pomc under the glucose loading condition caused by glucose feeding.According to the experimental results,it is speculated that orexin-A may maintain the body's blood glucose homeostasis by regulating glucose absorption,promoting glucose utilization,and inhibiting body feeding. |
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