The Effect Of Thyroid Function On Intestinal Metabolism And The Regulation Of The Major Energy-generating Transporters In Grass Carp Ctenopharyngodon Idellus

Thyroid functions in multiple physiological processes of fish.Various nutrients are utilized by transporters through the intestinal epithelial cells after the digestion in digestive tract.The metabolic activity of thyroid hormones(THs)can alter the process of nutrient transports.Some studies were focused on the primary investigation of various nutrient transporters in fish intestine.However,there are few studies on the mechanism of intestinal transporters regulated by THs.In this study,grass carp(Ctenopharyngodon idellus)were selected as the research object.We studied the molecular characteristics of intestinal nutrient transporter genes and explored the transporters activities regulated by THs.This work will help us to investigate the mechanism of intestinal nutrient absorption in fish.Meanwhile,these efforts can be a basic research on the mechanism of energy metabolism and efficient utilization of nutrients in grass carp.This doctoral dissertation consists of two parts.The first one was to analyze the molecular characteristics of intestinal transporter genes related to the energy metabolism by methods of gene cloning and bioinformatics.Subsequently,the experiment on different feeding drugs(T3 and PTU)were conducted.T3 and PTU were utilized to construct models of grass carp with different THs levels.All fish were fed for 40 days.After the breeding experiment,the samples were used for physiological and biochemical analysis,transcriptome and proteome analysis.These methods were utilized to investigate the intestinal nutrient transport and metabolism regulated by different thyroid functions in grass carp.Based on the results above,it is hoped to explore the relevant regulation mechanism of intestinal nutrients in fish.The main results obtained from the study are as follows: 1.Molecular characterization of intestinal nutrient transporters in grass carpIn this study,the sequences of intestinal nutrients(carbohydrates,fatty acids,and amino acids)transporter genes were cloned.The cloned intestinal sugar transporter genes include glut2,glut5,sglt1,and sglt4.The cloned glutamate and glutamine transporter genes include slc1a1,slc1a3,slc7a6,and slc7a7.And the cloned fatty acid transporter genes include fatp4,fabp1 a,fabp1b,fabp2,fabp6,fabppma,fabppmb,and acbp.The evolutionary analysis showed that the transporter in grass carp were all clustered closely with those of cyprinids but were distantly located from those of humans.The predicted secondary and tertiary structure of the transporters corresponding to different species(including grass carp)suggested the possible function in grass carp.The results of q RT-PCR showed that these intestinal nutrient transporters in grass carp were highly expressed in the intestine.These results verified that the nutrient transporter genes above were specific-expressed in the intestine,which provided a theoretical foundation for the research on the transport mechanism and function of intestinal nutrient transporters.2.The effects of thyroid hormone function on intestine nutrient transport,metabolism in grass carpThe growth was significantly inhibited in T3 treatment groups compared with that in control groups.The content of serum TSH and T4 was significantly decreased,while the content of T3 was significantly increased.In addition,the growth was also inhibited in PTU treatment groups compared with that in control groups.Differently,serum TSH and FT4 levels were significantly increased,while T3 levels were significantly decreased.The diets with T3 or PTU also affected the intestinal thyroid metabolism,nutrient metabolism,and immune activity in grass carp.The q RT-PCR results in T3 treatment groups showed that the expression levels of intestinal id3 was significantly increased and the expression levels of tr?2 was down-regulated.Meanwhile,the q RT-PCR results in PTU treatment groups showed that the expression levels of intestinal id1 was significantly increased,and the expression levels of tr?2 and tr? expression were significantly decreased.Combining with the results of omics analysis,we found that the expression levels of got1,mat1 a,cbsa,aldh1l1,and lacs was significantly up-regulated,while the expression levels of extracellular matrix receptor interaction pathway-related genes was significantly down-regulated in T3-fed fish intestine.These changes could induce cell energy expenditure and apoptosis and increase the risk of tumorigenesis in the intestine.On the other hand,the expression levels of rgn,fasn,agxta,cyp2p7,xdh,enpp7.1,gsto1 and g6 pd expression were significantly up-regulated,while the expression levels of amy2 a and pla2g1 b were significantly down-regulated in PTU-fed fish intestine.The changes could enhance carbohydrate and fat metabolism activities,promote fat synthesis,and easily induce oxidative stress to cause damage in the intestine.Therefore,nutrient metabolism and immune-related pathways in grass carp are different due to differential intestinal THs metabolism.The transcriptomics and proteomic results were enriched in the PPAR signaling pathway.According to the significant differential factors,it was shown that the diets of T3 would reduce the intestinal lipid metabolism and strengthen gluconeogenesis in grass carp,which were manifested by the significant down-regulation of fabp6,cyp7a1,cyp27a1,and cpt1?,and the significant up-regulation of acsbg2 and pck2.In addition,the diets of PTU could enhance intestinal lipid metabolism and weaken adipocyte differentiation in grass carp,which were manifested by the significant up-regulation of fabp3,fabp6,fabp7 a,ppar?,ppar?,and apoa1,and the significant decrease of sorbs1 expression.THs metabolism affects the activity of fatty acid transporters through the PPAR signaling pathway in grass carp.