| As a special kind of rodents,subterranean rodents live in a underground cave for live,so that they have evolved a series of adaptive ecological and physiological characteristics.Eospalax cansus is a typical subterranean rodent,making this species an excellent material for studying hypoxia tolerance.At present,many studies on the physiological and biochemical mechanisms of hypoxic tolerance has been reported,while few are researched about glucose metabolism of Eospalax cansus.Glucose stability affects the homeostasis.Insulin,a protein hormone secreted by ? cells in pancreatic tissue,is a key hormone to regulate glucolipid metabolism.Previous studies have shown that abnormal glucose metabolism often leads to abnormal glucose tolerance,diabetes,cardiovascular disease and obesity.However,impaired glucose tolerance in naked mole rats(Heterocephalus glaber)do not cause disease,even showing a series of surprising characteristics including longevity and cancer-free.Eospalax cansus also as a subterranean rodent,how about its glucose toleranace?Is there any abnormality in the regulation of insulin to glucose metabolism?Therefore,the purpose of this study was to investigate the insulin gene and regulation of glycometabolism by insulin in Eospalax cansus.In this study,The paraffin sections of pancreas in Eospalax cansus and SD rats,stained with he,and immunohistochemistry,were observed under light microscope.At the same time,fasting blood glucose,glucose tolerance,insulin tolerance and insulin release were compared between Eospalax cansus and SD rats.Then,based on the results of glucose tolerance test,the four time points which were 0min,15min,45min and 120min were selected,and the expression levels of insulin(Ins),insulin receptor(Insr),insulin receptor substrates 1(Irsl),insulin receptor substrates 2(Irs2),glucose transpoter 2(Glut2)and glucose transpoter 4(Glut4)mRNA in pancreas,liver and muscle of Eospalax cansus and SD rats were detected by quantitative Real-time PCR.Finally,Bioinformatics technology was used to analyse insulin gene of Eospalax cansus.The main results are as follows:1.Structure of the pancreas:In Eospalax cansus pancreas,the ? cells were distributed in the center core of the islet,and the number of ? cells were very few,which were distributed around.The ? cells of SD rats were located in the center core of the islet,and most of the ? cells were distributed around peripheral regions of islets,while a few were scattered inside the islet.The results showed the distribution of a and ? cells in Eospalax cansus were similar to that in SD rats.The number of capillaries and islet cells per unit area in the pancreas of the Eospalax cansus were higher than that of SD rats.It not only shortens the distance for transporting nutrients and oxygen,but also shortens the time for cells to secrete hormones into the blood circulation and reach target cells.2.Glucose tolerance,insulin tolerance and insulin release:After glucose load,the blood glucose of SD rats rose rapidly,reached the peak value at 15min,and then returned to pre-injection values about 1h.However,the blood glucose of Eospalax cansus reached a peak value 30-45min post-injection and remained high for a long time until it returned to pre-injection values 3h later,indicating that compared with SD rats,the glucose tolerance of Eospalax cansus was impaired.Under normal conditions,the urine glucose of Eospalax cansus was normal,and the fasting blood glucose of Eospalax cansus was significantly lower than that of SD rats,indicating that although the glucose tolerance of Eospalax cansus was impaired,they did not have diabetic pathology.After insulin load,the blood glucose of SD rats decreased rapidly and recovered quickly.However,the blood glucose of Eospalax cansus decreased rapidly to only one third of fasting blood glucose,and maintained for nearly 3 hours,then returned to the normal level gradually after about 8.5 hours.It showed that Eospalax cansus was sensitive to insulin.About insulin release,the insulin in the plasma of Eospalax cansus was significantly lower than that of SD rats.After 15min,the plasma insulin of Eospalax cansus increased slightly,but not significantly,and then reduced significantly.From the perspective of transcription level,the mRNA expression level of Ins gene in Eospalax cansus was significantly lower than that in SD rats at four time points.The results showed that the deficiency of insulin secretion led to the decrease of glucose tolerance.3.Regulation of glucose metabolism:Based on glucose tolerance,the four time points which were 0min,15min,45min and 120min,were selected to study the expression of genes about the regulation of insulin to glucose metabolism.The expression of Ins and Glut2 mRNA in pancreas.After glucose loading,the expression level of Ins mRNA in the pancreas of SD rats increased after 15min and then decreased after 45min,but it was not significant.The expression of Eospalax cansus increased significantly at 45min.Moreover,the expression level of Ins mRNA in Eospalax cansus was significantly lower than that of SD rats at four time points.The expression of Glut2 mRNA in the pancreas of SD rats and Eospalax cansus was similar to Ins.The peak value of Glut2 appeared in SD rats at 15min,while it appeared in Eospalax cansus at 45min.The results showed that insulin transcription level was low,time delay which resulted in the continuous hyperglycemia status of Eospalax cansus after glucose load.The expression of Irs1,Irs2 and Insr mRNA in the liver.After glucose loading,compared with SD rats,the expression levels of Irs1,Irs2 and Insr mRNA in Eospalax cansus liver all reached the peak at 15min,which was significantly higher than that of SD rats.Insr and Irs1 in the liver of SD rats showed a decreasing trend,and the expression of Irs2 is extremely low.The expression of Insr and Glut4 mRNA in muscle.The expression level of Insr mRNA in Eospalax cansus reached a peak at 15min and then decreased,while that in SD rats reached a peak at 45min,and the expression level of SD rats was lower than that in Eospalax cansus at four time points.Glut4 expression level of Eospalax cansus peaked at 45min,which was consistent with SD rats and even higher than SD rats,but there was no significant difference.Eospalax cansus showed compensation phenomenon in terms of insulin receptor and receptor substrates,and responded to hyperglycemia by up-regulating the expression of receptors and receptor substrates.4.Bioinformatics analysis of insulin gene in Eospalax cansus:The CDS region of the Eospalax cansus insulin gene was an open reading frame,which was 333 bp in length and encodes 110 amino acids.The molecular formula of the protein was C533H847N143O153S9,which was a hydrophobic stable protein.The secondary structure was dominated by loops and spirals,without folding structure.The sites 1 to 24 in sequence were a signal peptide,and there were three disulfide bonds connecting the two chains A and B together.Conserved site analysis showed that the conserved sites in the INS sequence of Eospalax cansus were mostly distributed on the two chains A and B.Mutations at some sites could cause diabetes,such as R55C,R46Q,and R6C.But in Eospalax cansus,these sites were more conserved than humans,mice or rat,indicating that the Eospalax cansus insulin sequence was more conserved to some extent.The selection pressure analysis showed that the Ins gene of Eospalax cansus was mainly subjected to purification selection pressure,and the possibility of non-synonymous substitution in the sequence was less than that of synonymous substitution,that is,the probability of harmful mutations was small.There were three positive selection sites,20N,64G,and 84S,all at variable amino acid positions.And in the INS sequence of blind mole rats(Nannospalax galili),SD rat,mice,and humans,this three sites were all different.The different types of amino acids of the same site indicated that these sites have undergone adaptive changes with the evolution of the Ins gene.The above results showed that,the distribution of ? cells and ? cells in Eospalax cansus were similar to that in SD rats.The ? cells were distributed in the center core of the islet,while the ? cells were distributed around.The capillaries in the pancreas of Eospalax cansus were densely distributed,and the density of endocrine cells in the islets was high.It not only shortens the distance for transporting nutrients and oxygen,but also shortens the time for cells to secrete hormones into the blood circulation and reach target cells.The insufficient insulin secretion of Eospalax cansus caused the persistent high blood glucose during the glucose tolerance test,that is,the phenomenon of glucose intolerance,but no diabetic pathology was evident.Of course,after the glucose load,Eospalax cansus showed compensation phenomenon in terms of insulin receptor and receptor substrates,and responded to hyperglycemia by up-regulating the expression of insulin receptor and receptor substrates. |
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