Dynamic Changes And Significance Of Rapamycin Complex 1 And Rapamycin Complex 2 In Gastric Smooth Muscle Of Diabetic Rats

Diabetic gastric motility disorder is a kind of syndrome complicated with diabetes mellitus,which is characterized by elimination of gastric mechanical obstruction and delayed gastric emptying.The pathogenesis of diabetic gastric motility disorder is not clear.Up to now,there are few researches on the pathogenesis of diabetic gastric motility disorder,especially on the energy metabolism of gastric smooth muscle cells.Rapamycin(mTOR)contains rapamycin complex 1(mTORC1)and rapamycin complex 2(mTORC1 1,mtorc2)two catalytic subunits can regulate cell growth,cycle,including energy metabolism and other physiological activities by integrating various extracellular signals such as growth factors,nutrition and energy status.The combination of mTORC1 and mtorc2 with diabetic gastric motility disorder has not been reported in literature.Objective:To explore the changes of two important complexes of rapamycin(mTORC1)and rapamycin complex 2(mTORC2)and their pathways in the pathogenesis of diabetic gastric motility disorder,and to elucidate their roles and mechanisms in the pathogenesis of diabetic gastric motility disorder.Methods:STZ induced diabetic rats were randomly divided into DM4w group,DM6w group and DM8w group.In vitro muscle strip experiment,the spontaneous contraction of smooth muscle of gastric antrum in each group was observed to determine the effect of diabetes on gastric smooth muscle movement;Western blot was used to observe the expression of Akt,p-Akt ser473,mTOR,p-mTOR ser2481,Raptor,GLUT4 and Rictor in gastric smooth muscle tissue of each group,and to determine the changes of mTORC1 and mTORC2 pathways.Results:1.Compared with the normal control group,the expression of p-AMPK thr172 in gastric smooth muscle tissue of DM6w group decreased(0.74±0.13 vs 1.03±0.12,p<0.01);compared with DM4w group,the expression of p-AMPK thr172 in gastric smooth muscle tissue of DM6w group decreased(0.74 ± 0.13 vs 1.00 ± 0.09,p<0.01);compared with DM6w group,the expression of p-AMPK thr172 in gastric smooth muscle tissue of DM8w group increased(0.92 ± 0.11 vs 0.74±0.13,p<0.01).Compared with the normal control group,the relative expression of mTOR protein in gastric smooth muscle of DM6W group increased(4.44 ± 0.26 vs 3.67 ±0.19,p<0.01);Compared with DM4w group,the relative expression of mTOR protein in DM6w group was increased(4.44±0.26 vs 3.75±0.20,p<0.01),the relative expression of mTOR in DM8w group was decreased(3.33± 0.25 vs 3.75 ± 0.20,p<0.05);Compared with DM6w group,the relative expression of mTOR in DM8w group decreased(3.33±0.25 vs 4.44±0.26,p<0.01).The relative content of raptor,the key protein of mTORC1,was 2.41 ± 0.16 in NC group,2.45 ± 0.12 in dm4w group,2.39±0.14 in dm6w group,2.39±0.20 in dm8w group.There was no significant difference in the relative ratio between normal group and diabetic group,and there was no significant difference in the content of raptor in all stages of diabetes(P>0.05).2.Compared with the normal control group,the content of Rictor protein in gastric smooth muscle tissue of diabetic rats increased,and increased with the development of the course,of diabetes.Compared with normal control group,Rictor protein content in DM4w group increased(4.30±0.13 vs 3.90±0.14,p<0.05),increased Rictor protein content in muscle strips of DM6w group(5.33±0.18 vs 3.90±0.14,p<0.01),increased Rictor protein content in muscle strips of DM8w group(5.87±0.11 vs 3.90±0.14,p<0.01);Compared with DM4w group,Rictor protein content of muscle strips in DM8w group increased(5.87±0.11 vs 3.90±0.14,p<0.01);Compared with DM6w group,Ricor protein content of muscle strip in DM8w group increased(5.87±0.11 vs 5.33±0.18,p<0.05).Compared with the normal control group,the phosphorylation level of mTOR ser2481 in gastric smooth muscle of diabetic rats in DM4w group decreased(1.17±0.08 vs 1.28±0.07,p<0.01),decreased phosphorylation of mTOR ser2481 in DM6w group(0.98±0.06 vs 1.28±0.07,p<0.01),phosphorylation level of mTOR ser2481 increased in DM8w group(1.51±0.11 vs 1.28 ±0.07,p<0.01);Compared with DM4w group,the phosphorylation level of mTOR ser2481 in DM6w group decreased(0.98±0.06 vs 1.17 ±0.08,p<0.01),the phosphorylation level of mTOR ser2481 increased in DM8w group(1.51±0.11 vs 1.17±0.08,p<0.01);Compared with DM6w group,the phosphorylation level of mTOR ser2481 in DM8w group was increased(1.51±0.11 vs 0.98±0.06,p<0.01).Compared with the normal control group,Akt ser473 phosphorylation level of gastric smooth muscle in DM4w group increased(1.80±0.23 vs 1.17±0.11,p<0.01),Akt ser473 phosphorylation decreased in DM8w group(0.99±0.19 vs 1.17±0.11,p<0.05);Compared with DM4w group,Akt ser473 phosphorylation level in DM6w group decreased(1.28±0.27 vs 1.80±0.23,p<0.01),Akt ser473 phosphorylation decreased in DM8w group(0.99±0.19 vs 1.80±0.23,p<0.01);Compared with DM6w group,Akt ser473 phosphorylation level in DM8w group decreased(0.99 ± 0.19 vs 1.28 ± 0.27,p<0.01).Compared with the nonnal control group,glucose transporter 4(GLUT4)on the surface of gastric smooth muscle cells in diabetic rats was higher than that in the cells,and the ratio in DM4w group was higher(1.29±0.08 vs 0.87±0.06,p<0.01);Compared with DM4w group,the GLUT4 ratio of DM6w group was lower than that of DM4w group(0.98±0.10 vs 1.29±0.08,p<0.01);Compared with DM4w group,the GLUT4 ratio on the surface of muscle strips in DM8w group was lower than that in DM4w group(0.88±0.08 vs 1.29±0.08,p<0.01);Compared with DM6w group,the GLUT4 ratio of DM8w group was lower than that of DM6w group(0.88±0.08 vs 0.98±0.10,p<0.05).3.Compared with the control.group,the contraction frequency of DM4w group decreased(6.63±0.26 vs 7.5±0.35,p<0.05),decrease of contraction frequency of muscle strips in DM6w group(5.8 ± 0.29 vs 7.5 ± 0.35,p<0.01),decrease of contraction frequency of muscle strips in DM8w group(4.4±0.41 vs 7.5±0.35,p<0.01);Compared with DM4w group,the contraction frequency of DM8w group decreased(4.4±0.41 vs 6.63 ±0.26,p<0.01);Compared with DM6w group,the contraction frequency of DM8w group decreased(4.4±0.41 vs 5.8±0.29,p<0.05).Compared with the normal control group,the contraction amplitude of DM6w group decreased(0.54±0.032 vs 0.85 ±0.026,p<0.01),decrease of muscle contraction amplitude in DM8w group(0.3 ± 0.045 vs 0.85 ± 0.026,p<0.01);Compared with DM4w group,the contraction frequency of DM8w group decreased(0.3 ±0.045 vs 0.82±0.022,p<0.01);Compared with DM6w group,the contraction frequency of DM8w group decreased(0.3±0.045 vs 0.54±0.032,p<0.01).Conclusion:1.mTORCl was not involved in the occurrence of diabetic gastric motility disorder.2.mTORC2 maybe involved in the development of diabetic gastric motility disorder by regulating Akt and GLUT4 activities.3.The frequency and amplitude of autonomic contraction of smooth muscle decreased with the course of diabetes.