| Objective To compare the changes of the amount of pancreas islets a cells and the function after GLP-1receptor agonist (Exendin-4) interfered IGT rats. To investigate Exendin-4suppress IGT pancreas islets a cells and on the mechanism of blood glucose regulation.Methods54male Wistar rats in4-5weeks (150g-170g) of clean grade were purchased in animal breeding center of Shan xi Medical University. After one week adaptive feeding (five/cage), they were divided in to the Conventional feed group (A group, n=18) and High sugar and fat diet fed group(B group, n=36) randomly. The A group rats were fed by normal diet (13.4kJ/g, which accounted for10.2%of calories, fat and protein accounted for23.3%respectively, carbohydrate accounted for66.5%). The B group rats were given high-sugar and high-fat feed(21.8kJ/g, which accounted for56.0%of calories, fat and protein accounted for7.0%respectively, carbohydrates accounted for37.0%) and free access to water, at room temperature controlled at18～22℃, relative humidity30%～70%. Two groups of rats twice a day morning and evening feeding, daily food in take for each rat to vote for3%of body weigt. When12weeks, do oral glucose tolerance test (OGTT):after fasting8h, cut the tail blood and rapid blood glucose meters measure fasting blood glucose (FBG), intragastric administration with50%glucose injection2g/kg body weight, after2h cut the tail blood again and measure2h postprandial blood glucose (2hPG). Building a successful model was7.8mmol/L≤2hPG<11.1mmol/L and continued for more than a week. The A group rats, blood glucose to normal, and being setted to the normal glucose tolerance control group(NGT group, n=16) and continue to give regular feed and freedom drinking water.62of the B group rats were into models, it has the making model suceess rate of over88%, and randomly divided the B group into two groups, the impaired glucose tolerance (IGT group, n=16), Exendin-4intervention in group (Ex group, n=16). To continue to give high-sugar high-fat feeding and freedom drinking water. Select randomly nine rats in NGT group, eight rats in IGT group and Ex group respectively, weighing, and then do oral glucose tolerance test measure FBG and2hPG. The next day, after fasting12-14h, anesthetized rats with5%chloral hydrate by intraperitoneal injection o.6ml/100g body weight, taken abdominal primary vein blood, rapid centrifugation of blood samples of serum specimens from70℃to save, the way of Radioimmunoassay under test glucagon, measure blood fat figures such as TG and TC. Quickly remove the pancreas after taking the blood, washed with ice PBS. Fixed the pancreas tissues by10%for malin, dehydrated, embedded in paraffin, regular slices, for HE staining, obverse the pancreas islets, measure the area percentage of pancreas islets α cells using immunohistochemical staining methods and determine the glucagon protein expression using immunofluorescence methods. The remaining rats in Ex group received Exendin-4(Eli Lilly and Company to provide)5ug/kg subcutaneously, twice daily, the NGT and IGT group were given equal volume of saline injection. When interventing4weeks, weighing, and then measure FBG and2hPG. The next day, anesthetized rats, take blood, and observe islets for HE staining, measure the area percentage and glucagon expression of pancreas islets a cells with immunohistochemical and immunofluorescence.All results were measured by (x±s), using SPSS13.0software package, multiple groups means were compared with single factor analysis of variance, LSDtest.P≤0.05was considered statistically significant.Results1The changes of general signs in each groups Before intervention, the rats in NGT group have moderate size and good spirit. They are agile and responsive to outside stimulation. The fur of the rats is shiny and the elasticity is good. Compared with NGT group, rats in the IGT group and Ex Group are obesity, especially abdominal obesity, apathetic, unresponsive to the outside, slow and hair less shiny, poor flexibility. After intervented with Exendin-44weeks, compared with the IGT group at the same period and the Ex group before intervented, Ex group shape is uniform, spirit improved clearly, sensitive to the outside, obviously agile, hair gloss and elasticity are improved markedly.2.The changes of clinical characteristics and biochemical indices between rats in each groups Before intervention, compared with the NGT group, the body weigh of rats and2hPG in the IGT group and Ex group were higher (448.67±12.90vs.514.13±13.41)(448.67±12.90vs.513.12±11.86) and the differences were also significant (both P<0.05). The2hPG of rats were higher (5.96±0.77vs.9.69±0.33)(5.96±0.77vs.9.26±0.38), the differences were also significant (both P<0.05). The FBG of rats were higher(4.79±0.55vs.5.05±0.72)(4.79±0.55vs.5.23±0.39), but the differences were not significant (both P>0.05). The TG of rats were higher (0.87±0.22vs.2.08±0.21)(0.87±0.22vs.2.12±0.21) and the differences were also significant (both P<0.05). The TC of rats were higher (0.84±0.19vs.1.91±0.16)(0.84±0.19vs.2.00±0.17), the differences were also significant (both P<0.05). Compared with the IGT group, the body weigh and the2hPG of rats in the Ex group was lower (514.13±13.41vs.513.12±11.86)(9.69±0.33vs.9.28±0.38); the FBG,TG and TC were higher(5.05±0.72vs.5.23±0.39)(2.08±0.21vs.2.12±0.21)(1.91±0.16vs.2.00±0.17), but the differences were not significant (P>0.05). After intervented with Exendin-44weeks, compared with the IGT group, the body weigh, the2hPG, the TG and TC of rats in the Ex group were decreased (520.75±18.42vs.445.88±20.29)(10.28±0.38vs.7.19±0.34)(2.22±0.14vs.1.06±0.13)、(2.09±0.18vs.1.02±0.09), the differences were significant (both P<0.05). The FBG in the Ex group were decreased (4.98±0.36vs.4.89±0.31), but the difference was not significant (P>0.05). Compared with the Ex group before intervented, the levels were decreased (520.75±18.42vs.445.88±20.29)(10.28±0.38vs.7.19±0.34)(2.12±0.21vs.1.06±0.13)(2.00±0.17vs.1.02±0.09), the differences were significant (both P<0.05). The FBG was also decreased (5.05±0.72vs.4.89±0.313), but the difference was not significant (P>0.05). Compared with the NGT group, the levels all above were higher (459±11.20vs.445.88±20.29)(4.93±0.39vs.4.89±0.31)(6.26±0.53vs.7.19±0.3)(0.89±0.23vs.1.06±0.13)(0.89±0.20vs.1.02±0.09), but the difference was not significant (P>0.05)3.The levels of blood glucagon in each groups Before intervention, compared with the NGT group, the blood glucagon in the IGT group and Ex group were higher (88.18±6.94vs.118.89±3.53)(88.18±6.94vs.116.12±3.37) and the differences were significant (both P<0.05). Compared with the IGT group, the blood glucagon in the Ex group was higher (118.89±3.53vs.116.12±3.37) but the difference was not significant (P>0.05). After intervented with Exendin-44weeks, compared with the IGT group at the same period, the blood glucagon in the Ex group was decreased (118.39±4.36vs.86.89±6.23), the differences was significant (P<0.05). Compared with the Ex group before intervented, the levels was also lower (116.12±3.37vs.86.89±6.23), the difference was significant (P<0.05). Compared with the NGT group, the level was decreased (88.07±7.34vs.86.89±6.23), but the difference was not significant (P>0.05).4. The configuration of rats pancreas by HE dyeing Taking pancreatic paraffin specimens, HE dyed, you can see the pancreas surface with a thin layer of connective tissue capsule, the pancreatic substance separated into many lobules by the connective tissue glands in depth. Pancreatic was made up of exocrine and endocrine. Acinar and ducts are composed of exocrine. Islet is located in the acinar, consisting of a spherical cell mass by endocrine cells. Cells are rich in fenestrated capillaries. After making sure the organization are pancreas islets, the remaining organizations will be taking immunohistochemical and immuno-fluorescence staining. Before intervention, compared with NGT group, the single islet area reduced in the IGT and Ex group. After intervented with Exendin-44weeks, the single islet area in the Ex group was increased compared with the IGT group at the same period and the Ex group before intervented.5. The changes of rats pancreas a cells with immunohistochemical staining(1)Location The expression of rats glucagon protein in NGT group was localization with immunhistochemical staining. Glucagon distribution in pancreatic islet cells is in a peripheral edge, less. Compared with NGT group, glucagon expression of rats in IGT group and Ex group increased, in addition to distribution in peripheral and central distribution, visible from the islet. After intervented with Exendin-44weeks, compared with IGT group at the same period, the expression of glucagon decreased and they are concentrated in the peripheral edge. All glucagon expressed in the cell cytoplasm.(2) a quantitative Before the intervention, compared with NGT group, the area percentage of a cells in rat islet in the IGT group and Ex group were increased (11.01±6.33vs.26.91±7.77)(11.01±6.33vs.24.00±9.88), the differences were significant (both P<0.05). Compared with the IGT group, pancreatic a cells area percentage of Ex group rat was not significant (26.91±7.77vs.24±9.88)(P>0.05). After intervented with Exendin-44weeks, compared with the IGT group at the same period, the area percentage of rat islet a cells in Ex group was reduced (29.21±5.66vs.15.09±3.35), the difference was significant (P<0.05). Compared with the Ex group before intervented, the area percentage was also reduced (24±9.88vs.15.09±3.35), the difference was significant (P<0.05). There wad no significant difference compared with the NGT group (12.03±5.55vs.15.09±3.35)(P>0.05)。6.The changes of rats pancreas a cells with immuno-fluorescence staining(l)Location The expression of rats glucagon protein in the NGT group was localization with immuno-fluorescence staining. Glucagon distribution in pancreatic islet cells was in a peripheral edge, less. Compared with the NGT group, glucagon expression and fluorescence of rats in the IGT group and Ex group were increased, in addition to distribution in peripheral and central distribution, visible from the islet. After intervented with Exendin-44weeks, compared with the IGT group at the same period, the expression of glucagon and fluorescence decreased and they are concentrated in the peripheral edge. All glucagon expressed in the cell cytoplasm.(2) a quantitative Before the intervention, compared with the NGT group, the glucagon of rat islet a cells in the IGT group and Ex group were increased (71.74±12.10vs.307.44± 71.85)(71.74±12.10vs.321.71±105.65) and the differences were significant (both P<0.05). Compared with the IGT group, the glucagon level of rat in the Ex group is not significant (307.44±71.85vs.321.71±105.65)(P>0.05). After intervented with Exendin-44weeks, compared with the IGT group at the same period, the expression of gluangon in rat pancreatic islet a cells was reduced in the Ex group (327.55±155.53vs.102.10±26.28), the difference was significant (P<0.05). Compared with the Ex group before intervented, the level was also reduced (327.55±155.53vs.102.10±26.28), the difference is significant (P<0.05). There was no significant difference compared with the NGT group (86.85±16.84vs.102.10±26.28)(P>0.05)。Conclusions1. In the stage of impaired glucose tolerance, a series of characteristics has occurrenced which included increase the amount of a cells and the secretion of glucadon. It was confirmed that the hyperplasia of pancreatic islet a cells was existed at this stage. The amount and function of pancreatic islet a cells is indeed involved in glucose metabolism, suggested that it was one of the pathogenesis of type2diabetes.2GLP-1receptor agonist reduced the amounts of pancreatic islet a cells in rats with impaired glucose tolerance and suppressed glucagon secretion. It may be one of the targets of decreasing blood glucose that reducing glucagon secretion through inhibiting hyperplasia of pancreatic islet a cells. |
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