| Objective To compare the change of ultrastructural of pancreas islet alpha cells and serum glucagon after GLP-1receptor agonist (Exendin-4) interfered IGT rats. To investigate the effect of Exendin-4on ultrastructure of pancreas islets a cells in IGT rats.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 into 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 was7.8mmo1/L≤2hPG<11.1mmol/L and continued for more than a week[1]. The A group rats, blood glucose is 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.32of the B group rats were into models, it has the making model suceess rate of over88%. 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. Quickly remove the pancreas after taking the blood, and randomly divided the B group into two groups, the impaired glucose tolerance (IGT group, n=16) and Exendin-4intervention in group (Ex group, n=16). Feeding remains high sugar high fat diet, drinking water is not limited. Feeding for one week, randomly selected three groups of1/2the number of rats were used to measure the body weight of rats, then underwent the OGTT test to detect the FBG and2hPG. The next day fasting14h, with5% chloral hydrate0.6m1/100g of the rat abdominal anesthesia, abdominal vein blood taken specimens rapidly centrifuged to obtain serum, placed in-70℃refrigerator be saved by radioimmunoassay Determination of serum glucagon (Glucagon). Quickly take the tail of the pancreas tissue, and cut into1mm3pieces, and placed in2%glutaraldehyde (1-4h) fixed, and fixed with1% osmium tetroxide, acetone series of dehydration, an epoxy resin618embedding, light microscopy to determine the tissue blocks containing islets, the production of ultra-thin slices (50nm), and slices through the electronic stained with uranyl acetate and lead citrate, JEM-1011in the Japan Electronics type transmission electron microscope and sliced observed islet a cytoplasmic endocrine granules, mitochondria, rough endoplasmic reticulum change. The remaining1/2the number of rats Ex group was given Exendin-45ug/kg injected subcutaneously twice daily. NGT group and IGT group were given an equal volume of saline injected subcutaneously at four weeks after the intervention were sacrificed, the detection of indicators.All results were measured by (x±s), using SPSS13.0software package, multiple groups means were compared with single factor analysis of variance, LSD test. P<0.05was considered statistically significant. Results1The rats fasting plasma glucose and2-hour postprandial blood glucose is as follows:Before the intervention, FBG:NGTvs.IGT(4.79±0.55vs.5.05±0.72), NGTvs.Ex:(4.79±0.55vs.5.23±0.39), Pwere greater than0.05, the difference was not statistically significant; IGTvs.EX(5.05±0.72vs.5.23±0.39), P<0.05, the difference was not statistically significant; Ex ago vs Ex:(5.05±0.72vs.4.89±0.313), P>0.05, the difference was not statistically significant.2hPG:NGTvs.IGT (5.96±0.77vs.9.69±0.33), NGTvs.Ex (5.96±0.77vs.9.26±0.38), all P<0.05, the difference was statistically significant; IGTvsEx(9.69±0.33vs.9.28±0.38), P<0.05, the difference was not statistically significant. Exendin-4after4weeks of intervention the FBG:Exvs.IGT (4.98±0.36vs.4.89±0.31), P>0.05, the difference was not statistically significant.2hPG:Exvs.IGT(10.28±0.38vs.7.19±0.34), P<0.05, difference was statistically significant. Ex ago vs.Ex after FBG:(5.05±0.72vs.4.89±0.313), P>0.05, the difference was not statistically significant. The the Ex vs.Ex2hPG:(9.28±0.38vs.7.19±0.34), P<0.05, the difference was statistically significant. Compared with the NGT group, the above-mentioned indicators differences were not statistically significant (4.93±0.39vs.4.89±0.31)(6.26±0.53vs.7.19±0.3),(all P>0.05).2. Groups of rat pancreatic tissue projection electron microscopy results Organizations take the tail of the pancreas, to produce ultra-thin slices (50nm) and transmission electron microscope JEM-1011type in the Japanese electronics and slice, you can see:NGT group rat islet a cells containing secretory granules with a halo, and full particleswithin the dense core of high electron density core round the nucleus round home base central nuclear membrane clear, evenly distributed nuclear chromatin and contents, mitochondria, endoplasmic reticulum are tightly packed, neatly. IGT group islet a cytoplasmic endocrine number of particles increases, can see the gap narrowing between the dense core and the limiting membrane, some disappear; mitochondrial swelling deformation, irregular nuclear membrane shrinkage, border irregularities, nuclear chromatin condensation endoplasmic reticulum arranged irregularly dispersed in cells, mitochondrial density decreases, the crest becomes short and shallow, the endoplasmic grana not clear off, organelle structure did not change significantly. Exendin-4group islet a cell cytoplasm endocrine particles reduce the number of gap between the dense core and the limiting membrane than IGT group widens reduced mitochondrial.3.The islet alpha cytoplasmic endocrine changes in the number of particles Specimens were the Japan Electronic JEM-1011type transmission electron microscope observation and radiography, each specimen were randomly selected five fields, each counting50alpha cells, count the number of each alpha cytoplasmic secretory granules, whichever isrepresented by the average number of secretory granules in the cytoplasm of the islet alpha cells in each group.Before the intervention, the IGT group, Ex group rat islet a cell secretory granules levels than the NGT group were higher (74.60±6.55vsl35.32±16.31)(74.60±6.55vsl29.64±12.78), there were significant differences(P<0.05). Ex group rat islet a-cell secretory granules was no significant difference compared with the IGT group (135.32±16.31vs.129.64±12.78)(P>0.05). Exendin-4four weeks after the intervention, compared with the same period in the IGT group Ex group rat islet a-cell secretory granules lower level (75.79±8.23vs135.32±16.31), the difference was statistically significant (P<0.05). And intervention before the Ex group decreased (75.79±6.55vs.129.64±12.78), the difference was statistically significant (P<0.05). No significant difference in comparison with the NGT group (75.79±8.23vs.74.60±6.55)(P>0.05).4.The levels of blood glucagon in each groups Before the intervention rat glucagon level comparison:the NGT group vs.IGT group (88.18±6.94vsll8.89±3.53), the NGT group vs. Ex group (88.18±6.94vs.116.12±3.37), P<0.05, difference statistically significant. The IGT group vs.Ex group (118.89±3.53vs.116.12±3.37), P>0.05, the difference was not statistically significant. Exendin-4intervention the rat glucagon level after4weeks, the IGT group vs. Ex group (118.39±4.36vs.86.89±6.23), P<0.05difference was statistically significant. Ex former vs.Ex of (116.12±3.37vs86.89±6.23), P<0.05, the difference was statistically significant. The NGT group vs.Ex group (88.07±7.34vs.86.89±6.23), P>0.05, the difference was not statistically significant.Conclusions1.Impaired glucose tolerance stage ultrastructural changes in islet a cells, the islet alpha cytoplasmic endocrine particles increased, an increase in the synthesis and secretion of glucagon, confirmed the ultrastructural changes of islet a cells at this stage could lead to isletabnormal alpha cell function involved in glucose metabolism process, suggesting that changes in islet a cell ultrastructure and secretion of functional enhancements for one of the pathogenesis of impaired glucose tolerance.2.Impaired glucose tolerance stage, the increase in the secretion of glucagon, blood glucose increased further, to enhanced synthesis and secretion of pancreatic alpha cells3-GLP-1receptor agonist Exendin-4can reduce impaired glucose tolerance islet alpha cytoplasmic secretory granules, mitochondria increase, an increase in the rough endoplasmic reticulum, can reduce the secretion of glucagon, which may improveultrastructure of pancreatic alpha cells while inhibiting the secretion of pancreatic alpha cells function, so that excessive glucagon secretion reduce, delay or prevent progress in IGT will become one of the targets. |
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