间歇性高血糖对胰岛β细胞功能和凋亡的影响

Effects of intermittent high glucose on islet IS-cell function and apoptosis in GK rats

目的观察间歇性高血糖和持续性高血糖对糖尿病大鼠胰岛β细胞功能和凋亡的影响。方法雄性GK大鼠22只，随机分为持续高血糖组（HG）、波动性高血糖组（FG），11只Wistar大鼠作为正常对照组（NG）。FG组每天定时腹腔注射超短效胰岛素类似物诺和锐并错时给予葡萄糖，造成1d中血糖浓度大幅度波动模型。HG和NG组给予生理盐水注射。制模6周后，进行葡萄糖耐量试验及胰岛素释放试验以评估胰岛β细胞的功能。以免疫组化染色及形态测量半定量分析胰岛素表达，TUNEL法观察胰岛细胞的凋亡并计数胰岛中β细胞凋亡率。结果（1）FG组空腹血糖及糖负荷后15、30、60、120min血糖均显著高于HG组（均P〈0．01），FG组葡萄糖曲线下面积（AUCg）也高于HG组[（1012．14±82．62对813．60±56．70）ng·ml^-1·h^-1·10^4，P〈0．01]；糖负荷后15、30、60、120min胰岛素水平、胰岛素曲线下面删葡萄糖曲线下面积（AUCi／AUCg）及15min胰岛素增值／血糖增值（△I15’／△G15’）较HG组降低[（0．55±0．18对0．95±0．28，0．43±0．17对0．85±0．21，0．47±0．11对0．76±0．16，0．58±0．13对1．08±0．26）ng／ml；（9．56±2．53对21．36±4．16）×10^-7；（3．95±3．45对27．02±8．62）×10^-7，均P〈0．05]。（2）FG组胰岛素染色阳性面积、胰岛素染色阳性率和积分光密度均低于HG组（均P〈0．05）。（3）FG组β细胞凋亡率显著高于HG组[（24．17±7．25对16．55±5．11）％，P〈0．01]。结论波动性高血糖可能较持续性高血糖对胰岛β细胞功能的损害更加明显，β细胞凋亡增加可能是血糖波动导致胰岛功能改变的机制之一。

Objective To compare the effects of intermittent high blood glucose and consistent high blood glucose on pancreatic islet β-cell function and β-cell apoptosis in GK rats. Methods Twenty-two male GK rats were randomly divided into 2 groups consisting of consistent high blood glucose group （HG） and intermittent high blood glucose group（FG）. Eleven male Wistar rats were used as normal glucose controls（NG）. The fluctuating high blood glucose animal model was induced by intraperitoneal injection of insulin and glucose at different time for six weeks. Intraperitoneal injection glucose tolerance test and insulin release test were performed. The area under curve of glncose（AUCg）, the area under carve of insulin （AUCi）/AUCg and the ratio of insulin increment to blood glucose increment 15 min after glucose load（△115＇/△G15＇） were calculated routinely. Then the pancreatic slides were stained with insulin antibody. The apoptotic β cells in islets were detected and quantified by the TUNEL technique. Results （1）The fasting plasma glucose and 15, 30, 60, and 120 rain plasma glucose levels after glucose loading in FG group were significantly higher than those in control group（ all P〈0.01 ） ,and AUCg was also markedly increased[ （ 1 012.14±82.62 vs 813.60±56.70） ng . ml^-1 . h^-1 . 10^4 ,P〈0. 01）. Insulin levels of FG group at 15, 30, 60, and 120 min after glucose loading were significantly lower than those in HG group[ （0.55± 0.18 vs 0. 95±0.28, 0.43±0.17 vs 0. 85±0.21, 0.47±0.11 vs 0. 76±0.16, 0.58±0.13 vs 1.08±0.26）ng/ml, P〈0.05 ] ,along with decreased AUCi/AUCg and △I15＇/△G15＇ [ （9.56±2.53 vs 21.36±4.16 ） × 10^-7; （3.95 ± 3.45 vs 27.02±8.62）× 10^-7 ,both P〈0.05 ]. （2）Image analysis of pancreatic islet immnnocytochemistry showed that the insulin staining positive area, area ratio and total density of insulin positive cells per islet were significantly lower in FG group than those in HG group （P〈0.05）. （3） The percentage of β-cell apoptosis in the FG group was statistically higher than that in the HG group [（24.17 ± 7. 25 vs 16. 55± 5.11）%, P 〈 0.01 ]. Conclusion Compared with the consistent high blood glucose, intermittent high glucose could lead to further impairment of β-cell function and increased β-cell apoptosis may partially contribute to this process.