高脂饮食对不同年龄小鼠胰岛素抵抗和胰岛β细胞功能的影响

发布时间：2018-09-17 08:13

【摘要】：目的:随着我国经济发展,饮食结构及生活方式发生巨大改变,肥胖及肥胖相关的谢性疾病如糖代谢紊乱逐年增多。代谢性疾病的发病年龄日趋年轻化,儿童和青少年肥胖及糖代谢紊乱的现象已引起广泛关注,然而,生物学年龄、饮食结构、肥胖、以及糖脂代谢之间的关系仍不十分清楚,不同年龄个体对高脂饮食所致代谢异常的敏感性有待明确。本研究的目的是通过观察高脂饮食对不同月龄小鼠胰岛素敏感性和胰岛β细胞功能的影响,加深对年龄因素在机体代谢应激反应中作用的理解,探索代谢应激时胰岛β细胞功能失代偿的早期标志,为深入研究代谢综合症和糖尿病发生发展过程中,胰岛β细胞功能衰竭的分子机制提供有益线索。方法:健康C57BL/6J雄性小鼠21天断乳组24只(平均体重12.66±0.74g),3月龄组24只(平均体重28.70±1.30g),6月龄组24只(平均体重33.16±2.74g),各年龄段小鼠随机分成两组,分别为21DHF/21DCD,3MHF/3MCD和6MHF/6MCD组,分别给予60%高脂饲料或正常饲料12周。每周称量体重、每三周测6小时空腹血糖、第12周灌胃法测量葡萄糖耐量、酶联免疫吸附法测空腹血清胰岛素、断颈法处死小鼠取胰腺、肝脏、脂肪组织进行组织学分析,胆管内穿刺内消化法提取小鼠胰岛并通过免疫印迹法进行胰岛素原的研究。结果:高脂饲料喂养12周后(1)各年龄高脂饮食组体重较正常饮食组体重明显增加(P0.01)(21DHF46.18±3.34g vs.21DCD28.51±1.66g;3MHF51.51±2.2g vs.3MCD32.99±1.26g;6MHF46.06±8.07g vs.6MCD36.87±4.67g)。21DHF小鼠体重增长量最大,但因高脂饲料而增加的体重在3MHF最明显。(2)各年龄段高脂组6小时空腹血糖均高于对照组(21DHF9.68±0.82 vs.21DCD6.60±0.86mmol/L;3MHF12.86±1.34 vs.3MCD6.30±1.42 mmol/L;6MHF9.68±1.32 vs.6MCD5.40±0.97mmol/L),差别均具有统计学意义(P0.05),3MHF空腹血糖最高且空腹血糖升高速率最大,3MHF较3MCD空腹血糖明显升高出现最早。(3)各年龄段高脂饮食组糖耐量曲线下面积较正常饮食组均有不同程度增高(21DHF32.18±10.45 vs.21DCD 20.91±1.05;3MHF 39.33±3.72 vs.3MCD16.74±1.70;6MHF 30.19±4.61 vs.6MCD 17.69±2.26),但21月龄组无统计学意义(P0.05),其余年龄组的差异均具有统计学意义(P0.05),3MHF组葡萄糖耐量最差。(4)胰岛素抵抗:各年龄高脂饮食组空腹血清胰岛素水平较正常饮食组明显升高,差别有统计学意义(P0.05),同年龄高脂饮食组与正常饮食组相比,HOMA-IR均明显升高,ISI均明显降低,差异均具有统计学意义(P0.05),且3MHF组胰岛素抵抗最明显,胰岛素敏感性最差。(5)胰岛素原占总胰岛素(胰岛素原PI+胰岛素I)的百分比[PI/(PI+I)x100%]:21DHF51.53±4.18%vs.21DCD28.20±6.20%,P0.01;3MHF 69.84±1.55%vs.3MCD 30.76±0.18%,P0.01;6MHF 46.65±2.49%vs.6MCD 39.64±1.28%,P0.01。3MHF与21DHF、6MHF胰岛素原所占百分比均有统计学差异(P0.05)。(6)组织学变化:各年龄高脂饮食组小鼠较正常饮食组胰岛平均面积均有不同程度的升高(21DHF14040±22274 um2 vs.21DCD 11061±7004 um2,P=0.62;3MHF 22759±36270um2 vs.3MCD 11191±12509 um2,P=0.02;6MHF 22072±30223um2 vs.6MCD 11424±17425um2,P=0.02);同年龄高脂饮食组较正常饮食组β细胞占胰岛面积的比例不同程度的增加(21DHF91.40±6.41%vs.21DCD 85.41±9.47%,P0.001;3MHF94.68±9.06%vs.3MCD 85.83±9.55%,P0.001;6MHF 88.87±7.72%vs.6MCD83.08±14.99%,P=0.04);同年龄高脂饮食组较正常饮食组α与β细胞比例均有不同程度降低(21DHF0.21±0.16 vs.21DCD 0.30±0.16,P0.05;3MHF 0.18±0.07vs.3MCD 0.28±0.16 P0.05;6MHF 0.22±0.09 vs.6MCD 0.27±0.13 P=0.09);β细胞平均横截面积在高脂组与正常饮食组间无统计学差异,胰岛的代偿性增生只要是β细胞代偿性增生所致,3MHF组β细胞代偿性增生最明显。不同年龄高脂鼠的胰岛内均可见不同程度的毛细血管的扩张,大量的血球堆积在胰岛内。高脂组肝脏均出现了脂肪肝,21DHF脂肪肝2级;3MHF脂肪肝3级;6MHF脂肪肝3级;各高脂组睾周脂肪组织内可见不同程度炎症细胞的浸润。结论:(1)60%高脂饲料喂养C57BL/6J雄性小鼠12周可以成功诱导小鼠肥胖、胰岛素抵抗和糖代谢异常。(2)与21天龄和6月龄小鼠相比,3月龄小鼠在高脂饲料喂养后体重增长最快,空腹血糖最高,糖耐量最差、胰岛素抵抗最严重,所以3月龄鼠对高脂饲料引起的代谢紊乱最敏感。做代谢模型时必须考虑实验动物的生物学年龄,本实验为其他基础研究实验动物的选择提供了一份参考证据。(3)高脂饲料喂养后各年龄小鼠均出现胰岛素抵抗,空腹血糖受损,糖耐量异常,胰岛内胰岛素原明显增多,胰岛代偿性增大(主要是β细胞代偿性增生),但胰岛的代偿性增生未能代偿糖代谢负荷,说明高脂饮食影响了β细胞功能。(4)本研究显示年龄在代谢应激反应中起到了重要的作用。明确年龄因素对β细胞代偿潜能的影响有助于揭示2型糖尿病的发病机制。(5)高脂饮食诱导的胰岛素原的增多可能是β细胞功能失代偿的早期标志。
[Abstract]:OBJECTIVE: With the development of economy in China, great changes have taken place in dietary structure and lifestyle. Obesity and obesity-related metabolic diseases such as glucose metabolism disorders are increasing year by year. The purpose of this study was to investigate the effects of high-fat diet on insulin sensitivity and islet beta cell function in mice of different months of age, and to deepen the effects of age on metabolic stress response in the body. To explore the early markers of islet beta cell decompensation during metabolic stress, and to provide useful clues for further study of the molecular mechanism of islet beta cell failure in the development of metabolic syndrome and diabetes mellitus. Methods: Twenty-four healthy C57BL/6J male mice (average weight 12.66.74 g) were weaned on 21 days, aged 3 months. Twenty-four mice in each age group were randomly divided into two groups: 21 DHF/21 DCD, 3 MHF/3 MCD and 6 MHF/6 MCD. The mice were given 60% high fat diet or normal diet for 12 weeks. The body weight was weighed weekly, fasting blood glucose was measured every three weeks for 6 hours, and glucose tolerance was measured by gavage at 12 weeks. Enzyme linked immunosorbent assay (ELISA) was used to measure fasting serum insulin, pancreas, liver and adipose tissue were taken from mice by neck-cutting method, pancreatic islets were extracted by biliary duct puncture and digestion, and proinsulin was detected by immunoblotting. Results: After 12 weeks of feeding with high-fat diet, the weight of the high-fat diet group was higher than that of the normal diet group. Obvious weight gain (P 0.01) (21DHF 46.18+3.34 g vs. 21DCD 28.51+1.66 g; 3MHF 51.51+2.2 g vs. 3MCD 32.99+1.26 g; 6MHF 46.06+8.07 g vs. 6MCD 36.87+4.67 g). The weight gain of 21DHF mice was the greatest, but the weight gain due to high-fat diet was the greatest at 3MHF. (2) The fasting blood glucose of the high-fat group at 6 hours was higher than that of the control group (21DHF 9.68+4.67 g). 82 vs. 21 DCD 6.60.86 mmol/L; 3MHF 12.86.34 vs. 3 MCD 6.30.42 mmol/L; 6MHF 9.68 1.32 vs. 6 MCD 5.40.97 mmol/L, the differences were statistically significant (P 0.05), 3MHF had the highest fasting blood glucose and the highest fasting blood glucose elevation rate, and 3MHF had the earliest glucose tolerance curve compared with 3MCD. Compared with the normal diet group, the area under the diet was increased in different degrees (21 DHF 32.18+10.45 vs.21 DCD 20.91+1.05; 3 MHF 39.33+3.72 vs.3 MCD 16.74+1.70; 6 MHF 30.19+4.61 vs.6 MCD 17.69+2.26), but there was no significant difference in 21-month-old group (P 0.05), the other age groups (P 0.05), and the glucose tolerance of 3 MHF group was the worst. Resistance to vegetarian: The fasting serum insulin level of the high-fat diet group was significantly higher than that of the normal diet group (P 0.05). Compared with the normal diet group, HOMA-IR was significantly higher and ISI was significantly lower in the high-fat diet group (P 0.05). Insulin resistance was the most obvious in the 3MHF group, and insulin resistance was the most obvious in the 3MHF group. (5) The percentage of proinsulin to total insulin (proinsulin PI + insulin I) [PI / (PI + I) x 100%]: 21DHF 51.53 (+ 4.18%) vs. 21DCD28.20 (+ 6.20%), P 0.01; 3MHF 69.84 (+ 1.55%) vs. 3MCD 30.76 (+ 0.18%), P 0.01; 6MHF 46.65 (+ 2.49%) vs. 6MCD 39.64 (+ 1.28%), P 0.01.3MHF vs. 21DHF, 6MHF, 6 MCD 30.76 (+ 0.18%). (P 0.05). (6) Histological changes: The average islet area of mice fed with high-fat diet at different ages was higher than those fed with normal diet (21DHF 14040 (+ 22274) Um2 vs. 21DCD 11061 (+ 7004) um2, P = 0.62; 3MHF 22759 (+ 36270) Um2 vs. 3MCD 11191 (+ 12509) um2, P = 0.02; 6MHF 22072 (+ 30223) Um2 vs. 6MCD 11424 (+) 174252, P = 0.02); and the same age group fed with high-fat diet was higher than that of mice fed with high- The proportion of beta cells to islet area in the diet group was higher than that in the normal diet group (21DHF 91.40+6.41% vs. 21DCD 85.41+9.47%, P 0.001; 3MHF 94.68+9.06% vs. 3MCD 85.83+9.55%, P 0.001; 6MHF 88.87+7.72% vs. 6MCD 83.08+14.99%, P = 0.04); the ratio of alpha cells to beta cells in the high-fat diet group was lower than that in the normal diet group at the same age (P = 0.04). (21DHF 0.21 + 0.16 vs. 21DCD 0.30 + 0.16, P 0.05; 3MHF 0.18 + 0.07 vs. 3 MCD 0.28 + 0.16 P 0.05; 6MHF 0.22 + 0.09 vs. 6 MCD 0.27 + 0.13 P = 0.09); the mean cross-sectional area of beta cells was not significantly different between the high-fat group and the normal diet group; as long as the compensatory proliferation of islets was caused by the compensatory proliferation of beta cells, the compensatory proliferation of beta cells in 3MHF group was not found. There were different degrees of telangiectasia and large amount of blood cells accumulated in the islets. Fatty liver appeared in the liver of high-fat group, 21DHF fatty liver grade 2; 3MHF fatty liver grade 3; 6MHF fatty liver grade 3; inflammatory cells infiltrated in the peritesticular adipose tissue of each high-fat group. C57BL/6J male mice fed with 0% high-fat diet for 12 weeks could successfully induce obesity, insulin resistance and abnormal glucose metabolism. (2) Compared with 21-day-old and 6-month-old mice, 3-month-old mice fed with high-fat diet had the fastest weight gain, the highest fasting blood glucose, the worst glucose tolerance and the most serious insulin resistance. Metabolic model must consider the biological age of experimental animals, this experiment provides a reference for the selection of other basic research experimental animals. (3) Insulin resistance, impaired fasting blood glucose, impaired glucose tolerance and proinsulin in islets of all ages after high-fat diet feeding mice are obvious. Many, islet compensatory enlargement (mainly beta-cell compensatory hyperplasia), but islet compensatory hyperplasia can not compensate for glucose metabolism load, indicating that high-fat diet affects beta-cell function. (4) This study shows that age plays an important role in metabolic stress response. (5) The increase of proinsulin induced by high fat diet may be an early marker of beta cell decompensation.
【学位授予单位】：天津医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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