组织蛋白酶K对高脂肪负荷小鼠胰岛素敏感性的影响研究

Inhibited cathepsinK improve insulin action inhigh-fat mice

目的探讨组织蛋白酶K对高脂肪负荷小鼠胰岛素敏感性的影响。方法 C57BL/6J雄性小鼠随机分为普通饲料+生理盐水组(control组)、普通饲料+组织蛋白酶K(CatK)抑制剂组(control+CatKⅡ组)、高脂肪食+生理盐水组(HFD+NS组)及高脂肪食+CatK抑制剂组(HFD+CatKⅡ组),每组6只。所有小鼠根据分组不同饲喂相应饲料,同时5 mg/(kg·d)腹腔注射CatK抑制剂或等量生理盐水2 w。测定各组空腹血糖、胰岛素及CatK浓度变化,采用高胰岛素-正常血糖钳夹技术评价胰岛素敏感性,蛋白质印迹法(Western blot)观察骨骼肌葡萄糖转运体4表达水平。结果到16 w时,HFD+NS组体重明显高于control组,组间比较差异具有统计学意义(F=28.55,P<0.05);HFD+CatKⅡ组与HFD+NS组比较体重略降低,但差异无统计学意义(F=3.49,P>0.05);高脂肪食饲养显著增加空腹血糖水平和空腹胰岛素浓度,HFD+NS组空腹血糖水平和胰岛素水平均明显高于control组(F=12.45、3.87,均P<0.05);CatKⅡ可显著降低高脂肪食引起的胰岛素水平增加,HFD+CatKⅡ组与HFD+NS组胰岛素水平比较差异具有统计学意义(F=1.58,P<0.05)。与control组比较,HFD+NS组血清CatK水平明显增加,组间比较差异有统计学意义(t=0.034,P<0.05);HFD+CatKⅡ组CatK水平为(5.69±0.82)ng/ml,与HFD+NS组的(9.19±0.85)ng/ml比较明显减少,差异具有统计学意义(t=0.021,P<0.05)。HFD+CatKⅡ组血糖水平变化情况与HFD+NS组类似,但组间比较差异无统计学意义(t=0.065,P>0.05)。高胰岛素-正常血糖钳夹实验显示,HFD+NS组空腹血糖/空腹胰岛素比(GIR)值明显低于control组,差异具有统计学意义(t=0.015,P<0.05);HFD+CatKⅡ组GIR值为(8.62±0.51)mg/(kg·ml)明显高于HFD+NS组的(4.08±0.34)mg/(kg·ml),差异具有统计学意义(t=0.027,P<0.05)。与普通饲料饲喂的小鼠比较,高脂肪食物饲喂小鼠GluT4蛋白表达水平明显降低,但HFD+NS组与HFD+CatKⅡ组GluT4蛋白表达水平没有明显差异(P>0.05)。结论高脂肪负荷可改变小鼠血浆CatK浓度,CatK抑制剂可改善高脂肪负荷小鼠的胰岛素敏感性。

Objective To investigate cathepsinK inhibitor improves insulin action of high-fat mice.Methods C57BL/6J male mice were randomly divided into common feed saline group ( control group) ,common feed cathepsin K ( CatK group)inhibitor group ( control CatKⅡ group) ,high fat food saline group ( HFD NS group) and high fat food CatK inhibitor group( HFD CatKⅡ group) ,six in each group. All mice were fed different diets,simultaneous 5 mg /( kg d) intraperitoneal injectionof CatK inhibitors or 2 w. of saline Determination of fasting blood glucose,insulin and CatK concentrations in eachgroup,high insulin - normal blood glucose clamp technique was used to evaluate insulin sensitivity,western blotting( Western blot) was used to observe the expression level of glucose transporter 4 in skeletal muscle.Results At 16 week,the weight of HFD NS group was significantly higher than that of control group,comparison between groups was statisticallysignificant ( F= 28.55,P<0.05);the weight of HFD CatKⅡ group and HFD NS group decreased slightly,but the differencewas not statistically significant ( F= 3.49,P>0.05);high fat feeding significantly increased fasting blood glucose levelsand fasting insulin concentrations,the levels of fasting blood glucose and insulin in HFD NS group were significantlyhigher than those in control group ( F= 12.45,3.87,all P<0.05);CatKⅡ can significantly reduce the increase in insulinlevels caused by high fat foods,there were significant differences in insulin levels between the HFD CatKⅡ group and theHFD NS group ( F= 1.58,P<0.05) . compared with control group,the serum CatK level in HFD NS group increased significantly,differences between groups were statistically significant ( t = 0.034,P<0.05);the CatK level of HFD CatKⅡ groupwas ( 5.69±0.82) ng /ml,there was a significant decrease compared with the ng /ml of the HFD NS group ( 9.19±0.85) ,differences were statistically significant ( t = 0.021,P<0.05) . the blood glucose levels in HFD CatKⅡ group were similar tothose in HFD NS group,but there was no statistically significant difference ( t = 0.065,P>0.05) . High insulin - normalblood glucose clamp test showed,the fasting blood glucose /fasting insulin ratio ( GIR) in HFD NS group was significantlylower than that in control group,differences were statistically significant ( t = 0.015,P<0.05);the GIR value of HFD CatKⅡ group was ( 8.62±0.51) mg /( kg·ml) significantly higher than that of HFD NS group ( 4.08±0.34) ( kg·ml) ,differenceswere statistically significant ( t = 0.027,P<0.05) . compared with the mice fed with common feed,High fat food feedingmice GluT4 protein expression levels decreased significantly,there was no significant difference in GluT4 protein expressionbetween HFD NS group and HFD CatKⅡ group ( P>0.05) .Conclusion High fat load can change plasma CatKconcentration in mice,CatK inhibitors improve insulin sensitivity in high-fat load mice.