利拉鲁肽对高脂喂养胰岛素抵抗大鼠肝脏脂质沉积的影响及相关机制的探讨

发布时间：2018-06-03 02:00

本文选题：利拉鲁肽 + 胰岛素抵抗　；参考：《承德医学院》2017年硕士论文

【摘要】：目的:利拉鲁肽是目前治疗2型糖尿病的新上市降糖药物,不仅可以促进胰岛素分泌,还可增加胰腺外组织对胰岛素作用的敏感性。胰岛素抵抗(insulin resistance,IR)是2型糖尿病及代谢综合征的发病机制之一。既往有研究发现利拉鲁肽可改善IR,但具体机制尚未明确。本实验通过使用不同浓度的利拉鲁肽对高脂饲料喂养的大鼠进行干预,观察其在IR大鼠肝脏的脂质沉积以及对胰岛素的敏感性的影响;本研究经检测肝脏内质网应激分子伴侣GRP78,内质网应激相关分子JNK,胰岛素信号传导通路相关分子GLUT-2、AKT表达的情况,探究利拉鲁肽改善IR,减少肝脏脂肪沉积的机制,为其在临床的应用提供理论支持。方法:Wistar雄性4月龄大鼠,体重250g左右,54只,经过3天适应性的喂养,随机将其分为对照组(NC,n=16)和高脂组(n=38),分别以普通和高脂饲料喂养。8周末,两组在随机下取5只大鼠在其清醒状态下进行高胰岛素-正葡萄糖钳夹试验,然后计算两组大鼠的葡萄糖输注率(glu cose infusion rate,GIR),判定其IR状态。判定造模成功后,将高脂大鼠分为三组即高脂对照组(HFD)、利拉鲁肽低剂量组(LL)、利拉鲁肽高剂量组(HL),每组11只。分别以0.5ml的灭菌生理盐水对NC组及HFD组进行皮下注射;以100ug/kg的利拉鲁肽对LL组日一次进行皮下注射;以200ug/kg的利拉鲁肽对HL组日一次行皮下注射。药物干预2周后,四组分别随机选取5只大鼠,后进行口服糖耐量(oral glucose tolerance test,OGTT)实验和钳夹试验,以算出大鼠葡萄糖曲线下面积(Area Un der Curve,AUC)及GIR。对各个组中剩余的6只大鼠分别称体重(body weight,BW),然后进行麻醉,腹主动脉取血,-80℃保存,测定甘油三酯(triglyceride,TG)、游离脂肪酸(free fatty acids,FFA)、高密度脂蛋白胆固醇(high density lipoprotein cholesterol,HDL-C)、总胆固醇(tot al cholesterol,TCH)以及低密度脂蛋白胆固醇(low density lipoprotein c holesterol,LDL-C)、肝转氨酶学改变。分别制作肝脏标本并保存,测定肝组织甘油三酯(LTG),经透电子显微镜察看肝细胞内质网结构是否发生改变,应用Western blot技术对肝脏中GRP78、GLUT-2、p-AKT、p-JN K蛋白表达情况进行检测,应用Realtime-PCR对GRP78、GLUT-2 m RNA表达情况进行测定。结果:1四组大鼠一般情况的比较与NC组相较,HFD组中BW、血FFA以及LTG均显著增高(P0.05)。与HFD组相比较,HL组中BW、血FFA、TG、LTG、肝转氨酶学指标均显著降低(P0.05);LL组中仅有FFA、肝转氨酶学指标的下降(P0.05)。与LL组相比,HL组血液指标均有明显改善,血FFA、TG、LTG也均有显著减少(P0.05)2四组大鼠GIR的比较喂养第8周时,高胰岛素-正葡萄糖钳夹实验结果示:与NC组相比,HFD组葡萄糖输注率GIR下降(30.21±2.51vs20.41±3.64mg/kg.min)(P0.05)。药物干预2周后,与HFD组相比,HL组GIR升高(18.83±1.95)vs(25.80±1.40)mg/kg.min(P0.05),LL组GIR升高(18.83±1.95)vs(21.55±2.46)mg/kg.min(P0.05);与LL组相比,HL组GIR亦升高(21.55±2.46)vs(25.80±1.40)mg/kg.min(P0.05)。3四组大鼠OGTT的比较与NC组相比较,HFD组大鼠血糖及葡萄糖曲线下面积AUC于0min、30min、60min、120min四个点处均显著升高(P0.05);与HFD组相比较,HL组大鼠血糖及AUC在四个点处均降低(P0.05),LL组大鼠虽四个点处血糖及AUC均有所降低,然而仅在120min时血糖变化有显著性差异(P0.05);与LL组相比较,HL组大鼠血糖及AUC于0min、120min两个点处均显著降低(P0.05)。4四组大鼠肝脏形态学变化(光学显微镜)在NC组中,肝细胞细胞壁及内部细胞器结构完整,肝小叶和中央静脉结构正常,可在中央静脉周围清晰观察到呈放射状排布的肝细胞索,HFD组肝细胞体积显著扩大,胞内存在大小不等的脂滴,细胞核移向细胞边缘,LL组肝细胞体积有所减小,胞内脂滴数目减少,体积缩小,HL组大鼠的肝细胞排列规整,胞内可见少量脂滴,其肝小叶及肝索结构完整。5四组大鼠肝细胞内质网变化(透射电子显微镜)与NC组相比,HFD组肝细胞核周围脂滴数量多,体积大,内质网明显扩张,脱颗粒明显;与HFD组相比,LL组肝细胞内质网轻度扩张,脱颗粒轻微缓解;与HFD组相比,HL组肝细胞核周围无明显脂滴存在,内质网扩张及脱颗粒得到显著缓解。6四组大鼠肝脏GRP78、GLUT-2 m RNA表达水平比较与NC组大鼠相比较,HFD组大鼠的GRP78 m RNA的表达显著增高,GLUT-2 m RNA表达显著减低(P0.05);与HFD组大鼠相比较,LL组及HL组大鼠GRP78 m RNA的表达均显著降低(P0.05),与LL组大鼠相比较,HL组减低更为明显(P0.05);与HFD组大鼠相比较,LL组及HL组GLUT-2 m RNA的表达均显著上调(P0.05),但HL组上调程度显著高于LL组(P0.05)。7四组大鼠肝GRP78、GLUT-2、p-AKT和p-JNK蛋白表达水平比较与NC组相比,HFD组GRP78蛋白表达上调,GLUT-2蛋白表达下调,磷酸化JNK(p-JNK)/JNK明显升高,磷酸化AKT(p-AKT)/AKT明显下降(P0.05)。与HFD组相比,HL组、LL组GRP78蛋白表达均下调,GLUT-2蛋白表达均上调,p-JNK/JNK均下降,p-AKT/AKT均升高(P0.05)。与LL组相比,HL组GRP78蛋白表达下调,GLUT-2蛋白表达上调,p-JNK/JNK下降,p-AKT/AKT增加(P0.05)。8相关分析研究结果:GIR与BW,血FFA、TG及LTG呈负相关(P0.05或P0.01)。GRP78蛋白表达量与血FFA、TG,LTG呈正相关(P0.05或P0.01),同时与GIR呈负相关(P0.01)。GLUT-2蛋白表达量与血FFA、TG,LTG呈负相关(P0.01),并且与GIR呈正相关(P0.01)。JNK磷酸化水平与血FFA、TG,LTG呈正相关(P0.01),且与GIR呈负相关(P0.01)。AKT磷酸化水平与与血FFA、TG,LTG呈负相关(P0.05或P0.01),且与GIR呈正相关(P0.01)。结论:1高脂饮食可使大鼠出现糖脂代谢紊乱,并增加肝脏脂肪沉积,出现糖耐量的降低及IR。2利拉鲁肽对改善糖脂代谢紊乱、减少肝脏脂质蓄积以及改善IR和糖耐量减低呈浓度依赖性。3利拉鲁肽呈浓度依赖性对GRP78/JNK/AKT/GLUT-2通路进行调控,与缓解肝脏内质网应激,减少肝脏脂质沉积,促进肝细胞对葡萄糖摄取,改善IR相关。
[Abstract]:Objective: it is a new hypoglycemic drug for the treatment of type 2 diabetes, which not only promotes insulin secretion, but also increases the sensitivity of external pancreatic tissue to insulin. Insulin resistance (insulin resistance, IR) is one of the pathogenesis of type 2 diabetes and metabolic syndrome. Good IR, but the specific mechanism is not clear. In this experiment, the rats fed with high fat diet were treated with different concentrations of lieragin, and the lipid deposition in the liver of IR rats and the effect on the sensitivity of insulin were observed. The study was conducted to detect the liver endoplasmic reticulum stress sub chaperone GRP78, the endoplasmic reticulum stress related molecule JNK, and the pancreas. The expression of GLUT-2 and AKT in the signal transduction pathway of ISO signal transduction pathway, explore the mechanism of liararu peptide to improve IR, reduce the liver fat deposition, and provide theoretical support for its clinical application. Methods: Wistar male 4 month old rats, weight 250g and 54, were randomly divided into control group (NC, n=16) and high fat group after 3 days of adaptive feeding. (n=38).8 weekend was fed with ordinary and high fat diet, and the two groups were randomly selected to take 5 rats at random to carry out high insulin positive glucose clamp test, and then calculate the glucose infusion rate (Glu cose infusion rate, GIR) in two groups of rats and determine their IR like state. After the model was successful, the high fat rats were divided into three groups, that is, high fat fat. The control group (HFD), the low dose group of Liraru peptide (LL), the high dose of Liraru peptide group (HL), 11 rats in each group, were subcutaneously injected with the NC and HFD group with 0.5ml sterilized saline, and a subcutaneous injection of the 100ug/kg's Liraru peptide to LL group daily, and a subcutaneous injection of the 200ug/kg's Liraru peptide to the HL group. In the four groups, 5 rats were randomly selected, then the oral glucose tolerance (oral glucose tolerance test, OGTT) experiment and clamp test were carried out to calculate the area of the glucose curve (Area Un der Curve, AUC) and GIR. on the remaining 6 rats in each group. Triglyceride, TG, free fatty acids (free fatty acids, FFA), high density lipoprotein cholesterol (high density lipoprotein cholesterol, HDL-C), total cholesterol (TOT), and low density lipoprotein cholesterol (LDL), liver transaminase change, were made respectively. The liver specimens were preserved, the liver tissue triglyceride (LTG) was measured, and the changes in the structure of the endoplasmic reticulum of the liver cells were examined through electron microscopy. The expression of GRP78, GLUT-2, p-AKT, p-JN K protein in the liver was detected by Western blot technique. The expression of GRP78 and GLUT-2 m was measured by Realtime-PCR. Results: 1 groups of four groups were large Compared with NC group, BW, blood FFA and LTG in group HFD were significantly higher (P0.05). Compared with group HFD, BW, FFA, TG, LTG, liver transaminase index in HL group were significantly decreased. Also significantly decreased (P0.05) 2 four groups of rats in the comparative feeding of GIR eighth weeks, the results of high insulin positive glucose clamp showed that compared with the NC group, the glucose infusion rate in the HFD group decreased (30.21 + 2.51vs20.41 + 3.64mg/kg.min) (P0.05). After 2 weeks of drug intervention, the GIR increased (18.83 + 1.95) vs (25.80 + 1.40) (25.80 + 1.40) compared with the HFD group. 05), GIR in group LL increased (18.83 + 1.95) vs (21.55 + 2.46) mg/kg.min (P0.05). Compared with group LL, HL group GIR also increased (21.55 + 2.46) vs (25.80 + 1.40) mg/kg.min (P0.05).3 four rats. Compared with group HL, blood glucose and AUC decreased at four points (P0.05). The blood glucose and AUC were decreased at four points in group LL rats, but there was a significant difference (P0.05) at 120min only (P0.05). Compared with group LL, the blood sugar and AUC of rats in group HL and 120min two points were significantly reduced in the four groups of rat liver morphology. In the NC group, the cell wall and internal organelle structure of the liver cells were complete, the hepatic lobule and the central vein were normal, and the hepatic cell cord was clearly observed around the central vein. The volume of the hepatocytes in the HFD group was significantly enlarged, the fat droplets of different sizes were found in the cell, the nucleus moved to the edge of the cell and the liver of the LL group. The cell volume decreased, the number of intracellular lipid droplets decreased and the volume was reduced. The liver cells in the HL group were arranged regularly and a small amount of lipid droplets were visible. The changes in the endoplasmic reticulum of the hepatocytes in the group of hepatic lobules and the complete.5 four groups of the hepatic cord (transmission electron microscope) were compared with that of the NC group. The number of lipid droplets around the nucleus of the liver of the group HFD was large and the endoplasmic reticulum was obvious. Compared with the HFD group, the endoplasmic reticulum in the LL group was slightly dilated and the degranulation was slightly relieved. Compared with the group HFD, there was no obvious lipid droplet around the nucleus of the liver in the group HL, and the endoplasmic reticulum dilation and degranulation significantly alleviated the GRP78 in the liver of.6 four rats, and the RNA expression level of GLUT-2 m was compared with the NC group rats, and G in HFD group rats. The expression of RP78 m RNA was significantly higher and the expression of GLUT-2 m RNA decreased significantly (P0.05). Compared with group HFD, the expression of GRP78 m in LL group and HL group decreased significantly. Compared with group NC, the expression level of GLUT-2, p-AKT and p-JNK protein in group.7 four was significantly higher than that of group.7 (P0.05). The expression level of GRP78 protein in HFD group was up, the expression of GLUT-2 protein was down, phosphorylation JNK was obviously increased and phosphorylation was obviously decreased. The expression of GLUT-2 protein increased, p-JNK/JNK decreased, and p-AKT/AKT increased (P0.05). Compared with the LL group, the expression of GRP78 protein in the HL group was down regulated, the expression of GLUT-2 protein was up, p-JNK/JNK decreased, and p-AKT/AKT increased (P0.05). TG, LTG was positively correlated (P0.05 or P0.01), and negative correlation with GIR (P0.01).GLUT-2 protein expression was negatively correlated with FFA, TG and LTG (P0.01). 01) and positive correlation with GIR (P0.01). Conclusion: 1 high fat diet can cause metabolic disorder of glycolipid in rats, increase liver fat deposition, decrease glucose tolerance, and IR.2 liololu peptide to improve glycolipid metabolic disorder, reduce liver lipid accumulation, and improve IR and impaired glucose tolerance in a concentration dependent.3 liololu peptide concentration dependence on G Regulation of RP78/JNK/AKT/GLUT-2 pathway is associated with alleviation of endoplasmic reticulum stress, reduction of hepatic lipid deposition, promotion of glucose uptake by hepatocytes, and improvement of IR.
【学位授予单位】：承德医学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R587.1

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