【作者】 宋璐璐；

【导师】 杨文英；

【作者基本信息】 北京协和医学院 ， 内科学， 2011， 博士

【摘要】 第一部分：尽早干预对db/db糖尿病小鼠胰岛p细胞保护的探讨目的：探讨在血糖正常期和糖尿病早期采取不同方式对db/db小鼠进行干预对胰岛p细胞的保护。方法：(1)选取血糖正常的雄性db/db小鼠98只,分别在4周龄(血糖正常)和6周龄(糖尿病早期)时进行干预,各分为4个干预组：利拉鲁肽(Liraglutide,LIRA).吡咯列酮(Pioglitazone,PIO)、限制饮食(Calories restriction,CR)和运动(Exercise,EX),另设1个对照组。入组前及干预结束时测定糖化血红蛋白(Glycosylated hemoglobin,HbAlc).每周监测小鼠空腹血糖(Fasting blood glucose,FBG).体重和进食量。小鼠12周龄时结束干预,分别进行腹腔注射糖耐量试验(Intraperitoneal glucose tolerance test,IPGTT)和胰岛素耐量试验(Insulin tolerance test,ITT)评价糖耐量、胰岛素释放反应及胰岛素敏感性。检测血浆甘油三酯(Triglyceride,TG).游离脂肪酸(free fatty acids,FFA).脂联素和胰高血糖素浓度。免疫组织化学方法检测胰岛p细胞质量、增殖(Brdu)和凋亡(TUNEL)；检测胰岛PDX-1和IRS-2表达变化。实时荧光定量PCR检测胰腺GRP78.CHOP mRNA.microRNA miR-34a.miR-375表达,普通PCR检测胰腺XBP-1/spliced XBP-1 mRNA表达.Western blot检测胰腺内质网应激标志物GRP78和CHOP蛋白表达。(2)35只8周龄db/db小鼠分为4周：对照组,PIO组,LIRA组,及PIO+LIRA联合治疗组,治疗4周后行IPGTT和ITT试验。测定血浆胰岛素、脂联素、游离脂肪酸和甘油三酯浓度。免疫组织化学方法检测胰岛β细胞质量、增殖(Brdu)和凋亡(TUNEL)水平。结果：1.(1)与未处理组相比,干预结束时各组血糖显著降低(HbA1c：对照组7.3±0.3%,4周龄干预组4.9±0.8%,6周龄干预组5.5士O.9％,P<0.001)；IPGTTl 20min血糖曲线下面积显著降低(AUCglu:对照组4568±190 mmol/1*min,4周龄干预组2306±727mmol/l*min,6周龄干预组3559士903 mmol/lmin,P<0.001)。不同干预方式之间存在差别(HbA1c：LIRA组4.9±0.5%,PIO组4.3±0.4%,CR组5.4±0.7%,EX组6.1±0.6%, P<O.OOl),糖耐量的改善与HbAlc一致(AUCglu:LIRA组2558±639mmol/l*min, PIO组1662±483 mmol/l*min, CR组3262±613mmol/l*min, EX组3904±610 mmol/l*min, P<0.001)。(2)空腹胰岛素较对照组升高(对照组：5.7±2.5ng/ml,4周龄干预组11.0±3.0ng/ml,6周龄干预组9.1±4.6ng/mlP<0.01),4周龄干预组较6周龄干预组轻度升高(P<0.05)。除LIRA (12.3±3.4ng/ml, p=0.001)以外,其它组小鼠空腹胰岛素水平的差别没有统计学显著意义。胰岛p细胞的胰岛素分泌反应得到不同程度的恢复,IPTGTT120min胰岛素曲线下面积(AUCglu:对照组799±186ng/ml*min,4周龄干预组1410±595ng/ml*min,6周龄干预组1216±347ng/ml*min, P<0.05)。对照组胰岛素曲线低平,LIRA组和PIO组在30min出现分泌高峰,升高幅度分别为2.0倍和2.1倍,二者没有统计学差异。CR组胰岛素分泌高峰延迟,EX组胰岛素分泌曲线和对照组类似。(3)血浆TG、FFA,胰高血糖素水平降低,4周龄干预组和6周龄干预组相比均没有统计学差异。PIO组脂联素水平升高(9.5±1.6vs.l5.3±3.9mg/l, P<0.05),其他组与对照组无显著差别。(4)胰岛免疫组织化学分析显示,对照组胰岛肥大,免疫组化显示胰岛β细胞减少,胰岛素染色变浅,呈严重脱颗粒。胰岛α细胞增多,分布紊乱,由胰岛周边向中心迁移,散在分布在整个胰岛。胰岛素染色面积明显增加(对照组27.0±1.5%,4周龄干预组50.8±6.4%,6周龄干预组44.5±8.1%,P<0.001)。与对照组相比,PIO组胰岛明显变小,胰岛p细胞增多,无脱颗粒现象,胰岛α细胞分布正常；LIRA组和CR组胰岛肥大,胰岛p细胞增多,脱颗粒较对照组减轻,胰岛a细胞分布改善；EX组胰岛肥大,p细胞数量和胰岛素含量轻度增多,脱颗粒及胰岛α细胞分布无明显改善。胰岛p细胞的质量和血糖的控制呈现一致的趋势。LIRA组和CR组胰岛p细胞增殖较对照组明显增多(P<0.05)。p细胞凋亡在LIRA、PIO和CR组受到不同程度的抑制。(5)胰岛PDX-1和IRS-2表达在LIRA组和PIO组均较对照组明显增加,其中LIRA组PDX-1表达较PIO组多,而PIO组IRS-2表达显著强于LIRA组。(6)LIRA组和PIO组胰腺内质网应激显著抑制,CR组和EX组没有明显变化。(7)LIRA降低miR-34a (71%, P<0.05)和miR-375 (51%, P<0.05)表达。2.各治疗组血糖显著降低,PIO+LIRA联合治疗组降糖效果优于单药组(糖化血红蛋白对照组：7.3±0.4%, PIO:4.9±0.6%, LIRA:5.5±0.4%, PIO+LIRA:4.5±0.6%);与对照组相比IPGTT120min血糖曲线下面积降低56%(P<0.001), IPGTT120min胰岛素曲线下面积增加91%(P<0.01)；脂联素升高95%(P<0.05)；游离脂肪酸降低29%(P<0.05),甘油三酯降低49%(P<0.01),均优于单药组；胰岛组织切片胰岛素阳性面积增加1.7倍(P<0.001),胰岛新生β细胞比例增加2倍(P<0.01),均优于单药组。联合治疗显著改善胰岛a细胞分布,促进胰岛β细胞增殖,恢复正常的胰岛形态。结论：1.尽早干预更有利于对胰岛β细胞及其功能的保护,提示2型糖尿病尽早干预的必要。2.利拉鲁肽直接保护胰岛β细胞及其功能,但只能部分代偿严重的胰岛素抵抗。3.吡格列酮联合利拉鲁肽治疗相比单药更好地改善db/db小鼠糖脂代谢和保护胰岛β细胞功能。第二部分：京尼平抑制饱和脂肪酸诱导的HepG2细胞凋亡和内质网应激目的：探讨京尼平减轻棕榈酸对HepG2细胞毒性的作用及机制。方法：HepG2细胞分为空白组、棕榈酸组、京尼平组和京尼平预处理的棕榈酸组,分别用牛血清白蛋白、棕榈酸(1mmol/1)、京尼平(20μmol/1)或京尼平(20μmol/1)预处理30mmin后棕榈酸(1mmol/1)孵育24h,检测细胞活力(MTT)及乳酸脱氢酶(LDH)释放；孵育16h后流式细胞术和Hoechst染色检测细胞凋亡；孵育6h后实时荧光定量PCR检测、GRP78、CHOP基因表达,PCR结合电泳检测XBP-1裂解。结果：和空白组相比,棕榈酸减低HepG2细胞活力、增加LDH释放(P<0.01),增加HepG2细胞凋亡(P<0.05),上调GRP-78和CHOP表达(P<0.05)及XBP-1裂解；和棕榈酸组相比,京尼平增加细胞活力(P<0.05)、减少LDH释放(P<0.05),显著抑制细胞凋亡(P<0.01),降低GRP-78和CHOP基因表达(p<0.01)及XBP-1裂解。结论：京尼平具有抗棕榈酸诱导的肝细胞凋亡的作用：可能与抑制内质网应激有关。第三部分：PPAR-γ和PPAR-α激动剂改善HepG2细胞胰岛素抵抗及机制研究目的：探讨过氧化物酶体增殖激活受体γ(PPAR-γ)或α(PPAR-α)在肝细胞糖代谢和胰岛素抵抗改善中扮演的角色。方法：体外培养人肝癌细胞系HepG2细胞并用高浓度棕榈酸处理建立胰岛素抵抗模型,分别给予PPAR-γ激动剂吡格列酮或高选择性PPAR-a激动剂WY 14643处理,酶法测定葡萄糖的消耗量和糖酵解产物乳酸、丙酮酸；实时荧光定量PCR测定PPAR-γ, PPAR-α,及糖酵解相关基因烯醇酶1 (ENO1)、肝型6-磷酸果糖激酶(PFKL)、磷酸甘油酯激酶(PGK1)和丙酮酸激酶M2 (PKM2) mRNA表达的变化。结果：吡格列酮增加胰岛素抵抗的HepG2细胞葡萄糖消耗,诱导糖酵解相关基因PFKL、PGK1和PKM2表达显著上调,糖酵解产物乳酸和丙酮酸生成增多。WY14643部分改善HepG2细胞胰岛素抵抗,对糖酵解相关基因表达及糖酵解产物生成没有显著影响。吡格列酮或WY 14643对PPAR-γ或PPAR-amRNA的表达没有影响。结论：PPAR-γ激动剂通过促进糖酵解改善HepG2细胞的胰岛素抵抗。更多还原

【Abstract】 Objectives:To explore the effects of different timings and intervention methods on the preservation of pancreaticβ-cells in db/db mice. To make comparison of effects ofβ-cell preservation between interventions on prediabetes and newly onset diabetes or cross-sectional comparison between different interventions.Methods:1. Ninety-eight male,3-week old db/db mice which were confirmed as euglycemia were randomly assigned into two intervention model, early and earlier intervention. The interventions started at age 4 week referred to earlier intervention (treatments starting from age 4 week, T4), whereas interventions started at age 6 week referred to early intervention (treatments starting from age 6 week, T6). The mice fed with normal chow set as control group. Four groups of mice received liraglutide (300μg/kg wt bid), pioglitazone (0.02%PIO in mice chow), calories restriction (70%of food available of control) and exercise treatment respectively. Glycosylated hemoglobin (HbAlc) were determined before and after interventions. Fasting blood glucose (FBG), body weight and 24h food intake were monitored weekly. All interventions ended when the mice were 12-week old and introperitoneai glucose tolerance lest (IPGTT) or insulin tolerance test (ITT) was performed before mice were sacrificed. Plasma TG, FFA, insulin, adiponectin, and glucagon were determined. Tissue slides from paraffin embedded pancreas were stained by HE or antibodies for insulin, glucagon, Brdu (for determiningβ-cell proliferation rate), PDX-1(a factor essential for insulin gene expression) and IRS-2 (a protein involved in insulin signal pathway ofβ-cells). Apoptosis of pancreaticβ-cells were detected by TUNEL assay. Expression of GRP78 and CHOP were quantified by real-time quantitative PCR and western blot for assessing endoplasmic reticulum stress (ERS). Gene expression of XBP-1/spliced XBP-1 was detected by PCR and electrophoresis. Expression of microRNA miR-34a and miR-375 were quantified by real-time quantitative PCR.2. Thirty five 8-week old male db/db mice were randomly assigned into 4 groups:control group, PIO group (0.02%PIO in mice chow), LIRA group (liraglutide 300μg/kg wt), and combined treatment group (liraglutide 300μg/kg wt+0.02%PIO in mice chow). The effects of combined interventions on glucose, lipid metabolism and pancreaticβ-cell preservation were assessed after 4 weeks intervention as described above. Results:1.1) HbA1c of mice (12 week of age) treated at 4 week of age were lower than mice treated at 6 week of age (control:7.3±0.3%, T4:4.9±0.8%, T6:5.5±0.9%, P<0.001 vs. control, P<0.001 vs. T6). AUCglu after IPGTT showed similar changes as HbA1c (control:4568±190 mmol/l*mi, T4:2306±727 mmol/l*mi, T6:3559±903 mmol/l*min, P<0.001 vs. control, P<0.001 vs. T6). There were differences among treatments in HbAlc:(LIRA4.9±0.5%, PIO 4.3±0.4%, CR 5.4±0.7%, EX 6.1±0.6%, P<0.001) and in AUCglu (LIRA:2558±639 mmol/l*min, PIO:1662±483 mmol/l*min, CR:3262±613 mmol/l*min; EX:3904±610 mmol/l*min, P<0.001).2) Fasting plasma insulin level were higher in treated groups (control:5.7±2.5ng/ml,T4 11.0±3.0ng/ml, T6: 9.1±4.6ng/ml P<0.01 vs. control, p<0.05 vs. T6). Regarding to various interventions, although fasting insulin levels in all intervention groups were slightly higher than control group, only a significant difference was found in mice with liraglutide intervention (P=0.001). Earlier intervention showed better preservation of glucose-stimulated insulin secretion assessed by AUCins by IPGTT (control:799±186 ng/ml*min, T4:1410±595 ng/ml*min, T6:1216±347 ng/ml*min, P<0.001 vs. control, P<0.05 vs. T6).3) The levels of plasma TG, FFA and glucagon were reduced by different magnitude with various interventions, while no differences were observed between time points that treatments started. Only pioglitazone increased adiponectin levels among interventions (9.5±1.6 vs.15.3±3.9mg/l, P<0.05).4) At 12 week of age, db/db mice without intervention showed hypertrophic islet with fewer and weaker insulin staining cells, and abnormally distributedα-cells. Interventions that started at 4 week of age had a better effect on the preservation of pancreaticβ-cells (Insulin stained area:control 27.0±1.5%, T4 50.8±6.4%, T6 44.5±8.1%, P<0.001). In PIO-treated mice, islets were smaller with well-stainedβ-cells and nearly normal distribution of a-cells; LIRA and CR failed to prevent islet from hypertrophy, but they preservedβ-cell mass and alleviated degranulation; EX only had modestly effects. Apoptosis were reduced with interventions except for EX-treated mice, in which LIRA shown the better effect. Increase of pancreaticβ-cell proliferation was observed in LIRA and CR groups.5) LIRA preserved PDX-1 expression more while PIO increased IRS-2 expression in islets.6) LIRA and PIO inhibited expression of pancreatic ERS markers GRP78 and CHOP.7) LIRA reduced pancreatic expression of miR-375 by 71% and miR-34a by 50%(P<0.05).(2) After 4 weeks, HbA1cwere decreased under all treatments (control:7.3±0.4, PIO:4.9±0.6, LIRA:5.5±0.4, combination:4.5±0.6) and combination therapy did better than PIO or LIRA alone. PIO in combination with LIRA improved glucose tolerance (reduction of area under curve of glucose by IPGTT of combination was 56%,P<0.001) and preserved insulin release response to glucose (augment of area under curve of insulin by IPGTT of combination was 91%, P<0.01), both of which were greater than either medicine alone. Combination treatment also reduced circulated FFA by 29%(PIO alone:22%; LIRA:no effect), TG by 49%(PIO alone:35%; LIRA alone:15%), and increased plasma adiponectin by 95%(PIO:80%; LIRA no effect) compared with control, more effectively than PIO or LIRA alone. Islet immunohistochemistry showed that insulin positive area were increased significantly by 1.7 folds of control (PIO:1.3 folds; LIRA:0.9 fold) and isletβ-cell proliferation rate were increased by 2 folds (PIO, no effect; LIRA:1.7 folds) in combination-treated group, which confirmed the greater preservation ofβ-cells by combination treatment of PIO and LIRA than either treatment alone.Conclusions:1. Earlier interventions when blood glucose is within normal range manifests greater effect on preservation of isletβ-cells than early interventions of type 2 diabetes.2. Liraglutide has dirrect effect on preservation of isletβ-cells but only partly compensates severe insulin resistance.3. Combined therapy improves glucose and lipid metabolism, preserves islet beta-cell function and stimulates beta-cell proliferation, which are greater than either liraglutide or pioglitazone treatment alone. Objectives.-To examine the protective effect of genipin from palmitate-induced cytotoxicity in HepG2 cells and investigate the underlying mechanism.Methods:HepG2 cells were treated respectively with bovine serum albumin (BSA), palmitate (lmmol/1), genipin(20μmol/l) or genipin+palmitate for 24h, the cell viability was assayed by Methyl thiazol tetrazolium (MTT) method. Lactate dehydrogenase enzyme (LDH) release was measured to assess cell damage. Flowcytometry (Annexin V-PI) and Hoechst staining were employed for determination of cell apoptosis after 16h-treatment. ERS markers GRP78 and CHOP mRNA expression were quantified by Real time PCR while XBP-1 splicing was showed by PCR and electrophoresis.Results:Compared with BSA, palmitate decreased cell viability while increased LDH release (P<0.05). It also significantly induced apoptosis of HepG2 cell (P<0.05). Expression of GRP78 and CHOP mRNA was up-regulated by palmitate (P<0.05), so was XBP-1 splicing. Compared with palmitate, genipin pretreatment increased cell viability (P<0.05), inhibited apoptosis (P<0.01), and reduced LDH release (P<0.01) of HepG2 cell. The expression of GRP78 and CHOP mRNA was decreased by genipin (P<0.01). Electrophoresis of XBP-1 PCR products showed less spliced XBP-1 in genipin pretreated cells than palmitate treated ones.Conclusions:The results suggest that genipin protects HepG2 cells from palmitate-induced cell apoptosis and death mediated by ER stress. Objectives:This study is aimed at characterizing the role of peroxisome proliferator activated receptors (PPAR) y or a in glucose metabolism and insulin resistance of human hepatoma cells.Methods:The model of insulin resistance was established with HepG2 cells cultured at high concentrations of palmitate. Insulin resistant HepG2 cells were treated with the PPAR-y agonist pioglitazone or the PPAR-a agonist WY14643. Quantification of glucose consumption and glycolysis products pyruvate and lactate were performed. Quantitative RT-PCR was employed to analyze mRNA expression of PPAR-γ/a and glycolysis related genes.Results:Palmitate treatment decreased glucose consumption of HepG2 cells. Pioglitazone increased glucose consumption of both normal and insulin resistant HepG2 cells. It induced an up-regulation of glycolysis gene expression and strongly increased glycolysis leading to an elevated pyruvate and lactate production. WY14643 slightly increased glucose consumption of insulin resistant HepG2 cells, but had no effects on glycolysis gene expression.Conclusions:Altogether these results show that PPAR-y or PPAR-a can enhance glucose metabolism in HepG2 cells through different mechanisms.更多还原