【作者】 王威；

【导师】 刘国良；

【作者基本信息】 中国医科大学 ， 内分泌与代谢病学， 2010， 博士

【摘要】 前言脂毒性目前被认为是2型糖尿病的主要发病机制之一。生理条件下,一定的糖和脂对于胰岛功能的维持是必不可少的。但是,慢性持续性的高糖和／或脂信号干预胰岛β细胞却可以引起胰岛素基因表达受损,胰岛素合成、分泌减少及p细胞凋亡。一系列的研究都证实了脂肪酸在高糖环境下可以诱导胰岛p细胞凋亡。丝氨酸-苏氨酸激酶Akt,也称作蛋白激酶B(PKB),在调节胰岛β细胞功能和结构方面扮演重要的角色。不断有证据显示,PKB蛋白激酶通过磷酸化激活后可以保护胰岛β细胞抵抗糖脂毒性的毒副作用。对于胰岛p细胞来说,PKB不仅是生长因子信号、细胞因子及其它细胞刺激的关键的下游蛋白,也是胰岛素信号系统的关键靶点。Ghrelin主要是由胃底X／A样细胞分泌的,由28个氨基酸组成的生物性多肽,在第3位丝氨酸上带有辛酰化基团。作为生长激素促泌剂受体的内源性配体,它能够促进生长激素的分泌和调节食物摄取。Ghrelin的生物作用广泛,其中包括调节能量和糖代谢稳态,调节多种正常和肿瘤细胞株的增殖、凋亡和分化等功能。近年来,Ghrelin和胰岛β细胞之间的关系越来越受到关注。有学者发现在胰岛边缘可以检测到单纯分泌ghrelin的ε细胞,提示ghrelin不仅可以通过内分泌还可能通过旁分泌途径作用于胰岛β细胞。不断有证据指出,Ghrelin对胰岛细胞增殖、凋亡和功能的调节作用是通过生长激素促泌剂受体1a依赖或非依赖的机制介导的。Ghrelin能够促进胰岛β细胞的增殖、抑制干扰素y(IFN-y)联合肿瘤坏死因子a(TNF-a)诱导的或用阿霉素诱导的胰岛β细胞株的凋亡。PI3K/PKB信号通路目前被认为是介导Ghrelin在1型糖尿病中发挥保护作用的主要通路。而对于2型糖尿病的主要机制之一,脂毒性诱导的胰岛β细胞凋亡,Ghrelin能否通过相近的信号通路发挥保护胰岛β细胞的作用,有待于进一步的研究。因此,本研究的目的主要就是探讨ghrelin能否保护胰岛β细胞拮抗脂毒性的毒副作用以及探索相关的机制。材料与方法一、实验材料胰岛p细胞株MIN6第10～30代；胃饥饿素(Ghrelin),不含游离脂肪酸的牛血清白蛋白(BSA),棕榈酸(PA),Hoechst33258, MTT, PI3K/PKB阻断剂LY294002,JNK阻断剂SP600125；实验使用的蛋白抗体有PKB及其第473位丝氨酸磷酸化一抗,JNK1／2及其磷酸化一抗为兔多抗；TUNEL原位末端POD标记检测试剂盒；Caspase3活性检测试剂盒；细胞甘油三脂含量检测试剂盒；AnnexinV-PI双染凋亡检测试剂盒；PCR引物和RT-PCR检测试剂。二、实验方法1、细胞培养小鼠胰岛β细胞株-MIN6在高糖DMEM培养基和15%胎牛血清中于37℃、5%CO2条件下进行培养,培养基中加入100 U／mL青霉素,100μg/mL链霉素,100μg/mL L-谷氨酰胺,5μL／Lβ-巯基乙醇。细胞单层生长达80-85%培养面积后,用于实验或传代。2、细胞活性检测收集对数期细胞,调整细胞悬液浓度,铺96孔板使待测细胞调密度至5000细胞／孔,过夜贴壁后,进行实验干预。细胞活性检测用MTT法。实验干预后,每孔加入20μL MTT溶液,继续在5%C02,370C条件下孵育4h后,小心吸去孔内培养液。每孔加入150gL二甲基亚砜,置摇床上低速振荡10min,使结晶物充分溶解。在酶联免疫检测仪570nm波长处测量各孔的吸光值。3、Caspase-3活性检测收集实验各组细胞(3×106／组),PBS洗二次,10,000 rpm/min离心1 min,去除PBS,沉淀细胞中加入100μL冰冷裂解液和1μL DTT,混匀。将细胞裂解物移入96孔板,每空加入5μL Caspase-3反应底物(DEVD-pNA),37℃孵育2h。分光光度计在405 nm波长测定其吸光值。4、TUNEL法检测细胞凋亡凋亡检测方法参照TUNEL原位末端POD标记检测试剂盒说明书进行,细胞爬片长至80%覆盖率后,进行实验处理。加入1 mL的4%甲醛溶液,4℃固定细胞10 min,弃去固定液,PBS洗二次,加入TUNEL反应混合液进行染色和50μLonverter-horse-radish peroxidase(POD);最后加入DAB反应底物和苏木素进行复染。光学显微镜下计数细胞凋亡。凋亡的细胞核呈现棕褐色或黑色；正常的细胞核用苏木素复染成蓝核。凋亡率=(凋亡细胞／全部细胞)×100%。5、Hoechst33258染色细胞爬片放置在6孔板中,加入1 mL的4%甲醛溶液,4℃固定细胞10 min,弃去固定液,PBS洗二次,滴加100μL Hoechst 33258(1 Oug/mL)工作液,室温染色10 min,流水冲净,于340nm波长的荧光显微镜下观察并计数细胞凋亡。凋亡细胞核染色质聚集或核碎裂。凋亡率=(凋亡细胞／全部细胞)×100%6、AnnexinV-PI双染,流式细胞仪分析收集细胞,用PBS轻轻重悬细胞并计数。取5万重悬的细胞,1000g离心5min,弃上清,加入195μL Annexin V-FITC结合液轻轻重悬细胞。加入5μLAnnexin V-FITC,轻轻混匀。室温(20-25℃)避光孵育10min。1000g离心5min,弃上清,加入190μL Annexin V-FITC结合液轻轻重悬细胞。加入10μL碘化丙啶(PI)染色液,轻轻混匀,随即进行流式细胞仪检测,Annexin V-FITC为绿色荧光,PI为红色荧光。7、电镜MIN6细胞在250mL培养瓶中长至80%,进行实验处理后,用2.5%戊二醛固定后收集细胞,制作电镜标本,进行观察。8、特殊染色法测细胞内甘油三脂含量应用甘油三酯检测试剂盒(GPO-POD)测定MIN6细胞胞浆甘油三脂含量。收集细胞,PBS冲洗两遍后在超声下裂解。将10 uL细胞裂解物或标准品移入96孔板。每孔加入200 uL甘油激酶,70 uL磷酸甘油氧化酶和显影剂。酶标仪500nm波长下测定吸光度值。绘制标准曲线,通过标准曲线计算胞浆甘油三酯含量。9、细胞总RNA的提取及RT-PCR检测应用逆转录多聚酶链式反应(RT-PCR)半定量测定SREBP1c、CHOP、BAX、BCL-2和β-actin等基因的表达。提取细胞总RNA,用逆转录酶和hexamers合成cDNA。使用Primer5软件设计引物,送试剂公司合成。PCR扩增目的基因,在1.5%琼脂糖凝胶电泳分离形成目的条带,溴化乙锭染色成像；测定电泳条带密度值；扫描成像用Scion Image软件分析灰度值。10、Western blot(免疫印记)应用细胞裂解液提取细胞蛋白,测定蛋白浓度。制备10%聚丙烯酰胺凝胶,进行电泳,转膜,杂交。磷酸化PKB和JNK上样量为100ug,总PKB和JNK的上样量为50ug。ECL发光法测定目的条带,目的条带的灰度值用Scion Image软件分析。11、统计学分析应用SPSS 13.0软件进行单因素方差(ANOVA)统计分析,结果用均数±标准差(x±s)表示,组间差异比较采用Dunnett’s检验,P<0.05差异有显著性。实验结果一、脂毒性对胰岛p细胞的细胞活性、微观结构和诱导细胞凋亡的影响研究1、棕榈酸产生的脂毒性能够浓度依赖性地降低胰岛β细胞的细胞活性。2、棕榈酸的毒性使胰岛p细胞形态严重受损(电镜),核皱缩,染色质凝聚,内质网扩张,线粒体肿胀,胞浆中的分泌颗粒减少或消失。3、棕榈酸能够浓度依赖性地诱导胰岛p细胞凋亡。二、Ghrelin对胰岛p细胞脂性凋亡和甘油三酯沉积的影响1、应用MTT法检测细胞活性的结果显示,0.4mM棕榈酸干预胰岛β细胞24h明显降低细胞活性21%(p<0.05),而加用ghrelin具有浓度依赖性地拮抗棕榈酸诱导的脂毒性的功效,100nM ghrelin的作用最强,可以显著提高细胞活性达34%(p<0.01)。Caspase3活性测定的结果显示,0.4mM棕榈酸可显著增加Caspase3活性61%,而加用100nM ghrelin可降低Caspase3活性27%(p<0.05)。Hoechst33258染色法评估细胞凋亡可以看到,棕榈酸作用MIN6细胞24h使细胞凋亡率达到47%,而联用100nM ghrelin可显著降低凋亡率16%(p<0.01)。2、和棕榈酸(p<0.01)的作用相反,Ghrelin能够浓度依赖性的降低胰岛β细胞胞浆甘油三酯的含量。无论是在基础状态下还是脂毒性条件下,100nM具有降低胞浆甘油三酯的最大功效(p<0.05)。三、Ghrelin保护胰岛p细胞拮抗脂毒性的分子机制研究1、0.4 mM棕榈酸干预MIN6细胞24h明显抑制蛋白激酶B的磷酸化(p<0.01),加入100nM ghrelin能够快速诱导蛋白激酶B的激活,作用30min达到最大作用高峰(p<0.01)。Ghrelin浓度依赖性地促进蛋白激酶B的磷酸化激活,100nM的浓度产生最大的效力(p<0.01)。Ghrelin诱导的蛋白激酶B的激活能够显著地被PI3K阻断剂-LY294002所阻断(p<0.05)。而且,TUNEL法和Hoechst染色法检测凋亡证明,LY294002能够阻断ghrelin对胰岛β细胞脂性凋亡的保护作用。2、MIN6细胞用棕榈酸干预24h能够引起JNK磷酸化的激活(p<0.01),100nMghrelin能够在作用30min后引起JNK磷酸化显著意义地降低(p<0.05),而该降低的过程能够被PI3K阻断剂-LY294002所阻断(p<0.05)。JNK的阻断剂-SP600125,能够显著抑制棕榈酸诱导的凋亡。凋亡实验显示,联合应用SP600125和ghrelin能够产生最大保护MIN6细胞、防止凋亡的作用。3、同Caspase 3活性测定结果相一致,棕榈酸显著上调BAX、SREBP1c和CHOP-10的mRNA表达而下调BCL-2mRNA的表达；100nM ghrelin下调BAX、SREBP1c和CHOP-10的mRNA表达,但对BCL-2 mRNA的表达没有统计学意义上的影响。结论1、棕榈酸产生的脂毒性降低MIN6胰岛β细胞的细胞活性,引起细胞微观结构改变,浓度依赖性的诱导细胞凋亡。2、Ghrelin促进胰岛β细胞生长,提高细胞活性,抑制脂毒性诱导的细胞凋亡。3、Ghrelin保护MIN6胰岛β细胞株抵抗脂毒性和PI3K／PKB信号通路有关。4、Ghrelin减弱棕榈酸诱导的JNK磷酸化激活,JNK特异阻断剂-SP600125增强ghrelin保护MIN6胰岛β细胞株抗凋亡的作用。5、脂毒性条件下,Ghrelin的抗凋亡作用涉及线粒体途径。6、脂毒性条件下,Ghrelin减少胞浆内甘油三酯含量,抗脂毒性作用和内质网应激途径有关。更多还原

【Abstract】 Lipotoxicity plays an important role in underlying mechanism of type 2 diabetes. In principle, physiologic levels of glucose and lipids are not toxic but essential to normalβ-cell function. However, the prolonged exposure of pancreatic (3-cells to elevated levels of glucose or/and fat signal is associated with impairment of insulin gene expression,inhibition of insulin synthesis and secretion and induction ofβ-cell apoptosis.. A number of studies have shown that fatty acids can induceβ-cell apoptosis in the presence of high glucose. The serine/threonine kinase Akt, also known as protein kinase B (PKB), plays a vital role in regulating mass and function of pancreaticβ-cell. There are growing evidences that activation of PKB phosphorylation is able to prevent pancreatic P-cell from lipotoxicity. To pancreatic P-cells, PKB is not an unique central node in cell signaling downstream of growth factors and cytokines, but a key target of insulin signal system as well.Ghrelin is a 28-amino acid peptide acylated at the serine 3 position with an octanoyl group, and is mainly secreted from X/A like cells of gastric fungus, as a natural endogenous ligand of the orphan growth hormone secretagogue receptor type la (GHS-Rla), through which it acts as a growth hormone releasing peptide and food intake modulator. The effects of ghrelin are considered to be broadly including energy and glucose homeostasis, regulation of proliferation, apoptosis and differentiation of various normal and neoplastic cells lines, et.al.. Recently, the relationship between ghrelin and pancreaticβ-cells has attracted much attention. Some studies have showed ghrelin-secreting cells namedεcells could be detected in the verges of pancreatic islets, suggesting that ghrelin affects pancreaticβ-cells via both endocrine and paracrine pathway. It becomes increasingly clear that the effect of ghrelin on the regulation of pancreatic P-cells proliferation, apoptosis and function, are mediated by mechanisms dependent and independent of GHS-Rla. Ghrelin could promote cell proliferation and inhibit pancreaticβ-cells apoptosis induced by interferon-r/tumor necrosis factor-a (IFN-r /TNF-a) synergism, as well as doxorubicin-inducedβ-cells apoptosis. PI3K/PKB is supposed as the main signal pathway that mediated protective effect of ghrelin in type 1 diabetes. However, it remains to be elucidated whether ghrelin protects P cells against lipotoxicity-induced apoptosis in type 2 diabetes.Therefore, the aim of the present study is to investigate whether ghrelin preventsβ-cells from lipotoxicity and explore the mechanism behind.Materials and methodsMaterialsMIN6 cells, a widely used pancreaticβ-cell line(passages 11-30); Ghrelin, Fatty acid-free bovine serum albumin(BSA, fraction V), Palmitate, Hoechst33258, MTT, LY294002, SP600125; Antibodies used were anti-phospho-ser473-PKB, anti-total PKB, anti-phospho-JNK1/2, anti-total JNK; TUNEL (in situ cell death detection kit, POD) kit; Caspase-3 activity assay kit; TG GPO-POD assay kit; AnnexinV-PI apoptosis detection kit; The primer of PCR and the agent of demi-quantitate PT-PCR detection.Methods1、Cell cultureMIN6 cells, a widely used pancreaticβ-cell line(passages 11-30), were cultured in Dulbecco’s modified eagle’s medium (DMEM) containing 25 mM glucose, with 15% fetal bovine serum (FBS),100 U/mL penicillin,100μg/mL streptomycin,100μg/mL L-glutamine,5μL/Lβ-mercaptoethanol in humidified 5% CO2,95%air at 37℃. As grew to 80-85%, the cells were utilized for experiment or passage.2、Cell viability assay Cells were seeded on 96-well plates at a density of 5000 cells per well. For detection, cells were incubated with 5mg/mL MTT for approximately 4h under 5%CO2,37℃. The medium was removed and the formazan product was solubilized with 150μL dimethylsulfoxide. The plates were vibrated on swing bed for 10 min. Viability was assessed by spectrophotometry at 570nm absorbance using a 96-well plate reader.3、Caspase-3 activity assayBriefly, harvested cells were centrifuged at 10,000 rpm/min for 1 min after washed twice by PBS, followed by the addition of 1μL DTT and 100μL lysis buffer. Cell lysates in a 96 well microplate were incubated at 37℃with 5μL of Caspase-3 colorimetric substrate (DEVD-pNA) for 2 h. Absorbances were read by a microplate reader at 405 nm wavelength.4、TUNEL assayTUNEL staining was performed according to the manufacturer’s protocol with few modifications, Cell slides were fixed in 4% paraformaldehyde and stained with TUNEL reaction mixture and 50μL converter-horse-radish peroxidase (POD), followed by adding DAB substrate and hematoxylin. Apoptosis indexes were assessed by counting TUNEL positive cells (apoptotic nucleus was brown-stained or black-stained) through light microscope. Apoptosis ratio was TUNEL positive cells over the whole cells5、Hoechst33258 stainingHoechst staining was performed by fixing the cells in 4% paraformaldehyde for 10 min under 4℃and exposing the cell slides to lOug/mL Hoechst 33258 for 10 min at room temperature. Apoptosis indexes were assessed by counting Hoechst positive cells (chromatin condensation or fragmented nuclear membrane) through fluorescent microscope. Apoptosis ratio was Hoechst positive cells over the whole cells.6、AnnexinV-PI staining and Flow cytometry assayHarvested cells were mixed with PBS and counted number.5×104 cells were collected and centrifuged at 1000 r/min for 5 min, followed by adding 195μl Annexin V-FITC binding fluid and 5μl Annexin V-FITC. After recentrifuged at 1000 r/min for 5 min, the cells sample were mixed with 190μl Annexin V-FITC binding fluid and 10μl PI. Apoptosis index were detected and assessed by flow cytometry. Annexin V-FITC and PI respectively present green and red fluorescence.7、Electron microscope analysisAs grown to 80% in 250ml culture flask, the MIN6 cells were fixed with 2.5% Glutaral. Cells sample were assessed by electron microscope.8、Cytoplasmic triglyceride assayCytoplasmic TG was detected with a TG GPO-POD assay kit according to the manufacturer’s protocol. In brief, after two washes in PBS, cells were harvested,6×106 cells/mL were lysed by ultrasound, and 10-uL aliquots of cell lysates or standard samples were added to 96-well plates, followed by the addition of 200μL glycerokinase and 70μL glycerophosphate oxidase and developer. Absorbance at 500 nm was measured with a microplate reader. The TG concentration was calculated according to a standard curve.9、RNA isolation and Reverse-transcription polymerase chain reactionReverse-transcription polymerase chain reaction was performed to semi-quantify mRNA expression of SREBPlc, CHOP, BAX, BCL-2 andβ-actin gene.Total RNA was isolated and first strand cDNA synthesized using MMLV reverse transcriptase and hexamers from MIN6 cells. The primer sequences were devised by primer5 software.cDNA(9μL) was amplified by PCR in a 50μL volume with amplitaq gold polymerase and the PCR products were separated by 1.5% agarose gel electrophoresis and visualized by ethidiumbromide staining. The resulting images were analyzed by Scion Image software.10、Western blotBriefly, protein was extracted with a cell lysis buffer. Protein samples (100μg for p-PKB (ser473) and p-JNK1/2 or 50ug for total PKB, JNK) were separated by SDS-electrophoresis through either 10% gradient polyacrylamide gels and transferred to nitrocellulose membranes, followed by immunoblotting using all primary antibodies according to the manufacturer’s instructions. Immuno-detection was developed with ECL advance, and the resulting images were analyzed by Scion Image software. 11、Statistical analysisData are presented as means±SE. Statistical analyses were performed with SPSS using ANOVA. Differences between groups were assessed for significance by Dunnett’s test. A p value of less than 0.05 was considered significant.Results1.The effects of lipotoxicity on cells viabiltiy, microstructure and cells apoptosis in pancreaticβ-cells(1) Palmitate-induced lipotoxicity could concentration-dependently decrease cells viability of pancreaticβ-cells.(2)Lipotoxicity of palmitate damaged serevely cell morphous of pancreatic P-cells: nuclear crenation, chromatin condensation, distension of endoplasmic reticulum, engorgement of chondrosome, decreased or disappeared secretory granule in cytoplasm.(3) Palmitate concentration-dependently induced apoptosis of pancreatic P-cells.2.The effects of ghrelin on lipotoxicity-induced apoptosis and deposition of triglyceride in pancreaticβ-cells(1) Cell viability assessed by MTT was decreased by 21% (p<0.05) after 0.4mM palmitate (PA) treatment. Treatment with AG dose-dependently prevented PA-induced toxicity, most effectively by 34% at 100nM (p＜0.01). Cell apoptosis evaluated by caspase3 activity assay was significantly increased in PA group by 61%, while PA /AG (at 100nM) synergism decreased caspase 3 activity by 27% (p<0.05). Apoptosis evaluated by hoechst33258 staining showed, the percentage of apoptosis reached to 47% after PA treatment for 24 hours, while treatment with AG at 100nM reversed apoptosis rate by 16%(p＜0.01).(2)As opposed to the effects of palmitate (p＜0.01), ghrelin significantly decreased the cytoplasmic TG level in BSA-treated or palmitate-treated MIN6 cells in a concentration-dependent manner. A maximal effect was observed at 100nM(p＜0.05). 3.Molecular mechanism of ghrelin protective actions on pancreaticβ-cells against lipotoxicity(1)Exposure of the cells to 0.4mM palmitate for 24 hours caused significant inhibition of PKB phosphorylation (p<0.01), while ghrelin at 100nM induced rapid activation of PKB and reached the maximal effect at 30 min (p<0.01). Ghrelin dose-dependently stimulated activation of PKB phosphorylation and treatment at 100nM produced most effective potentiation(p<0.01). Ghrelin-induced activation of PKB was markedly blocked by PI3K inhibitor, LY294002(p<0.05).Moreover, LY294002 abolished ghrelin cytoprotective activity against palmitate-induced apoptosis,.which evaluated by TUNEL assay and hoechst33258 staining.(2)MIN6 cells were incubated with PA for 24 hours and produced activation of JNK phosphorylation (p<0.01), while ghrelin at 100nM resulted in a transient decrease at 30 min (p<0.05). ghrelin-induced inhibition of JNK phosphorylation was blocked by PI3K inhibitor, LY294002 (p<0.05). SP600125 alone can significantly prevent palmitate induced apoptosis. Combination of SP600125 with AG at 100nM could enhance ghrelin’s antiapoptotic effect in MIN6 cells.(3)Consistent with caspase 3 activity assay, palmitate significantly up-regulated BAX, SREBP1C, and CHOP-10 and down-regulatesd BCL-2. However, ghrelin at 100nM downregulated BAX, SREBP1C, and CHOP-10 mRNA expression,but did not affect BCL-2 mRNA expression.Conclusions1.Palmitate-produced lipotoxicity decreases cells viability, changes cells microstructure and induces concentration-dependently in MIN6 pancreaticβ-cells.2.Ghrelin promote pancreatic P-cells growth, increases cells viability and inhibits lipotoxicity-induced cell apoptosis.3.Ghrelin prevents MIN6 cells from apoptosis induced by lipotoxicity via PI3K/PKB pathway.4. Ghrelin attenuates activation of palmitate-induced JNK phosphorylation and JNK inhibitor, SP600125, enhances antiapoptotic effects of ghrelin in MIN6 cells.5. Ghrelin antiapoptosis effect involve mitochondrial pathways under lipotoxic state.6.Ghrelin decreases the cytoplasmic TG level and its antilipotoxicity is associated with endoplasmic reticulum stress pathways.更多还原