TRB3在游离脂肪酸诱导胰岛β细胞凋亡中的作用及机制研究
摘要
研究背景
在世界范围内,糖尿病已成为继心脑血管疾病、癌症之后危害人类健康的第三大非传染性疾病,糖尿病已成为严重威胁人类健康的世界性公共卫生问题。近十年来,我国人口糖尿病发病率激增,到2010年,中国成年人糖尿病发病率已达11.6%。糖尿病引起的糖代谢紊乱导致一系列并发症,严重影响患者的生命和健康。因此,阐明糖尿病的发病机制以开发有效的防治方法成为当前医学研究的热点。
糖尿病患者中90%以上是以胰岛素抵抗和胰岛β细胞衰竭为特征的2型糖尿病。其中胰岛p细胞凋亡导致胰岛p细胞数量减少是2型糖尿病发展的关键环节。因此,如何保护和防止胰岛p细胞功能衰竭和细胞凋亡是目前2型糖尿病的防治的关键之一。p细胞凋亡的机制与脂毒性密切相关,2型糖尿病患者中约有80-90%属于肥胖或超重,其体内脂肪组织分解产生的游离脂肪酸(free fatty acids, FFA)显著增加,大量FFA损害胰岛β细胞功能,加速和促进p细胞凋亡。脂毒性致使胰岛p细胞凋亡的具体分子机制还不是十分清楚。针对这一过程相关分子的干预可能遏制或延缓β细胞的凋亡,从而有助于延缓2型糖尿病的发生与发展。
Tribbles同源蛋白家族(TRBs)成员TRB3是一种应激相关蛋白,通过与胰岛素信号通路中的重要分子Akt结合,作为内源性Akt抑制剂,阻断其磷酸化并干扰Akt向细胞膜的转位,影响Akt的功能,从而抑制胰岛素信号细胞内的传递途径,导致外周组织胰岛素抵抗。目前研究显示TRB3在不同的细胞中细胞具有促进或抑制细胞凋亡的作用,存在明显争议。由于该分子不仅参与调控营养物质代谢,而且介导应激反应,推测其可能与胰岛β细胞的脂毒性相关。TRB3是否参与脂毒性情况下的胰岛p细胞凋亡,国内外还未见报道。本研究利用基因过表达和基因敲降技术,在体外细胞模型和体内胰岛β细胞移植瘤模型中首次证明了TRB3在脂毒性诱导胰岛p细胞凋亡中的作用,并对其介导β细胞凋亡相关分子机制进行了进一步的研究。
研究目的
1.验证游离脂肪酸对胰岛β细胞TRB3表达的调节作用,并探讨其可能的分子机制；
2.研究TRB3与脂毒性导致的胰岛β细胞凋亡的关系,并探讨TRB3介导脂毒性所致的β细胞凋亡的分子机制。
研究方法
第一部分游离脂肪酸对胰岛β细胞TRB3表达的调节作用
体内实验
1.C57BL/6小鼠连续7天腹腔注射高浓度游离脂肪酸(Palmitate, PA),采集小鼠血清并检测血清游离脂肪酸浓度,分离纯化小鼠胰岛,采用Caspase-Glo3/7蛋白酶活性试剂盒检测小鼠胰岛凋亡情况。
2.应用Real-time PCR和Western blot检测小鼠胰岛TRB3及内质网应激标志物CHOP和ATF4mRNA及蛋白的表达情况。
体外实验
1.构建胰岛β细胞脂性凋亡模型,应用TUNEL染色法检测游离脂肪酸对INS-1细胞凋亡的影响：为观察不同浓度PA刺激对INS-1细胞凋亡的影响,我们分别给予0,0.2,0.4及0.8mM的PA孵育INS-1细胞24小时；为观察不同作用时间PA刺激对INS-1细胞凋亡的影响,采用0.2mM的PA孵育INS-1细胞0,12,24,48小时。
2.应用Real-time PCR和Western blot检测游离脂肪酸诱导的TRB3与内质网应激标志物CHOP和ATF4的mRNA及蛋白表达水平的关系。
3.应用siRNA干扰技术敲降INS-1细胞的CHOP基因,应用TUNEL染色检测CHOP基因沉默对游离脂肪酸致INS-1细胞凋亡的影响,应用Western blot检测敲降CHOP对INS-1细胞TRB3表达水平的影响。
第二部分TRB3参与调控游离脂肪酸诱导的胰岛β细胞凋亡
体外实验：
1.利用Tet-on系统构建可诱导性表达TRB3的胰岛β细胞系,采用Real-time PCR Western blot和细胞免疫荧光染色验证所构建的β细胞TRB3基因的可诱导性。
2.应用Caspase-3活性检测、TUNEL染色及流式细胞仪检测AnnexinV-FITC/PI双染分析TRB3过表达对游离脂肪酸诱导的β细胞凋亡的影响,
3.应用RNA干扰技术在INS-1细胞及TRB3细胞中敲降TRB3,并应用TUNEL染色检测TRB3基因沉默对游离脂肪酸致β细胞凋亡的影响。
4.应用Western blot检测TRB3过表达或敲降对AKT、PKCδ的磷酸化水平及PKCδ核转位的影响,并采用TUNEL染色检测PKCδ抑制剂或阻断PKCδ核转位对TRB3过表达所致p细胞凋亡的影响。
体内实验：
1.建立肾包膜下β细胞移植瘤动物模型：首先,用STZ处理免疫缺陷小鼠,建立糖尿病模型。待血糖显著升高后,将TRB3细胞移植入模型动物肾包膜下。当血糖开始降低后按照实验分组分别给予PA/Dox腹腔注射8天处理。在实验终点分别留取小鼠血清检测各组小鼠血胰岛素水平。
2.采用免疫组织化学染色观察移植瘤中TRB3表达差异,并用TUNEL染色比较移植瘤中β细胞的凋亡差异。
3.用PKCδ抑制剂rottlerin验证在体内PKCδ是否参与TRB3调控的胰岛β细胞脂性凋亡：β细胞移植瘤模型小鼠在给予PA和/或Dox之前给予rottlerin预处理,用TUNEL染色分析比较PKCδ抑制剂对移植瘤中β细胞凋亡的影响。
结果
第一部分游离脂肪酸对胰岛p细胞TRB3表达的调节作用
1.高浓度血清游离脂肪酸诱导小鼠胰岛细胞凋亡
连续7天腹腔注射PA后,小鼠血清游离脂肪酸(FFA)相较于对照组显著升高,Caspase3/7蛋白酶活性增加(P<0.01)。
2.游离脂肪酸诱导小鼠胰岛TRB3及内质网应激标志物表达上调
Real-time PCR结果显示游离脂肪酸处理组小鼠TRB3及CHOP、ATF4mRNA表达水平相较于对照组均显著增加(P<0.01)；Western blot检测结果显示TRB3及CHOP蛋白表达上调。
3.游离脂肪酸诱导INS-1细胞凋亡呈现时间及浓度梯度依赖性
TUNEL染色结果显示游离脂肪酸诱导INS-1细胞凋亡具有明显的剂量依赖性及时间依赖性关系。
4.游离脂肪酸诱导INS-1细胞TRB3、CHOP、ATF4的表达上调Real-timePCR检测结果显示,PA孵育时间超过12小时后,TRB3、CHOP、ATF4的mRNA表达水平随诱导时间及PA浓度的增加而逐渐升高。同时,我们采用Western blot检测了TRB3及CHOP的蛋白表达水平,TRB3、CHOP的蛋白表达水平随诱导时间及PA浓度的增加而上调。
5.敲降CHOP基因可以抑制游离脂肪酸刺激引起的TRB3上调及p细胞凋亡
采用siRNA干扰技术沉默了CHOP基因的表达,Western blot结果显示siCHOP显著地抑制了由PA诱导的CHOP及TRB3蛋白的表达上调。TUNEL染色显示,敲降CHOP后,由游离脂肪酸诱导的细胞凋亡显著减轻。
第二部分TRB3参与调控游离脂肪酸诱导的胰岛p细胞凋亡
1.可诱导的TRB3细胞的建立鉴定
利用Tet-on系统成功构建可诱导表达TRB3的胰岛p细胞系。经Real-time PCR、 Western blot及细胞免疫荧光染色证实,该细胞系由强力霉素500ng/ml诱导48小时后,TRB3表达水平显著升高。
2.TRB3过表达引起胰岛β细胞凋亡并加重了游离脂肪酸引起的β细胞凋亡
Caspase-3活性检测、TUNEL染色及AnnexinV-FITC/PI双染结果显示,TRB3细胞经Dox诱导后,其细胞凋亡率增加。同时,TRB3的过表达加重了PA刺激引起的p细胞凋亡。
3.敲降TRB3基因可以明显抑制游离脂肪酸诱导的胰岛β细胞凋亡
采用siRNA干扰技术敲降INS-1细胞及TRB3细胞的TRB3基因,Caspase-3活性检测及TUNEL染色结果显示敲降TRB3基因可以明显减少游离脂肪酸诱导的胰岛p细胞凋亡。
4.TRB3通过PKC8通路介导了游离脂肪酸诱导的β细胞凋亡
伴随着TRB3的过表达,PKCδ磷酸化水平增加其核转位显著增加,而敲降TRB3后,PKCδ活性及核蓄积降低。用PKCδ抑制剂或阻断PKCδ的核转位都显著地降低了过表达TRB3引起的细胞凋亡,而对TRB3的表达水平无显著影响。由此,我们认为TRB3通过PKCδ通路介导了游离脂肪酸诱导的β细胞凋亡。
5.在体内,TRB3通过PKCδ通路介导了游离脂肪酸诱导的β细胞凋亡
建立免疫缺陷糖尿病小鼠肾包膜下胰岛β细胞移植瘤模型,在该模型上证实,体内TRB3过表达诱导了胰岛β细胞凋亡,同时TRB3过表达加重了胰岛β细胞的脂性凋亡。应用该动物模型,证实PKCδ特异性抑制剂1ottlerin能够显著降低由TRB3过表达引起的细胞凋亡。
结论
1.游离脂肪酸通过CHOP/ATF4途径诱导胰岛β细胞中TRB3上调表达。
2.TRB3通过PKCδ通路参与调控游离脂肪酸诱导的胰岛β细胞凋亡。
研究意义
本研究首次阐明TRB3介导了脂毒性导致的胰岛β细胞凋亡。我们发现在脂毒性存在的情况下,游离脂肪酸通过内质网应激而上调TRB3的表达。上调的TRB3通过促进PKCδ的活化及核转位导致了胰岛β细胞的凋亡。而抑制TRB3/PKCδ通路可以明显缓解游离脂肪酸引起的胰岛β细胞凋亡。本研究为阐明2型糖尿病胰岛β细胞凋亡的分子机制提供了线索和实验依据,为寻找保护胰岛β细胞的治疗提供了新的靶点。
Backgrounds:
Diabetes, as one of the most common noncommunicable chronic diseases, has become the leading cause of disease and mortality burden worldwide. In2010, the estimated prevalence of diabetes Chinese adults was11.6%. The prevalence of diabetes has increased significantly in the recent decade and is now becoming epidemic proportions in China. Therefore, the pathogenesis, prevention and treatment of diabetes has become the research hotspot. More than90%of patients with diabetes is type2diabetes. Type2diabetes is characterized by insulin resistance and pancreatic β-cell failure,while the latter is the central link of type2diabetes. Therefore, how to protect and prevent islet β-cell dysfunction and apoptosis is critical prevention of type2diabetes.
Epidemiological studies have shown that, about80to90percent patients with type2diabetes are obese or overweight. Accumulating evidence indicate that FFA are chronically elevated in obesity-linked T2DM. Numerous in vivo and in vitro studies have demonstrated the destructive effects of chronic exposure of FFA on β cell survival resulting in defective glucose-induced insulin secretion and β cell dysfunction and loss through apoptosis.
The mechanisms underlying FFAs-induced β cell apoptosis and dysfunction may be associated with oxidative stress, endoplasmic reticulumstress, ceramide pathway, peroxisome proliferator-activated receptor pathway, hydrogen peroxide produced by peroxisomes, G protein-coupled receptors and so on. Recently, researchers discovered a new series of molecules, which could play an important role in pancreatic β-cell apoptosis induced by FFA. These molecules could serve as targets for therapeutic intervention, which may deter or delay pancreatic β-cell lipoapoptosis. Although the overall picture is known, mechanisms of lipoapoptosis in β cells are still very fragmentary and the details remain to be further examined.
TRB3, the best studied member of the mammalian Tribbles family, coordinates crucial cellular processes, including adipocyte differentiation, lipid metabolism, regulation of collagen expression, and modulation of tumorigenesis. Several studies have described that TRB3promotes apoptosis, while others have revealed TRB3to possess an anti-apoptotic role. In diabetes mellitus, TRB3was involved in impairing insulin action in peripheral tissues by binding and inhibiting AKT/PKB phosphorylation. The potential significance of the regulation of TRB3function to FFAs-induced β cell apoptosis deserves further investigation.
Aims
1. Identify whether TRB3could be upregulated by the free fatty acids in vivo and in vitro; investigated the potential mechanism involved in the process.
2. Identify the importance of TRB3in lipotoxicity induced β cell apoptosis and investigate the relevant mechanisms underlying TRB3's activity in β cells.
Methords
Part I:TRB3expression is upregulated by free fatty acid
1. C57BL/6mice were injected intraperitoneally with or without palmitate for7days. Mice serum was separated the FFA concentrations were assayed following a modified version of the manufacturer's protocol by the nonesterified fatty acid assay kit. Then pancreatic islets were isolated. Apoptosis of islets was determined by Caspase3/7activity assays.
2. Quantitative real-time PCR and Western blot were utilized to test the mRNA and protein levels of TRB3、CHOP and ATF4in mice islets.
3. To investigate the time-course of apoptosis induced by palmitate, INS-1cells were cultured in the presence of0.2mM palmitate for0,12,24,48h. To study the trend of apoptosis with changing concentrations of palmitate, cells were cultured with0,0.2,0.4,0.8mM palmitate for24h. Apoptosis was assessed by TUNEL staining.
4. Quantitative real-time PCR and Western blot were utilized to test the mRNA and protein levels of TRB3、CHOP and ATF4.
5. The siRNA of CHOP or negative control were introduced into INS-1cells by Lipofectamine2000, then TUNEL staining was utilized to test the apoptotic effect. The effects of CHOP silencing onTRB3expression was detected by Western blot.
Part Ⅱ:TRB3is involved in free fatty acid-induced β cell apoptosis
1. Establishment of INS-1derived cell lines, allowing inducible expression of TRB3by Tet-on system. The inducible TRB3cells were cultured in RPMI1640medium with G418, hygromycin, and10%FCS. TRB3cells were induced with500ng/ml Dox for48h. The induction of TRB3expression in the beta cell line was confirmed by Quantitative real-time PCR and Western blot and immunofluorescence staining.
2. Caspase-3activity、TUNEL staining and Annexin V/PI staining were utilized to analysis the cell apoptosis in the normal and palmitate conditions with or without TRB3overexpression.
3. The siRNAs of TRB3or negative control were introduced into INS-1cells or TRB3cells by Lipofectamine2000, then TUNEL staining and Caspase-3activity were utilized to test the apoptotic effect. The effects of on TRB3knockdown was detected by Western blot.
4. TRB3cells were treated with or without500ng/ml Dox for48h to induce TRB3expression, and then incubated with or without0.2mM palmitate. The nuclear accumulation and activation of PKCδ was determined by western blot. TRB3cells were pretreated with U73122or rottlerin to block the activation or nuclear accumulation and then incubated with Dox followed by palmitate, cell apoptosis was assessed by TUNEL staining.
5. Establish subrenal capsular transplantation animal model:Firstly, nu/nu mice were irreversibly induced to be diabetic by intraperitoneal injection with a relatively high single dose of STZ. At day6, TRB3cells (5×106) were transplanted into the left kidney of STZ-treated diabetic mice to establish the subrenal capsular animal model. The fasting blood glucose began to decline significantly18days post-transplantation, and from then on, we injected STZ intraperitoneally once daily with or without palmitate and Dox for8days. All mice were sacrificed at day32. Sections of the kidneys were then subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry. The different effect on apoptosis between groups was tested by TUNEL staining.
6. Using the animal model described above, TUNEL staining was utilized assessed whether blocking PKC8activation by rottlerin could inhibit TRB3-mediated β cell apoptosis under high plasma FFAs conditions in vivo.
Results
PartⅠ
1. High level serum free fatty acid resulted in an increased cell apoptosis in mice islets.
We injected palmitate into mice intraperitoneally once daily for7days, and serum free fatty acid increased markedly. In addition, caspase-3/7activity in the isolated islets was increased in palmitate-injected mice (P<0.01).
2. Free fatty acid up regulated the expression of TRB3、CHOP、ATF4in islets.
Using quantitative real-time PCR, we found that TRB3、CHOP、ATF4mRNA in palmitate-injected mice were significantly increased. In addition, Western blot analysis revealed that TRB3and CHOP were upregulated in palmitate-injected mice.
3. FFA induced apoptosis in INS-1cells in duration-and dose-dependent manner.
TUNEL assay was used to investigate the time-course and dose-couse of apoptosis induced by palmitate, we found that palmitate induced apoptosis in INS-1cells in a duration-and dose-dependent manner.
4. TRB3, CHOP, ATF4were upregulated in INS-1cell by free fatty acid.
Results of real-time PCR showed that, mRNA levels of TRB3, CHOP and ATF4were increased as the duration and concentrations increased. Meanwhile, Western blot analysis conformed that TRB3and CHOP was upregulated by palmitate in INS-1cells in duration-and dose-dependent manner.
5. CHOP knockdown reduced the upregulation of TRB3and apoptotic effect induced by palmitate
According to the result of western blot, CHOP knockdown reduced the upregulation of TRB3and CHOP induced by palmitate. In addition, TUNEL assay showed that CHOP silencing offset the apoptotic effect of palmitate in INS-1cells.
Part II
1. The inducible TRB3cell line was established by Tet-on system.
In order to investigate the exact role of TRB3in palmitate-induced β cell apoptosis, we established a stable cell line capable of an inducible expression of TRB3by tet-on system, termed TRB3cells. According to the results of real-time PCR、Western blot and immunofluorescence analysis, induced with500ng/ml of doxycycline (Dox) for48h, TRB3expression was upregulated in TRB3cells.
2. Overexpression of TRB3induced apoptosis and exacerbated lipoapoptosis.
According to the results of Caspase-3activity analysis、TUNEL staining and AnnexinV/PI analysis, cell apoptosis was induced by TRB3overexpression and exacerbated by the addition of palmitate.
3. TRB3knockdown significantly inhibited palmitate-induced βcell apoptosis.
TUNEL staining showed that TRB3silencing largely offset the apoptotic effect of palmitate in TRB3cells and INS-1cells.
4. TRB3mediated FFAs-induced apoptosis via PKCδ pathway.
Accompanied with TRB3upregulation, PKC8was activated and accumulated in the nuclei, and these events were enhanced by the addition of palmitate. In contrast, TRB3silencing largely decreased the activity and nuclear accumulation. The inhibitor or preventing nuclear accumulation of PKC8reduced the apoptosis restricted to TRB3overexpression significantly, without altering the expression of TRB3. These results indicated that PKCδ is a novel and key downstream mediator of TRB3effects in lipoapoptosis.
5. TRB3mediated FFAs-induced INS-1-derived cell apoptosis in vivo.
Subrenal capsular transplantation animal model was used to validate our in vitro results. About2weeks post-transplantation, the fasting blood glucose of the mice began to decline gradually. Then, the mice were intraperitoneally injected once daily with Dox to induce TRB3overexpression and with palmitate to increase the plasma FFAs. After8days, the groups treated with Dox or palmitate exhibited increased blood glucose levels, which were further enhanced in the group treated with both Dox and palmitate. We assessed the apoptosis in TRB3cell xenografts by TUNEL staining for each treatment group. TRB3overexpression exacerbated FFAs-induced apoptosis.
6.Blocking PKCS activation prevented TRB3-mediated β cell apoptosis under high plasma FFA conditions in vivo.
We further assessed whether blocking PKCδ activation by rottlerin could inhibit TRB3-mediated β cell apoptosis under high plasma FFAs conditions in vivo. Injections of rottlerin inhibited PKCδ phosphorylation, which significantly prevented TRB3-mediated β cell apoptosis under high plasma FFA conditions. These results indicate that TRB3could activate PKCδ and mediate FFAs-induced INS-1-derived cell apoptosis in vivo.
Significance
These studies provide novel insights into the central role of TRB3in lipoapoptosis in pancreatic β cells. TRB3expression is upregulated by free fatty acid via CHOP/ATF4 pathway in pancreatic β cells. We demonstrated that TRB3expression may explain FFAs-induced (3cell apoptosis and implicated the PKCδ pathway in this process for the first time. Inhibition of the TRB3/PKCδ axis could be relevant for preservation of β cell mass and function in conditions associated with increased serum FFA levels. These findings can be incorporated into a new potential therapeutic approach for the treatment of T2DM.
在世界范围内,糖尿病已成为继心脑血管疾病、癌症之后危害人类健康的第三大非传染性疾病,糖尿病已成为严重威胁人类健康的世界性公共卫生问题。近十年来,我国人口糖尿病发病率激增,到2010年,中国成年人糖尿病发病率已达11.6%。糖尿病引起的糖代谢紊乱导致一系列并发症,严重影响患者的生命和健康。因此,阐明糖尿病的发病机制以开发有效的防治方法成为当前医学研究的热点。
糖尿病患者中90%以上是以胰岛素抵抗和胰岛β细胞衰竭为特征的2型糖尿病。其中胰岛p细胞凋亡导致胰岛p细胞数量减少是2型糖尿病发展的关键环节。因此,如何保护和防止胰岛p细胞功能衰竭和细胞凋亡是目前2型糖尿病的防治的关键之一。p细胞凋亡的机制与脂毒性密切相关,2型糖尿病患者中约有80-90%属于肥胖或超重,其体内脂肪组织分解产生的游离脂肪酸(free fatty acids, FFA)显著增加,大量FFA损害胰岛β细胞功能,加速和促进p细胞凋亡。脂毒性致使胰岛p细胞凋亡的具体分子机制还不是十分清楚。针对这一过程相关分子的干预可能遏制或延缓β细胞的凋亡,从而有助于延缓2型糖尿病的发生与发展。
Tribbles同源蛋白家族(TRBs)成员TRB3是一种应激相关蛋白,通过与胰岛素信号通路中的重要分子Akt结合,作为内源性Akt抑制剂,阻断其磷酸化并干扰Akt向细胞膜的转位,影响Akt的功能,从而抑制胰岛素信号细胞内的传递途径,导致外周组织胰岛素抵抗。目前研究显示TRB3在不同的细胞中细胞具有促进或抑制细胞凋亡的作用,存在明显争议。由于该分子不仅参与调控营养物质代谢,而且介导应激反应,推测其可能与胰岛β细胞的脂毒性相关。TRB3是否参与脂毒性情况下的胰岛p细胞凋亡,国内外还未见报道。本研究利用基因过表达和基因敲降技术,在体外细胞模型和体内胰岛β细胞移植瘤模型中首次证明了TRB3在脂毒性诱导胰岛p细胞凋亡中的作用,并对其介导β细胞凋亡相关分子机制进行了进一步的研究。
研究目的
1.验证游离脂肪酸对胰岛β细胞TRB3表达的调节作用,并探讨其可能的分子机制；
2.研究TRB3与脂毒性导致的胰岛β细胞凋亡的关系,并探讨TRB3介导脂毒性所致的β细胞凋亡的分子机制。
研究方法
第一部分游离脂肪酸对胰岛β细胞TRB3表达的调节作用
体内实验
1.C57BL/6小鼠连续7天腹腔注射高浓度游离脂肪酸(Palmitate, PA),采集小鼠血清并检测血清游离脂肪酸浓度,分离纯化小鼠胰岛,采用Caspase-Glo3/7蛋白酶活性试剂盒检测小鼠胰岛凋亡情况。
2.应用Real-time PCR和Western blot检测小鼠胰岛TRB3及内质网应激标志物CHOP和ATF4mRNA及蛋白的表达情况。
体外实验
1.构建胰岛β细胞脂性凋亡模型,应用TUNEL染色法检测游离脂肪酸对INS-1细胞凋亡的影响：为观察不同浓度PA刺激对INS-1细胞凋亡的影响,我们分别给予0,0.2,0.4及0.8mM的PA孵育INS-1细胞24小时；为观察不同作用时间PA刺激对INS-1细胞凋亡的影响,采用0.2mM的PA孵育INS-1细胞0,12,24,48小时。
2.应用Real-time PCR和Western blot检测游离脂肪酸诱导的TRB3与内质网应激标志物CHOP和ATF4的mRNA及蛋白表达水平的关系。
3.应用siRNA干扰技术敲降INS-1细胞的CHOP基因,应用TUNEL染色检测CHOP基因沉默对游离脂肪酸致INS-1细胞凋亡的影响,应用Western blot检测敲降CHOP对INS-1细胞TRB3表达水平的影响。
第二部分TRB3参与调控游离脂肪酸诱导的胰岛β细胞凋亡
体外实验：
1.利用Tet-on系统构建可诱导性表达TRB3的胰岛β细胞系,采用Real-time PCR Western blot和细胞免疫荧光染色验证所构建的β细胞TRB3基因的可诱导性。
2.应用Caspase-3活性检测、TUNEL染色及流式细胞仪检测AnnexinV-FITC/PI双染分析TRB3过表达对游离脂肪酸诱导的β细胞凋亡的影响,
3.应用RNA干扰技术在INS-1细胞及TRB3细胞中敲降TRB3,并应用TUNEL染色检测TRB3基因沉默对游离脂肪酸致β细胞凋亡的影响。
4.应用Western blot检测TRB3过表达或敲降对AKT、PKCδ的磷酸化水平及PKCδ核转位的影响,并采用TUNEL染色检测PKCδ抑制剂或阻断PKCδ核转位对TRB3过表达所致p细胞凋亡的影响。
体内实验：
1.建立肾包膜下β细胞移植瘤动物模型：首先,用STZ处理免疫缺陷小鼠,建立糖尿病模型。待血糖显著升高后,将TRB3细胞移植入模型动物肾包膜下。当血糖开始降低后按照实验分组分别给予PA/Dox腹腔注射8天处理。在实验终点分别留取小鼠血清检测各组小鼠血胰岛素水平。
2.采用免疫组织化学染色观察移植瘤中TRB3表达差异,并用TUNEL染色比较移植瘤中β细胞的凋亡差异。
3.用PKCδ抑制剂rottlerin验证在体内PKCδ是否参与TRB3调控的胰岛β细胞脂性凋亡：β细胞移植瘤模型小鼠在给予PA和/或Dox之前给予rottlerin预处理,用TUNEL染色分析比较PKCδ抑制剂对移植瘤中β细胞凋亡的影响。
结果
第一部分游离脂肪酸对胰岛p细胞TRB3表达的调节作用
1.高浓度血清游离脂肪酸诱导小鼠胰岛细胞凋亡
连续7天腹腔注射PA后,小鼠血清游离脂肪酸(FFA)相较于对照组显著升高,Caspase3/7蛋白酶活性增加(P<0.01)。
2.游离脂肪酸诱导小鼠胰岛TRB3及内质网应激标志物表达上调
Real-time PCR结果显示游离脂肪酸处理组小鼠TRB3及CHOP、ATF4mRNA表达水平相较于对照组均显著增加(P<0.01)；Western blot检测结果显示TRB3及CHOP蛋白表达上调。
3.游离脂肪酸诱导INS-1细胞凋亡呈现时间及浓度梯度依赖性
TUNEL染色结果显示游离脂肪酸诱导INS-1细胞凋亡具有明显的剂量依赖性及时间依赖性关系。
4.游离脂肪酸诱导INS-1细胞TRB3、CHOP、ATF4的表达上调Real-timePCR检测结果显示,PA孵育时间超过12小时后,TRB3、CHOP、ATF4的mRNA表达水平随诱导时间及PA浓度的增加而逐渐升高。同时,我们采用Western blot检测了TRB3及CHOP的蛋白表达水平,TRB3、CHOP的蛋白表达水平随诱导时间及PA浓度的增加而上调。
5.敲降CHOP基因可以抑制游离脂肪酸刺激引起的TRB3上调及p细胞凋亡
采用siRNA干扰技术沉默了CHOP基因的表达,Western blot结果显示siCHOP显著地抑制了由PA诱导的CHOP及TRB3蛋白的表达上调。TUNEL染色显示,敲降CHOP后,由游离脂肪酸诱导的细胞凋亡显著减轻。
第二部分TRB3参与调控游离脂肪酸诱导的胰岛p细胞凋亡
1.可诱导的TRB3细胞的建立鉴定
利用Tet-on系统成功构建可诱导表达TRB3的胰岛p细胞系。经Real-time PCR、 Western blot及细胞免疫荧光染色证实,该细胞系由强力霉素500ng/ml诱导48小时后,TRB3表达水平显著升高。
2.TRB3过表达引起胰岛β细胞凋亡并加重了游离脂肪酸引起的β细胞凋亡
Caspase-3活性检测、TUNEL染色及AnnexinV-FITC/PI双染结果显示,TRB3细胞经Dox诱导后,其细胞凋亡率增加。同时,TRB3的过表达加重了PA刺激引起的p细胞凋亡。
3.敲降TRB3基因可以明显抑制游离脂肪酸诱导的胰岛β细胞凋亡
采用siRNA干扰技术敲降INS-1细胞及TRB3细胞的TRB3基因,Caspase-3活性检测及TUNEL染色结果显示敲降TRB3基因可以明显减少游离脂肪酸诱导的胰岛p细胞凋亡。
4.TRB3通过PKC8通路介导了游离脂肪酸诱导的β细胞凋亡
伴随着TRB3的过表达,PKCδ磷酸化水平增加其核转位显著增加,而敲降TRB3后,PKCδ活性及核蓄积降低。用PKCδ抑制剂或阻断PKCδ的核转位都显著地降低了过表达TRB3引起的细胞凋亡,而对TRB3的表达水平无显著影响。由此,我们认为TRB3通过PKCδ通路介导了游离脂肪酸诱导的β细胞凋亡。
5.在体内,TRB3通过PKCδ通路介导了游离脂肪酸诱导的β细胞凋亡
建立免疫缺陷糖尿病小鼠肾包膜下胰岛β细胞移植瘤模型,在该模型上证实,体内TRB3过表达诱导了胰岛β细胞凋亡,同时TRB3过表达加重了胰岛β细胞的脂性凋亡。应用该动物模型,证实PKCδ特异性抑制剂1ottlerin能够显著降低由TRB3过表达引起的细胞凋亡。
结论
1.游离脂肪酸通过CHOP/ATF4途径诱导胰岛β细胞中TRB3上调表达。
2.TRB3通过PKCδ通路参与调控游离脂肪酸诱导的胰岛β细胞凋亡。
研究意义
本研究首次阐明TRB3介导了脂毒性导致的胰岛β细胞凋亡。我们发现在脂毒性存在的情况下,游离脂肪酸通过内质网应激而上调TRB3的表达。上调的TRB3通过促进PKCδ的活化及核转位导致了胰岛β细胞的凋亡。而抑制TRB3/PKCδ通路可以明显缓解游离脂肪酸引起的胰岛β细胞凋亡。本研究为阐明2型糖尿病胰岛β细胞凋亡的分子机制提供了线索和实验依据,为寻找保护胰岛β细胞的治疗提供了新的靶点。
Backgrounds:
Diabetes, as one of the most common noncommunicable chronic diseases, has become the leading cause of disease and mortality burden worldwide. In2010, the estimated prevalence of diabetes Chinese adults was11.6%. The prevalence of diabetes has increased significantly in the recent decade and is now becoming epidemic proportions in China. Therefore, the pathogenesis, prevention and treatment of diabetes has become the research hotspot. More than90%of patients with diabetes is type2diabetes. Type2diabetes is characterized by insulin resistance and pancreatic β-cell failure,while the latter is the central link of type2diabetes. Therefore, how to protect and prevent islet β-cell dysfunction and apoptosis is critical prevention of type2diabetes.
Epidemiological studies have shown that, about80to90percent patients with type2diabetes are obese or overweight. Accumulating evidence indicate that FFA are chronically elevated in obesity-linked T2DM. Numerous in vivo and in vitro studies have demonstrated the destructive effects of chronic exposure of FFA on β cell survival resulting in defective glucose-induced insulin secretion and β cell dysfunction and loss through apoptosis.
The mechanisms underlying FFAs-induced β cell apoptosis and dysfunction may be associated with oxidative stress, endoplasmic reticulumstress, ceramide pathway, peroxisome proliferator-activated receptor pathway, hydrogen peroxide produced by peroxisomes, G protein-coupled receptors and so on. Recently, researchers discovered a new series of molecules, which could play an important role in pancreatic β-cell apoptosis induced by FFA. These molecules could serve as targets for therapeutic intervention, which may deter or delay pancreatic β-cell lipoapoptosis. Although the overall picture is known, mechanisms of lipoapoptosis in β cells are still very fragmentary and the details remain to be further examined.
TRB3, the best studied member of the mammalian Tribbles family, coordinates crucial cellular processes, including adipocyte differentiation, lipid metabolism, regulation of collagen expression, and modulation of tumorigenesis. Several studies have described that TRB3promotes apoptosis, while others have revealed TRB3to possess an anti-apoptotic role. In diabetes mellitus, TRB3was involved in impairing insulin action in peripheral tissues by binding and inhibiting AKT/PKB phosphorylation. The potential significance of the regulation of TRB3function to FFAs-induced β cell apoptosis deserves further investigation.
Aims
1. Identify whether TRB3could be upregulated by the free fatty acids in vivo and in vitro; investigated the potential mechanism involved in the process.
2. Identify the importance of TRB3in lipotoxicity induced β cell apoptosis and investigate the relevant mechanisms underlying TRB3's activity in β cells.
Methords
Part I:TRB3expression is upregulated by free fatty acid
1. C57BL/6mice were injected intraperitoneally with or without palmitate for7days. Mice serum was separated the FFA concentrations were assayed following a modified version of the manufacturer's protocol by the nonesterified fatty acid assay kit. Then pancreatic islets were isolated. Apoptosis of islets was determined by Caspase3/7activity assays.
2. Quantitative real-time PCR and Western blot were utilized to test the mRNA and protein levels of TRB3、CHOP and ATF4in mice islets.
3. To investigate the time-course of apoptosis induced by palmitate, INS-1cells were cultured in the presence of0.2mM palmitate for0,12,24,48h. To study the trend of apoptosis with changing concentrations of palmitate, cells were cultured with0,0.2,0.4,0.8mM palmitate for24h. Apoptosis was assessed by TUNEL staining.
4. Quantitative real-time PCR and Western blot were utilized to test the mRNA and protein levels of TRB3、CHOP and ATF4.
5. The siRNA of CHOP or negative control were introduced into INS-1cells by Lipofectamine2000, then TUNEL staining was utilized to test the apoptotic effect. The effects of CHOP silencing onTRB3expression was detected by Western blot.
Part Ⅱ:TRB3is involved in free fatty acid-induced β cell apoptosis
1. Establishment of INS-1derived cell lines, allowing inducible expression of TRB3by Tet-on system. The inducible TRB3cells were cultured in RPMI1640medium with G418, hygromycin, and10%FCS. TRB3cells were induced with500ng/ml Dox for48h. The induction of TRB3expression in the beta cell line was confirmed by Quantitative real-time PCR and Western blot and immunofluorescence staining.
2. Caspase-3activity、TUNEL staining and Annexin V/PI staining were utilized to analysis the cell apoptosis in the normal and palmitate conditions with or without TRB3overexpression.
3. The siRNAs of TRB3or negative control were introduced into INS-1cells or TRB3cells by Lipofectamine2000, then TUNEL staining and Caspase-3activity were utilized to test the apoptotic effect. The effects of on TRB3knockdown was detected by Western blot.
4. TRB3cells were treated with or without500ng/ml Dox for48h to induce TRB3expression, and then incubated with or without0.2mM palmitate. The nuclear accumulation and activation of PKCδ was determined by western blot. TRB3cells were pretreated with U73122or rottlerin to block the activation or nuclear accumulation and then incubated with Dox followed by palmitate, cell apoptosis was assessed by TUNEL staining.
5. Establish subrenal capsular transplantation animal model:Firstly, nu/nu mice were irreversibly induced to be diabetic by intraperitoneal injection with a relatively high single dose of STZ. At day6, TRB3cells (5×106) were transplanted into the left kidney of STZ-treated diabetic mice to establish the subrenal capsular animal model. The fasting blood glucose began to decline significantly18days post-transplantation, and from then on, we injected STZ intraperitoneally once daily with or without palmitate and Dox for8days. All mice were sacrificed at day32. Sections of the kidneys were then subjected to hematoxylin and eosin (H&E) staining and immunohistochemistry. The different effect on apoptosis between groups was tested by TUNEL staining.
6. Using the animal model described above, TUNEL staining was utilized assessed whether blocking PKC8activation by rottlerin could inhibit TRB3-mediated β cell apoptosis under high plasma FFAs conditions in vivo.
Results
PartⅠ
1. High level serum free fatty acid resulted in an increased cell apoptosis in mice islets.
We injected palmitate into mice intraperitoneally once daily for7days, and serum free fatty acid increased markedly. In addition, caspase-3/7activity in the isolated islets was increased in palmitate-injected mice (P<0.01).
2. Free fatty acid up regulated the expression of TRB3、CHOP、ATF4in islets.
Using quantitative real-time PCR, we found that TRB3、CHOP、ATF4mRNA in palmitate-injected mice were significantly increased. In addition, Western blot analysis revealed that TRB3and CHOP were upregulated in palmitate-injected mice.
3. FFA induced apoptosis in INS-1cells in duration-and dose-dependent manner.
TUNEL assay was used to investigate the time-course and dose-couse of apoptosis induced by palmitate, we found that palmitate induced apoptosis in INS-1cells in a duration-and dose-dependent manner.
4. TRB3, CHOP, ATF4were upregulated in INS-1cell by free fatty acid.
Results of real-time PCR showed that, mRNA levels of TRB3, CHOP and ATF4were increased as the duration and concentrations increased. Meanwhile, Western blot analysis conformed that TRB3and CHOP was upregulated by palmitate in INS-1cells in duration-and dose-dependent manner.
5. CHOP knockdown reduced the upregulation of TRB3and apoptotic effect induced by palmitate
According to the result of western blot, CHOP knockdown reduced the upregulation of TRB3and CHOP induced by palmitate. In addition, TUNEL assay showed that CHOP silencing offset the apoptotic effect of palmitate in INS-1cells.
Part II
1. The inducible TRB3cell line was established by Tet-on system.
In order to investigate the exact role of TRB3in palmitate-induced β cell apoptosis, we established a stable cell line capable of an inducible expression of TRB3by tet-on system, termed TRB3cells. According to the results of real-time PCR、Western blot and immunofluorescence analysis, induced with500ng/ml of doxycycline (Dox) for48h, TRB3expression was upregulated in TRB3cells.
2. Overexpression of TRB3induced apoptosis and exacerbated lipoapoptosis.
According to the results of Caspase-3activity analysis、TUNEL staining and AnnexinV/PI analysis, cell apoptosis was induced by TRB3overexpression and exacerbated by the addition of palmitate.
3. TRB3knockdown significantly inhibited palmitate-induced βcell apoptosis.
TUNEL staining showed that TRB3silencing largely offset the apoptotic effect of palmitate in TRB3cells and INS-1cells.
4. TRB3mediated FFAs-induced apoptosis via PKCδ pathway.
Accompanied with TRB3upregulation, PKC8was activated and accumulated in the nuclei, and these events were enhanced by the addition of palmitate. In contrast, TRB3silencing largely decreased the activity and nuclear accumulation. The inhibitor or preventing nuclear accumulation of PKC8reduced the apoptosis restricted to TRB3overexpression significantly, without altering the expression of TRB3. These results indicated that PKCδ is a novel and key downstream mediator of TRB3effects in lipoapoptosis.
5. TRB3mediated FFAs-induced INS-1-derived cell apoptosis in vivo.
Subrenal capsular transplantation animal model was used to validate our in vitro results. About2weeks post-transplantation, the fasting blood glucose of the mice began to decline gradually. Then, the mice were intraperitoneally injected once daily with Dox to induce TRB3overexpression and with palmitate to increase the plasma FFAs. After8days, the groups treated with Dox or palmitate exhibited increased blood glucose levels, which were further enhanced in the group treated with both Dox and palmitate. We assessed the apoptosis in TRB3cell xenografts by TUNEL staining for each treatment group. TRB3overexpression exacerbated FFAs-induced apoptosis.
6.Blocking PKCS activation prevented TRB3-mediated β cell apoptosis under high plasma FFA conditions in vivo.
We further assessed whether blocking PKCδ activation by rottlerin could inhibit TRB3-mediated β cell apoptosis under high plasma FFAs conditions in vivo. Injections of rottlerin inhibited PKCδ phosphorylation, which significantly prevented TRB3-mediated β cell apoptosis under high plasma FFA conditions. These results indicate that TRB3could activate PKCδ and mediate FFAs-induced INS-1-derived cell apoptosis in vivo.
Significance
These studies provide novel insights into the central role of TRB3in lipoapoptosis in pancreatic β cells. TRB3expression is upregulated by free fatty acid via CHOP/ATF4 pathway in pancreatic β cells. We demonstrated that TRB3expression may explain FFAs-induced (3cell apoptosis and implicated the PKCδ pathway in this process for the first time. Inhibition of the TRB3/PKCδ axis could be relevant for preservation of β cell mass and function in conditions associated with increased serum FFA levels. These findings can be incorporated into a new potential therapeutic approach for the treatment of T2DM.
引文
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