GLP-1对IL-1β诱导INS-1细胞损伤的保护作用及机制初探
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摘要
[背景]
随着世界经济的发展,人们生活水平的提高,全球人类健康面临的糖尿病威胁正日益增加,已成为严重威胁人类健康的世界性公共卫生问题,也给个人、家庭和社会带来了极大的经济负担。早期预防、早期干预无疑是解决这类问题的理想措施。
2型糖尿病(Type 2 diabetes mellitus,T2DM)患者占全部糖尿病患者的绝大多数,其发病机制复杂,至今未被完全阐明。T2DM的发病基础是胰岛素抵抗和胰岛素分泌缺陷,但谁起主导作用仍存争议。过去认为胰岛素抵抗占主导地位,近来,越来越多研究提示胰岛素分泌缺陷在T2DM的发生发展中起重要作用。前期研究提示高糖、高脂能破坏胰岛β细胞的分泌功能和减少胰岛β细胞量,所以与胰岛素分泌缺陷关系密切。
近期研究提示2型糖尿病是一种慢性低度炎症性疾病。作为炎症过程的重要调节因子,白细胞介素-1β(interleukin-1β,IL-1β)与2型糖尿病的关系日益引起重视。IL-1β不仅是由脂肪细胞、巨噬细胞、内皮细胞产生和分泌的,而且胰岛β细胞自身具有分泌IL-1β的功能。
IL-1β具有广泛的生物学效应,在介导β细胞损伤中起着重要作用。一方面IL-1β抑制胰岛素的合成与释放；另一方面IL-1β促进胰岛β细胞的死亡。IL-1β不但介导白细胞间的相互作用,还参与了其他细胞的调节,并与其他细胞因子相互影响,相互制约,构成一个复杂的细胞因子调节网络,进一步加重胰岛β细胞的损伤。
因此,在目前T2DM的治疗过程中,抑制IL-1β对胰岛β细胞的损伤作用对维持一定数量有功能的胰岛p细胞,延缓糖尿病的发生发展十分重要。
胰高血糖素样肽-1(GLP-1)是1983年在分析胰高血糖素的前体基因--胰高血糖素原(proglucagon, GP)基因序列时被发现的。该基因可在胰腺的α细胞、肠道的L细胞内表达。GLP-1由30个氨基酸组成,主要的生物活性型是GLP-1(7-36)。它的受体广泛分布于全身各个组织器官,被激活后产生多种生物学效应。
GLP-1受体激动剂对胰腺本身产生的生物学效应：一方面增加胰岛素的分泌和合成功能,另一方面可以增加胰岛β细胞量,主要表现为增加胰岛β细胞的增殖、增加胰岛β细胞的新生、抑制胰岛β细胞的凋亡
近期研究提示,GLP-1可以抑制高糖诱导的人胰岛β细胞和大鼠胰岛β细胞株INS832／13细胞的凋亡,其机制主要是GLP-1激活的PI-3K/PKB通路,诱导抗凋亡基因IAP-2(inhibitor of apoptosis protein-2)和BCL-2的表达增加。
T2DM患者胰岛β细胞内IL-1β的表达显著高于正常对照组,进一步研究发现高糖和IL-1β自身能增加胰岛内IL-1β的表达,且与NF-κB的激活有关,而IL-1受体拮抗剂(IL-1 receptor antagonist, IL-1Ra)能显著抑制IL-1β的表达。提示高糖和IL-1β自身可以刺激胰岛β细胞生成和分泌IL-1β。
研究表明,表儿茶精(epicatechin)能通过阻断NF-κB信号通路的激活,抑制IL-1β对胰岛β细胞系RINm5F和大鼠胰岛的细胞毒性。另外,通过包含IκB抑制剂的腺病毒转染β细胞系、大鼠及人的胰岛均能降低由IL-1β引起的细胞凋亡。IL-1β可以激活大鼠胰岛NF-κB信号通路,诱导COX-2和前列腺素受体3(EP3)基因的表达增加,导致胰岛β细胞的分泌功能下降,而水杨酸钠可以通过抑制IL-1β对NF-κB信号通路的激活,恢复胰岛β细胞的分泌功能。说明NF-κB通路的激活与IL-1β诱导的胰岛β细胞损伤关系密切。
前述提示,T2DM是一个慢性低度炎症性疾病,其胰岛内局部IL-1β表达增加,这可能与高糖和胰岛自身分泌的IL-1β诱导有关,而NF-κB通路的激活与IL-1β诱导的β细胞损伤关系密切。GLP-1能抑制高糖对胰岛p细胞的损伤作用,这是否与GLP-1抑制IL-1β对胰岛β细胞的损伤作用有关呢?其机制是否与抑制IL-1β诱导NF-κB的激活有关呢?目前这方面的研究较少。
本课题以大鼠胰岛细胞瘤系INS-1细胞为研究对象,观察GLP-1对IL-1β诱导INS-1细胞损伤的保护作用并探讨其部分保护机制。以期为GLP-1在2型糖尿病治疗中的应用提供更多的理论支持。
具体研究内容分为以下两个部分：
第一章GLP-1对IL-1β诱导INS-1细胞损伤的保护作用
[目的]
观察GLP-1对IL-1β诱导INS-1细胞损伤的保护作用。
[研究对象和方法]
1、以大鼠胰岛细胞瘤株INS-1细胞为实验对象；
2、实验分组
IL-1β浓度梯度分组(4组)
第1组：0ng/ml rIL-1β干预INS-1细胞组,简称0组；
第2组：1ng/ml rIL-1β干预INS-1细胞组,简称1组；
第3组：5ng/ml rIL-1β干预INS-1细胞组,简称5组；
第4组：lOng/ml rIL-1β干预INS-1细胞组,简称10组。
各干预组分组(3组)
第1组：正常对照组(Untreated group),以下简称对照组；
第2组：lOng/ml rIL-1β干预INS-1细胞组,以下简称IL-1β组；
第3组：在lOng/ml rIL-1β干预INS-1细胞前,予以hGLP-1预处理18小时,且间隔12小时追加一次相同剂量的hGLP-1,以下简称GLP-1+IL-1β组。
3、胰岛素放免试法检测各组INS-1细胞胰岛素量：在干预结束后,各组细胞先用0mM糖KRHB缓冲液冲洗两遍,再依次用含0mM糖、2.5mM糖、15mM糖的KRHB缓冲液分别孵育30min、1h、1h。收集上清用于胰岛素放免测定。测定各组蛋白含量,用于均衡各组胰岛素分泌量。
4、四氮唑蓝(MTT)法检测各组细胞活力：各组细胞干预结束后,分别经MTT、二甲亚砜处理,酶标仪检测各孔490nm处的光吸收值(OD值)。结果以正常对照组0D值标化余各组OD值,即设定正常对照组细胞活力为100%,由此计算余各组细胞活力百分比。
5、统计学处理：采用SPSS13.0统计软件进行分析,实验数据以均数±标准差(x±s)表示,多组资料比较采用独立样本t检验、单向方差分析(One-way ANOVA)和协方差分析,进一步多重比较采用LSD法(Least-significant difference test),方差不齐性时,用Welch法校正,当P<0.05时用Games-Howell法作多重比较。P<0.05被认为差异具有统计学意义。
[结果]
1、各组分泌功能的比较
在2.5mmol／L葡萄糖KRHB溶液中,与对照组相比,INS-1细胞的分泌功能较其它组略有下降,但各组间和组内比较均无统计学差异(P>0.05)
在15mmol/L葡萄糖KRHB溶液刺激下,与对照组(29.806±1.173 ng/mg/h)相比,IL-1β组的胰岛素分泌功能(16.335±0.724 ng/mg/h)显著降低,差异具有统计学意义(P<0.05)；与IL-1β组(16.335±0.72432 ng/mg/h)相比,GLP-1+IL-1β组的胰岛素分泌功能(26.335±2.609 ng／mg／h)显著增强,差异具有统计学意义(P<0.05和P<0.01 ng/mg／h)
2、不同浓度IL-1β和干预时间对INS-1细胞活力的影响
不同浓度IL-1β干预INS-1细胞24小时后,与对照组相比,5 ng/ml组、10ng/ml组细胞活力分别下降18%、22%,差异均具有统计学意义(P<0.001),但5ng／ml组和10ng/ml组相比无统计学意义(P>0.05)；不同浓度IL-1β干预INS-1细胞48小时后,与对照组相比,1ng/ml组、5 ng/ml组、lOng/ml组细胞活力分别下降14%、25%、34%,差异具有统计学意义(P<0.001)；与1ng/m1组相比,5 ng／ml组细胞活力下降11%,差异具有统计学意义(P<0.05),,10ng/ml组细胞活力下降19%,差异具有统计学意义(P<0.01)；与5ng／ml组相比,10ng／ml组细胞活力下降8%,差异具有统计学意义(P<0.05)。与24小时10ng／ml组相比,48小时10ng／ml组细胞活力下降了11%,差异有统计学意义(P<0.05)。
3、各组细胞活力的比较
实验终点测定细胞活力,与对照组相比,IL-1β组细胞活力下降29%,差异具有统计学意义(P<0.001)；与IL-1β组相比,GLP-1+IL-1β组上升了30%,差异具有统计学意义(P<0.001)。
[结论]
1、IL-1β能够显著的抑制INS-1细胞的分泌功能,减少INS-1细胞的数量,且呈剂量依赖性和时间依赖性。提示IL-1β对INS-1细胞存在着显著的损伤作用。
2、GLP-1能显著改善IL-1β对INS-1细胞功能的抑制,增加INS-1细胞的数量。提示GLP-1能够减轻IL-1β对INS-1细胞的损伤作用。
第二章GLP-1保护IL-1β诱导INS-1细胞损伤的机制初探
[目的]
初步探讨GLP-1对IL-1β损伤INS-1细胞的保护机制。
[研究对象和方法]
1、以大鼠胰岛细胞瘤株INS-1细胞为实验对象；
2、实验分组
第1组：正常对照组(Untreated gloup),以下简称对照组；
第2组：10ng/ml rIL-1β干预INS-1细胞组,以下简称IL-1β组；
第3组：在10ng/ml rIL-1β干预INS-1细胞前,予以hGLP-1预处理18小时,且间隔12小时追加一次相同剂量的hGLP-1,以下简称GLP-1+IL-1β组；3、采用荧光定量(real time) PCR法检测各组IKKβ和PDX-1 mRNA水平
4、免疫印迹法(Western blot)检测NF-KB蛋白在细胞核内表达
5、统计学处理：采用SPSS13.0统计软件进行分析,实验数据以均数±标准差(x±s)表示,多组资料比较采用单向方差分析(One-way ANOVA),进一步多重比较采用LSD法(Least-significant difference test),方差不齐性时,用Welch法校正,当P<0.05时用Games-Howell去作多重比较。P<0.05被认为差异具有统计学意义
[结果]
1、各组细胞IKKβmRNA的表达
与对照组相比,IL-1β组的IKKβmRNA表达显著增加(1.967±0.091 VS 1±0),差异具有统计学意义(P<0.01)；与IL-1β组比,GLP-1+IL-1β组的IKKβmRNA表达显著降低(1.967±0.091 VS 1.287±0.084),差异具有统计学意义(P<0.01)
2、各组细胞核内NF-κB蛋白表达水平
与对照组相比,IL-1β组核内NF-κB蛋白水平显著增高(1.166±0.056 VS0.042±0.007),差异具有统计学意义(P<0.01)；与IL-1β组比,GLP-1+IL-1β组核内NF-κB蛋白水平显著减少(0.648±0.050 VS 1.166±0.056),差异具有统计学意义(P<0.01)
[结论]
GLP-1能够抑制IL-1β诱导INS-1细胞IKKβ的表达,同时减少转入细胞核内的NF-κB蛋白量。提示抑制NF-κB通路可能是GLP-1减轻IL-1β诱导INS-1细胞损伤的机制之一。
Background
With the development of world economy and improvement of people's material living standard,the threat of diabetes faced by global human health are increasingly growing and diabetes has become one of public problem that severely threaten the health of the people worldwide, It also brings a great financial burden for individuals, families and society. Early prevention, early intervention is the ideal measures to solve such problems.
Type 2 diabetes mellitus (T2DM) is the vast majority of the total DM, its pathogenesis is complex and has not been fully understood. T2DM is based on insulin resistance and inadequate insulin secretion, but there is a controversy which is the lead.Insulin resistance is the lead in the past,But more and more papers suggest that the inadequate insulin secretion plays an important role in the pathogenesis of T2DM. Early studies suggest that high glucose and high fat can damage insulin secretion of the pancreaticβcell and reduce the amount of pancreaticβcells, so it is closely related with the inadequate insulin secretion.
Recent studies suggest that type 2 diabetes is a chronic low-grade inflammatory disease. As an important regulator of inflammation, the relationship of interleukin-1β(IL-1β) and type 2 diabetes increasingly receive attention. IL-1βis not only secreted by fat cells, macrophages and endothelial cells,but also secreted by the isletβcell.
IL-1βhas a wide biological function,it plays an important role in mediating P-cell injury. On the one hand,IL-1βinhibits insulin synthesis and release;on the other hand,IL-1βpromotesβ-cell death.IL-1βnot only mediates the interaction among leukocytes,but also takes part in the regulation of other cells and interact with other cytokines,constitutes a complex network of cytokine regulation, further increase the isletβcell damage.
Therefore, the treatment of T2DM at the present,the inhibition of cytokines on islet P-cell damage is very important to delay the development of diabetes by maintenaning a number of functional pancreatic isletβcells,.
Glucagon-like peptide-1 (GLP-1) has been found when the precursor gene of glucagon--proglucagon gene (GP) sequences are analyzed in 1983.this gene is expressed in a cells and L cells,the protein composes of 30 amino acids, the main type of biological activity is GLP-1 (7-36).Its receptor is widely distributed in every tissue and organ, producing a variety of biological functions when it is activated.
GLP-1 receptor agonists can produce the biological effects on the pancreas itself:on the one hand, it can increase insulin secretion and synthesis;on the other hand, it can increase the amount of isletβcells, mainly by increasing the isletβcell proliferation, increasingβ-cell The neogenesis, inhibiting the apoptosis of isletβcells.
Recent researches suggest that GLP-1 can inhibit the apoptosis of human isletβcells and rat pancreaticβcell line INS832/13 induced by high glucose and high fat, it mainly activated PI-3K/PKB pathway to increase the expression induce anti-apoptotic gene-inhibitor of apoptosis protein-2(IAP-2) and BCL-2.
The expression of IL-1βin T2DM patients isletβcells is significantly higher than in the normal control group, further research found that high glucose and IL-1βitself can increase the expression of IL-1βin islet and depend on the activation of NF-κB, but IL-1 receptor antagonist (IL-1Ra) can significantly inhibite IL-1βexpression. This shows that high glucose and IL-1βitself can stimulate the production and secretion of IL-1βin islet P cells.
The studies show that epicatechin can inhibit the toxicity of IL-1βon isletβcell line RINm5F and primary rat islet cells by blocking NF-κB signaling pathway. In addition, it could reduce the apoptosis induced IL-1βby transfecting adenovirus including inhibitors of IκB to aβcell line, rat and human islets.IL-1βcan induced COX-2 and prostaglandin receptor 3 (EP3) gene expression by activating NF-κB signaling pathway in pancreatic islets,leading to pancreaticβcell function decreased,but sodium salicylate can inhibit NF-κB signaling pathway to restore P-cell function.those show that the activation of NF-κB pathway and IL-1β-induced P-cell damage is closely related.
As mentioned before, T2DM is a low degree of inflammatory disease, local expression of IL-1βincreased in the islet, which may be related to high glucose and islet self-secreted IL-1β, whereas the NF-κB pathway activation and IL-1β-inducingβ-cell damage are closely related.GLP-1 can inhibit the damage of high glucose on isletβcell, is it related to the effect of GLP-1 to IL-1β?is the mechanism related to the inhibition of the activation of NF-κB? There are few researches concerning these aspects at present.
This study aims at studying that Glucagon-like peptide-1 protects INS-1 cells from IL-1β-induced damage and its mechanism. We wish to provide more theoretical support for GLP-1 in the intervention of T2DM.
Two Specific contents are as follows:
Chapter 1 Glucagon-like peptide-1 protects INS-1 cells from IL-1β-induced damage
[Objective]
To observe the effect of GLP-1 on INS-1 cells damage induced by IL-1β
[Methods]
1. Cell culture:The experiment object is rat's islet cell lines---INS-1 cells Goups
Accord to Concentration gradient of IL-1β(4 Groups)
(1). 0ng/ml rIL-1βintervents INS-1 cells,0 group
(2). 1ng/ml rIL-1βintervents INS-1 cells,1 group
(3).5ng/ml rIL-1βintervents INS-1 cells,5 group
(4).10ng/ml rIL-1βintervents INS-1 cells,10 group
There were three groups according to intervention reagents:
(1) The normal control group (control or untreated group);
(2) INS-1 cells were exposed to 10ng/ml rIL-1βfor 48h(IL-1βgroup);
(3) Pretreat with 20nmol/L hGLP-1 for 18h before cells were exposed to rIL-1β,add the same dose of hGLP-1 at the interval of 12 hours (GLP-1+IL-1βgroup).
2. insulin were measured by Rat insulin Ria Kit:After treatment with IL-1 P for 48h, the cells were washed twice with KRHB buffer containing OmM glucose,then ordinally incubated with the same KRHB buffer for 30min and incubated in KRHB containing 2.5mM glucose and 15mM glucose for 1 h, aliquots of the media were collected for insulin radioimmunoassay kit. measure total protein content of each group. Insulin secretion was normalized based on the corresponding protein in each group
3. Cell viabilities were evaluated by MTT assay:The optical density (OD) value was read at 490nm in a plate reader. The reduction in optical density was used as a measurement of cell viability, normalized to cells incubated in normal control group, which were considered 100% viable.
4. Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. All data were expressed as the mean±SD. Statistical significance of differences among groups was evaluated by independent-Samples t test, one-way ANOVA and ANCOVA. multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. multiple comparisons was analyzed by Games-Howell method when P values less than 0.05,P<0.05 was considered as significant.
[Results]
1. Comparison insulin(ng/mg/h) secretion of in each group
Incubated with KRHB buffer containing 2.5 mM glucose, In comparison with the Utreated group, the insulin secretion of the IL-1βgroup were decreased,but the difference is not significant(p>0.05),and there were no statistical difference in each group and all groups(p>0.05).
Incubated with KRHB buffer containing 2.5 mM glucose, In comparison with the Utreated group (29.806±1.173), the insulin secretion of the IL-1βgroup (16.335±0.724) were decreased significantly(p<0.05); In comparison with IL-1βgroup, the insulin secretion of GLP-1+IL-1β(26.335±2.609) group was increased significantly (P<0.05)
2. Effect of different concentrations of IL-1βand the intervention time on the viability in INS-1 cells
INS-1 cells have been exposed to different concentrations of IL-1βfor 24 hours, In comparison with the Utreated group, the cell viabilities of 5 ng/ml group and lOng/ml group decreased 18%、22% respectively (P<0.01),but the comparision of 5ng/ml group and 10ng/ml group have no significant difference; INS-1 cells have been exposed to different concentrations of IL-1βfor 48 hours, In comparison with the Utreated group,the cell viabilities of 1 ng/ml group、5 ng/ml group and lOng/ml 14%、25%、34% respectively (P<0.01), In comparison with 1ng/ml group, the cell viabilities of 5ng/ml group decreased 11%(P<0.05), the cell viabilities of 5ng/ml group decreased 19%(P<0.01), In comparison with 5ng/ml group, the cell viabilities of lOng/ml group decreased 8%(P<0.05).in comparision of lOng/ml group for 24 hours, he cell viabilities of 10ng/ml group for 48 hours decreased 11%(P<0.05).
3. Comparison of cell viability(%) in each group
In comparison with the Utreated group, the cell viabilities of the IL-1βgroup were decreased by 29%(P<0.01), In comparison with the IL-1βgroup, the cell viabilities of GLP-1+IL-1βgroup was increased by 30%(P<0.01)
[Conclusion]
1. IL-1βcan significantly inhibit the insulin secretion of INS-1 cells and decrease the number of INS-1 cells in concentration--dependent and time—dependent manner. It shows that IL-1βcan significantly damage INS-1 cells.
2. GLP-1 can significantly improve the inhibition of insulin secretion of INS-1 cells induced by IL-1βand increase the number of INS-1 cells.It shows that GLP-1 can improve the damage of INS-1 cells induced by IL-1β.
Chapter 2 mechanisms of GLP-1 pretection in IL-1β-induced INS-1 cells damage
[Objective]
To investigate the mechanisms included the effect of GLP-1 on IL-1β-induced INS-1 cells damage.
[Methods]
1、Cell culture and groups is like the first chapter;
2、Groups
(1) The normal control group (control or untreated group);
(2) INS-1 cells were exposed to 10ng/ml rIL-1βfor 48h(IL-1(3 group);
(3) Pretreat with 20nmol/L hGLP-1 for 18h before cells were exposed to rIL-1β,add the same dose of hGLP-1 at the interval of 12 hours (GLP-1+IL-1βgroup).
3、Real time PCR was performed to assay the expression of IKKP mRNA
4、The expressions of NF-κB protein were detected by western blot.
5、Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. All data were expressed as the mean±SD. Statistical significance of differences among groups was evaluated by one-way ANOVA. multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. multiple comparisons was analyzed by Games-Howell method when P values less than 0.05,P<0.05 was considered as significant.
[Results]
1、the expression of IKKβmRNA in each group
In comparison with the Utreated group, IL-1βgroup increased the expression of (1.967±0.091 VS 1±0) significantly(p<0.05); In comparison with IL-1βgroup,the expression of IKKβmRNA in GLP-1+IL-1βgroup (1.967±0.091 VS 1.287±0.084) were decreasd significantly (p<0.05)
2、The expression of NF-κB protein in the INS-1 cell nuclear
In comparison with the Utreated group, the NF-κB protein expressions in IL-1βgroup were increased (1.166±0.056 VS 0.042±0.007) significantly (P<0.01); In comparison with IL-1βgroup, the expression of the NF-κB protein in GLP-1+IL-1βgroup (0.648±0.050 VS 1.166±0.056) was decreased significantly(p<0.05)
[Conclusions]
GLP-1 can inhibit the expression of IKKβmRNA and reduce NF-κB protein transferred into nucleus induced by IL-1βin INS-1 cells.this indicates that the inhibition of NF-κB pathway may be one of the mechanisms which GLP-1 inhibited IL-1β-inducedβ-cell damage.
随着世界经济的发展,人们生活水平的提高,全球人类健康面临的糖尿病威胁正日益增加,已成为严重威胁人类健康的世界性公共卫生问题,也给个人、家庭和社会带来了极大的经济负担。早期预防、早期干预无疑是解决这类问题的理想措施。
2型糖尿病(Type 2 diabetes mellitus,T2DM)患者占全部糖尿病患者的绝大多数,其发病机制复杂,至今未被完全阐明。T2DM的发病基础是胰岛素抵抗和胰岛素分泌缺陷,但谁起主导作用仍存争议。过去认为胰岛素抵抗占主导地位,近来,越来越多研究提示胰岛素分泌缺陷在T2DM的发生发展中起重要作用。前期研究提示高糖、高脂能破坏胰岛β细胞的分泌功能和减少胰岛β细胞量,所以与胰岛素分泌缺陷关系密切。
近期研究提示2型糖尿病是一种慢性低度炎症性疾病。作为炎症过程的重要调节因子,白细胞介素-1β(interleukin-1β,IL-1β)与2型糖尿病的关系日益引起重视。IL-1β不仅是由脂肪细胞、巨噬细胞、内皮细胞产生和分泌的,而且胰岛β细胞自身具有分泌IL-1β的功能。
IL-1β具有广泛的生物学效应,在介导β细胞损伤中起着重要作用。一方面IL-1β抑制胰岛素的合成与释放；另一方面IL-1β促进胰岛β细胞的死亡。IL-1β不但介导白细胞间的相互作用,还参与了其他细胞的调节,并与其他细胞因子相互影响,相互制约,构成一个复杂的细胞因子调节网络,进一步加重胰岛β细胞的损伤。
因此,在目前T2DM的治疗过程中,抑制IL-1β对胰岛β细胞的损伤作用对维持一定数量有功能的胰岛p细胞,延缓糖尿病的发生发展十分重要。
胰高血糖素样肽-1(GLP-1)是1983年在分析胰高血糖素的前体基因--胰高血糖素原(proglucagon, GP)基因序列时被发现的。该基因可在胰腺的α细胞、肠道的L细胞内表达。GLP-1由30个氨基酸组成,主要的生物活性型是GLP-1(7-36)。它的受体广泛分布于全身各个组织器官,被激活后产生多种生物学效应。
GLP-1受体激动剂对胰腺本身产生的生物学效应：一方面增加胰岛素的分泌和合成功能,另一方面可以增加胰岛β细胞量,主要表现为增加胰岛β细胞的增殖、增加胰岛β细胞的新生、抑制胰岛β细胞的凋亡
近期研究提示,GLP-1可以抑制高糖诱导的人胰岛β细胞和大鼠胰岛β细胞株INS832／13细胞的凋亡,其机制主要是GLP-1激活的PI-3K/PKB通路,诱导抗凋亡基因IAP-2(inhibitor of apoptosis protein-2)和BCL-2的表达增加。
T2DM患者胰岛β细胞内IL-1β的表达显著高于正常对照组,进一步研究发现高糖和IL-1β自身能增加胰岛内IL-1β的表达,且与NF-κB的激活有关,而IL-1受体拮抗剂(IL-1 receptor antagonist, IL-1Ra)能显著抑制IL-1β的表达。提示高糖和IL-1β自身可以刺激胰岛β细胞生成和分泌IL-1β。
研究表明,表儿茶精(epicatechin)能通过阻断NF-κB信号通路的激活,抑制IL-1β对胰岛β细胞系RINm5F和大鼠胰岛的细胞毒性。另外,通过包含IκB抑制剂的腺病毒转染β细胞系、大鼠及人的胰岛均能降低由IL-1β引起的细胞凋亡。IL-1β可以激活大鼠胰岛NF-κB信号通路,诱导COX-2和前列腺素受体3(EP3)基因的表达增加,导致胰岛β细胞的分泌功能下降,而水杨酸钠可以通过抑制IL-1β对NF-κB信号通路的激活,恢复胰岛β细胞的分泌功能。说明NF-κB通路的激活与IL-1β诱导的胰岛β细胞损伤关系密切。
前述提示,T2DM是一个慢性低度炎症性疾病,其胰岛内局部IL-1β表达增加,这可能与高糖和胰岛自身分泌的IL-1β诱导有关,而NF-κB通路的激活与IL-1β诱导的β细胞损伤关系密切。GLP-1能抑制高糖对胰岛p细胞的损伤作用,这是否与GLP-1抑制IL-1β对胰岛β细胞的损伤作用有关呢?其机制是否与抑制IL-1β诱导NF-κB的激活有关呢?目前这方面的研究较少。
本课题以大鼠胰岛细胞瘤系INS-1细胞为研究对象,观察GLP-1对IL-1β诱导INS-1细胞损伤的保护作用并探讨其部分保护机制。以期为GLP-1在2型糖尿病治疗中的应用提供更多的理论支持。
具体研究内容分为以下两个部分：
第一章GLP-1对IL-1β诱导INS-1细胞损伤的保护作用
[目的]
观察GLP-1对IL-1β诱导INS-1细胞损伤的保护作用。
[研究对象和方法]
1、以大鼠胰岛细胞瘤株INS-1细胞为实验对象；
2、实验分组
IL-1β浓度梯度分组(4组)
第1组：0ng/ml rIL-1β干预INS-1细胞组,简称0组；
第2组：1ng/ml rIL-1β干预INS-1细胞组,简称1组；
第3组：5ng/ml rIL-1β干预INS-1细胞组,简称5组；
第4组：lOng/ml rIL-1β干预INS-1细胞组,简称10组。
各干预组分组(3组)
第1组：正常对照组(Untreated group),以下简称对照组；
第2组：lOng/ml rIL-1β干预INS-1细胞组,以下简称IL-1β组；
第3组：在lOng/ml rIL-1β干预INS-1细胞前,予以hGLP-1预处理18小时,且间隔12小时追加一次相同剂量的hGLP-1,以下简称GLP-1+IL-1β组。
3、胰岛素放免试法检测各组INS-1细胞胰岛素量：在干预结束后,各组细胞先用0mM糖KRHB缓冲液冲洗两遍,再依次用含0mM糖、2.5mM糖、15mM糖的KRHB缓冲液分别孵育30min、1h、1h。收集上清用于胰岛素放免测定。测定各组蛋白含量,用于均衡各组胰岛素分泌量。
4、四氮唑蓝(MTT)法检测各组细胞活力：各组细胞干预结束后,分别经MTT、二甲亚砜处理,酶标仪检测各孔490nm处的光吸收值(OD值)。结果以正常对照组0D值标化余各组OD值,即设定正常对照组细胞活力为100%,由此计算余各组细胞活力百分比。
5、统计学处理：采用SPSS13.0统计软件进行分析,实验数据以均数±标准差(x±s)表示,多组资料比较采用独立样本t检验、单向方差分析(One-way ANOVA)和协方差分析,进一步多重比较采用LSD法(Least-significant difference test),方差不齐性时,用Welch法校正,当P<0.05时用Games-Howell法作多重比较。P<0.05被认为差异具有统计学意义。
[结果]
1、各组分泌功能的比较
在2.5mmol／L葡萄糖KRHB溶液中,与对照组相比,INS-1细胞的分泌功能较其它组略有下降,但各组间和组内比较均无统计学差异(P>0.05)
在15mmol/L葡萄糖KRHB溶液刺激下,与对照组(29.806±1.173 ng/mg/h)相比,IL-1β组的胰岛素分泌功能(16.335±0.724 ng/mg/h)显著降低,差异具有统计学意义(P<0.05)；与IL-1β组(16.335±0.72432 ng/mg/h)相比,GLP-1+IL-1β组的胰岛素分泌功能(26.335±2.609 ng／mg／h)显著增强,差异具有统计学意义(P<0.05和P<0.01 ng/mg／h)
2、不同浓度IL-1β和干预时间对INS-1细胞活力的影响
不同浓度IL-1β干预INS-1细胞24小时后,与对照组相比,5 ng/ml组、10ng/ml组细胞活力分别下降18%、22%,差异均具有统计学意义(P<0.001),但5ng／ml组和10ng/ml组相比无统计学意义(P>0.05)；不同浓度IL-1β干预INS-1细胞48小时后,与对照组相比,1ng/ml组、5 ng/ml组、lOng/ml组细胞活力分别下降14%、25%、34%,差异具有统计学意义(P<0.001)；与1ng/m1组相比,5 ng／ml组细胞活力下降11%,差异具有统计学意义(P<0.05),,10ng/ml组细胞活力下降19%,差异具有统计学意义(P<0.01)；与5ng／ml组相比,10ng／ml组细胞活力下降8%,差异具有统计学意义(P<0.05)。与24小时10ng／ml组相比,48小时10ng／ml组细胞活力下降了11%,差异有统计学意义(P<0.05)。
3、各组细胞活力的比较
实验终点测定细胞活力,与对照组相比,IL-1β组细胞活力下降29%,差异具有统计学意义(P<0.001)；与IL-1β组相比,GLP-1+IL-1β组上升了30%,差异具有统计学意义(P<0.001)。
[结论]
1、IL-1β能够显著的抑制INS-1细胞的分泌功能,减少INS-1细胞的数量,且呈剂量依赖性和时间依赖性。提示IL-1β对INS-1细胞存在着显著的损伤作用。
2、GLP-1能显著改善IL-1β对INS-1细胞功能的抑制,增加INS-1细胞的数量。提示GLP-1能够减轻IL-1β对INS-1细胞的损伤作用。
第二章GLP-1保护IL-1β诱导INS-1细胞损伤的机制初探
[目的]
初步探讨GLP-1对IL-1β损伤INS-1细胞的保护机制。
[研究对象和方法]
1、以大鼠胰岛细胞瘤株INS-1细胞为实验对象；
2、实验分组
第1组：正常对照组(Untreated gloup),以下简称对照组；
第2组：10ng/ml rIL-1β干预INS-1细胞组,以下简称IL-1β组；
第3组：在10ng/ml rIL-1β干预INS-1细胞前,予以hGLP-1预处理18小时,且间隔12小时追加一次相同剂量的hGLP-1,以下简称GLP-1+IL-1β组；3、采用荧光定量(real time) PCR法检测各组IKKβ和PDX-1 mRNA水平
4、免疫印迹法(Western blot)检测NF-KB蛋白在细胞核内表达
5、统计学处理：采用SPSS13.0统计软件进行分析,实验数据以均数±标准差(x±s)表示,多组资料比较采用单向方差分析(One-way ANOVA),进一步多重比较采用LSD法(Least-significant difference test),方差不齐性时,用Welch法校正,当P<0.05时用Games-Howell去作多重比较。P<0.05被认为差异具有统计学意义
[结果]
1、各组细胞IKKβmRNA的表达
与对照组相比,IL-1β组的IKKβmRNA表达显著增加(1.967±0.091 VS 1±0),差异具有统计学意义(P<0.01)；与IL-1β组比,GLP-1+IL-1β组的IKKβmRNA表达显著降低(1.967±0.091 VS 1.287±0.084),差异具有统计学意义(P<0.01)
2、各组细胞核内NF-κB蛋白表达水平
与对照组相比,IL-1β组核内NF-κB蛋白水平显著增高(1.166±0.056 VS0.042±0.007),差异具有统计学意义(P<0.01)；与IL-1β组比,GLP-1+IL-1β组核内NF-κB蛋白水平显著减少(0.648±0.050 VS 1.166±0.056),差异具有统计学意义(P<0.01)
[结论]
GLP-1能够抑制IL-1β诱导INS-1细胞IKKβ的表达,同时减少转入细胞核内的NF-κB蛋白量。提示抑制NF-κB通路可能是GLP-1减轻IL-1β诱导INS-1细胞损伤的机制之一。
Background
With the development of world economy and improvement of people's material living standard,the threat of diabetes faced by global human health are increasingly growing and diabetes has become one of public problem that severely threaten the health of the people worldwide, It also brings a great financial burden for individuals, families and society. Early prevention, early intervention is the ideal measures to solve such problems.
Type 2 diabetes mellitus (T2DM) is the vast majority of the total DM, its pathogenesis is complex and has not been fully understood. T2DM is based on insulin resistance and inadequate insulin secretion, but there is a controversy which is the lead.Insulin resistance is the lead in the past,But more and more papers suggest that the inadequate insulin secretion plays an important role in the pathogenesis of T2DM. Early studies suggest that high glucose and high fat can damage insulin secretion of the pancreaticβcell and reduce the amount of pancreaticβcells, so it is closely related with the inadequate insulin secretion.
Recent studies suggest that type 2 diabetes is a chronic low-grade inflammatory disease. As an important regulator of inflammation, the relationship of interleukin-1β(IL-1β) and type 2 diabetes increasingly receive attention. IL-1βis not only secreted by fat cells, macrophages and endothelial cells,but also secreted by the isletβcell.
IL-1βhas a wide biological function,it plays an important role in mediating P-cell injury. On the one hand,IL-1βinhibits insulin synthesis and release;on the other hand,IL-1βpromotesβ-cell death.IL-1βnot only mediates the interaction among leukocytes,but also takes part in the regulation of other cells and interact with other cytokines,constitutes a complex network of cytokine regulation, further increase the isletβcell damage.
Therefore, the treatment of T2DM at the present,the inhibition of cytokines on islet P-cell damage is very important to delay the development of diabetes by maintenaning a number of functional pancreatic isletβcells,.
Glucagon-like peptide-1 (GLP-1) has been found when the precursor gene of glucagon--proglucagon gene (GP) sequences are analyzed in 1983.this gene is expressed in a cells and L cells,the protein composes of 30 amino acids, the main type of biological activity is GLP-1 (7-36).Its receptor is widely distributed in every tissue and organ, producing a variety of biological functions when it is activated.
GLP-1 receptor agonists can produce the biological effects on the pancreas itself:on the one hand, it can increase insulin secretion and synthesis;on the other hand, it can increase the amount of isletβcells, mainly by increasing the isletβcell proliferation, increasingβ-cell The neogenesis, inhibiting the apoptosis of isletβcells.
Recent researches suggest that GLP-1 can inhibit the apoptosis of human isletβcells and rat pancreaticβcell line INS832/13 induced by high glucose and high fat, it mainly activated PI-3K/PKB pathway to increase the expression induce anti-apoptotic gene-inhibitor of apoptosis protein-2(IAP-2) and BCL-2.
The expression of IL-1βin T2DM patients isletβcells is significantly higher than in the normal control group, further research found that high glucose and IL-1βitself can increase the expression of IL-1βin islet and depend on the activation of NF-κB, but IL-1 receptor antagonist (IL-1Ra) can significantly inhibite IL-1βexpression. This shows that high glucose and IL-1βitself can stimulate the production and secretion of IL-1βin islet P cells.
The studies show that epicatechin can inhibit the toxicity of IL-1βon isletβcell line RINm5F and primary rat islet cells by blocking NF-κB signaling pathway. In addition, it could reduce the apoptosis induced IL-1βby transfecting adenovirus including inhibitors of IκB to aβcell line, rat and human islets.IL-1βcan induced COX-2 and prostaglandin receptor 3 (EP3) gene expression by activating NF-κB signaling pathway in pancreatic islets,leading to pancreaticβcell function decreased,but sodium salicylate can inhibit NF-κB signaling pathway to restore P-cell function.those show that the activation of NF-κB pathway and IL-1β-induced P-cell damage is closely related.
As mentioned before, T2DM is a low degree of inflammatory disease, local expression of IL-1βincreased in the islet, which may be related to high glucose and islet self-secreted IL-1β, whereas the NF-κB pathway activation and IL-1β-inducingβ-cell damage are closely related.GLP-1 can inhibit the damage of high glucose on isletβcell, is it related to the effect of GLP-1 to IL-1β?is the mechanism related to the inhibition of the activation of NF-κB? There are few researches concerning these aspects at present.
This study aims at studying that Glucagon-like peptide-1 protects INS-1 cells from IL-1β-induced damage and its mechanism. We wish to provide more theoretical support for GLP-1 in the intervention of T2DM.
Two Specific contents are as follows:
Chapter 1 Glucagon-like peptide-1 protects INS-1 cells from IL-1β-induced damage
[Objective]
To observe the effect of GLP-1 on INS-1 cells damage induced by IL-1β
[Methods]
1. Cell culture:The experiment object is rat's islet cell lines---INS-1 cells Goups
Accord to Concentration gradient of IL-1β(4 Groups)
(1). 0ng/ml rIL-1βintervents INS-1 cells,0 group
(2). 1ng/ml rIL-1βintervents INS-1 cells,1 group
(3).5ng/ml rIL-1βintervents INS-1 cells,5 group
(4).10ng/ml rIL-1βintervents INS-1 cells,10 group
There were three groups according to intervention reagents:
(1) The normal control group (control or untreated group);
(2) INS-1 cells were exposed to 10ng/ml rIL-1βfor 48h(IL-1βgroup);
(3) Pretreat with 20nmol/L hGLP-1 for 18h before cells were exposed to rIL-1β,add the same dose of hGLP-1 at the interval of 12 hours (GLP-1+IL-1βgroup).
2. insulin were measured by Rat insulin Ria Kit:After treatment with IL-1 P for 48h, the cells were washed twice with KRHB buffer containing OmM glucose,then ordinally incubated with the same KRHB buffer for 30min and incubated in KRHB containing 2.5mM glucose and 15mM glucose for 1 h, aliquots of the media were collected for insulin radioimmunoassay kit. measure total protein content of each group. Insulin secretion was normalized based on the corresponding protein in each group
3. Cell viabilities were evaluated by MTT assay:The optical density (OD) value was read at 490nm in a plate reader. The reduction in optical density was used as a measurement of cell viability, normalized to cells incubated in normal control group, which were considered 100% viable.
4. Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. All data were expressed as the mean±SD. Statistical significance of differences among groups was evaluated by independent-Samples t test, one-way ANOVA and ANCOVA. multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. multiple comparisons was analyzed by Games-Howell method when P values less than 0.05,P<0.05 was considered as significant.
[Results]
1. Comparison insulin(ng/mg/h) secretion of in each group
Incubated with KRHB buffer containing 2.5 mM glucose, In comparison with the Utreated group, the insulin secretion of the IL-1βgroup were decreased,but the difference is not significant(p>0.05),and there were no statistical difference in each group and all groups(p>0.05).
Incubated with KRHB buffer containing 2.5 mM glucose, In comparison with the Utreated group (29.806±1.173), the insulin secretion of the IL-1βgroup (16.335±0.724) were decreased significantly(p<0.05); In comparison with IL-1βgroup, the insulin secretion of GLP-1+IL-1β(26.335±2.609) group was increased significantly (P<0.05)
2. Effect of different concentrations of IL-1βand the intervention time on the viability in INS-1 cells
INS-1 cells have been exposed to different concentrations of IL-1βfor 24 hours, In comparison with the Utreated group, the cell viabilities of 5 ng/ml group and lOng/ml group decreased 18%、22% respectively (P<0.01),but the comparision of 5ng/ml group and 10ng/ml group have no significant difference; INS-1 cells have been exposed to different concentrations of IL-1βfor 48 hours, In comparison with the Utreated group,the cell viabilities of 1 ng/ml group、5 ng/ml group and lOng/ml 14%、25%、34% respectively (P<0.01), In comparison with 1ng/ml group, the cell viabilities of 5ng/ml group decreased 11%(P<0.05), the cell viabilities of 5ng/ml group decreased 19%(P<0.01), In comparison with 5ng/ml group, the cell viabilities of lOng/ml group decreased 8%(P<0.05).in comparision of lOng/ml group for 24 hours, he cell viabilities of 10ng/ml group for 48 hours decreased 11%(P<0.05).
3. Comparison of cell viability(%) in each group
In comparison with the Utreated group, the cell viabilities of the IL-1βgroup were decreased by 29%(P<0.01), In comparison with the IL-1βgroup, the cell viabilities of GLP-1+IL-1βgroup was increased by 30%(P<0.01)
[Conclusion]
1. IL-1βcan significantly inhibit the insulin secretion of INS-1 cells and decrease the number of INS-1 cells in concentration--dependent and time—dependent manner. It shows that IL-1βcan significantly damage INS-1 cells.
2. GLP-1 can significantly improve the inhibition of insulin secretion of INS-1 cells induced by IL-1βand increase the number of INS-1 cells.It shows that GLP-1 can improve the damage of INS-1 cells induced by IL-1β.
Chapter 2 mechanisms of GLP-1 pretection in IL-1β-induced INS-1 cells damage
[Objective]
To investigate the mechanisms included the effect of GLP-1 on IL-1β-induced INS-1 cells damage.
[Methods]
1、Cell culture and groups is like the first chapter;
2、Groups
(1) The normal control group (control or untreated group);
(2) INS-1 cells were exposed to 10ng/ml rIL-1βfor 48h(IL-1(3 group);
(3) Pretreat with 20nmol/L hGLP-1 for 18h before cells were exposed to rIL-1β,add the same dose of hGLP-1 at the interval of 12 hours (GLP-1+IL-1βgroup).
3、Real time PCR was performed to assay the expression of IKKP mRNA
4、The expressions of NF-κB protein were detected by western blot.
5、Statistical analysis:Statistical analyses were performed using the SPSS 13.0 software package. All data were expressed as the mean±SD. Statistical significance of differences among groups was evaluated by one-way ANOVA. multiple comparisons were carried out using the Least-significant Difference (LSD) method. Welch method was used when equal variances not assumed. multiple comparisons was analyzed by Games-Howell method when P values less than 0.05,P<0.05 was considered as significant.
[Results]
1、the expression of IKKβmRNA in each group
In comparison with the Utreated group, IL-1βgroup increased the expression of (1.967±0.091 VS 1±0) significantly(p<0.05); In comparison with IL-1βgroup,the expression of IKKβmRNA in GLP-1+IL-1βgroup (1.967±0.091 VS 1.287±0.084) were decreasd significantly (p<0.05)
2、The expression of NF-κB protein in the INS-1 cell nuclear
In comparison with the Utreated group, the NF-κB protein expressions in IL-1βgroup were increased (1.166±0.056 VS 0.042±0.007) significantly (P<0.01); In comparison with IL-1βgroup, the expression of the NF-κB protein in GLP-1+IL-1βgroup (0.648±0.050 VS 1.166±0.056) was decreased significantly(p<0.05)
[Conclusions]
GLP-1 can inhibit the expression of IKKβmRNA and reduce NF-κB protein transferred into nucleus induced by IL-1βin INS-1 cells.this indicates that the inhibition of NF-κB pathway may be one of the mechanisms which GLP-1 inhibited IL-1β-inducedβ-cell damage.
引文
[1]Wild S, Roglic G, Green A, Sicree R, King H. Global prevalence of diabetes: estimates for the year 2000 and projections for 2030. Diabetes Care.2004; 27: 1047-1053.
[2]Chalmers J, Cooper ME. UKPDS and the legacy effect. N Engl J Med.2008; 359:1618-1620.
[3]Mathis D, Vence L, Benoist C. beta-Cell death during progression to diabetes. Nature.2001; 414:792-798.
[4]Maedler K, Donath MY. Beta-cells in type 2 diabetes:a loss of function and mass. Horm Res.2004; 62 Suppl 3:67-73.
[5]Maclean N, Ogilvie RE Quantitative estimation of the pancreatic islet tissue in diabetic subjects. Diabetes.1955; 4:367-376.
[6]Hou ZQ, Li HL, Gao L, Pan L, Zhao JJ, Li GW. Involvement of chronic stresses in rat islet and INS-1 cell glucotoxicity induced by intermittent high glucose. Mol Cell Endocrinol.2008; 291:71-78.
[7]Federici M, Hribal M, Perego L, et al. High glucose causes apoptosis in cultured human pancreatic islets of Langerhans:a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program. Diabetes.2001; 50: 1290-1301.
[8]Lupi R, Dotta F, Marselli L, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets:evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes.2002; 51:1437-1442.
[9]Piro S, Anello M, Di Pietro C, et al. Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets:possible role of oxidative stress. Metabolism.2002; 51:1340-1347.
[10]Schmidt MI, Duncan BB, Sharrett AR, et al. Markers of inflammation and prediction of diabetes mellitus in adults (Atherosclerosis Risk in Communities study): a cohort study. Lancet.1999; 353:1649-1652.
[11]Crook MA, Tutt P, Simpson H, Pickup JC. Serum sialic acid and acute phase proteins in type 1 and type 2 diabetes mellitus. Clin Chim Acta.1993; 219:131-138.
[12]Pickup JC, Chusney GD, Thomas SM, Burt D. Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes. Life Sci.2000; 67:291-300.
[13]Yuan M, Konstantopoulos N, Lee J, et al. Reversal of obesity-and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science.2001; 293:1673-1677.
[14]Hundal RS, Petersen KF, Mayerson AB, et al. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest.2002; 109: 1321-1326.
[15]Andersson AK, Borjesson A, Sandgren J, Sandler S. Cytokines affect PDX-1 expression, insulin and proinsulin secretion from iNOS deficient murine islets. Mol Cell Endocrinol.2005; 240:50-57.
[16]Oetjen E, Blume R, Cierny I, et al. Inhibition of MafA transcriptional activity and human insulin gene transcription by interleukin-lbeta and mitogen-activated protein kinase kinase kinase in pancreatic islet beta cells. Diabetologia.2007; 50: 1678-1687.
[17]Veluthakal R, Jangati GR, Kowluru A. IL-1beta-induced iNOS expression, NO release and loss in metabolic cell viability are resistant to inhibitors of ceramide synthase and sphingomyelinase in INS 832/13 cells. JOP.2006; 7:593-601.
[18]Eizirik DL, Mandrup-Poulsen T. A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia.2001; 44:2115-2133.
[19]Siegel RM, Frederiksen JK, Zacharias DA, et al. Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science.2000; 288:2354-2357.
[20]Curtin JF, Cotter TG. Live and let die:regulatory mechanisms in Fas-mediated apoptosis. Cell Signal.2003; 15:983-992.
[21]Donath MY, Storling J, Maedler K, Mandrup-Poulsen T. Inflammatory mediators and islet beta-cell failure:a link between type 1 and type 2 diabetes. J Mol Med.2003; 81:455-470.
[22]Maedler K, Sergeev P, Ris F, et al. Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest.2002; 110:851-860.
[23]Donath MY, Ehses JA, Maedler K, et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes.2005; 54 Suppl 2:S108-113.
[24]Bell GI, Santerre RF, Mullenbach GT. Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature.1983; 302:716-718.
[25]Turton MD, O'Shea D, Gunn I, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature.1996; 379:69-72.
[26]Gonzalez C, Beruto V, Keller G, Santoro S, Di Girolamo G. Investigational treatments for Type 2 diabetes mellitus:exenatide and liraglutide. Expert Opin Investig Drugs.2006; 15:887-895.
[27]Zander M, Madsbad S, Madsen JL, Hoist JJ. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes:a parallel-group study. Lancet.2002; 359:824-830.
[28]Kolterman OG, Buse JB, Fineman MS, et al. Synthetic exendin-4 (exenatide) significantly reduces postprandial and fasting plasma glucose in subjects with type 2 diabetes. J Clin Endocrinol Metab.2003; 88:3082-3089.
[29]Dillon JS, Lu M, Bowen S, Homan LL. The recombinant rat glucagon-like peptide-1 receptor, expressed in an alpha-cell line, is coupled to adenylyl cyclase activation and intracellular calcium release. Exp Clin Endocrinol Diabetes.2005; 113:182-189.
[30]Fehmann HC, Hering BJ, Wolf MJ, et al. The effects of glucagon-like peptide-I (GLP-I) on hormone secretion from isolated human pancreatic islets. Pancreas.1995; 11:196-200.
[31]Holz GG, Leech CA, Heller RS, Castonguay M, Habener JF. cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic beta-cells. A Ca2+signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7-37). JBiol Chem.1999; 274:14147-14156.
[32]Skoglund G, Hussain MA, Holz GG. Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element. Diabetes.2000; 49:1156-1164.
[33]Abraham EJ, Leech CA, Lin JC, Zulewski H, Habener JF. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology.2002; 143:3152-3161.
[34]Tourrel C, Bailbe D, Meile MJ, Kergoat M, Portha B. Glucagon-like peptide-1 and exendin-4 stimulate beta-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes.2001; 50:1562-1570.
[35]Li Y, Hansotia T, Yusta B, Ris F, Halban PA, Drucker DJ. Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem.2003; 278: 471-478.
[36]Buteau J, El-Assaad W, Rhodes CJ, Rosenberg L, Joly E, Prentki M. Glucagon-like peptide-1 prevents beta cell glucolipotoxicity. Diabetologia.2004; 47: 806-815.
[37]Boni-Schnetzler M, Thorne J, Parnaud G, et al. Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta-cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation. J Clin Endocrinol Metab.2008; 93:4065-4074.
[38]Kim MJ, Ryu GR, Kang JH, et al. Inhibitory effects of epicatechin on interleukin-1beta-induced inducible nitric oxide synthase expression in RINm5F cells and rat pancreatic islets by down-regulation of NF-kappaB activation. Biochem Pharmacol.2004; 68:1775-1785.
[39]Giannoukakis N, Rudert WA, Trucco M, Robbins PD. Protection of human islets from the effects of interleukin-1beta by adenoviral gene transfer of an Ikappa B repressor. J Biol Chem.2000; 275:36509-36513.
[40]Iran PO, Gleason CE, Robertson RP. Inhibition of interleukin-1beta-induced COX-2 and EP3 gene expression by sodium salicylate enhances pancreatic islet beta-cell function. Diabetes.2002; 51:1772-1778.
[41]Farilla L, Bulotta A, Hirshberg B, et al. Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology.2003; 144:5149-5158.
[42]刘礼斌,王燕萍,潘晓东,姜苏原,陈洲.Exendin-4体外通过抑制NF-KB-iNOS-NO信号减轻氧化应激诱导的小鼠MIN6胰岛β细胞凋亡.药学学报2008：0513-4870.
[43]Li L, El-Kholy W, Rhodes CJ, Brubaker PL. Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis:role of protein kinase B. Diabetologia.2005; 48:1339-1349.
[44]Li LX, Yoshikawa H, Egeberg KW, Grill V. Interleukin-1beta swiftly down-regulates UCP-2 mRNA in beta-cells by mechanisms not directly coupled to toxicity. Cytokine.2003; 23:101-107.
[45]Tellez N, Montolio M, Biarnes M, Castano E, Soler J, Montanya E. Adenoviral overexpression of interleukin-1 receptor antagonist protein increases beta-cell replication in rat pancreatic islets. Gene Ther.2005; 12:120-128.
[46]Ferdaoussi M, Abdelli S, Yang JY, et al. Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway. Diabetes.2008; 57:1205-1215.
[47]Drucker DJ. The biology of incretin hormones. Cell Metab.2006; 3:153-165.
[48]Hui H, Nourparvar A, Zhao X, Perfetti R. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A-and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology.2003; 144:1444-1455.
[49]Jhala US, Canettieri G, Screaton RA, et al. cAMP promotes pancreatic beta-cell survival via CREB-mediated induction of IRS2. Genes Dev.2003; 17:1575-1580.
[50]Withers DJ, Gutierrez JS, Towery H, et al. Disruption of IRS-2 causes type 2 diabetes in mice. Nature.1998; 391:900-904.
[51]Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev.2004; 18: 2195-2224.
[2]Chalmers J, Cooper ME. UKPDS and the legacy effect. N Engl J Med.2008; 359:1618-1620.
[3]Mathis D, Vence L, Benoist C. beta-Cell death during progression to diabetes. Nature.2001; 414:792-798.
[4]Maedler K, Donath MY. Beta-cells in type 2 diabetes:a loss of function and mass. Horm Res.2004; 62 Suppl 3:67-73.
[5]Maclean N, Ogilvie RE Quantitative estimation of the pancreatic islet tissue in diabetic subjects. Diabetes.1955; 4:367-376.
[6]Hou ZQ, Li HL, Gao L, Pan L, Zhao JJ, Li GW. Involvement of chronic stresses in rat islet and INS-1 cell glucotoxicity induced by intermittent high glucose. Mol Cell Endocrinol.2008; 291:71-78.
[7]Federici M, Hribal M, Perego L, et al. High glucose causes apoptosis in cultured human pancreatic islets of Langerhans:a potential role for regulation of specific Bcl family genes toward an apoptotic cell death program. Diabetes.2001; 50: 1290-1301.
[8]Lupi R, Dotta F, Marselli L, et al. Prolonged exposure to free fatty acids has cytostatic and pro-apoptotic effects on human pancreatic islets:evidence that beta-cell death is caspase mediated, partially dependent on ceramide pathway, and Bcl-2 regulated. Diabetes.2002; 51:1437-1442.
[9]Piro S, Anello M, Di Pietro C, et al. Chronic exposure to free fatty acids or high glucose induces apoptosis in rat pancreatic islets:possible role of oxidative stress. Metabolism.2002; 51:1340-1347.
[10]Schmidt MI, Duncan BB, Sharrett AR, et al. Markers of inflammation and prediction of diabetes mellitus in adults (Atherosclerosis Risk in Communities study): a cohort study. Lancet.1999; 353:1649-1652.
[11]Crook MA, Tutt P, Simpson H, Pickup JC. Serum sialic acid and acute phase proteins in type 1 and type 2 diabetes mellitus. Clin Chim Acta.1993; 219:131-138.
[12]Pickup JC, Chusney GD, Thomas SM, Burt D. Plasma interleukin-6, tumour necrosis factor alpha and blood cytokine production in type 2 diabetes. Life Sci.2000; 67:291-300.
[13]Yuan M, Konstantopoulos N, Lee J, et al. Reversal of obesity-and diet-induced insulin resistance with salicylates or targeted disruption of Ikkbeta. Science.2001; 293:1673-1677.
[14]Hundal RS, Petersen KF, Mayerson AB, et al. Mechanism by which high-dose aspirin improves glucose metabolism in type 2 diabetes. J Clin Invest.2002; 109: 1321-1326.
[15]Andersson AK, Borjesson A, Sandgren J, Sandler S. Cytokines affect PDX-1 expression, insulin and proinsulin secretion from iNOS deficient murine islets. Mol Cell Endocrinol.2005; 240:50-57.
[16]Oetjen E, Blume R, Cierny I, et al. Inhibition of MafA transcriptional activity and human insulin gene transcription by interleukin-lbeta and mitogen-activated protein kinase kinase kinase in pancreatic islet beta cells. Diabetologia.2007; 50: 1678-1687.
[17]Veluthakal R, Jangati GR, Kowluru A. IL-1beta-induced iNOS expression, NO release and loss in metabolic cell viability are resistant to inhibitors of ceramide synthase and sphingomyelinase in INS 832/13 cells. JOP.2006; 7:593-601.
[18]Eizirik DL, Mandrup-Poulsen T. A choice of death--the signal-transduction of immune-mediated beta-cell apoptosis. Diabetologia.2001; 44:2115-2133.
[19]Siegel RM, Frederiksen JK, Zacharias DA, et al. Fas preassociation required for apoptosis signaling and dominant inhibition by pathogenic mutations. Science.2000; 288:2354-2357.
[20]Curtin JF, Cotter TG. Live and let die:regulatory mechanisms in Fas-mediated apoptosis. Cell Signal.2003; 15:983-992.
[21]Donath MY, Storling J, Maedler K, Mandrup-Poulsen T. Inflammatory mediators and islet beta-cell failure:a link between type 1 and type 2 diabetes. J Mol Med.2003; 81:455-470.
[22]Maedler K, Sergeev P, Ris F, et al. Glucose-induced beta cell production of IL-1beta contributes to glucotoxicity in human pancreatic islets. J Clin Invest.2002; 110:851-860.
[23]Donath MY, Ehses JA, Maedler K, et al. Mechanisms of beta-cell death in type 2 diabetes. Diabetes.2005; 54 Suppl 2:S108-113.
[24]Bell GI, Santerre RF, Mullenbach GT. Hamster preproglucagon contains the sequence of glucagon and two related peptides. Nature.1983; 302:716-718.
[25]Turton MD, O'Shea D, Gunn I, et al. A role for glucagon-like peptide-1 in the central regulation of feeding. Nature.1996; 379:69-72.
[26]Gonzalez C, Beruto V, Keller G, Santoro S, Di Girolamo G. Investigational treatments for Type 2 diabetes mellitus:exenatide and liraglutide. Expert Opin Investig Drugs.2006; 15:887-895.
[27]Zander M, Madsbad S, Madsen JL, Hoist JJ. Effect of 6-week course of glucagon-like peptide 1 on glycaemic control, insulin sensitivity, and beta-cell function in type 2 diabetes:a parallel-group study. Lancet.2002; 359:824-830.
[28]Kolterman OG, Buse JB, Fineman MS, et al. Synthetic exendin-4 (exenatide) significantly reduces postprandial and fasting plasma glucose in subjects with type 2 diabetes. J Clin Endocrinol Metab.2003; 88:3082-3089.
[29]Dillon JS, Lu M, Bowen S, Homan LL. The recombinant rat glucagon-like peptide-1 receptor, expressed in an alpha-cell line, is coupled to adenylyl cyclase activation and intracellular calcium release. Exp Clin Endocrinol Diabetes.2005; 113:182-189.
[30]Fehmann HC, Hering BJ, Wolf MJ, et al. The effects of glucagon-like peptide-I (GLP-I) on hormone secretion from isolated human pancreatic islets. Pancreas.1995; 11:196-200.
[31]Holz GG, Leech CA, Heller RS, Castonguay M, Habener JF. cAMP-dependent mobilization of intracellular Ca2+ stores by activation of ryanodine receptors in pancreatic beta-cells. A Ca2+signaling system stimulated by the insulinotropic hormone glucagon-like peptide-1-(7-37). JBiol Chem.1999; 274:14147-14156.
[32]Skoglund G, Hussain MA, Holz GG. Glucagon-like peptide 1 stimulates insulin gene promoter activity by protein kinase A-independent activation of the rat insulin I gene cAMP response element. Diabetes.2000; 49:1156-1164.
[33]Abraham EJ, Leech CA, Lin JC, Zulewski H, Habener JF. Insulinotropic hormone glucagon-like peptide-1 differentiation of human pancreatic islet-derived progenitor cells into insulin-producing cells. Endocrinology.2002; 143:3152-3161.
[34]Tourrel C, Bailbe D, Meile MJ, Kergoat M, Portha B. Glucagon-like peptide-1 and exendin-4 stimulate beta-cell neogenesis in streptozotocin-treated newborn rats resulting in persistently improved glucose homeostasis at adult age. Diabetes.2001; 50:1562-1570.
[35]Li Y, Hansotia T, Yusta B, Ris F, Halban PA, Drucker DJ. Glucagon-like peptide-1 receptor signaling modulates beta cell apoptosis. J Biol Chem.2003; 278: 471-478.
[36]Buteau J, El-Assaad W, Rhodes CJ, Rosenberg L, Joly E, Prentki M. Glucagon-like peptide-1 prevents beta cell glucolipotoxicity. Diabetologia.2004; 47: 806-815.
[37]Boni-Schnetzler M, Thorne J, Parnaud G, et al. Increased interleukin (IL)-1beta messenger ribonucleic acid expression in beta-cells of individuals with type 2 diabetes and regulation of IL-1beta in human islets by glucose and autostimulation. J Clin Endocrinol Metab.2008; 93:4065-4074.
[38]Kim MJ, Ryu GR, Kang JH, et al. Inhibitory effects of epicatechin on interleukin-1beta-induced inducible nitric oxide synthase expression in RINm5F cells and rat pancreatic islets by down-regulation of NF-kappaB activation. Biochem Pharmacol.2004; 68:1775-1785.
[39]Giannoukakis N, Rudert WA, Trucco M, Robbins PD. Protection of human islets from the effects of interleukin-1beta by adenoviral gene transfer of an Ikappa B repressor. J Biol Chem.2000; 275:36509-36513.
[40]Iran PO, Gleason CE, Robertson RP. Inhibition of interleukin-1beta-induced COX-2 and EP3 gene expression by sodium salicylate enhances pancreatic islet beta-cell function. Diabetes.2002; 51:1772-1778.
[41]Farilla L, Bulotta A, Hirshberg B, et al. Glucagon-like peptide 1 inhibits cell apoptosis and improves glucose responsiveness of freshly isolated human islets. Endocrinology.2003; 144:5149-5158.
[42]刘礼斌,王燕萍,潘晓东,姜苏原,陈洲.Exendin-4体外通过抑制NF-KB-iNOS-NO信号减轻氧化应激诱导的小鼠MIN6胰岛β细胞凋亡.药学学报2008：0513-4870.
[43]Li L, El-Kholy W, Rhodes CJ, Brubaker PL. Glucagon-like peptide-1 protects beta cells from cytokine-induced apoptosis and necrosis:role of protein kinase B. Diabetologia.2005; 48:1339-1349.
[44]Li LX, Yoshikawa H, Egeberg KW, Grill V. Interleukin-1beta swiftly down-regulates UCP-2 mRNA in beta-cells by mechanisms not directly coupled to toxicity. Cytokine.2003; 23:101-107.
[45]Tellez N, Montolio M, Biarnes M, Castano E, Soler J, Montanya E. Adenoviral overexpression of interleukin-1 receptor antagonist protein increases beta-cell replication in rat pancreatic islets. Gene Ther.2005; 12:120-128.
[46]Ferdaoussi M, Abdelli S, Yang JY, et al. Exendin-4 protects beta-cells from interleukin-1 beta-induced apoptosis by interfering with the c-Jun NH2-terminal kinase pathway. Diabetes.2008; 57:1205-1215.
[47]Drucker DJ. The biology of incretin hormones. Cell Metab.2006; 3:153-165.
[48]Hui H, Nourparvar A, Zhao X, Perfetti R. Glucagon-like peptide-1 inhibits apoptosis of insulin-secreting cells via a cyclic 5'-adenosine monophosphate-dependent protein kinase A-and a phosphatidylinositol 3-kinase-dependent pathway. Endocrinology.2003; 144:1444-1455.
[49]Jhala US, Canettieri G, Screaton RA, et al. cAMP promotes pancreatic beta-cell survival via CREB-mediated induction of IRS2. Genes Dev.2003; 17:1575-1580.
[50]Withers DJ, Gutierrez JS, Towery H, et al. Disruption of IRS-2 causes type 2 diabetes in mice. Nature.1998; 391:900-904.
[51]Hayden MS, Ghosh S. Signaling to NF-kappaB. Genes Dev.2004; 18: 2195-2224.