蛋白酪氨酸磷酸酶1B对β细胞胰岛素分泌功能及其胰岛素信号转导通路的影响
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摘要
[背景]肥胖、2型糖尿病目前已成为世界流行病。据统计:到2007年,全世界超重者已超过11亿,其中3.12亿人口为肥胖者。糖尿病也已成为危害全世界人民健康的重大问题,到2030年,预计全世界糖尿病患病人数将从2000年的1.71亿发展为3.66亿。肥胖,尤其是腹型肥胖,是导致胰岛素抵抗(Insulin resistance)的重要因素。而2型糖尿病的主要病理生理特征亦为胰岛素抵抗。存在胰岛素抵抗的人群较一般人群发生糖尿病的风险增高5倍,心血管风险增高2倍。因此,如何减轻肥胖诱导的胰岛素抵抗,成为预防和治疗糖尿病、减少心血管风险的关键问题。
目前对胰岛素抵抗的研究主要集中在对传统意义上的胰岛素敏感组织如脂肪、肌肉、肝脏、下丘脑等的研究。初步认为:增加运动、生活方式改善可增加脂肪消耗、提高肌肉对葡萄糖的利用,改善外周组织胰岛素抵抗。但人们发现:β细胞存在胰岛素抵抗,它表现为β细胞胰岛素信号转导障碍,导致β细胞分泌胰岛素减少。β细胞胰岛素抵抗这一概念的提出对胰岛素抵抗的概念做了更进一步的补充,β细胞成为又一胰岛素敏感组织,而重新被人们认识。因此,对胰岛β细胞胰岛素抵抗的研究是治疗肥胖和2型糖尿病的又一有益补充。
既往文献报道:β细胞胰岛素受体基因条件性敲除(conditional knockout)(βIRKO)小鼠可出现与人2型糖尿病初期相似的临床特征:即葡萄糖刺激的胰岛素第一相分泌(GSIS)丧失85%以上,βIRKO于2月龄出现糖耐量受损,并且糖耐量进行性恶化。这说明β细胞的胰岛素一相分泌需要胰岛素受体的存在,胰岛素信号通路障碍使β细胞对胰岛素也发生了抵抗,表现为β细胞分泌异常。因此,人们首次认识到胰岛β细胞胰岛素抵抗。这种β细胞胰岛素抵抗可诱导糖耐量异常和2型糖尿病的发生。然而,对于β细胞胰岛素抵抗的研究,尚处于起步阶段,相关文献报道甚少。
在分子水平,胰岛素的信号是以胰岛素受体(insulin receptor, IR)的激活导致自身磷酸化开始,然后激活的受体引起几个底物的酪氨酸磷酸化,包括胰岛素受体底物1和2(insulin receptor substrate-1and 2, IRS-1, IRS-2)等。IRS-1和IRS-2酪氨酸磷酸化后,会激活磷脂酰肌醇(-3)激酶(phosphatidylinositol 3 kinase, PI3-K) ,激活的PI3-K引起蛋白激酶B(PKB/Akt)丝氨酸磷酸化,从而刺激葡萄糖转运到肝脏、肌肉和脂肪等组织,在肝脏和肌肉组织中刺激糖原合成,在脂肪组织中促进脂肪合成。目前认为:胰岛细胞上已存在胰岛素信号通路,但该通路在胰岛细胞如何发挥作用,受哪些因素影响,机制如何尚需要进一步研究阐明。对于传统意义上的胰岛素抵抗,近年来很多研究显示:蛋白酪氨酸磷酸酶1B(Protein Tyrosine Phosphatase-1B, PTP-1B)可能在胰岛素抵抗中发挥了重要的作用。它是胰岛素信号转导中一个十分关键的分子。通过去磷酸化信号分子,从而终止胰岛素信号转导。PTP-1B缺失小鼠,其表型及寿命都正常,同时胰岛素和瘦素的敏感性明显增加,能够抵抗饮食诱导的肥胖的发生。许多研究也进一步证实PTP-1B可以负性调节胰岛素和瘦素的信号转导。但在胰岛细胞胰岛素抵抗中,PTP-1B是否也发挥着类似于其他胰岛素敏感组织中的作用,需要进一步探讨。
第一部分蛋白酪氨酸磷酸酶1B在高糖/高脂诱导β细胞胰岛素抵抗中的作用
[目的]研究胰岛β细胞胰岛素抵抗的诱发因素及其与蛋白酪氨酸磷酸酶1B(PTP-1B)的关系。
[方法]传代培养处于对数生长期的胰岛beta细胞MIN6细胞株分为四组,①高糖组:分别加入终浓度为33.3mmol/L、40mmol/L葡萄糖,孵育24 h;②高脂组:分别加入终浓度125umol/L、250umol/L软脂酸,孵育24h;③无水乙醇对照组:与高脂组相对应,加入相同体积无水乙醇,孵育24h;④正常对照组:不加葡萄糖或软脂酸或无水乙醇,其他培养条件相同。应用Trizol试剂盒分别提取高糖、高脂、无水乙醇及对照组的细胞总RNA。采用realtime-PCR法检测各组PTP-1B基因表达水平。免疫细胞化学染色观察胰岛beta细胞PTP-1B的表达。Western-blot免疫印迹法检测PTP-1B蛋白表达水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果]高糖40mmol/L孵育可成功诱导β细胞胰岛素抵抗,胰岛细胞分泌功能显著下降。与相应的正常对照组相比,当葡萄糖浓度为40mmol/L时,基线胰岛素分泌水平下降(P<0.05);高糖刺激后胰岛素分泌明显下降(P<0.01)。但当葡萄糖浓度为33.3mmol/L时,β细胞胰岛素分泌水平基线时较正常对照组略升高(P=0.05),而高糖刺激时与正常对照组相比无明显差异(P>0.05)。
2、软脂酸亦可诱导β细胞胰岛素抵抗,胰岛细胞分泌功能下降。
当软脂酸浓度为125umol/L时,β细胞胰岛素分泌水平基线时与正常对照组相似(P>0.05),而高糖刺激时与正常对照组相比明显降低(P<0.01);当软脂酸浓度为250umol/L时,基线胰岛素分泌较正常对照组升高(P<0.05),高糖刺激后明显下降,降幅与正常对照组的降幅相比,具有统计学差异(P<0.05)。无水乙醇组与高脂B组胰岛素分泌结果相似,与正常对照组亦无显著差异(P>0.05)。
3、高糖、高脂均可诱导β细胞PTP-1B表达增高
(1)免疫细胞化学:不同处理因素与对照组相比,各处理因素PTP-1B的表达均存在非常显著性差异(P=0.000)。高糖33.3mmol/L组与正常对照组PTP-1B表达无明显差异,高脂250umol/L组与正常对照组相比,PTP-1B表达亦无显著性差异,但仍有表达增高趋势。正常组与高脂125umol/L组间PTP-1B表达存在显著差异(P=0.000),正常组与高糖40mmol/L组间PTP-1B表达也存在显著差异(P=0.000);高糖40mmol/L组PTP-1B表达较高脂125umol/L组略增高,但差异无显著意义(P=0.211)。
(2)Realtime-PCR提示高糖、高脂诱导后,PTP-1B基因表达水平增高高糖40mmol/L组、高脂125umol/L组诱导胰岛β细胞PTP-1B基因表达增高PTP-1B/β-actin基因的溶解曲线;显示呈单峰,无明显引物二聚体,峰值与目的扩增片段Tm一致;各组PTP-1B/β-actin基因的表达量及其相对比值可见:高糖和高脂诱导组PTP-1B基因的表达水平均显著高于对照组(P<0.05),分别升高50倍及25倍,高糖40mmol/L组PTP-1B基因的表达水平较高脂125umol/L组高约1倍。
(3)Western-blot提示高糖、高脂作用于β细胞,使PTP-1B蛋白表达增高。高糖40mmol/L、高脂125umol/L组PTP-1B表达均显著高于对照组(P<0.05),其中,高糖组PTP-1B表达又显著高于高脂组(P<0.05)。
[结论]高糖、高脂均可诱导β细胞葡萄糖刺激的胰岛素分泌减少,发生胰岛β细胞胰岛素抵抗,但二者对β细胞的分泌功能的影响略有差异,β细胞胰岛素抵抗的同时,PTP-1BmRNA、蛋白表达均升高。高糖、高脂等外源性因素可能通过PTP-1B表达升高,抑制胰岛素的信号转导。PTP-1B在高糖、高脂诱导的β细胞胰岛素抵抗中发挥着重要的作用。
第二部分蛋白酪氨酸磷酸酶1B在慢性炎症因子诱导β细胞胰岛素抵抗中的作用
[目的]研究胰岛β细胞胰岛素抵抗的诱发因素及其与蛋白酪氨酸磷酸酶1B(PTP-1B)的关系。
[方法]传代培养处于对数生长期的胰岛beta细胞MIN6细胞株分为四组,①TNF-αA组:加入终浓度为1.0 ug/L TNF-α,孵育24 h;②TNF-αB组:加入终浓度10 ug/L TNF-α,孵育24h;③TNF-αC组:加入终浓度20 ug/L TNF-α,孵育24h;④正常对照组:不加TNF-α,其他培养条件相同。应用Trizol试剂盒分别提取各组的细胞总RNA。采用RT-PCR法检测各组PTP-1B基因表达水平。Western-blot免疫印迹法检测PTP-1B蛋白表达水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果] 1.当不同浓度TNF-α(1ug/L、10ug/L、20ug/L)分别诱导胰岛细胞胰岛素抵抗时,可以发现:MIN6细胞胰岛素分泌能力明显下降,但各干预组之间并未呈现剂量依赖性变化趋势(P<0.01)。各组基线水平的胰岛素释放均较正常组降低,高糖刺激后胰岛素释放也较正常对照组明显减低,但TNF-α10ug/L、20ug/L组间无明显差异(P>0.05)。
2.TNF-α诱导β细胞PTP-1B基因表达变化:各组TNF-α均诱导β细胞PTP-1B基因表达增多,其中20ug/L、1ug/L干预组PTP-1B表达明显增多(P<0.01),10ug/L干预组PTP-1B表达亦明显增加(P<0.05)。
3.TNF-α诱导β细胞PTP-1B蛋白水平变化:免疫印迹检测结果显示,经内参照β-Actin蛋白校正后,TNF-α干预各组较正常对照组,PTP-1B表达增加(P<0.05)。
[结论] TNF-α可诱导β细胞葡萄糖刺激的胰岛素分泌减少,发生胰岛β细胞胰岛素抵抗,但各干预组之间并未呈现剂量依赖性变化趋势。TNF-α通过PTP-1B表达升高,抑制胰岛素的信号转导。PTP-1B在慢性炎症诱导的β细胞胰岛素抵抗中发挥着重要的作用。
第三部分过表达蛋白酪氨酸磷酸酶1B对胰岛β细胞分泌功能的影响
[目的]明确蛋白酪氨酸磷酸酶1B对胰岛β细胞胰岛素分泌功能的影响。
[方法]传代培养处于对数生长期的HEK293细胞,扩增Ad-PTP-1B腺病毒,氯化铯密度梯度离心纯化病毒并测定病毒滴度;传代培养处于对数生长期的胰岛beta细胞MIN6细胞株转染MIN6细胞株,分三组:①转染Ad-PTP-1B胰岛β细胞组;②转染Ad-EGFP胰岛β细胞组;③胰岛β细胞正常对照组。应用Trizol试剂盒分别提取各组的细胞总RNA。采用RT-PCR法检测各组PTP-1B基因表达水平。Western-blot免疫印迹法检测PTP-1B蛋白表达水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果] 1、氯化铯密度梯度离心法纯化病毒后,取病毒液1:1稀释后测得A260为0.029,根据公式计算病毒滴度为:3.19×1010pfu/ml。
2、重组腺病毒PCR鉴定:经RT-PCR对病毒DNA鉴定,在大约85bp处可见一清晰条带。鉴定PTP-1B病毒。
3.以MOI 1:10,1:50,1:100分别感染MIN6细胞,western-blot检测可见:随着感染复数的升高,PTP-1B转染效率逐渐升高,其中,以MOI 1:100感染效率最高,故在后续的实验中拟用MOI 1:100感染MIN6细胞。
4.PTP-1B过表达MIN6细胞,胰岛素释放试验结果:Ad-PTP-1B转染MIN6细胞24小时后,可见转染PTP-1B组的MIN6细胞胰岛素分泌显著(P<0.01),而转染绿色荧光蛋白对照病毒的MIN6细胞胰岛素分泌Ad-PTP-1B组相比,明显升高(P<0.05)。与正常组相比,亦有增高趋势,但差异无统计学意义。
5.重组腺病毒感染MIN6细胞,PTP-1B蛋白表达结果:PTP-1B感染MIN6细胞后,表达明显增强,与GFP和空白对照均有显著性差异(P<0.05)。
[结论] PTP-1B在胰岛β细胞表达升高后,可诱导β细胞胰岛素分泌功能降低。提示:PTP-1B直接作用于胰岛β细胞,引发胰岛β细胞胰岛素抵抗。PTP-1B在胰岛β细胞胰岛素抵抗中发挥着重要的作用。
第四部分PTP-1B诱导β细胞胰岛素抵抗胰岛素信号途径变化
[目的]观察PTP-1B诱发β细胞胰岛素抵抗的可能机制及其对胰岛素信号的影响。
[方法]传代培养处于对数生长期的HEK293细胞,扩增Ad-PTP-1B腺病毒,氯化铯密度梯度离心纯化病毒并测定病毒滴度;传代培养处于对数生长期的胰岛beta细胞MIN6细胞株转染MIN6细胞株,分三组:①转染Ad-PTP-1B胰岛β细胞组;②转染Ad-EGFP胰岛β细胞组;③胰岛β细胞正常对照组。应用Trizol试剂盒分别提取各组的细胞总RNA。采用RT-PCR法检测各组PTP-1B基因表达水平。Western-blot免疫印迹法检测胰岛素受体β亚单位(IR-β)、胰岛素受体底物-1(IRS-1)蛋白表达水平,免疫沉淀法检测IR-β、IRS-1磷酸化水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果] 1.转染PTP-1B的β细胞在无胰岛素刺激时,胰岛素受体β亚单位表达与正常对照组无明显差异,但受到胰岛素刺激后,IRβ磷酸化明显减弱。而转染GFP对照病毒组与正常对照组无明显差异。提示:PTP-1B表达增高,可引发IRβ去磷酸,阻碍胰岛素信号转导;
2.转染PTP-1B的β细胞在无胰岛素刺激时,胰岛素受体底物1(IRS-1)表达较正常对照组略偏低,但差异不显著。当受到胰岛素刺激后,IRS-1磷酸化明显减弱。而转染GFP对照病毒组与正常对照组无明显差异。提示:PTP-1B表达增高,可引发IRS-1去磷酸,阻碍胰岛素信号转导。
[结论]胰岛细胞过表达PTP-1B后,胰岛素受体β亚单位(IRβ)、胰岛素底物1(IRS-1)磷酸化程度明显减弱,提示PTP-1B通过负调控胰岛素信号通路上IRβ、IRS-1磷酸化程度,使胰岛素信号转导发生障碍,从而诱导胰岛细胞胰岛素抵抗。
Introduction
Obetsity and Type 2 Diabetes become the most popular diseases all over the world. The epidemic data showed that there had been 1,100,000,000 people who were overweight, and even 312,000,000 of them had suffered from obesity. Type 2 Diabetes is also popular and harmful to people. It is estimated that the population of type 2 DM will up to 366,000,000 in 2030. As we know, the common pathogenesis of obesity and type 2 DM is insulin resistance and data showed that people with insulin resistance had 5-fold higher risk of type 2 Diabetes as well as 2-fold higher risk of cardiovascular events than people without insulin resistantance. Insulin resistance become a key point in type 2 diabetes and obesity treatment. Most studies focused on insulin resistance of the typical insulin-sensitive tissue such as adipose, muscle, liver and hypothalamus.These studies concluded that increasing exercises, modifying daily-life style can increase fat comsuptoin, improve glucose utilization of muscle and thus ameliorate peripheral insulin resistance. However, beta cell also had insulin resistance which was characterized as impaired insulin secretion by glucose stimulated insulin secretion test (GSIS) and insulin signaling obstruction. The new concept of beta-cell insulin resistance act as a supplement of the theory of typical insulin resistance.
Previous study indicate that insulin receptor-βsubunit conditional knockout mice(βIRKO) lost more than 85% insulin secretion by GSIS and moved on to impaired glucose torlence (IGT) in 2 -month -years old. This demonstrate that insulin signaling is essential toβcell itself for insulin secretion. However, the exact mechanism ofβcell insulin resistance remained unclear.At the molecular level, the insulin signaling begins with activation of insulin receptor (IR) resulted in tyrosine phosphorylation of serials of substrates, including the IR substrate 1 (IRS-1) and IRS-2. After tyrosine phosphorylation of IRS-1 and IRS-2, they bind and activate the enzyme- phosphatidylinositol 3-kinase (PI3-K). The activation of PI3-K increases serine phosphorylation of protein kinase B (Akt), which in turn stimulates the glucose transport in the muscle and adipose tissue, glycogen synthesis in the liver and muscle, and lipogenesis in the adipose tissue. Recent studies suggest that protein tyrosine phosphatase-1B (PTP-1B) was an important negative regulator of insulin and leptin signal transduction. However, whether PTP-1B has a similar effect on beta cell insulin resistance remained unclear.
Part One: Expression of PTP-1B in beta cell insulin resistance induced by glucose and palmitic acid.
Objective
In this study, we investigated the causative factors ofβcell insulin resistance and the expression of PTP-1B. We also observed the relationship between PTP-1B andβcell insulin secreting function.
Methods
MIN6 cells under serial subcultivation were divided into four groups:①glucose treatment: the final concentration of glucose was 33.3mmol/L and 40mmol/L, respectively; incubated for 24 hours;②palmitic acid treatment (PA group): the final concentration of PA was 125umol/L, 250umol/L, respectively; incubated for 24 hours;③dehydrated-alcohol control group: added identical volume of dehydrated alcohol compared with PA group; incubated for 24 hours;④control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase.Realtime-PCR was used to detect the expression of PTP-1B mRNA level.Immunocytochemical stain and western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1. glucose concentration of 40mmol/L treatment induce beta cell insulin resistance successfully and the glucose stimulated insulin secretion reduced significantly.Compared with the control group, the basic insulin secretion decreased significantly(P<0.05) while insulin levels were much lower in high glucose concentration(P<0.01).However, this phenomenon was not seen in group of 33.3mmol/L glucose treatment . In contrast, the base line of insulin secretion in this group increased slightly and no differences of insulin secretion were seen comparing with control group.
2. Palmitic acid treatment also induce beta cell insulin resistance, resulted in the decrease of insulin secretion. Although the base line insulin releasing was similar to control group in PA of 125umol/L,the high glucose stimulated insulin secretion was much lower than control group. In group of 250umol/L PA,decreasing insulin secretion was only seen in high glucose stimulation(P<0.05). And there no differences of insulin secretion between dehydrated-alcohol control group and normol control.
3. The expressions of protein tyrosine phosphatase-1B were much higher in either glucose or PA group than in control group.
(1) Immunocytochemical stain showed that the expressions of PTP-1B obviously increased in glucose treatment (40mmol/L) and PA (125umol/L) group, respectively (P=0.000).Furthermore, the expression of PTP-1B was much higher in glucose treatment than in PA group.However, Neither 33.3mmol/L glucose treatment nor 250umol/L PA group had increased expression of PTP-1B,compared with control group.
(2) Realtime- Polymerase Chain Reaction (Realtime-PCR) is a simple and powerful method to amplify PTP-1B gene of a tiny amount. Amplified products are monitored in real time.In both glucose treatment (40mmol/L) and PA (125umol/L) group, the ratio of PTP-1B /β-actin were higher.Compared with control, the expression of PTP-1B mRNA level increased 50 fold in glucose treatment and 25 fold in PA group.
(3)Western-bloting showed that the expression of PTP-1B protein in each group was much higher than control.And there were similar results consistent wiht realtime-PCR results.
Conclusion:
Both glucose concentration of 40mmol/L treatment and palmitic acid treatment induce beta cell insulin resistance, resulted in the decrease of insulin secretion. The expressions of protein tyrosine phosphatase-1B were much higher in these groups than in control group.
PTP-1B may be an important regulator in the development of beta cell insulin resistance induced by glucose or PA.
Part Two: Expression of PTP-1B in beta cell insulin resistance induced by chronic stimulation of inflammatory factor.
Objective : In this study, we investigated the causative factors ofβcell insulin resistance and the expression of PTP-1B. We also observed the relationship between PTP-1B andβcell insulin secreting function.
Methods
MIN6 cells under serial subcultivation were divided into four groups:①Tumor necrosis factor-α(TNF-α)treatment group A :with final concentration of 1.0 ug/L; incubated for 24 hours;②TNF-αtreatment group B :with final concentration of 10 ug/L; incubated for 24 hours;③TNF-αtreatment group C :with final concentration of 20 ug/L ;incubated for 24 hours;④control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1.Different concentrations of TNF-αinduced beta cell insulin resistance and the insulin secretion reduced sinificantly in these three groups vs control group. However, TNF-αdid not have concentration-dependent effects on beta cell. Both TNF-αB group and A group showed the same effects on the severity of beta cell dysfunction.
2. The expression of PTP-1B mRNA increased in MIN6 cells induced by TNF-α.
3.The expression of PTP-1B protein increased in MIN6 cells induced by TNF-α. Western-bloting showed that PTP-1B protein expression was much higher in TNF-αintervention group.
Conclusion:
TNF-αinduce beta cell insulin resistance, resulted in the decrease of insulin secretion. However, TNF-αdid not have concentration-dependent effects on beta cell.Meanwhile, the expressions of protein tyrosine phosphatase-1B were much higher in TNF-αtreatment groups than in control group. PTP-1B may be an important regulator in the development of beta cell insulin resistance induced by stimulation of inflammatory factor.
Part Three: Effects of overexpression of PTP-1B on beta cell insulin secretion.
Objective :
In this part, we investigated the effects of overexpression of PTP-1B on beta cell insulin secretion.
Methods:
HEK293 cell line were serial subcultivated to amplify Ad-PTP-1B adenovirus. Cesium chloride centrifugal purification was performed to purify Ad-PTP-1B adenovirus and virus titre was detected. MIN6 cells under serial subcultivation were divided into 3 groups:①MIN6 cells transfected with Ad-PTP-1B;②MIN6 cells transfected with Ad-EGFP;③control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1. Virus titre of Ad-PTP-1B was 3.19×1010pfu/ml.
2.MOI 1:100 was the most effective concentration for transfection.
3.Overexpression of PTP-1B in MIN6 cells resulted in the siginificant decrease of insulin secretion by GSIS vs. control. And there were no effects on MIN6 cell insulin secretion by transfected with Ad-EGFP.
Conclusions:
PTP-1B had direct negative effects on beta cell function and induced beta cell insulin resistance.Therefore, PTP-1B plays an important role in the development of beta cell insulin resistance.
Part Four: Effects of overexpression of PTP-1B on beta cell insulin signaling.
Objective:
In this part, we investigated the effects of overexpression of PTP-1B on beta cell insulin signaling.
Methods
HEK293 cell lines were serial subcultivated to amplify Ad-PTP-1B adenovirus. Cesium chloride centrifugal purification was performed to purify Ad-PTP-1B adenovirus and virus titre was detected. MIN6 cells under serial subcultivation were divided into 3 groups:①M IN6 cells transfected with Ad-PTP-1B;②MIN6 cells transfected with Ad-EGFP;③control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of IR-βand IRS-1. The tyrosine phosphorylations of IRS-1, IRβwere detected by immuno-precipitation. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1. The insulin-stimulated tyrosine phosphorylation of IRβin MIN6 cells in Ad-PTP-1B group were significantly lower than that in control group (P < 0.05).
2. The insulin-stimulated tyrosine phosphorylation of IRS-1 in MIN6 cells in Ad-PTP-1B group were significantly lower than that in control group (P < 0.05).
Conclusions:
The expression of PTP-1B increased to suppress the insulin signal transduction in beta cell and insulin-stimulated tyrosine phosphorylation of both IRβand IRS-1 decreased in beta cell.According to these results, it is supposed that the signal transduction pathway of insulin is the key part of beta cell insulin resistance induced by PTP-1B.
目前对胰岛素抵抗的研究主要集中在对传统意义上的胰岛素敏感组织如脂肪、肌肉、肝脏、下丘脑等的研究。初步认为:增加运动、生活方式改善可增加脂肪消耗、提高肌肉对葡萄糖的利用,改善外周组织胰岛素抵抗。但人们发现:β细胞存在胰岛素抵抗,它表现为β细胞胰岛素信号转导障碍,导致β细胞分泌胰岛素减少。β细胞胰岛素抵抗这一概念的提出对胰岛素抵抗的概念做了更进一步的补充,β细胞成为又一胰岛素敏感组织,而重新被人们认识。因此,对胰岛β细胞胰岛素抵抗的研究是治疗肥胖和2型糖尿病的又一有益补充。
既往文献报道:β细胞胰岛素受体基因条件性敲除(conditional knockout)(βIRKO)小鼠可出现与人2型糖尿病初期相似的临床特征:即葡萄糖刺激的胰岛素第一相分泌(GSIS)丧失85%以上,βIRKO于2月龄出现糖耐量受损,并且糖耐量进行性恶化。这说明β细胞的胰岛素一相分泌需要胰岛素受体的存在,胰岛素信号通路障碍使β细胞对胰岛素也发生了抵抗,表现为β细胞分泌异常。因此,人们首次认识到胰岛β细胞胰岛素抵抗。这种β细胞胰岛素抵抗可诱导糖耐量异常和2型糖尿病的发生。然而,对于β细胞胰岛素抵抗的研究,尚处于起步阶段,相关文献报道甚少。
在分子水平,胰岛素的信号是以胰岛素受体(insulin receptor, IR)的激活导致自身磷酸化开始,然后激活的受体引起几个底物的酪氨酸磷酸化,包括胰岛素受体底物1和2(insulin receptor substrate-1and 2, IRS-1, IRS-2)等。IRS-1和IRS-2酪氨酸磷酸化后,会激活磷脂酰肌醇(-3)激酶(phosphatidylinositol 3 kinase, PI3-K) ,激活的PI3-K引起蛋白激酶B(PKB/Akt)丝氨酸磷酸化,从而刺激葡萄糖转运到肝脏、肌肉和脂肪等组织,在肝脏和肌肉组织中刺激糖原合成,在脂肪组织中促进脂肪合成。目前认为:胰岛细胞上已存在胰岛素信号通路,但该通路在胰岛细胞如何发挥作用,受哪些因素影响,机制如何尚需要进一步研究阐明。对于传统意义上的胰岛素抵抗,近年来很多研究显示:蛋白酪氨酸磷酸酶1B(Protein Tyrosine Phosphatase-1B, PTP-1B)可能在胰岛素抵抗中发挥了重要的作用。它是胰岛素信号转导中一个十分关键的分子。通过去磷酸化信号分子,从而终止胰岛素信号转导。PTP-1B缺失小鼠,其表型及寿命都正常,同时胰岛素和瘦素的敏感性明显增加,能够抵抗饮食诱导的肥胖的发生。许多研究也进一步证实PTP-1B可以负性调节胰岛素和瘦素的信号转导。但在胰岛细胞胰岛素抵抗中,PTP-1B是否也发挥着类似于其他胰岛素敏感组织中的作用,需要进一步探讨。
第一部分蛋白酪氨酸磷酸酶1B在高糖/高脂诱导β细胞胰岛素抵抗中的作用
[目的]研究胰岛β细胞胰岛素抵抗的诱发因素及其与蛋白酪氨酸磷酸酶1B(PTP-1B)的关系。
[方法]传代培养处于对数生长期的胰岛beta细胞MIN6细胞株分为四组,①高糖组:分别加入终浓度为33.3mmol/L、40mmol/L葡萄糖,孵育24 h;②高脂组:分别加入终浓度125umol/L、250umol/L软脂酸,孵育24h;③无水乙醇对照组:与高脂组相对应,加入相同体积无水乙醇,孵育24h;④正常对照组:不加葡萄糖或软脂酸或无水乙醇,其他培养条件相同。应用Trizol试剂盒分别提取高糖、高脂、无水乙醇及对照组的细胞总RNA。采用realtime-PCR法检测各组PTP-1B基因表达水平。免疫细胞化学染色观察胰岛beta细胞PTP-1B的表达。Western-blot免疫印迹法检测PTP-1B蛋白表达水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果]高糖40mmol/L孵育可成功诱导β细胞胰岛素抵抗,胰岛细胞分泌功能显著下降。与相应的正常对照组相比,当葡萄糖浓度为40mmol/L时,基线胰岛素分泌水平下降(P<0.05);高糖刺激后胰岛素分泌明显下降(P<0.01)。但当葡萄糖浓度为33.3mmol/L时,β细胞胰岛素分泌水平基线时较正常对照组略升高(P=0.05),而高糖刺激时与正常对照组相比无明显差异(P>0.05)。
2、软脂酸亦可诱导β细胞胰岛素抵抗,胰岛细胞分泌功能下降。
当软脂酸浓度为125umol/L时,β细胞胰岛素分泌水平基线时与正常对照组相似(P>0.05),而高糖刺激时与正常对照组相比明显降低(P<0.01);当软脂酸浓度为250umol/L时,基线胰岛素分泌较正常对照组升高(P<0.05),高糖刺激后明显下降,降幅与正常对照组的降幅相比,具有统计学差异(P<0.05)。无水乙醇组与高脂B组胰岛素分泌结果相似,与正常对照组亦无显著差异(P>0.05)。
3、高糖、高脂均可诱导β细胞PTP-1B表达增高
(1)免疫细胞化学:不同处理因素与对照组相比,各处理因素PTP-1B的表达均存在非常显著性差异(P=0.000)。高糖33.3mmol/L组与正常对照组PTP-1B表达无明显差异,高脂250umol/L组与正常对照组相比,PTP-1B表达亦无显著性差异,但仍有表达增高趋势。正常组与高脂125umol/L组间PTP-1B表达存在显著差异(P=0.000),正常组与高糖40mmol/L组间PTP-1B表达也存在显著差异(P=0.000);高糖40mmol/L组PTP-1B表达较高脂125umol/L组略增高,但差异无显著意义(P=0.211)。
(2)Realtime-PCR提示高糖、高脂诱导后,PTP-1B基因表达水平增高高糖40mmol/L组、高脂125umol/L组诱导胰岛β细胞PTP-1B基因表达增高PTP-1B/β-actin基因的溶解曲线;显示呈单峰,无明显引物二聚体,峰值与目的扩增片段Tm一致;各组PTP-1B/β-actin基因的表达量及其相对比值可见:高糖和高脂诱导组PTP-1B基因的表达水平均显著高于对照组(P<0.05),分别升高50倍及25倍,高糖40mmol/L组PTP-1B基因的表达水平较高脂125umol/L组高约1倍。
(3)Western-blot提示高糖、高脂作用于β细胞,使PTP-1B蛋白表达增高。高糖40mmol/L、高脂125umol/L组PTP-1B表达均显著高于对照组(P<0.05),其中,高糖组PTP-1B表达又显著高于高脂组(P<0.05)。
[结论]高糖、高脂均可诱导β细胞葡萄糖刺激的胰岛素分泌减少,发生胰岛β细胞胰岛素抵抗,但二者对β细胞的分泌功能的影响略有差异,β细胞胰岛素抵抗的同时,PTP-1BmRNA、蛋白表达均升高。高糖、高脂等外源性因素可能通过PTP-1B表达升高,抑制胰岛素的信号转导。PTP-1B在高糖、高脂诱导的β细胞胰岛素抵抗中发挥着重要的作用。
第二部分蛋白酪氨酸磷酸酶1B在慢性炎症因子诱导β细胞胰岛素抵抗中的作用
[目的]研究胰岛β细胞胰岛素抵抗的诱发因素及其与蛋白酪氨酸磷酸酶1B(PTP-1B)的关系。
[方法]传代培养处于对数生长期的胰岛beta细胞MIN6细胞株分为四组,①TNF-αA组:加入终浓度为1.0 ug/L TNF-α,孵育24 h;②TNF-αB组:加入终浓度10 ug/L TNF-α,孵育24h;③TNF-αC组:加入终浓度20 ug/L TNF-α,孵育24h;④正常对照组:不加TNF-α,其他培养条件相同。应用Trizol试剂盒分别提取各组的细胞总RNA。采用RT-PCR法检测各组PTP-1B基因表达水平。Western-blot免疫印迹法检测PTP-1B蛋白表达水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果] 1.当不同浓度TNF-α(1ug/L、10ug/L、20ug/L)分别诱导胰岛细胞胰岛素抵抗时,可以发现:MIN6细胞胰岛素分泌能力明显下降,但各干预组之间并未呈现剂量依赖性变化趋势(P<0.01)。各组基线水平的胰岛素释放均较正常组降低,高糖刺激后胰岛素释放也较正常对照组明显减低,但TNF-α10ug/L、20ug/L组间无明显差异(P>0.05)。
2.TNF-α诱导β细胞PTP-1B基因表达变化:各组TNF-α均诱导β细胞PTP-1B基因表达增多,其中20ug/L、1ug/L干预组PTP-1B表达明显增多(P<0.01),10ug/L干预组PTP-1B表达亦明显增加(P<0.05)。
3.TNF-α诱导β细胞PTP-1B蛋白水平变化:免疫印迹检测结果显示,经内参照β-Actin蛋白校正后,TNF-α干预各组较正常对照组,PTP-1B表达增加(P<0.05)。
[结论] TNF-α可诱导β细胞葡萄糖刺激的胰岛素分泌减少,发生胰岛β细胞胰岛素抵抗,但各干预组之间并未呈现剂量依赖性变化趋势。TNF-α通过PTP-1B表达升高,抑制胰岛素的信号转导。PTP-1B在慢性炎症诱导的β细胞胰岛素抵抗中发挥着重要的作用。
第三部分过表达蛋白酪氨酸磷酸酶1B对胰岛β细胞分泌功能的影响
[目的]明确蛋白酪氨酸磷酸酶1B对胰岛β细胞胰岛素分泌功能的影响。
[方法]传代培养处于对数生长期的HEK293细胞,扩增Ad-PTP-1B腺病毒,氯化铯密度梯度离心纯化病毒并测定病毒滴度;传代培养处于对数生长期的胰岛beta细胞MIN6细胞株转染MIN6细胞株,分三组:①转染Ad-PTP-1B胰岛β细胞组;②转染Ad-EGFP胰岛β细胞组;③胰岛β细胞正常对照组。应用Trizol试剂盒分别提取各组的细胞总RNA。采用RT-PCR法检测各组PTP-1B基因表达水平。Western-blot免疫印迹法检测PTP-1B蛋白表达水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果] 1、氯化铯密度梯度离心法纯化病毒后,取病毒液1:1稀释后测得A260为0.029,根据公式计算病毒滴度为:3.19×1010pfu/ml。
2、重组腺病毒PCR鉴定:经RT-PCR对病毒DNA鉴定,在大约85bp处可见一清晰条带。鉴定PTP-1B病毒。
3.以MOI 1:10,1:50,1:100分别感染MIN6细胞,western-blot检测可见:随着感染复数的升高,PTP-1B转染效率逐渐升高,其中,以MOI 1:100感染效率最高,故在后续的实验中拟用MOI 1:100感染MIN6细胞。
4.PTP-1B过表达MIN6细胞,胰岛素释放试验结果:Ad-PTP-1B转染MIN6细胞24小时后,可见转染PTP-1B组的MIN6细胞胰岛素分泌显著(P<0.01),而转染绿色荧光蛋白对照病毒的MIN6细胞胰岛素分泌Ad-PTP-1B组相比,明显升高(P<0.05)。与正常组相比,亦有增高趋势,但差异无统计学意义。
5.重组腺病毒感染MIN6细胞,PTP-1B蛋白表达结果:PTP-1B感染MIN6细胞后,表达明显增强,与GFP和空白对照均有显著性差异(P<0.05)。
[结论] PTP-1B在胰岛β细胞表达升高后,可诱导β细胞胰岛素分泌功能降低。提示:PTP-1B直接作用于胰岛β细胞,引发胰岛β细胞胰岛素抵抗。PTP-1B在胰岛β细胞胰岛素抵抗中发挥着重要的作用。
第四部分PTP-1B诱导β细胞胰岛素抵抗胰岛素信号途径变化
[目的]观察PTP-1B诱发β细胞胰岛素抵抗的可能机制及其对胰岛素信号的影响。
[方法]传代培养处于对数生长期的HEK293细胞,扩增Ad-PTP-1B腺病毒,氯化铯密度梯度离心纯化病毒并测定病毒滴度;传代培养处于对数生长期的胰岛beta细胞MIN6细胞株转染MIN6细胞株,分三组:①转染Ad-PTP-1B胰岛β细胞组;②转染Ad-EGFP胰岛β细胞组;③胰岛β细胞正常对照组。应用Trizol试剂盒分别提取各组的细胞总RNA。采用RT-PCR法检测各组PTP-1B基因表达水平。Western-blot免疫印迹法检测胰岛素受体β亚单位(IR-β)、胰岛素受体底物-1(IRS-1)蛋白表达水平,免疫沉淀法检测IR-β、IRS-1磷酸化水平。每组分别加入含2.8、16.7mmol/L葡萄糖的KRB缓冲液,37℃分别孵育1 h,进行胰岛素释放试验;放射免疫法测定胰岛素水平。
[结果] 1.转染PTP-1B的β细胞在无胰岛素刺激时,胰岛素受体β亚单位表达与正常对照组无明显差异,但受到胰岛素刺激后,IRβ磷酸化明显减弱。而转染GFP对照病毒组与正常对照组无明显差异。提示:PTP-1B表达增高,可引发IRβ去磷酸,阻碍胰岛素信号转导;
2.转染PTP-1B的β细胞在无胰岛素刺激时,胰岛素受体底物1(IRS-1)表达较正常对照组略偏低,但差异不显著。当受到胰岛素刺激后,IRS-1磷酸化明显减弱。而转染GFP对照病毒组与正常对照组无明显差异。提示:PTP-1B表达增高,可引发IRS-1去磷酸,阻碍胰岛素信号转导。
[结论]胰岛细胞过表达PTP-1B后,胰岛素受体β亚单位(IRβ)、胰岛素底物1(IRS-1)磷酸化程度明显减弱,提示PTP-1B通过负调控胰岛素信号通路上IRβ、IRS-1磷酸化程度,使胰岛素信号转导发生障碍,从而诱导胰岛细胞胰岛素抵抗。
Introduction
Obetsity and Type 2 Diabetes become the most popular diseases all over the world. The epidemic data showed that there had been 1,100,000,000 people who were overweight, and even 312,000,000 of them had suffered from obesity. Type 2 Diabetes is also popular and harmful to people. It is estimated that the population of type 2 DM will up to 366,000,000 in 2030. As we know, the common pathogenesis of obesity and type 2 DM is insulin resistance and data showed that people with insulin resistance had 5-fold higher risk of type 2 Diabetes as well as 2-fold higher risk of cardiovascular events than people without insulin resistantance. Insulin resistance become a key point in type 2 diabetes and obesity treatment. Most studies focused on insulin resistance of the typical insulin-sensitive tissue such as adipose, muscle, liver and hypothalamus.These studies concluded that increasing exercises, modifying daily-life style can increase fat comsuptoin, improve glucose utilization of muscle and thus ameliorate peripheral insulin resistance. However, beta cell also had insulin resistance which was characterized as impaired insulin secretion by glucose stimulated insulin secretion test (GSIS) and insulin signaling obstruction. The new concept of beta-cell insulin resistance act as a supplement of the theory of typical insulin resistance.
Previous study indicate that insulin receptor-βsubunit conditional knockout mice(βIRKO) lost more than 85% insulin secretion by GSIS and moved on to impaired glucose torlence (IGT) in 2 -month -years old. This demonstrate that insulin signaling is essential toβcell itself for insulin secretion. However, the exact mechanism ofβcell insulin resistance remained unclear.At the molecular level, the insulin signaling begins with activation of insulin receptor (IR) resulted in tyrosine phosphorylation of serials of substrates, including the IR substrate 1 (IRS-1) and IRS-2. After tyrosine phosphorylation of IRS-1 and IRS-2, they bind and activate the enzyme- phosphatidylinositol 3-kinase (PI3-K). The activation of PI3-K increases serine phosphorylation of protein kinase B (Akt), which in turn stimulates the glucose transport in the muscle and adipose tissue, glycogen synthesis in the liver and muscle, and lipogenesis in the adipose tissue. Recent studies suggest that protein tyrosine phosphatase-1B (PTP-1B) was an important negative regulator of insulin and leptin signal transduction. However, whether PTP-1B has a similar effect on beta cell insulin resistance remained unclear.
Part One: Expression of PTP-1B in beta cell insulin resistance induced by glucose and palmitic acid.
Objective
In this study, we investigated the causative factors ofβcell insulin resistance and the expression of PTP-1B. We also observed the relationship between PTP-1B andβcell insulin secreting function.
Methods
MIN6 cells under serial subcultivation were divided into four groups:①glucose treatment: the final concentration of glucose was 33.3mmol/L and 40mmol/L, respectively; incubated for 24 hours;②palmitic acid treatment (PA group): the final concentration of PA was 125umol/L, 250umol/L, respectively; incubated for 24 hours;③dehydrated-alcohol control group: added identical volume of dehydrated alcohol compared with PA group; incubated for 24 hours;④control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase.Realtime-PCR was used to detect the expression of PTP-1B mRNA level.Immunocytochemical stain and western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1. glucose concentration of 40mmol/L treatment induce beta cell insulin resistance successfully and the glucose stimulated insulin secretion reduced significantly.Compared with the control group, the basic insulin secretion decreased significantly(P<0.05) while insulin levels were much lower in high glucose concentration(P<0.01).However, this phenomenon was not seen in group of 33.3mmol/L glucose treatment . In contrast, the base line of insulin secretion in this group increased slightly and no differences of insulin secretion were seen comparing with control group.
2. Palmitic acid treatment also induce beta cell insulin resistance, resulted in the decrease of insulin secretion. Although the base line insulin releasing was similar to control group in PA of 125umol/L,the high glucose stimulated insulin secretion was much lower than control group. In group of 250umol/L PA,decreasing insulin secretion was only seen in high glucose stimulation(P<0.05). And there no differences of insulin secretion between dehydrated-alcohol control group and normol control.
3. The expressions of protein tyrosine phosphatase-1B were much higher in either glucose or PA group than in control group.
(1) Immunocytochemical stain showed that the expressions of PTP-1B obviously increased in glucose treatment (40mmol/L) and PA (125umol/L) group, respectively (P=0.000).Furthermore, the expression of PTP-1B was much higher in glucose treatment than in PA group.However, Neither 33.3mmol/L glucose treatment nor 250umol/L PA group had increased expression of PTP-1B,compared with control group.
(2) Realtime- Polymerase Chain Reaction (Realtime-PCR) is a simple and powerful method to amplify PTP-1B gene of a tiny amount. Amplified products are monitored in real time.In both glucose treatment (40mmol/L) and PA (125umol/L) group, the ratio of PTP-1B /β-actin were higher.Compared with control, the expression of PTP-1B mRNA level increased 50 fold in glucose treatment and 25 fold in PA group.
(3)Western-bloting showed that the expression of PTP-1B protein in each group was much higher than control.And there were similar results consistent wiht realtime-PCR results.
Conclusion:
Both glucose concentration of 40mmol/L treatment and palmitic acid treatment induce beta cell insulin resistance, resulted in the decrease of insulin secretion. The expressions of protein tyrosine phosphatase-1B were much higher in these groups than in control group.
PTP-1B may be an important regulator in the development of beta cell insulin resistance induced by glucose or PA.
Part Two: Expression of PTP-1B in beta cell insulin resistance induced by chronic stimulation of inflammatory factor.
Objective : In this study, we investigated the causative factors ofβcell insulin resistance and the expression of PTP-1B. We also observed the relationship between PTP-1B andβcell insulin secreting function.
Methods
MIN6 cells under serial subcultivation were divided into four groups:①Tumor necrosis factor-α(TNF-α)treatment group A :with final concentration of 1.0 ug/L; incubated for 24 hours;②TNF-αtreatment group B :with final concentration of 10 ug/L; incubated for 24 hours;③TNF-αtreatment group C :with final concentration of 20 ug/L ;incubated for 24 hours;④control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1.Different concentrations of TNF-αinduced beta cell insulin resistance and the insulin secretion reduced sinificantly in these three groups vs control group. However, TNF-αdid not have concentration-dependent effects on beta cell. Both TNF-αB group and A group showed the same effects on the severity of beta cell dysfunction.
2. The expression of PTP-1B mRNA increased in MIN6 cells induced by TNF-α.
3.The expression of PTP-1B protein increased in MIN6 cells induced by TNF-α. Western-bloting showed that PTP-1B protein expression was much higher in TNF-αintervention group.
Conclusion:
TNF-αinduce beta cell insulin resistance, resulted in the decrease of insulin secretion. However, TNF-αdid not have concentration-dependent effects on beta cell.Meanwhile, the expressions of protein tyrosine phosphatase-1B were much higher in TNF-αtreatment groups than in control group. PTP-1B may be an important regulator in the development of beta cell insulin resistance induced by stimulation of inflammatory factor.
Part Three: Effects of overexpression of PTP-1B on beta cell insulin secretion.
Objective :
In this part, we investigated the effects of overexpression of PTP-1B on beta cell insulin secretion.
Methods:
HEK293 cell line were serial subcultivated to amplify Ad-PTP-1B adenovirus. Cesium chloride centrifugal purification was performed to purify Ad-PTP-1B adenovirus and virus titre was detected. MIN6 cells under serial subcultivation were divided into 3 groups:①MIN6 cells transfected with Ad-PTP-1B;②MIN6 cells transfected with Ad-EGFP;③control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of PTP-1B protein. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1. Virus titre of Ad-PTP-1B was 3.19×1010pfu/ml.
2.MOI 1:100 was the most effective concentration for transfection.
3.Overexpression of PTP-1B in MIN6 cells resulted in the siginificant decrease of insulin secretion by GSIS vs. control. And there were no effects on MIN6 cell insulin secretion by transfected with Ad-EGFP.
Conclusions:
PTP-1B had direct negative effects on beta cell function and induced beta cell insulin resistance.Therefore, PTP-1B plays an important role in the development of beta cell insulin resistance.
Part Four: Effects of overexpression of PTP-1B on beta cell insulin signaling.
Objective:
In this part, we investigated the effects of overexpression of PTP-1B on beta cell insulin signaling.
Methods
HEK293 cell lines were serial subcultivated to amplify Ad-PTP-1B adenovirus. Cesium chloride centrifugal purification was performed to purify Ad-PTP-1B adenovirus and virus titre was detected. MIN6 cells under serial subcultivation were divided into 3 groups:①M IN6 cells transfected with Ad-PTP-1B;②MIN6 cells transfected with Ad-EGFP;③control group: nothing treated. We performed trizol kit to extract total RNA of each group and synthesized cDNA from RNA using reverse transcriptase. PCR was used to detect the expression of PTP-1B mRNA level.Western-bloting were performed to observe the expression of IR-βand IRS-1. The tyrosine phosphorylations of IRS-1, IRβwere detected by immuno-precipitation. We also investigated MIN6 cell insulin secreting function by performing the insulin releasing test and serum levels of insulin were determined by RIA assay.
Results:
1. The insulin-stimulated tyrosine phosphorylation of IRβin MIN6 cells in Ad-PTP-1B group were significantly lower than that in control group (P < 0.05).
2. The insulin-stimulated tyrosine phosphorylation of IRS-1 in MIN6 cells in Ad-PTP-1B group were significantly lower than that in control group (P < 0.05).
Conclusions:
The expression of PTP-1B increased to suppress the insulin signal transduction in beta cell and insulin-stimulated tyrosine phosphorylation of both IRβand IRS-1 decreased in beta cell.According to these results, it is supposed that the signal transduction pathway of insulin is the key part of beta cell insulin resistance induced by PTP-1B.
引文
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37 Tseng YH, Ueki K, Kriauciunas KM, et al. Differential roles of insulin receptor substrates in the anti-apoptotic function of insulin-like growth factor-1 and insulin. J Biol Chem. 2002, 277(35);31601-31611.
38 Seo C, Sohn JH, et al. Usimines A-C, Bioactive Usnic Acid Derivatives from the Antarctic Lichen Stereocaulon alpinum. J Nat Prod. 2008 , Feb , 21.
39 Miao Wang(王淼), Dajin Zou, Jiong Hou. Expression of tyrosine protein phosphatase 1B in adipose tissues of newly diagnosed diabetic patients . Academic Journal of Second Military Medical University(English), 2005, 26(6);648-650.
40 Zabolotny JM, Haj FG, Kim YB, et al. Transgenic overexpression of protein-tyrosine phosphatase 1B in muscle causes insulin resistance, but overexpression with leukocyte antigen-related phosphatase does not additively impair insulin action. J Biol Chem. 2004 Jun 4; 279(23);24844-24851.
41 Lalli CA, Pauli JR, Prada PO, Cintra DE, Ropelle ER, Velloso LA, Saad MJ. Statin modulates insulin signaling and insulin resistance in liver and muscle of rats fed a high-fat diet. Metabolism. 2008 Jan; 57(1);57-65.
42 Christopher D. Morrison1, Christy L. White, et al. Increased hypothalamic PTP-1B contributes to leptin resistance with age. Endocrinology. Jan 2007 ; 148; 433 - 440.
43 Koizumi M, Takagi-Sato M, Okuyama R, Araki K, Sun W, Nakai D. In vivo antisense activity of ENA oligonucleotides targeting PTP-1B mRNA in comparison of that of 2'-MOE-modified oligonucleotides. Nucleic Acids Symp Ser (Oxf). 2007; (51);111-2
44 Paty Karoll Picardi, Vivian Cristine Calegari, Patrícia de Oliveira Prada, et al. Reduction of hypothalamic PTP-1B improves insulin and leptin resistance in dietinduced obese rats Endocrinology. First published ahead of print May 8, 2008.
45 Liu F, Dallas-Yang Q, Castriota G, Fischer P, Santini F, Ferrer M, Li J, Akiyama TE, Berger JP, Zhang BB, Jiang G. Development of a novel GLUT4 translocation assay for identifying potential novel therapeutic targets for insulin sensitization. Biochem J. 2009 Mar 1; 418(2);413-20.
46 Uysal KT, Wlesbrock SM, Marino MW, et al. Protection from obesity-induced insulin resistance in mice lacking TNF2αfunction. Nature , 1997 , 389 ;6102614.
47鲁谨,邹大进,张家庆.肿瘤坏死因子2α致胰岛素抵抗.中国病理生理杂志, 1999 , 15 ;114621148.
48 Hotamisligil GS , Spiegelman BM. Tumor necrosis factor2α; a key component of obe2 sity2diabetes links. Diabetes , 1994 , 43 ; 127121278.
49金朝晖,邹大进.游离脂肪酸致胰岛素抵抗的机制.中华内分泌代谢杂志, 1999, 15, 4;247
50陈诗书,汤雪明主编, 2004,医学细胞与分子生物学。科学出版社。
51 Ortis F, Cardozo AK, Crispim D, et al. Cytokine-induced proapoptotic gene expression in insulin-producing cells is related to rapid, sustained, and nonoscillatory nuclear factor-kappaB activation. Mol Endocrinol. 2006, 20;1867-1879.
52 Jorns A, Gunther A, Hedrich HJ, et al. Immune cell infiltration, cytokine expression, and beta-cell apoptosis during the development of type 1 diabetes in the spontaneously diabetic LEW. 1AR1/Ztm-iddm rat. Diabetes, 2005 , 54;2041-2052.
53 Kim JK, et al. Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance . Proceedings of the National Academy of Sciences(USA). 2001 ; 98;7522-7527
54胡中波,仲照东,张友山,等.一种重组腺病毒载体产生及操作的新方法。华中科技大学学报(医学版), 2003, 32(4);409.
55 Parvez Hossain, Bisher Kawar, and Meguid El Nahas. Obesity and Diabetes in the Developing World—A Growing Challenge. N Engl J Med 356;213, January 18, 2007.
56 Cornier MA, Dabelea D, Hernandez TL, Lindstrom RCThe metabolic syndrome. Endocr Rev. 2008 Dec; 29(7);777-822.
57 Wajchenberg BL. Subcutaneous and visceral adipose tissue; their relation to the metabolic syndrome. Endocr Rev. 2000; 21;697-738.
58 Caserta F, Tchkonia T, Civelek VN, et al. Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes. Am J Physiol Endocrinol Metab 2001; 280; E238-E247.
59 Nieto-Vazquez I, Fernández-Veledo S, de Alvaro C, Lorenzo M Dual role of interleukin-6 in regulating insulin sensitivity in murine skeletal muscle. Diabetes. 2008 Dec; 57(12);3211-21.
60 Picardi PK, Calegari VC, Prada Pde O, Moraes JC, Araújo E, Marcondes MC, Ueno M, Carvalheira JB, Velloso LA, Saad MJ. Reduction of hypothalamic protein tyrosine phosphatase improves insulin and leptin resistance in diet-induced obese rats. Endocrinology. 2008 Aug; 149(8);3870-80.
61 Christy L. White, Amy Whittington, Maria J. Barnes, Zhong Wang, George A. Bray, and Christopher D. Morrison. HF diets increase hypothalamic PTP-1B and induce leptin resistance through both leptin-dependent and -independent mechanisms. Am J Physiol Endocrinol Metab 2009; 296; E291-E299.
62 Dadke S, Kusari J, and Chernoff J. Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region. J Biol Chem. 2000, 275(31);23642-23647.
62 Salmeen A, Andersen JN, Myers MP, et al. Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B. Mol Cell. 2000 , 6(6);1401-1412.
63 Salmeen A, Andersen JN, Myers MP, et al. Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B. Mol Cell. 2000 , 6(6);1401-1412. .
64 Da Silva Xavier G, Varadi A, Ainscow WK. Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase. J Biol Chem. 2000, 275(46);36269-36277.
65 Leibiger IB, Leibiger B, Moede T, et al. Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic beta cells. Mol Cell. 2001, 7(3);559-570.
66 Schwartz MW, Woods SC, Porte D Jr, et al. Central nervous system control of food intake. Nature. 2000 Apr 6; , 404(6778);661-671.
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6 Fu Mao, Li Xiu jun, et al. Increased expression of neuropeptide Y and its mRNA in STZ rats. Chin Med J, 2002, 115;690-695
7 Kulkarni RN, Bruning JC, Winnay JN, et al. Tissue - specific knockout of the insulin receptor in pancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell, 1999, 96(3);329-339.
8 Karaskov E, et al. Chronic palmitate but not oleate exposure induces endoplasmic reticulum stress, which may contribute to INS-1 pancreatic beta-cell apoptosis. Endocrinology, 2006 Jul; 147(7);3398-407.
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14 Asante-Appiah E, Kennedy BP. Protein tyrosine phosphatases; the quest for negative regulators of insulin action. Am J Physiol Endocrinol Metab. 2003, 284(4);E663-670.
15 Goldstein BJ, Bittner-Kowalczyk A, White MF, et a1. Tyrosine dephosphorylation and deactivation of insulin receptor substrate-1 by protein-tyrosine phosphatase 1B. Possible facilitation by the formation of a ternary complex with the Grb2 adaptor protein. J Biol Chem. 2000, 275(6);4283-4289.
16 Cheung A, Kusari J, Jansen D, et al. Marked impairment of protein tyrosine phosphatase 1B activity in adipose tissue of obese subjects with and without type 2 diabetes mellitus. J Lab Clin Med. 1999, 134(2);115-123.
17 Zabolotny JM, Haj FG, Kim YB, et al. Transgenic overexpression of protein-tyrosine phosphatase 1B in muscle causes insulin resistance, but overexpression with leukocyte antigen-related phosphatase does not additively impair insulin action. J Biol Chem. 2004, 279(23);24844-24851.
18 Clampit JE, Meuth JL, Smith HT, et a1. Reduction of protein-tyrosine phosphatase-1B increases insulin signaling in FAO hepatoma cells. Biochem Biophys Res Commun. 2003, 300(2);261-267.
19 Fawaz G. Haj, Janice M, et al. Liver-specific Protein-tyrosine Phosphatase 1B (PTP-1B) Re-expression Alters Glucose Homeostasis of PTP-1B–/–Mice Biol. Chem. 2005, April; 280(15) 15038-15046.
20 Christopher D. Morrison1, Christy L. White, et al. Increased hypothalamic PTP-1B contributes to leptin resistance with age. Endocrinology. Jan 2007 ; 148; 433 - 440.
21 Koizumi M, Takagi-Sato M, Okuyama R, Araki K, Sun W, Nakai D. In vivo antisense activity of ENA oligonucleotides targeting PTP-1B mRNA in comparison of that of 2'-MOE-modified oligonucleotides. Nucleic Acids Symp Ser (Oxf). 2007; (51);111-2.
22 Lund IK, Hansen JA, Andersen HS, et al. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling. J Mol Endocrinol. 2005, 34(2);339-351.
23 Zabolotny JM, Bence-Hanulec KK, Stricker-Krongrad A, et al. PTP-1B regulates leptin signal transduction in vivo. Dev Cell. 2002, 2(4);489-495.
24 Cheng A , Vetani N, Simoncic PD, et al. Attenuation of leptin action and regulation of obesity by protein tyrosine phosphatase 1B. Dev Cell. 2002, 2(4);497-503.
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27 Ford ES, Williamson DF, Liu S. Weight change and diabetes incidence; findings from a national cohort of US adults. Am J Epidemiol. 1997, 146(3);214-222.
28邬云红,李秀钧,李宏亮等.高脂饮食肥胖大鼠胰岛细胞胰岛素抵抗机理的探讨.中华医学杂志2005, 85(27);1907-1910.
29张丽娟、邹大进等.高脂饮食诱导的肥胖大鼠胰腺蛋白酪氨酸磷酸酶1B表达增高.中华内分泌代谢杂志2007, 23(5);437-441
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32张芳林,李果,刘优萍,等. 2型糖尿病大鼠模型的建立及其糖代谢特征分析.中国实验动物学报, 2002, 10(1);16-20.
33 Gremlich S, Bonny C, Waeber G, et al. Fatty acids decrease IDX-1 expression in rat pancreatic islets and reduce GLUT2, glucokinase, insulin, and somatostatin levels. J Biol Chem, 1997, 272;30261-30269.
34 Aspinwall CA, Lakey JR, Kennedy RT. Insulin-stimulated Insulin Secretion in Single Pancreatic Beta Cells. J Biol Chem. 1999 Mar 5; 274(10);6360-6365.
35 Leibiger IB, Leibiger B, Berggren PO. Insulin feedback action on pancreatic beta-cell function. FEBS Lett. 2002, 532(1-2);1-6.
36 Kulkarni RN, Bruning JC, Winnay JN, et al. Tissue-specific knockout of the insulin receptor inpancreatic beta cells creates an insulin secretory defect similar to that in type 2 diabetes. Cell. 1999, 96(3);329-339.
37 Tseng YH, Ueki K, Kriauciunas KM, et al. Differential roles of insulin receptor substrates in the anti-apoptotic function of insulin-like growth factor-1 and insulin. J Biol Chem. 2002, 277(35);31601-31611.
38 Seo C, Sohn JH, et al. Usimines A-C, Bioactive Usnic Acid Derivatives from the Antarctic Lichen Stereocaulon alpinum. J Nat Prod. 2008 , Feb , 21.
39 Miao Wang(王淼), Dajin Zou, Jiong Hou. Expression of tyrosine protein phosphatase 1B in adipose tissues of newly diagnosed diabetic patients . Academic Journal of Second Military Medical University(English), 2005, 26(6);648-650.
40 Zabolotny JM, Haj FG, Kim YB, et al. Transgenic overexpression of protein-tyrosine phosphatase 1B in muscle causes insulin resistance, but overexpression with leukocyte antigen-related phosphatase does not additively impair insulin action. J Biol Chem. 2004 Jun 4; 279(23);24844-24851.
41 Lalli CA, Pauli JR, Prada PO, Cintra DE, Ropelle ER, Velloso LA, Saad MJ. Statin modulates insulin signaling and insulin resistance in liver and muscle of rats fed a high-fat diet. Metabolism. 2008 Jan; 57(1);57-65.
42 Christopher D. Morrison1, Christy L. White, et al. Increased hypothalamic PTP-1B contributes to leptin resistance with age. Endocrinology. Jan 2007 ; 148; 433 - 440.
43 Koizumi M, Takagi-Sato M, Okuyama R, Araki K, Sun W, Nakai D. In vivo antisense activity of ENA oligonucleotides targeting PTP-1B mRNA in comparison of that of 2'-MOE-modified oligonucleotides. Nucleic Acids Symp Ser (Oxf). 2007; (51);111-2
44 Paty Karoll Picardi, Vivian Cristine Calegari, Patrícia de Oliveira Prada, et al. Reduction of hypothalamic PTP-1B improves insulin and leptin resistance in dietinduced obese rats Endocrinology. First published ahead of print May 8, 2008.
45 Liu F, Dallas-Yang Q, Castriota G, Fischer P, Santini F, Ferrer M, Li J, Akiyama TE, Berger JP, Zhang BB, Jiang G. Development of a novel GLUT4 translocation assay for identifying potential novel therapeutic targets for insulin sensitization. Biochem J. 2009 Mar 1; 418(2);413-20.
46 Uysal KT, Wlesbrock SM, Marino MW, et al. Protection from obesity-induced insulin resistance in mice lacking TNF2αfunction. Nature , 1997 , 389 ;6102614.
47鲁谨,邹大进,张家庆.肿瘤坏死因子2α致胰岛素抵抗.中国病理生理杂志, 1999 , 15 ;114621148.
48 Hotamisligil GS , Spiegelman BM. Tumor necrosis factor2α; a key component of obe2 sity2diabetes links. Diabetes , 1994 , 43 ; 127121278.
49金朝晖,邹大进.游离脂肪酸致胰岛素抵抗的机制.中华内分泌代谢杂志, 1999, 15, 4;247
50陈诗书,汤雪明主编, 2004,医学细胞与分子生物学。科学出版社。
51 Ortis F, Cardozo AK, Crispim D, et al. Cytokine-induced proapoptotic gene expression in insulin-producing cells is related to rapid, sustained, and nonoscillatory nuclear factor-kappaB activation. Mol Endocrinol. 2006, 20;1867-1879.
52 Jorns A, Gunther A, Hedrich HJ, et al. Immune cell infiltration, cytokine expression, and beta-cell apoptosis during the development of type 1 diabetes in the spontaneously diabetic LEW. 1AR1/Ztm-iddm rat. Diabetes, 2005 , 54;2041-2052.
53 Kim JK, et al. Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance . Proceedings of the National Academy of Sciences(USA). 2001 ; 98;7522-7527
54胡中波,仲照东,张友山,等.一种重组腺病毒载体产生及操作的新方法。华中科技大学学报(医学版), 2003, 32(4);409.
55 Parvez Hossain, Bisher Kawar, and Meguid El Nahas. Obesity and Diabetes in the Developing World—A Growing Challenge. N Engl J Med 356;213, January 18, 2007.
56 Cornier MA, Dabelea D, Hernandez TL, Lindstrom RCThe metabolic syndrome. Endocr Rev. 2008 Dec; 29(7);777-822.
57 Wajchenberg BL. Subcutaneous and visceral adipose tissue; their relation to the metabolic syndrome. Endocr Rev. 2000; 21;697-738.
58 Caserta F, Tchkonia T, Civelek VN, et al. Fat depot origin affects fatty acid handling in cultured rat and human preadipocytes. Am J Physiol Endocrinol Metab 2001; 280; E238-E247.
59 Nieto-Vazquez I, Fernández-Veledo S, de Alvaro C, Lorenzo M Dual role of interleukin-6 in regulating insulin sensitivity in murine skeletal muscle. Diabetes. 2008 Dec; 57(12);3211-21.
60 Picardi PK, Calegari VC, Prada Pde O, Moraes JC, Araújo E, Marcondes MC, Ueno M, Carvalheira JB, Velloso LA, Saad MJ. Reduction of hypothalamic protein tyrosine phosphatase improves insulin and leptin resistance in diet-induced obese rats. Endocrinology. 2008 Aug; 149(8);3870-80.
61 Christy L. White, Amy Whittington, Maria J. Barnes, Zhong Wang, George A. Bray, and Christopher D. Morrison. HF diets increase hypothalamic PTP-1B and induce leptin resistance through both leptin-dependent and -independent mechanisms. Am J Physiol Endocrinol Metab 2009; 296; E291-E299.
62 Dadke S, Kusari J, and Chernoff J. Down-regulation of insulin signaling by protein-tyrosine phosphatase 1B is mediated by an N-terminal binding region. J Biol Chem. 2000, 275(31);23642-23647.
62 Salmeen A, Andersen JN, Myers MP, et al. Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B. Mol Cell. 2000 , 6(6);1401-1412.
63 Salmeen A, Andersen JN, Myers MP, et al. Molecular basis for the dephosphorylation of the activation segment of the insulin receptor by protein tyrosine phosphatase 1B. Mol Cell. 2000 , 6(6);1401-1412. .
64 Da Silva Xavier G, Varadi A, Ainscow WK. Regulation of gene expression by glucose in pancreatic beta -cells (MIN6) via insulin secretion and activation of phosphatidylinositol 3'-kinase. J Biol Chem. 2000, 275(46);36269-36277.
65 Leibiger IB, Leibiger B, Moede T, et al. Selective insulin signaling through A and B insulin receptors regulates transcription of insulin and glucokinase genes in pancreatic beta cells. Mol Cell. 2001, 7(3);559-570.
66 Schwartz MW, Woods SC, Porte D Jr, et al. Central nervous system control of food intake. Nature. 2000 Apr 6; , 404(6778);661-671.