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亨廷顿蛋白相关蛋白1调节胰岛β细胞胰岛素的分泌
中文摘要

 亨廷顿蛋白相关蛋白1(Huntingtin-associated protein 1,HAP1)是一个功能未明的新蛋白,至少具有2种同工异构型,即HAP1A和HAP1B。最初的研究表明, 2种HAP1均广泛分布在神经系统,以在下丘脑的表达水平最高。下丘脑是内分泌系统的调节中枢,下丘脑许多神经元具有内分泌功能,HAP1在下丘脑内的高水平表达提示HAP1可能与内分泌功能有关。随后原位杂交研究显示,垂体、生殖腺中有HAP1 mRNA表达,肾上腺髓质嗜铬细胞的瘤细胞系PC12细胞表达高水平的HAP1,提示HAP1可能也在内分泌细胞中表达。本实验室最近的研究证明,在大鼠内分泌系统中,HAP1选择性表达于包括胰岛内分泌细胞在内的分泌含氮类激素的内分泌细胞中。已有研究显示,HAP1与神经细胞的微管、突触囊泡密切相关,参与神经细胞内突触囊泡的运输和神经递质受体的再循环,并可能参与调节递质的释放过程。分泌含氮激素的内分泌细胞在激素的合成、转运、分泌等方面均与神经细胞内递质的合成、转运、释放等过程十分相似,然而HAP1是否参与内分泌细胞激素的分泌,目前尚未见报道。本研究在证明HAP1选择性分布于胰岛β细胞的基础上,观察了禁食、腹腔注射葡萄糖对大鼠胰岛内HAP1表达的影响,利用来自非肥胖型糖尿病(NOD)转基因小鼠的胰岛β细胞系NIT-1细胞检测了高糖刺激对HAP1表达的影响,并观察了不同HAP1表达水平对NIT-1细胞中胰岛素表达和分泌的影响,第一次证明了HAP1能调节胰岛β细胞系胰岛素的分泌。 一、HAP1选择性地表达于胰岛β细胞 本室以前曾用免疫组织化学染色发现,HAP1选择性地表达于大鼠胰腺内分泌部——胰岛内。为了明确HAP1表达于胰岛何种内分泌细胞,首先应用免疫组织化学ABC法证实了HAP1也特异性在小鼠胰岛中表达。在腹腔注射链脲佐菌素诱导的实验型糖尿病小鼠胰腺中,随着胰岛的逐渐萎缩,胰岛中的HAP1免疫反应阳性细胞和胰岛素免疫反应阳性细胞均逐渐减少,在注射链脲佐菌素第7天,胰岛中仅偶见HAP1和胰岛素阳性细胞,提示胰岛中的HAP1可能主要表达于链脲佐菌素敏感的β细胞内。免疫荧光双标显示,小鼠胰岛中HAP1阳性细胞呈胰岛素免疫反应阳性,但呈胰高血糖素和生长抑素阴性;所有非肥胖型糖尿病(NOD)转基因小鼠来源的胰腺β细胞系NIT-1细胞均为HAP1和胰岛素双标细胞,表明HAP1在胰岛中选择性表达于分泌胰岛素的β细胞,而与分泌胰高血糖素的α细胞和分泌生长抑素的δ细胞无关。Western blotting分析显示,NIT-1细胞中的HAP1也具有HAP1A和HAP1B 2种类型,二者的分子大小分别与小鼠脑中的HAP1A和HAP1B相同。HAP1在胰岛β细胞的选择性表达提示,HAP1可能参与胰岛β细胞的功能调节,如可能与胰岛素的合成、运输、分泌等过程的调节有关。2种HAP1均在NIT-1细胞中表达,表明NIT-1细胞可作为研究HAP1是否调节胰岛素的合成、运输、分泌等过程的有用细胞模型。 二、胰岛素分泌状态改变对HAP1表达的影响 为了了解HAP1是否参与调解胰岛素的分泌过程,应用免疫组织化学ABC法观察了禁食及腹腔注射葡萄糖对小鼠胰岛的HAP1免疫反应性的影响,应用免疫印迹和RT-PCR技术检测高葡萄糖刺激对NIT-1细胞内HAP1表达水平的影响。 免疫组织化学研究显示,禁食2~4天的小鼠胰岛内HAP1免疫反应性较正常进食小鼠明显增强;腹腔注射葡萄糖后,小鼠胰岛HAP1免疫反应性较腹腔注射生理盐水小鼠明显减弱。禁食或腹腔注射葡萄糖引起胰岛β细胞胰岛素分泌改变后,HAP1在胰岛内表达水平的变化提示HAP1可能参与了胰岛素的分泌过程。禁食可使β细胞胰岛素分泌减少时,此时β细胞内HAP1水平升高,可能与HAP1利用减少有关。腹腔注射葡萄糖则使β细胞胰岛素分泌增加,此时β细胞内HAP1水平降低,可能与HAP1参与胰岛素分泌活动而被大量消耗或代谢有关。 免疫荧光、免疫印迹和RT-PCR分析显示,在用高浓度(16.5mmol/L)葡萄糖刺激NIT-1细胞不同时间后,细胞内HAP1水平先(刺激15min)明显降低,但随后逐渐回升,4h后上升到高于基础糖浓度(5.5mmol/L)组细胞。在此过程中,HAP1的mRNA水平无明显变化。但在高葡萄糖(11.1mmol/L和16.5mmol/L)刺激NIT-1细胞24h后,HAP1及其mRNA表达明显升高,糖浓度越高,升高越明显。HAP1 水平在高糖刺激之初的降低可能与HAP1参与胰岛素分泌活动而被大量消耗或代谢有关;HAP1水平在高糖刺激30min后逐渐回升,在刺激4h后高于正常水平,但其mRNA水平无明显变化,可能与HAP1的翻译增加有关。在刺激24h后,HAP1的蛋白和mRNA水平均明显升高,说明HAP1基因的转录和翻译均明显增加。 当细胞外葡萄糖浓度升高时,胰岛的胰岛素分泌呈二相性。第一相大约持续10分钟,在此期间分泌率上升到峰值,然后下降;第二相分泌迟缓而稳定。NIT-1细胞在糖刺激15分钟内,HAP1水平明显降低,提示HAP1可能参与了胰岛素的第一相快速分泌过程。在高糖刺激引起的胰岛素第一相分泌后,HAP1的水平持续升高,提示了HAP1亦参与胰岛素的第二相分泌过程。 三、不同HAP1表达水平对NIT-1细胞胰岛素分泌的影响 为了明确HAP1是否具有调节胰岛素分泌的功能,应用HAP1基因过表达和基因沉默技术观察了不同HAP1表达水平对NIT-1细胞胰岛素表达和分泌的影响。 在对NIT-1 细胞分别转染pEGFP-HAP1A、pEGFP-HAP1B、HAP1 siRNA及相应的对照质粒后,免疫荧光和RT-PCR检测显示,与转染相应对照质粒的NIT-1细胞比较,过表达HAP1A或HAP1B或沉默HAP1的细胞中胰岛素的表达水平无明显变化。对细胞裂解物中胰岛素的放射免疫分析(RIA)表明,无论是在基础糖浓度下还是在高糖刺激下,过表达HAP1A或HAP1B或HAP1被沉默的细胞内胰岛素含量与转染相应对照质粒的细胞内胰岛素含量无明显差别。由此表明,胰岛β细胞HAP1的表达水平对胰岛素的表达无明显影响。 用RIA对分泌至培养基中的胰岛素进行检测发现,无论在基础糖浓度条件下还是在高糖刺激时,过表达HAP1A或HAP1B的NIT-1细胞其胰岛素的分泌量无明显变化,表明过多的HAP1并不影响胰岛素的分泌。而用SiRNA沉默HAP1表达后的NIT-1细胞,其基础胰岛素分泌显著减少;在高糖刺激时,HAP1沉默的NIT-1细胞在刺激10min和90min后,其分泌的胰岛素分别减少16%和23%,即胰岛素的第一相分泌和第二相分泌均被明显抑制,由此进一步提示HAP1对胰岛素的第一、二相分泌过程均有调节作用。 糖刺激通过K〓通道依赖途径和K〓通道非依赖途径分别引起胰岛素的第一相和第二相分泌,碳纤电极安培法检测可反映通过K〓通道依赖途径产生的胰岛素释放。碳纤电极安培法检测显示,当用SiRNA干扰NIT-1细胞HAP1表达后,在高钾刺激下,记录到的电流峰信号频率明显降低,但电流峰幅度和电流峰容量无明显变化,即沉默HAP1后,NIT-1细胞分泌颗粒的量子释放速度降低,但每个分泌颗粒所含有的分泌物量未发生改变。因此,HAP1可通过影响K〓通道依赖途径而调节糖刺激引起的胰岛素第一相分泌,但其具体环作用环节有待进一步研究。 关键词 亨廷顿蛋白相关蛋1(HAP1);胰岛;β细胞;NIT-1细胞系;葡萄糖刺激;胰岛素分泌。

英文摘要

 Huntingtin-associated protein 1 (HAP1) is a novel protein which function is not very clear. It can be classified to, at least, two isoforms, HAP1A and HAP1B. Some initial studies indicated HAP1 was widely distributed in the rat/mouse brain, but this distribution had regional difference. Hypothalamus exhibited the most intense staining of HAP1 immunoreactivity. Hypothalamus is a critical area modulating endocrine secretion. HAP1 high expression in hypothalamus suggested that HAP1 was probably associated with endocrine. The following study by hybridization insitu showed that HAP1 expressed in hypophysis, genital glands and high expressed in the neoplastic cell line PC12 derived from the chromaffin cells in the medulla of adrenal gland, which suggested that HAP1 perhaps expressed in endocrine system. Our recent study found HAP1 selective distribution in nitrogenous hormone-secreting endocrine cells in rat endocrine system. Some initial studies indicated that HAP1 was associated with microbule and synaptic vesicle, and participated in the vesicle transportation, substance receptor recycle and substance releasing in neurons. Nitrogenous hormone-secreting endocrine cells were similar to neurons in the processes of substance synthesis, transportation and releasing. Whereas, whether HAP1 participated in hormone secretion in endocrine cells was not be reported. In this paper, on the basis of HAP1 selective distribution in pancreatic islet β cells, we observed the influence of fasting or intraperitoneal injection of glucose on expression of HAP1 in the mouse pancreatic islet, and detected the impact of high glucose stimulation on HAP1 in NIT-1 cells from non-obese diabetic (NOD) transgenic mice, and observed the effect of different HAP1 expression level on insulin expression and secretion. The study proved HAP1 modulated insulin secretion in β cell line for the first time. 1.Selective expression of HAP1 in the pancreatic islet β cells We ever found HAP1 expressed in rat pancreatic islets by immunohistochemistry method. In order to illuminate which kind of cell of islet HAP1 expressed in, we used immunohistochemical ABC method to approve that HAP1 selectively expressed in the mouse pancreatic islet β cells. We replicated mouse model of diabetes through selectively destroying β cells of pancreas islet by one peritoneal injection of large dose of streptozotocin, and found HAP1-immunoreactive cells or insulin-immunoreactive cells decreased with the progressive atrophy of islet in the diabetic mice. Only a few of HAP1 positive cells or insulin positive cells remained in islets 7 days after injection of streptozotocin, which suggested that HAP1 mainly located in streptozotocin-sensitive β cells in the pancreas islet. Immunofluorecent double staining showed that HAP1-immunoreactive cells were almost immunoreactive with insulin in the pancreatic islet, and NIT-1 cells from non-obese diabetic (NOD) transgenic mice were double staining cells of HAP1 and insulin. Western blotting assay demonstrated that HAP1A and HAP1B existed in NIT-1 cells and their molecular weight was the same as that of mouse brain. Selective distribution of HAP1 in pancreatic islet β cells suggested that HAP1 probably participated in function activity of pancreatic islet β cells, such as synthesis, transportation and releasing of insulin. The data that two isoforms of HAP1 expressed in NIT-1 cells suggested the cells can act as a useful cell model in the study of whether HAP1 was associated with synthesis, transportation and releasing of insulin. 2.Influence of change in insulin secretion on HAP1 expression In order to elucidate whether HAP1 took part in the process of insulin secretion, we used immunohistochemical ABC method to observe the influence of fasting or intraperitoneal injection of glucose on expression of HAP1 in the mouse pancreatic islet, and used western blotting method and PT-PCR to detect the effect of high glucose stimulation on HAP1 in the NIT-1 cells. The result of immunohistochemistry showed that fasting (2-4d) caused an increase in the level of HAP1 compared to control (mice fed normal chow). Conversely, intraperitoneal injection of glucose reduced expression of HAP1 in the pancreatic islet compared to intraperitoneal injection of saline. When fasting or intraperitoneal injection of glucose caused the change of insulin secretion in the β cells, the expression of HAP1 in the cells changed. These results suggested that HAP1 probably involved in the process of insulin secretion. In the research, expression of HAP1 in the β cells increased when insulin secretion in the β cells decreased in the situation of fasting, which was probably associated with the decline of HAP 1 utilization. Whereas, expression of HAP1 in the β cells decreased when insulin secretion in the β cells increased after intraperitoneal injection of glucose, which suggested that HAP1 probably were consumed or metabolized in the function activity of insulin secretion. The influence of glucose stimulation on HAP1 in the NIT-1 cells was observed by using western blotting method and PT-PCR. We used 16.5mM high glucose to stimulate NIT-1 cells in different time, including 15min, 30min, 90min, 4h, and detected protein and mRNA level of HAP1 compared with 5.5mM glucose. And we found HAP1 expression level decreased dramatically in the 15min after glucose stimulation, subsequently, HAP1 increased gradually with the time, and exceeded normal level in 4h after glucose stimulation. During the process, no change of HAP1 mRNA was detected. Twenty four hours after high glucose (11.1mM and 16.5mM) stimulated NIT-1 cells, expression of HAP1 and its mRNA increased, and the more high glucose concentration, the more increase. The detected increase of HAP1 expression was likely to relate to the raise in translation level of HAP1 in the short time (15min-4h), and be involved in the increase of transcription and translation in the longer time(24h). In vivo and in vitro, pancreatic islets respond to increase in extracellular glucose with a biphasic pattern of insulin release. The first phase lasts about ten minutes, during which the secretory rate rised to a peak and then declined. In mouse, the second phase consisted of steady secretion at slower rate. Expression of HAP1 dramatically declined in the 15min after glucose stimulation, which suggested HAP1 probably participated in the rapid first phase of insulin secretion. HAP1 continued to change after the first phase of insulin secretion, which showed HAP1 probably took part in the second phase. 3. Influence of different HAP1 expression level on insulin secretion in NIT-1 cells In order to elucidate whether HAP1 modulated insulin secretion, overexpression and silencing of HAP1 were applied to detect the influence of different expression level of HAP1 on insulin secretion . We transfected pEGFP-HAP1A/HAP1B, HAP1siRNA and their corresponding control plasmid into NIT-1 cells, and observed expression level of insulin was no change in the situation of overexpression or silencing of HAP1 compared to corresponding control plasmid by immunofluorecent method and RT-PCR. We used radioimmunoassay(RIA) to detect insulin content in cell lysates , and found there was no any significant change in overexpression(HAP1A/HAP IB) group or silencing group compared with corresponding control group under basal condition or high glucose stimulation, which showed that HAP1 expression level had no effect on insulin expression. Insulin secreted into the media was quantitated by RIA, and we found insulin secretion of HAP1A-overexpreesed cells or HAP1B-overexpressed cells had no obvious change under basal condition or high glucose stimulation, which suggested excess HAP1 had no effect on insulin secretion. However, silencing of HAP1 resulted in an obvious decrease of insulin secretion with basal 5.5mM glucose; under the condition of 16.5mM high glucose stimulation, the HAP1-silencing NIT-1 cells displayed a 16% decline by 10min, a 26% decrease by 90min, compared with the corresponding control groups (1min/90min) transfected with HK. These data showed the first phase and the second phase of insulin secretion were restrained, which further suggested that HAP1 participated in the regulation of biphasic process of insulin release. Glucose-stimulated biphasic insulin secretion involves at least two signaling pathways, the K〓 channel-dependent and K〓 channel-independent pathways, respectively. The carbon fiber electrode measurement can reflect the insulin secretion through K〓 channel-dependent pathways. The carbon fiber electrode measurement detected that silencing of HAP1 induced an obvious decrease in frequency of the ampere spikes, no change in amplitude and charge of the spikes. Namely, after HAP1 was silenced, quantum release rate of secretory granules in NIT-1 cells declined, but secretory content of every secretory granule had no change. So HAP1 probably regulated the first phase of insulin secretion through K〓 channel-dependent pathways. The detailed mechanism need to be further investigated. Key words: Huntingtin-associate protein 1; pancreatic islet; β cells; NIT-1 cell line;glucose stimulate; insulin secretion.

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