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Study for pathophysiological significance of insulin-like growth factor II

胰岛素样生长因子II的病理生理意义研究

基本信息

批准号：
10671043
负责人：
HIZUKA Naomi
金额：
$ 2.05万
依托单位：
Tokyo Women's Medical University, School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
1998
资助国家：
日本
起止时间：
1998 至 2000
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-10671043/
关键词：
IGF-II tumor hypoglycemia non-islet cell tumor hypoglycemia (NICIH)

项目摘要

In this study, we have investigated pathophysiological significance of IGF-II as follows.1) Mechanism of hypoglycemia in non-islet cell tumor hypoglycemia (NICTH) : NICTH is one of the major causes of fasting hypoglycemia. It has been suggested that the hypoglycemia in the NICTH is related to the secretion of big IGF-II, but the mechanism of hypoglycemia is still unknown. In this syndrome, serum big IGF-II increases and the big IGF-II circulates with IGF binding proteins (IGFBPs) as a binary complex but not as the ternary complex (150 kDa) of big IGF-II-IGFBP-3-acid-labile subunit (ALS). The impaired formation of 150 kDa complex of big IGF-II could be one of the major causes of hypoglycemia. In the present study, we measured serum ALS levels by RIA, and also characterized serum ALS by Western immunoblot (WIB) to analyze the size and the quantity in 33 patients with NICTH.Serum ALS levels were significantly lower than those in normal subjects (3.5±0.5 vs 16.3±0.5 mg/L). In six patients, … More the ALS levels increased after successful tumor resection. Serum ALS in the patients with NICTH was detected as 80/78kDa duplet by WIB as same as in normal subjects, suggesting that the glycosylation of ALS was not different from that for normal subjects. These data demonstrate that the molecular weight sizes of ALS in the NICTH were the same as those in the normal subjects, and the decreased serum ALS levels could be one of the causes of impaired formation of 150kDa complex in the NICTH.To characterize big IGF-II, we studied big IGF-II in patients with NICTH by WIB using specific antibody against E10-21 (Ab E10-21) and E71-89 (Ab E71-89) of pro-IGF-II.The 14〜18 kDa protein was detected in sera from NICTH by WIB using Ab E10-21 and IGF-II antibody, respectively. When analyzed by Western immunoblot using Ab E71〜89, the 〜10 kDa protein was detected. As Ab E71-89 did not cross react with mature IGF-II, detected 10-kDa protein was thought to be C-terminal fragment of pro-IGF-II.These data suggest that big IGF-II in NICTH is not intact pro-IGF-II, but pro-IGF-II- (E1-21) generated from abnormal processing, and the C-terminal fragment is also present in the circulation.2) Clinical application of Western immunoblot (WIB) of insulin-like growth factor II using unextrated seium : To investigate big IGF-II, WIB technique has been used. In our previous study, acid-ethanol extraction of IGF-II from serum was required for electrophoresed sample. Recently, we simplified the method, and have directly used unextracted serum sample for electrophoresed sample and Mini-Gel System. Using this method, the results were the same as those by WIB using acid-ethanol extracted serum. Furthermore, time required to analysis of IGF-II was shortened (2 days vs 5 days). Thus, this WIB system enabled us to analyze easily molecular heterogeneity of IGF-II.3) Usefulness of allele-specific polymerase chain (AS-PCR) method to study for loss of imprinting of insulin-like growth factor II gene in tumors : We have examined IGF2 expression in tumors from ten NICTH using the AS-PCR method. IGF2 was expressed in all examined tumor tissues, and was expressed biallelically (LOI) in five of seven informative tumors. These data were concordant with those by Apa I digestion method. In a case of NICTH (gastric cancer) whose tumor tissues (primary and metastatic tumors) showed biallelic expression of IGF2, IGF-2 was monoallelic expressed in the non-tumor tissues by AS-PCR, but was not clearly shown by Apa digestion method. The present study show that the AS-PCR is simple and useful method to study LOI of IGF2. Less

在本研究中，我们对IGF-II的病理生理学意义进行了如下研究。1）非胰岛细胞瘤低血糖（NICTH）的低血糖机制：NICTH是空腹低血糖的主要原因之一。有人提出NICTH低血糖与大IGF-II的分泌有关，但低血糖的机制尚不清楚。在这种综合征中，血清大 IGF-II 增加，大 IGF-II 与 IGF 结合蛋白 (IGFBP) 作为二元复合物而不是大 IGF-II-IGFBP-3-酸不稳定亚基 (ALS) 的三元复合物 (150 kDa) 一起循环。大 IGF-II 150 kDa 复合物的形成受损可能是低血糖的主要原因之一。在本研究中，我们采用放射免疫法（RIA）测定血清ALS水平，并通过免疫印迹（WIB）对血清ALS进行表征，分析33例NICTH患者的血清ALS大小和数量。血清ALS水平显着低于正常受试者（3.5±0.5 vs 16.3±0.5 mg/L）。成功切除肿瘤后，六名患者的 ALS 水平有所升高。 WIB检测NICTH患者血清ALS为80/78kDa双联体，与正常人相同，提示ALS糖基化与正常人无差异。这些数据表明 NICTH 中 ALS 的分子量大小与正常受试者相同，血清 ALS 水平降低可能是 NICTH 中 150kDa 复合物形成受损的原因之一。为了表征大 IGF-II，我们通过 WIB 使用针对 E10-21 (Ab E10-21) 和 E71-89 的特异性抗体研究了 NICTH 患者中的大 IGF-II。 (Ab E71-89) pro-IGF-II。分别使用 Ab E10-21 和 IGF-II 抗体通过 WIB 检测来自 NICTH 的血清中的 14〜18 kDa 蛋白。当使用 Ab E71〜89 通过蛋白质免疫印迹分析时，检测到〜10 kDa 的蛋白质。由于 Ab E71-89 不与成熟的 IGF-II 发生交叉反应，因此检测到的 10 kDa 蛋白被认为是 pro-IGF-II 的 C 端片段。这些数据表明 NICTH 中的大 IGF-II 并不是完整的 pro-IGF-II，而是异常加工产生的 pro-IGF-II- (E1-21)，并且 C 端片段也存在于 2) 使用未提取的硒对胰岛素样生长因子 II 进行蛋白质免疫印迹 (WIB) 的临床应用：为了研究大 IGF-II，已使用 WIB 技术。在我们之前的研究中，电泳样品需要从血清中用酸-乙醇提取IGF-II。最近，我们简化了方法，直接使用未提取的血清样品进行电泳样品和Mini-Gel系统。使用该方法，结果与使用酸-乙醇提取血清的WIB结果相同。此外，分析 IGF-II 所需的时间也缩短了（2 天 vs 5 天）。因此，该 WIB 系统使我们能够轻松分析 IGF-II 的分子异质性。3) 等位基因特异性聚合酶链 (AS-PCR) 方法在研究肿瘤中胰岛素样生长因子 II 基因印记丢失的有用性：我们使用 AS-PCR 方法检查了来自 10 个 NICTH 的肿瘤中 IGF2 的表达。 IGF2 在所有检查的肿瘤组织中都有表达，并且在七个信息性肿瘤中的五个中以双等位基因 (LOI) 表达。这些数据与 Apa I 消化法的数据一致。在一例肿瘤组织（原发性肿瘤和转移性肿瘤）显示IGF2双等位基因表达的NICTH（胃癌）病例中，AS-PCR显示IGF-2在非肿瘤组织中呈单等位基因表达，但Apa消化法未明确显示IGF-2。本研究表明AS-PCR是研究IGF2 LOI的简单且有用的方法。较少的