Study for pathophysiological significance of insulin-like growth factor II

在本研究中，我们从以下方面探讨了IGF-II的病理生理意义。1)非胰岛细胞肿瘤低血糖（NICTH）的低血糖机制：NICTH是导致空腹低血糖的主要原因之一。已有研究认为NICTH的低血糖与大IGF-II的分泌有关，但低血糖的机制尚不清楚。在该综合征中，血清大IGF- ii升高，大IGF- ii与IGF结合蛋白（igfbp）作为二元复合物循环，而不是作为大IGF- ii - igfbp -3酸不稳定亚基（ALS）的三元复合物（150 kDa）循环。150 kDa大IGF-II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RIA法测定了33例NICTH患者的血清ALS水平，并采用Western免疫印迹（WIB）对血清ALS进行了表征，分析了其大小和数量。血清ALS水平明显低于正常人（3.5±0.5 vs 16.3±0.5 mg/L）。在成功切除肿瘤后，6例患者的肌萎缩侧索硬化症水平升高。与正常人一样，NICTH患者血清中ALS的WIB检测结果为80/78kDa双链，提示ALS的糖基化与正常人无异。这些数据表明，NICTH中ALS的分子量大小与正常人相同，血清ALS水平的降低可能是NICTH中150kDa复合物形成受损的原因之一。为了表征大IGF-II，我们使用针对促IGF-II的E10-21 （Ab E10-21）和E71-89 （Ab E71-89）的特异性抗体，通过WIB研究了NICTH患者的大IGF-II。采用Ab E10-21和IGF-II抗体，用WIB法分别检测NICTH血清中14 ~ 18kda蛋白。用Ab E71 ~ 89进行Western免疫印迹分析，检测到~ 10 kDa蛋白。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蛋白被认为是促IGF-II的c端片段。这些数据提示NICTH中的大IGF-II不是完整的pro-IGF-II，而是由异常加工产生的pro-IGF-II- (E1-21)，并且c端片段也存在于循环中。2)胰岛素样生长因子ⅱ（insulin-like growth factor II）未提取硒Western immunoblot （WIB）的临床应用：为了研究大的IGF-II，采用了WIB技术。在我们之前的研究中，电泳样品需要从血清中酸乙醇提取IGF-II。最近，我们简化了方法，将未提取的血清样品直接用于电泳样品和Mini-Gel系统。该方法与酸-乙醇提取血清WIB法测定结果一致。此外，分析IGF-II所需的时间缩短了（2天比5天）。因此，该WIB系统使我们能够轻松地分析IGF-II的分子异质性。3)等位基因特异性聚合酶链（AS-PCR）方法在肿瘤中研究胰岛素样生长因子II基因印迹缺失的有效性：我们使用AS-PCR方法检测了10例NICTH肿瘤中IGF2的表达。IGF2在所有检查的肿瘤组织中均有表达，并且在7个信息性肿瘤中有5个呈双等位表达（LOI）。这些数据与Apa - 1消化法的结果一致。1例NICTH（胃癌）肿瘤组织（原发和转移肿瘤）显示IGF2双等位基因表达，AS-PCR显示IGF-2在非肿瘤组织中为单等位基因表达，但Apa消化法未清晰显示。本研究表明，AS-PCR是研究IGF2 LOI的一种简便易行的方法。少

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