Study for biological effect of IGF-II and the Pathological significance.

在本研究中，我们研究了IGF-II的生物学作用和病理意义如下。1)正常血清中胰岛素样生长因子II （IGF-II）的主要形态为7.5Da，少量IGF-II为11kDa。相比之下，在非胰岛细胞肿瘤低血糖（NICTH）患者中，大多数IGF-II在11-18kDa检测到。我们研究了NICTH患者的大IGF-II是否被o糖基化，如果是，糖块的大小在不同的病例中是否不同。在正常受试者和肢端肥大症患者中，使用O-Glycanase后，IGF-II的大小为11kDa，降至9.5kDa。在11例NICTH患者中，IGF-II的11- 18kda大小降至与正常人相同的9.5kDa。这些数据表明NICTH患者的大IGF-II是o糖基化的，并且糖块的大小在不同病例之间有所不同。来自NICTH的大IGF-II的胰岛素样作用与脂肪细胞中真正的IGF-II没有区别。人类IGF-II基因（IGF2）是主要的印迹基因，通常只表达IGF2的父系等位基因。最近，IGF2的印迹缺失（LOI）在一些胚胎性肿瘤中被报道，如Wilms'肿瘤和横纹肌肉瘤，这表明该基因的双等位基因表达导致IGF-II肽的过度表达和有丝分裂活性的增加。我们利用IGF2第9外显子的Apa I限制性内切酶片段长度多态性（RELP）检测了NICTH中IGF2的等位基因表达。IGF2在6个信息性肿瘤中的5个呈双等位表达（LOI），提示LOI可能导致IGF-II过表达。2)在胰岛素抵抗小鼠中，胰岛素没有降低血糖水平，但注射IGF-1和IGF-II后血糖水平降低。IGF-II突变体对IGF-II受体不具有亲和力，但对IGF- i和胰岛素受体具有与IGF-II相同的亲和力，导致与IGF-II相同的低血糖。然而，对胰岛素和IGF- i受体的亲和力明显降低的IGF-II突变体没有降低血糖水平，而对胰岛素和IGF-II受体的亲和力略有降低的IGF-II突变体略有降低血糖水平。这些数据表明，IGF-II的降糖作用主要通过胰岛素和/或IGF-I受体起作用，而不是通过IGF-II受体起作用。此外，数据表明IGF-II也可能对治疗胰岛素抵抗状态有用。3) igfbp干扰RIA中IGF-II的测定。我们建立了不受igfbp干扰直接检测IGF-II的点印迹法，并用该方法测定了脑脊液中IGF-II的含量。我们发现该点印迹系统可用于评估脑脊液中IGF-II的值。少

In this study, we have investigated biological effect of IGF-II and the pathological significance as follows. 1) In normal serum, major form of insulin-like growth factor II (IGF-II) was 7.5Da and small amount of 11kDa IGF-II was found. By contrast, in patients with non-islet cell tumor hypoglycemia (NICTH), most of IGF-II was detected at 11-18kDa. We investigated whether the big IGF-II from patients with NICTH was O-glycosylated, and if so, whether the size of sugar moiety is different among the cases. In normal subjects, and patients with acromegaly, size of 11kDa IGF-II was reduced to 9.5kDa after O-Glycanase. In 11 patients with NICTH,size of 11-18kDa IGF-II was reduced to 9.5kDa as same as normal subjects. These data indicated that big IGF-II from patients with NICTH was O-glycosylated, and the size of sugar moiety was different among cases. Insulin-like efect of the big IGF-II from NICTH did not differ from that of authentic IGF-II in adipocytes. Human IGF-II gene (IGF2) is mater … More nally imprinted, and only the paternal allele of IGF2 is usually expressed. Recently, loss of imprinting (LOI) of IGF2 has been reported in some embryonal tumors, such as Wilms' tumor and rhabdomyosarcoma, suggesting that biallelic expression of the gene leads to overexpression of IGF-II peptide and increased mitogenic activity. We examined allelic gene expression of IGF2 in NICTH using the Apa I restriction nzyme fragment length polymorphism (RELP) in exon 9 of IGF2. IGF2 was expressed bialleically (LOI) in five of six informative tumors, suggesting that LOI might lead overexpression of IGF-II.2) In the insulin resistant mice, insulin did not decrease the blood glucose levels, but, the blood glucose levels decreased after IGF-1 and IGF-II injection. The IGF-II mutant without affinity for IGF-II receptor but with affinities for both IGF-I and insulin receptors as same as IGF II,caused hypoglycemia as same as IGF-II.However, the IGF-II mutant with markedly decreased affinities for both insulin and IGF-I receptors did not decrease blood glucose levels, and the IGF-II mutant with slightly decreased affinities for both insulin and IGF II receptors slightly decreased blood glucose levels. These data indicate that the hypoglycemic effect of IGF-II is acted through mainly insulin and/or IGF-I receptor but not IGF-II receptor. Furthermore, the data suggest that IGF-II might be also useful for treatment of insulin resistant status. 3) IGFBPs interfere with the measurement of IGF-II in RIA.We developed dot blot method to detect directly IGF-II without interference by IGFBPs and measured IGF-II in cerebrospinal fluid (CSF) by this method. We found that this dot blot system is useful to evaluate IGF-II values in CSF. Less