| Research Abstract |
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,
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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
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