Study of serum big-insulin-like growth factor (IGF)-II and IGF binding proteins in two patients with extrapancreatic tumor hypoglycemia, using a combination of Western blotting methods

Abstract

Extrapancreatic tumor hypoglycemia (EPTH) is associated with increased amounts of high-molecularweight precursor forms of insulin-like growth factor (IGF)-II ('big-IGF-II') that have a primary role in the pathophysiology of hypoglycemia. In the present study, using Western ligand and immunoblotting methods, we investigated IGF-binding proteins (IGFBPs), IGFBP-3 proteolysis and big-IGF-II in pre-and postoperative serum from two patients with EPTH due to benign pleural fibroma. In the preoperative serum, IGFBP-3 was reduced and IGFBP-2 was increased compared with that from an age-matched healthy control. IGFBP-3 proteolysis was dramatically reduced in one patient, whereas no major alteration was observed in the other (9% and 120% of control serum, respectively). IGFBPs progressively returned to a subnormal pattern in postoperative serum, whereas IGFBP-3 proteolysis remained greater than in preoperative serum in both patients at days 14 and 90 after surgery. High-molecular-weight forms of IGF-II predominate in EPTH serum (65% and 57% of total IGF-II immunoreactivity in patients 1 and 2, respectively, compared with 25% in control serum). Two forms, of molecular mass 10 and 12 kDa ('standard big-IGF-II') were present in both EPTH and control sera, whereas two additionnal forms, of molecular mass 15 and 18 kDa ('big big-IGF-II') were observed in EPTH sera only. Big big-IGF-II represented 72% and 55% of total high-molecular-weight forms of IGF-II in the two EPTH sera, respectively. All big forms of IGF-II disappeared from the serum as early as 6 h after surgery. This study shows that combination of simple Western blotting methods, available routinely in most laboratories, should prove useful in providing reliable physiopathological information in EPTH.

Myelination Is Altered in Insulin-Like Growth Factor-I Null Mutant Mice

“…The ϳ7.5 kDa comigrates with recombinant IGF-II and represents mature IGF-II, whereas the more abundant bands migrating at ϳ12 kDa likely represent precursor forms. Precursor IGF-II forms of 10 -18 kDa have been reported in multiple tumor cells lines (Schmitt et al, 1997;Bae et al, 1998) and in the serum of normal and tumor patients (Hoeflich et al, 1995;Bae et al, 1998;Christofilis et al, 1998). The expression of IGF-II in developing and adult rodent CNS has been well studied using a variety of methods, including immunohistochemistry (Logan et al, 1994;Walter et al, 1999), in situ hybridization (Cavallaro et Logan et al, 1994;Walter et al, 1999), and RNA protection assay (Rotwein et al, 1988); however, only a few studies have documented its molecular forms in brain (Haselbacher et al, 1985).…”

“…The expression of IGF-II in developing and adult rodent CNS has been well studied using a variety of methods, including immunohistochemistry (Logan et al, 1994;Walter et al, 1999), in situ hybridization (Cavallaro et Logan et al, 1994;Walter et al, 1999), and RNA protection assay (Rotwein et al, 1988); however, only a few studies have documented its molecular forms in brain (Haselbacher et al, 1985). Although the physiologic significance of the expressed IGF-II precursor is not clear, the precursor derived from tumors has been shown to have biologic activity, including promotion of angiogenesis and hexosaminidase secretion (Hoeflich et al, 1995;Bae et al, 1998;Christofilis et al, 1998). We speculate that the increase in IGF-II during rapid myelination (2-4 weeks of age) and in early adulthood is responsible for the recovery of myelin-specific protein expression in adult IGF-I KO mice.…”

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2

“…Moreover, the previous studies 7 , 10 , 16 – 19 that demonstrated an abnormal occurrence of pro- and big-IGF2s have mostly been unable to distinguish between the exact proforms of IGF2. This was, and still is, challenging due to a varying degree of glycosylation affecting the molecular weight of IGF2 proforms 30 . In this study, we produced all three IGF2 proforms in their non-glycosylated forms that are suspected of having stronger proliferative properties than their glycosylated counterparts.…”

Non-glycosylated IGF2 prohormones are more mitogenic than native IGF2

“…These unprocessed forms of pro-IGF2(156) result likely from a defective glycosylation of the IGF2 prohormone which prevents the cleavage of this precursor to the mature protein by prohormone convertase (probably pro-protein convertase subtilisin/kexin type 4 (PCSK4)) (Kawai, 2018). However, the molecular mechanisms of these effects and the impact of individual IGF2-derived proforms are still unknown, and – importantly – previous studies have been unable to distinguish between the exact proforms of the IGF2 (De Groot, 2007; (Dynkevich, 2013; Khosla, 2002; Ono, 2021; Qiu, 2010; Zapf, 1992), due to degree of glycosylation affecting the molecular weight of these IGF2 polypeptides (Christofilis, 1998).…”