Role for membrane and secreted insulin-like growth factor-binding protein-2 in the regulation of insulin-like growth factor action in lung tumors

The insulin-like growth factors (IGFs) have been implicated in the autocrine and/or paracrine growth of a number of tumor types, including lung tumors. Importantly, insulin-like growth factor-binding proteins (IGFBPs), which both enhance and inhibit the physiological and biological actions of the IGFs, have been shown to be secreted in vitro by a wide range of tumors. In particular, IGFBP-2 is frequently produced by human tumor cells, suggesting that this protein may be an important determinant of IGF action in tumors. In the present study, we investigated IGFBP-2 effects in lung tumor cells by examining the influence of IGFBP-2 on IGF-receptor interaction and the biological actions of IGF-I and IGF-II. Affinity cross-linking studies demonstrated expression of type-I and type-II IGF receptors on small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC) cells and the presence of abundant membrane-associated IGFBP in SCLC cells but not in NSCLC cells. An antiserum specific for IGFBP-2 was used in immunoprecipitation and immunoblotting studies which demonstrated that the membrane-associated IGFBP identified by affinity cross-linking in SCLC cells is IGFBP-2. In NSCLC cells, both IGF-I and IGF-II bound predominantly to IGF-I receptors, whereas in SCLC cells binding was principally to surface-associated IGFBP-2. SCLC cells failed to respond to IGF-I and -II stimulation in a DNA synthesis assay. For NSCLC cells, IGF-II was a more potent stimulator of DNA synthesis than IGF-I. Soluble IGFBP-2 inhibited the binding of radiolabeled IGF-I and -II to both SCLC and NSCLC cells in a concentration-dependent manner and inhibited IGF-stimulated DNA synthesis in NSCLC cells. These observations indicate that both soluble and membrane-associated IGFBP-2 compete with IGF receptors for ligand binding and, thus, are likely to be important determinants of IGF responsiveness. The findings of the present study also indicate that the type-I receptor on NSCLC cells contains a high-affinity binding site for IGF-II which presumably mediates the biological effects of IGF-II in these cells, thereby implicating IGF-II in the autocrine/paracrine growth of NSCLC.     

Histamine-stimulated expression of insulin-like growth factors in human glioma cells

Glioma tumour growth is associated with the expression of insulin-like growth factors I and II (IGFs) and of both type I and type II IGF receptors. It has also been shown that IGFs can stimulate proliferation of cultured glioma cells. We previously reported that histamine too can stimulate the growth of glioma cells in vitro. In this report, we study whether the histamine-induced growth of G47 glioma cells is mediated by the IGFs. We found that histamine stimulates the expression of both IGF-I and IGF-II mRNAs, as determined by a semiquantitative in situ hybridization analysis. Furthermore, incubation of G47 cells with histamine also induced cellular immunostaining for IGF-II. It could be shown that IGF-I-stimulated proliferation is inhibited by IGFBP-3, which decreases the availability of IGFs for binding to the IGF receptors, and by beta-galactosidase, which may decrease IGF binding to the type II IGF receptor, but is not inhibited by the anti-type I IGF receptor monoclonal antibody alphaIR3. However, neither IGFBP-3 nor beta-galactosidase nor alphaIR3 inhibited the histamine-induced proliferation. These results show that the growth-stimulatory effect of histamine is accompanied by the induction of IGFs. This histamine-induced growth stimulation is not mediated by activation of cell surface IGF receptors, although intracrine activation of type II IGF receptors may be involved.     

Growth advantage of human leiomyoma cells compared to normal smooth-muscle cells due to enhanced sensitivity toward insulin-like growth factor I

Human uterine leiomyomas exhibit increased IGF-I binding compared to myometrium, while both tissues show IGF-I gene expression. In this study we have examined the functional importance of these findings by testing the presence of IGF-I in 15 leiomyoma biopsies and in 18 myometrium biopsies and the capacity of smooth-muscle cells cultured from these tissues to react to IGF-I. The mean IGF-I peptide concentration in leiomyomas was 3 times higher than in myometrium. This resulted from increased IGF-I uptake in leiomyomas rather than from increased synthesis, as these tissues contain higher concentrations of type-I IGF receptors, as detected by immunohistochemistry, and equal levels of IGF-I mRNA. Blocking IGF-I transport with cytochalasin-B and with the type-I IGF receptor blocking antibody alpha IR3 in cultured cells induced decreased immunostaining intensity for IGF-I in most myometrium and leiomyoma cultures, indicating that the detected IGF-I is internalized. Depending on the culture conditions, IGF-I administration yielded increased survival or a higher proliferation rate in leiomyoma cultures than in myometrium cultures, indicating the increased importance of exogenous IGF-I for the growth of transformed smooth-muscle cells. We conclude that the increased concentrations of type-I IGF receptors in leiomyoma compared to myometrial smooth-muscle cells are functional with respect to the enhanced internalization of IGF-I and that they provide these tumor cells with a growth advantage compared to their normal counterparts.     

Cellular distribution and ontogeny of insulin-like growth factors (IGFs) and IGF binding protein messenger RNAs and peptides in developing rat pancreas

To determine the role of insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) in the development of the pancreas, and specifically of the islets of Langerhans, we have examined the cellular distribution and developmental changes in the expression of IGFs and IGFBPs in the pancreas of the fetal and neonatal rat between 19.5 days of gestation and postnatal day 28. This represents a period of substantial growth and restructuring of the beta cell component in islets of this species. IGF-I, IGF-II, and IGFBPs-1 to -6 mRNAs were localized by in situ hybridization, and peptides by immunohistochemistry, in histological sections. IGF-II mRNA was highly expressed in islet cells and some ductal epithelial cells in late fetal and early neonatal life, but was barely detectable by postnatal day 28. IGF-II peptide showed a similar distribution. IGF-I mRNA was barely detected in the fetus or neonate and was localized predominantly in the ductal and acinar tissues after postnatal day 7. IGF-I immunoreactivity was associated with some islet cells in the fetus and neonate, suggesting an endocrine rather than a paracrine source. We performed co-localization studies to assess whether the distribution of IGFs within the pancreas might be due to a sequestration by locally produced IGFBPs. The presence of mRNAs for both IGFBPs-1 and -2 was minimal in the pancreas prior to postnatal day 7, although subsequently IGFBP-1 mRNA was seen in islet cells, while IGFBP-2 mRNA was localized in both islets and acinar tissues. In contrast, both IGFBPs-1 and -2 immunoreactivities were identified in islets from late fetal life, suggesting a circulatory source for these IGFBPs during early pancreatic development. IGFBPs-3 to -5 mRNAs and immunoreactivities were identified within islet cells throughout fetal and neonatal life, with IGFBPs-3 and -5 being mainly associated with the alpha cell-rich islet mantle. The results show a compartmentalization of IGFs within pancreatic tissue, reflecting both paracrine and endocrine sources. The localization and action of IGFs in pancreas likely involves sequestration and distribution by endogenous as well as circulating IGFBPs.     

Structure of the IGF-binding domain of the insulin-like growth factor-binding protein-5 (IGFBP-5): implications for IGF and IGF-I receptor interactions

Binding proteins for insulin-like growth factors (IGFs) IGF-I and IGF-II, known as IGFBPs, control the distribution, function and activity of IGFs in various cell tissues and body fluids. Insulin-like growth factor-binding protein-5 (IGFBP-5) is known to modulate the stimulatory effects of IGFs and is the major IGF-binding protein in bone tissue. We have expressed two N-terminal fragments of IGFBP-5 in Escherichia coli; the first encodes the N-terminal domain of the protein (residues 1-104) and the second, mini-IGFBP-5, comprises residues Ala40 to Ile92. We show that the entire IGFBP-5 protein contains only one high-affinity binding site for IGFs, located in mini-IGFBP-5. The solution structure of mini-IGFBP-5, determined by nuclear magnetic resonance spectroscopy, discloses a rigid, globular structure that consists of a centrally located three-stranded anti-parallel beta-sheet. Its scaffold is stabilized further by two inside packed disulfide bridges. The binding to IGFs, which is in the nanomolar range, involves conserved Leu and Val residues localized in a hydrophobic patch on the surface of the IGFBP-5 protein. Remarkably, the IGF-I receptor binding assays of IGFBP-5 showed that IGFBP-5 inhibits the binding of IGFs to the IGF-I receptor, resulting in reduction of receptor stimulation and autophosphorylation. Compared with the full-length IGFBP-5, the smaller N-terminal fragments were less efficient inhibitors of the IGF-I receptor binding of IGFs.     

Follicular fluid insulin-like growth factor-I and insulin-like growth factor-binding protein-1 and -3 vary as a function of ovarian reserve and ovarian stimulation

PURPOSE: Follicular fluid concentrations of insulin-like growth factor (IGF)-I, IGF-II, IGF-binding protein (BP)-1, and IGFBP-3 in 57 women undergoing in vitro fertilization and embryo transfer were examined to determine whether levels reflected differences in patients' exposure to gonadotropin stimulation and a diminished ovarian reserve. METHODS: Preovulatory follicular fluid was obtained from both gonadotropin-stimulated and unstimulated cycles. Subjects were grouped according to normal or decreased ovarian reserve and whether or not they received gonadotropin stimulation. RESULTS: The mean follicular fluid concentrations of IGF-I and IGFBP-1 were significantly lower in the "decreased" ovarian reserve group compared with the "normal" ovarian reserve group, with no change in estradiol or IGF-II levels. This resulted in a decreased molar IGF-I: BP ratio and an increased molar IGF-II:IGFBP-1 ratio. In unstimulated cycles, mean follicular fluid concentrations of IGFs did not differ significantly compared with those in stimulated cycles, whereas concentrations of IGFBP-1 and IGFBP-3 were significantly lower, leading to higher molar ratios of the IGFs to the binding proteins. CONCLUSIONS: Follicular fluid IGF and binding proteins vary as a function of ovarian reserve and gonadotropin stimulation. This may reflect either differences in oocyte quality or a suboptimal follicular fluid environment.     

Role of the insulin-like growth factor system in uterine function and placental development in ruminants

Maternal nutrition during pregnancy influences fetal and placental weights. The insulin-like growth factors (IGF) are also important determinants of fetal size. Furthermore, the expression of several components of the IGF system is regulated by nutrition. Effects of nutrition on fetal growth could therefore be mediated by the IGF system in the uterus and placenta. The oviductal mucosa produces IGF-I, which may influence oviductal secretions or act directly on embryonic type 1 IGF receptors. In the uterus, IGF-I mRNA is localized to the stroma surrounding the endometrial glands, which contain high concentrations of IGF type 1 receptors. Uterine IGF-I concentrations fall during pregnancy; therefore, glandular activity is more likely influenced by systemic than local IGF-I production. The IGF-II mRNA is present in both caruncles and fetal placental mesoderm, but concentrations are much higher in the latter. The actions of IGF-I and IGF-II on the endometrium and placenta are influenced by IGF-binding proteins. In the ewe, mRNAs for IGF binding protein-1 and -5 are located in the luminal and glandular epithelia, IGF binding proteins-2 and -4 are produced in the subepithelial stroma, and IGF binding protein-4 is also in the placentome capsule; IGF binding protein-3 is more widely expressed in both maternal and fetal tissues. The IGF binding proteins, therefore, form a major barrier to the passage of IGF between the fetal and maternal circulatory systems.     

Biosensor measurement of the binding of insulin-like growth factors I and II and their analogues to the insulin-like growth factor-binding protein-3

Most insulin-like growth factor (IGF) molecules in the circulation are found in a 150-kDa complex containing IGF-binding protein-3 (IGFBP-3) and an acid-labile subunit, which does not itself bind IGF. Affinities (Kd values) between 0.03 and 0.5 nM have been reported for IGF-I/IGFBP-3 binding, but no kinetic data are available. In this study we measured the high affinity binding of unlabeled IGFs and IGF analogues to recombinant unglycosylated IGFBP-3, using a BIAcoretrade mark instrument (Pharmacia Biosensor AB). IGF-I binding showed fast association and slow non-first-order dissociation kinetics, and an equilibrium Kd of 0.23 nM. IGF-II had similar kinetics with slightly higher affinity. Analogues with mutations in the first 3 amino acids of the B-region (des(1-3) IGF-I and long IGF-I) showed 25 and 50 times lower affinity than IGF-I. Replacement of residues 28-37 by Gly-Gly-Gly-Gly or deletion of residues 29-41 in the C-region had little effect on the kinetic parameters, contrasting with the markedly impaired binding of these analogues to the IGF-I receptor. Swapping of the disulfide bridges in IGF-I and the C-region mutants decreased the affinity dramatically for IGFBP-3, primarily by decreasing the association rate. Insulin had approximately 1000 times lower affinity than IGF-I.     

Insulin-like growth factors and ovarian physiology

OBJECTIVE: To review the available information regarding the roles of insulin-like growth factor (IGF)-IGF binding protein (IGFBP) system in ovarian physiology. DESIGN: Studies that specifically relate to the roles of ovarian folliculogenesis, oocyte maturation, and ovulation were identified through the literature and Medline searches. RESULTS: Numerous actions of the IGFs have been demonstrated in the ovary, including an enhancement of cell proliferation, aromatase activity, and progesterone biosynthesis. The ovarian IGF system, comprised of IGF-I and IGF-II peptides, IGFBPs and IGF receptors, plays a significant role in the process of follicular development. In addition, IGF-I stimulates the meiotic maturation of follicle-enclosed oocytes in vitro via the IGF-I receptors. IGFBP-3 significantly inhibit gonadotropin-induced ovulation and oocyte maturation by neutralizing endogenously produced IGF-I. Thus, the intraovarian IGF-IGFBP system play a significant role in the processes of follicular development, oocyte maturation, and ovulation. CONCLUSION: IGF-IGFBP systems have autocrine/paracrine regulatory actions in ovarian physiology. The disturbance of the IGF-IGFBP system in human ovaries may lead to an ovulation, disorders of androgen excess, and infertility.     

Cancellous bone structure in the growing and aging lumbar spine in a historic Nubian population

CONCLUSION: Serum levels of IGF-I, IGF-II, and IGFBP-3 are not elevated in pancreatic cancer and do not appear to have a significant role in glucose homeostasis in this group of patients. BACKGROUND: The insulin-like growth factors (IGFs) and their binding proteins (IGFBPs) have been implicated recently in the pathogenesis of pancreatic cancer, and increased serum levels of IGF-I or IGF-II have been reported previously in a number of other gastrointestinal malignancies. METHODS: Serum levels of IGF-I, IGF-II, and IGFBP-3 were measured by RIA in 20 patients with pancreatic cancer and 20 age-matched healthy control subjects and correlated with serum glucose, C-peptide, and glucose tolerance. RESULTS: No significant difference was observed in serum levels of IGF-I (13 vs 17 nmol/L, respectively), IGF-II (0.67 vs 0.91 U/mL), or IGFBP-3 (2.3 vs 2.3 mg/L) between the two groups of patients. Twelve (60%) patients had impaired glucose tolerance, but no correlation was observed between the serum levels of the IGFs and glucose tolerance.     

Effects of growth hormone and pregnancy on expression of growth hormone receptor, insulin-like growth factor-I, and insulin-like growth factor binding protein-2 and -3 genes in bovine uterus, ovary, and oviduct

The effects of growth hormone (GH) and pregnancy on insulin-like growth factor (IGF)-I, IGF binding protein (IGFBP)-2, and IGFBP-3 mRNA in reproductive tissues were studied in cattle. Lactating dairy cows were inseminated at estrus and treated with 25 mg/day GH (n = 8) or saline (n = 8) for 16 days. Corpus luteum (CL), ovary (CL removed), oviduct, endometrium, and myometrium were collected at the end of treatment. Messenger RNA for GH receptor, IGF-I, IGFBP-2, IGFBP-3, and actin were measured by nuclease protection assays. The CL contained more GH receptor mRNA than the other reproductive tissues examined. Expression of IGF-I mRNA was highest in myometrium, with lower amounts found in endometrium; the CL expressed the least amount of IGF-I mRNA. The IGFBP-2 mRNA was most abundant in endometrium and least abundant in CL. Expression of IGFBP-3 mRNA was detected in all reproductive tissues examined. However, endometrium, a tissue that expressed the most IGFBP-2 mRNA, had the lowest amount of IGFBP-3 mRNA. The GH receptor mRNA was decreased in cows treated with GH whereas the mRNA for IGF-I, IGFBP-2, or IGFBP-3 was not changed. In the reproductive tissues evaluated, cows that contained a conceptus at tissue collection (pregnant) had higher amounts of IGF-I mRNA than did nonpregnant cows. In summary, the level of mRNA encoding GH receptor, IGF-I, IGFBP-2, and IGFBP-3 varied within the tissues examined, suggesting that these genes may play a variety of roles in the bovine female reproductive tract. Supplemental GH failed to change the expression of IGF-I, IGFBP-2, and IGFBP-3 mRNA, possibly because of low GH receptor mRNA levels in tissues other than CL. A direct action of GH on IGF-I, IGFBP-2, or IGFBP-3 gene expression within cow reproductive tissues was not supported because the amount of IGF-I, IGFBP-2, or IGFBP-3 mRNA was not altered by GH.     

Insulin-like growth factor binding protein production in bovine retinal endothelial cells

Retinopathy is the most frequent microangiopathic complication in diabetes. Many circulating hormones and locally produced mitogenic factors have been involved. Bovine retinal endothelial cells (BRECs) were cultured to investigate if insulin, insulin-like growth factors (IGFs), IGF binding proteins (IGFBPs), and a chronic high-glucose condition could control endothelial cell growth. Specific IGF-I receptors with two binding sites with high (Kd 0.03 nmol/L) and low (Kd 1.3 nmol/L) affinity were found when analyzing families of displacement curves between IGF-I versus IGF-I and IGF-I versus insulin. However, IGFs failed to be mitogenic factors in these cells. This could be explained by an inhibitory effect due to the presence of specific IGFBPs with a molecular weight between 24 and 43 kd. Using Western blot and immunoblot analysis, Northern blot study, and specific radioimmunoassay (RIA), these IGFBPs have been identified as IGFBP-3, -2, -5, and -4. Insulin, which does not bind to IGFBPs, was a potent mitogenic factor in these cells at a high concentration (10 nmol/L), suggesting a cross-reaction to IGF-I receptor. These IGFBPs, except the 24-kd form (IGFBP-4), were modulated by both IGF-I and IGF-II, with a maximum effect at 100 and 10 nmol/L, respectively. This regulation on IGFBPs was IGF-I receptor-independent. In fact, (1) IGFBP mRNA levels were not modified after stimulation with 100 nmol/L IGF-I, (2) 100 nmol/L IGF plus an equimolar concentration of alpha IR3 did not affect IGFBP production, (3) Des(1-3)IGF-I had no effect on IGFBP modulation, whereas at 10 nmol/L it enhanced BREC thymidine cell incorporation, and (4) 100 nmol/L insulin, which at this concentration can cross-react with the IGF-I receptor, did not modify the IGFBP pattern. Chronic exposure (4 weeks) of BRECs to 25 mmol/L glucose had no effect on cell growth. However, after 3 weeks, we observed a decreased IGFBP detection, and addition of 100 nmol/L IGF-I did not change IGFBP levels and did not modify cell growth. Conversely, BRECs grown in regular medium for 4 weeks showed increased IGFBP production. In conclusion, we showed that conditions mimicking hyperinsulinemia, rather than high levels of IGFs, could regulate BREC growth and that the IGF-I analog, Des(1-3), even with reduced affinity for IGFBPs but in part capable of binding to IGFBP-3, significantly stimulated BRECs growth only at 10 nmol/L. IGF actions are modulated by locally produced endothelial IGFBPs, and in turn, these endothelial IGFBPs are regulated, via in IGF-I receptor-independent mechanism, by the presence of IGFs. The autoregulatory IGF system together with the direct glucose modulation of IGFBPs could contribute in diabetic subjects to the retinal endothelial cell growth and metabolism through local changes in IGF bioavailability.     

Microalbuminuria in IDDM is associated with increased expression of monocyte procoagulant activity

Insulin-like growth factor II (IGF-II) plays a key role in mammalian growth, influencing foetal cell division and differentiation and possibly metabolic regulation. The mature 67 amino acid peptide shares sequence homology with both insulin and IGF-I. The liver is the main endocrine source of IGFs, but autocrine/paracrine activity is found in most tissues. The type 1 receptor mediates most of the biological effects of IGF-I and IGF-II; the type 2 receptor is involved with IGF-II degradation. Binding proteins may both localise IGFs to the receptors and regulate their activities. The IGF2 gene is maternally imprinted in mouse and human. Relaxation of IGF2 imprinting occurs in the Beckwith-Wiedemann syndrome of somatic overgrowth, sporadic Wilms' tumour and a number of other cancers. In the general adult population, the IGF2-INS gene cluster may also influence body weight, in which case IGF-II function could become a target for therapeutic intervention in obesity.     

Insulin-like growth factor system abnormalities in hepatitis C-associated osteosclerosis. Potential insights into increasing bone mass in adults

Hepatitis C-associated osteosclerosis (HCAO) is a rare disorder characterized by a marked increase in bone mass during adult life. Despite the rarity of HCAO, understanding the mediator(s) of the skeletal disease is of great interest. The IGFs-I and -II have potent anabolic effects on bone, and alterations in the IGFs and/or IGF-binding proteins (IGFBPs) could be responsible for the increase in bone formation in this disorder. Thus, we assayed sera from seven cases of HCAO for IGF-I, IGF-II, IGF-IIE (an IGF-II precursor), and IGFBPs. The distribution of the serum IGFs and IGFBPs between their ternary ( approximately 150 kD) and binary (approximately 50 kD) complexes was also determined to assess IGF bioavailability. HCAO patients had normal serum levels of IGF-I and -II, but had markedly elevated levels of IGF-IIE. Of the IGFBPs, an increase in IGFBP-2 was unique to these patients and was not found in control hepatitis C or hepatitis B patients. IGF-I and -II in sera from patients with HCAO were carried, as in the case of sera from control subjects, bound to IGFBP-3 in the approximately 150-kD complex, which is retained in the circulation. However, IGF-IIE was predominantly in the approximately 50-kD complex in association with IGFBP-2; this complex can cross the capillary barrier and access target tissues. In vitro, we found that IGF-II enhanced by over threefold IGFBP-2 binding to extracellular matrix produced by human osteoblasts and that in an extracellular matrix-rich environment, the IGF-II/IGFBP-2 complex was as effective as IGF-II alone in stimulating human osteoblast proliferation. Thus, IGFBP-2 may facilitate the targeting of IGFs, and in particular IGF-IIE, to skeletal tissue in HCAO patients, with a subsequent stimulation by IGFs of osteoblast function. Our findings in HCAO suggest a possible means to increase bone mass in patients with osteoporosis.     

The insulin-like growth factors (IGF) and IGF type I receptor during postnatal growth of the murine mammary gland: sites of messenger ribonucleic acid expression and potential functions

The goals of this study were to determine the cellular sites of insulin-like growth factor (IGF) and IGF type-I receptor (IGF-IR) expression and to begin to elucidate functional roles for the IGFs during postnatal development of the murine mammary gland. Using in situ hybridization analyses, we determined that IGF-I, IGF-II, and IGF-IR messenger RNAs were expressed in the highly proliferative terminal end buds during pubertal ductal growth. Consistent with these data, IGF-I (in combination with mammogenic hormones) promoted ductal growth in pubertal stage mammary glands cultured in vitro. During postpubertal and pregnancy stages, IGF-II and IGF-IR continued to be expressed in ductal epithelium. Expression of IGF-II in ductal and alveolar epithelium correlated with the pattern of rapidly proliferating cells, as determined by incorporation of 5-Bromo-2'-deoxyuridine, suggesting a potential autocrine or paracrine role for IGF-II as a mitogen for ductal epithelial cells. IGF-I expression was reinitiated in mammary epithelium in the differentiated alveoli at the end of pregnancy, suggesting an additional role for this factor in maintenance of the alveoli during lactation. Taken together, these data support an in vivo role for locally-produced IGFs in promoting ductal growth during puberty and suggest that IGF-I and IGF-II may have distinct functions during pregnancy-induced alveolar development.     

Circulating and ovarian IGF binding proteins: potential roles in normo-ovulatory cycles and in polycystic ovarian syndrome

IGFs function as co-gonadotropins in the ovary, facilitating steroidogenesis and follicle growth. IGFBP-1 to -5 are expressed in human ovary and mostly inhibit IGF action in in vitro ovarian cell culture systems. In the clinical disorder of polycystic ovarian syndrome (PCOS), which is characterized by hyperandrogenemia, polycystic ovaries and anovulation, follicles have a higher androgen: estradiol (A : E2) content and growth is arrested at the small antral stage. In the PCOS follicle, follicle stimulating hormone (FSH) and IGF levels are in the physiologic range, and even in the face of abundant androstenedione (AD) substrate, aromatase activity and E2 production are low. When PCOS granulosa are removed from their ovarian environment, they respond normally or hyperrespond to FSH. It has been postulated that an inhibitor of IGF's synergistic actions with FSH on aromatase activity may be one (or more) of the IGFBPs, which contributes to the arrested state of follicular development commonly observed in this disorder. High levels of IGFBP-2 and IGFBP-4 are present in follicular fluid (FF) from androgen-dominant follicles (FFa) from normally cycling women and in women with PCOS. This is in marked contrast to the near absence of these IGFBPs in estrogen-dominant FF (FFe), determined by Western ligand blotting. Regulation of granulosa-derived IGFBPs is effected by gonadotropins and insulin-like peptides. In addition, an IGFBP-4 metallo-serine protease is present in FFe, but not in FFa in ovaries from normally cycling women and those with PCOS, although the IGFBP-4 protease is present in PCOS follicles hyperstimulated for in vitro fertilization. Recent studies demonstrate that IGF-II in FFe is higher than in FFa' whereas IGF-I, IGFBP-3 and IGFBP-1 levels do not differ, underscoring the importance of local IGF-II production by the granulosa and the importance of IGFBP-4 and IGFBP-2 in regulation of IGF-II action within the follicle during its developmental pathway as an E2- or A-dominant follicle. In the androgen-treated female-to-male transsexual (TSX) model for PCOS, IGF-I, IGF-II, IGFBP-3 and IGFBP-1 levels do not differ.     

Insulin-like growth factor (IGF)-binding protein 5 forms an alternative ternary complex with IGFs and the acid-labile subunit

Up to 90% of circulating insulin-like growth factors (IGF-I and IGF-II) are carried in heterotrimeric complexes with a binding protein (IGFBP) and a liver-derived glycoprotein known as the acid-labile subunit. IGFBP-3 is considered unique among the six well characterized IGFBPs in its ability to complex with the acid-labile subunit. However, a basic carboxyl-terminal domain of IGFBP-3, known to be involved in its interaction with the acid-labile subunit, is shared by IGFBP-5, suggesting the possibility of ternary complexes containing IGFBP-5. We now demonstrate using three independent methods that human IGFBP-5, when occupied by IGF-I or IGF-II, forms ternary complexes of approximately 130 kDa with the acid-labile subunit. IGFBP-3 competes with approximately twice the potency of IGFBP-5 for the formation of such complexes. No other IGFBP complexes with the acid-labile subunit itself or competes with IGFBP-5 for complex formation. As observed for IGFBP-3, ternary complexes containing IGFBP-5 form preferentially in the presence of IGF-I, even though IGFBP-5 has a preferential affinity for IGF-II over IGF-I. By size fractionation chromatography, serum IGFBP-5 co-elutes predominantly with ternary complexes. The demonstration of IGFBP-5-containing ternary complexes indicates an unrecognized form of IGF transport in the circulation and an additional mechanism for regulating IGF bioavailability.     

Free and total insulin-like growth factors and insulin-like growth factor binding proteins during 14 days of growth hormone administration in healthy adults

The objective was to investigate the effect of growth hormone (GH) administration on circulating levels of free insulin-like growth factors (IGFs) in healthy adults. Eight healthy male subjects were given placebo and two doses of GH (3 and 6 IU/m2 per day) for 14 days in a double-blind crossover study. Fasting blood samples were obtained every second day. Free IGF-I and IGF-II were determined by ultrafiltration of serum. Total IGF-I and IGF-II were measured after acid-ethanol extraction. In addition, GH, insulin, IGF binding protein 1 (IGFBP-1) and IGFBP-3 were measured. Serum-free and total IGF-I increased in a dose-dependent manner during the 14 days of GH administration. After 14 days, serum-free IGF-I values were 610 +/- 100 ng/l (mean +/- SEM) (placebo), 2760 +/- 190 ng/l (3 IU/ m2) and 3720 +/- 240 ng/l (6 IU/m2) (p = 0.0001 for 3 and 6 IU/m2 vs placebo; p = 0.004 for 3 IU/m2 vs 6 IU/m2). Total IGF-I values were 190 +/- 10 micrograms/l (placebo), 525 +/- 10 (3 IU/m2), and 655 +/- 40 micrograms/l (6 IU/m2) (p < 0.0001 for 3 and 6 IU/m2 vs placebo; p = 0.04 for 3 IU/m2). There were no differences in the levels of free or total IGF-II during the three study periods. Insulin-like growth factor binding protein 1 was decreased during GH administration (p = 0.04 for placebo vs 3 IU/m2; p = 0.006 for placebo vs 6 IU/m2). In conclusion, fasting serum free IGF-I increased dose dependently during GH administration and free IGF-I increased relatively more than total IGF-I. This may partly be due to the decrease in IGFBP-1.