BMN 250, a fusion of lysosomal alpha-N-acetylglucosaminidase with IGF2, exhibits different patterns of cellular uptake into critical cell types of Sanfilippo syndrome B disease pathogenesis.

Sanfilippo syndrome type B (Sanfilippo B; Mucopolysaccharidosis type IIIB) occurs due to genetic deficiency of lysosomal alpha-N-acetylglucosaminidase (NAGLU) and subsequent lysosomal accumulation of heparan sulfate (HS), which coincides with devastating neurodegenerative disease. Because NAGLU expressed in Chinese hamster ovary cells is not mannose-6-phosphorylated, we developed an insulin-like growth factor 2 (IGF2)-tagged NAGLU molecule (BMN 250; tralesinidase alfa) that binds avidly to the IGF2 / cation-independent mannose 6-phosphate receptor (CI-MPR) for glycosylation independent lysosomal targeting. BMN 250 is currently being developed as an investigational enzyme replacement therapy for Sanfilippo B. Here we distinguish two cellular uptake mechanisms by which BMN 250 is targeted to lysosomes. In normal rodent-derived neurons and astrocytes, the majority of BMN250 uptake over 24 hours reaches saturation, which can be competitively inhibited with IGF2, suggestive of CI-MPR-mediated uptake. Kuptake, defined as the concentration of enzyme at half-maximal uptake, is 5 nM and 3 nM in neurons and astrocytes, with a maximal uptake capacity (Vmax) corresponding to 764 nmol/hr/mg and 5380 nmol/hr/mg, respectively. Similar to neurons and astrocytes, BMN 250 uptake in Sanfilippo B patient fibroblasts is predominantly CI-MPR-mediated, resulting in augmentation of NAGLU activity with doses of enzyme that fall well below the Kuptake (5 nM), which are sufficient to prevent HS accumulation. In contrast, uptake of the untagged recombinant human NAGLU (rhNAGLU) enzyme in neurons, astrocytes and fibroblasts is negligible at the same doses tested. In microglia, receptor-independent uptake, defined as enzyme uptake resistant to competition with excess IGF2, results in appreciable lysosomal delivery of BMN 250 and rhNAGLU (Vmax = 12,336 nmol/hr/mg and 5469 nmol/hr/mg, respectively). These results suggest that while receptor-independent mechanisms exist for lysosomal targeting of rhNAGLU in microglia, BMN 250, by its IGF2 tag moiety, confers increased CI-MPR-mediated lysosomal targeting to neurons and astrocytes, two additional critical cell types of Sanfilippo B disease pathogenesis.

Direct repression of IGF2 is implicated in the anti-angiogenic function of microRNA-210 in human retinal endothelial cells.

Pathological angiogenesis leads to the development of retinal vasculopathies and causes severe vision impairment. Increased understanding of the mechanisms underlying the angiogenic behavior of retinal endothelial cells helps provide new insights for developing treatment of retinal vasculopathies. Pro-angiogenic function of miR-210 has previously been identified. However, the functional implication of miR-210 in retinal endothelial cells remains unknown. Human retinal microvascular endothelial cells (HRECs) were employed to investigate the impact of miR-210 on the angiogenic capacity of retinal endothelial cells. It was observed that without affecting the viability of HRECs, miR-210 significantly suppressed the migration and capillary-like tube formation in HRECs. Moreover, pro-angiogenic insulin growth factor 2 (IGF2) was newly identified as a direct target of miR-210 in HRECs. MiR-210 decreased the expression of IGF2 at both mRNA and protein levels in HRECs. IGF2-simulated activation of p38 MAPK was attenuated by miR-210 in HRECs. Recombinant IGF2 protein rescued miR-210-induced impairment of tube formation in HRECs. Therefore, in contrast to the previously reported pro-angiogenic function of miR-210, the current work reveals novel anti-angiogenic activity of miR-210 in HRECs. Furthermore, IGF2 is identified for the first time as a direct target of miR-210 in HRECs, adding new mechanistic insights into the expression regulation of pro-angiogenic IGF2 in human retinal endothelial cells. The current work helps increase the understanding of regulatory mechanisms underlying retinal endothelial cell physiology, justifying further evaluation for the therapeutic implications of miR-210/IGF2 interaction in the treatment of related retinal vasculopathies.

IGF2 actions on trophoblast in human placenta are regulated by the insulin-like growth factor 2 receptor, which can function as both a signaling and clearance receptor.

Insulin-like growth factor 2 (IGF2) enhances proliferation and survival of human first-trimester cytotrophoblasts (CTB) by signaling through the insulin-like growth factor 1 receptor (IGF1R). However, the role of the IGF2 receptor (IGF2R) in regulating trophoblast kinetics is unclear: It could act as a clearance receptor for trafficking excess ligand to lysosomes for degradation and/or directly mediate IGF2 signaling. We used an IGF2R knockdown strategy in BeWo cells and placental villous explants to investigate trophoblast proliferation and survival in response to stimulation by IGF. Both IGF1 and IGF2 significantly (P < 0.001) increased mitosis and reduced apoptosis in serum-starved BeWo cells. Small interfering RNA (siRNA)-mediated knockdown of IGF2R further enhanced IGF2-stimulated mitosis (P < 0.01), and IGF2-mediated rescue of apoptosis (P < 0.001) in these cells. Leu(27)IGF2, an IGF2 analogue that binds to IGF2R but not IGF1R, also protected IGF2R-expressing BeWo cells from apoptosis but did not increase mitosis. IGF treatment of term placental villous explants with reduced syncytial expression of IGF2R increased CTB proliferation (P < 0.001) and decreased apoptosis (P < 0.01) compared to untreated controls. Moreover, IGF2-mediated rescue of CTB apoptosis was significantly greater than that in tissue with normal IGF2R expression. Leu(27)IGF2 promoted mitogenesis and survival only in explants with intact IGF2R expression. Given that altered CTB turnover is observed in pregnancies complicated by fetal growth restriction, the development of strategies to manipulate the IGF2R signaling axis in the syncytiotrophoblast may provide a therapeutic avenue for treating this condition.

Decreased expression of the mannose 6-phosphate/insulin-like growth factor-II receptor promotes growth of human breast cancer cells.

BACKGROUND: Loss or mutation of the mannose 6-phosphate/insulin-like growth factor-II receptor (M6P/IGF2R) has been found in breast cancer. However, whether or not decreased levels of functional M6P/IGF2R directly contribute to the process of carcinogenesis needs to be further verified by functional studies. METHODS: In this study, using viral and ribozyme strategies we reduced the expression of M6P/IGF2R in human breast cancer cells and then examined the effect on growth and apoptosis of these cells. RESULTS: Our results showed that infection of MCF-7 cells with the adenovirus carrying a ribozyme targeted against the M6P/IGF2R mRNA dramatically reduced the level of transcripts and the functional activity of M6P/IGF2R in these cells. Accordingly, cells treated with a ribozyme exhibited a higher growth rate and a lower apoptotic index than control cells (infected with a control vector). Furthermore, decreased expression of M6P/IGF2R enhanced IGF-II-induced proliferation and reduced cell susceptibility to TNF-induced apoptosis. CONCLUSIONS: These results suggest that M6P/IGF2R functions as a growth suppressor and its loss or mutation may contribute to development and progression of cancer. This study also demonstrates that adenoviral delivery of the ribozyme provides a useful tool for investigating the role of M6P/IGF2R in regulation of cell growth.

Characterization of pituitary IGF-I receptors: modulation of prolactin and growth hormone.

There have been no studies in any vertebrate that have localized insulin-like growth factor (IGF)-I receptors in prolactin (PRL) cells or that have correlated pituitary binding to the potency of IGF-I in regulating both PRL and growth hormone (GH) secretion. We show that IGF-I binds with high affinity and specificity to the pituitary gland of hybrid striped bass (Morone saxatilis x M. chrysops). IGF-I and IGF-II were equipotent in inhibiting saturable (125)I-IGF-I binding, whereas insulin was ineffective. IGF-I binds with similar affinity to the rostral pars distalis (>95% PRL cells) as the whole pituitary gland and immunohistochemistry colocalizes IGF-I receptors and PRL in this same region. Des(1-3)IGF-I, a truncated analog of IGF-I that binds with high affinity to IGF-I receptors but weakly to IGF-I binding proteins (IGFBPs), showed a similar inhibition of saturable (125)I-IGF-I binding, but it was more potent than IGF-I in stimulating PRL and inhibiting GH release. These results are the first to localize IGF-I receptors to PRL cells, correlate IGF-I binding to its efficacy in regulating GH and PRL secretion, as well as demonstrate that IGFBPs may play a significant role in modulating the disparate actions of IGF-I on PRL and GH secretion.

Target-derived cardiotrophin-1 and insulin-like growth factor-I promote neurite growth and survival of developing oculomotor neurons.

Several trophic factors support the survival of developing motoneurons, but it is not known whether these factors act in a retrograde fashion from the motoneuron target muscle or are derived from other sources. Cardiotrophin-1 (CT-1) and the insulin-like growth factors (IGFs) are candidate target-derived motoneuron survival factors as both are expressed in muscle during naturally occurring motoneuron death and, applied systemically, support the survival of developing motoneurons. By using the embryonic chick oculomotor system, we show that CT-1 and IGF-I promote neurite outgrowth from E13-derived oculomotor explants and are retrogradely transported from muscle to nerve cell body in vivo, and injection of CT-1 or IGF-I into eye muscles increases motoneuron survival by 20 and 30%, respectively, as evidenced by calibrated stereological counting techniques. Pharmacological depletion of endogenous target-derived IGF-I in vivo reduces oculomotor neuron survival by up to 30% in a dose-dependent manner. These results significantly extend previous studies using systemic administration of trophic factors and are the first to demonstrate a target-derived retrograde mechanism of developing motoneuron survival factors.

Uptake of circulating insulin-like growth factors (IGFs) into cerebrospinal fluid appears to be independent of the IGF receptors as well as IGF-binding proteins.

Peripheral administration of human insulin-like growth factor (hIGF) results in both uptake of hIGF into the cerebrospinal fluid (CSF) and amelioration of brain injury. We tested the hypotheses that IGF uptake into CSF is independent of IGF receptors and IGF-binding proteins (IGFBP). Adult rats were injected sc with various concentrations of hIGF-I or structural analogs, and serum and CSF were withdrawn for assay 90 min later. An enzyme-linked immunoassay was used that detected immunoreactive hIGF-I and its analogs, but not rat IGF-I, IGF-II, or insulin. Plasma hIGF-I levels increased linearly (r = 0.97) with hIGF-I dose between 25-300 microgram/rat. By contrast, uptake into CSF reached saturation above 100 microgram, suggesting carrier-mediated uptake. hIGF-II reduced the uptake of hIGF-I into CSF (P < 0.02). Des(1-3)hIGF-I is a hIGF-I analog missing the N-terminal tripeptide, resulting in greatly reduced affinity for IGFBP-1, -3, -4, and -5. Nevertheless, des(1-3)hIGF-I was taken up into CSF. [Leu(24)]hIGF-I and [Leu(60)]hIGF-I have 20- to 85-fold reduced affinity for the type I IGF receptor, yet both were taken up into CSF in amounts similar to hIGF-I. In addition, hIGF-I and des(1-3)hIGF-I were taken up into CSF, although binding to the type II receptor is extremely weak. These data suggest that uptake of circulating IGF-I into CSF is independent of the type I or II IGF receptors as well as IGF sequestration to IGFBP-1, -3, -4, or -5.

Absorption of milk-borne insulin-like growth factor-I into portal blood of suckling rats.

BACKGROUND: Insulin-like growth factors (IGFs) are potent mitogens that have been implicated in control of growth and development during the perinatal period. These hormones are also present in biologically significant quantities in mammalian milks. Although one site of action of these IGFs may be at the intestinal level, current information about whether they pass intact into the circulation is conflicting. METHODS: To test the hypothesis that milk-borne IGFs are absorbed into blood in receptor-active form, suckling rats were given either recombinant human (rh)125I-IGF-I or -II (4 x 10(6) counts per minute [cpm]), and the activity present in portal and cardiac blood was examined at 5, 10, 20, and 30 minutes after ingestion for presence of appropriate molecular weight peptides in these samples. In selected samples, purified radioactive samples were tested for their ability to bind competitively to crude membranes bearing IGF receptors. RESULTS: The results of these studies indicate that rh125I-IGF-I is absorbed in receptor-active form into the portal circulation and that maximal amounts are present 20 to 30 minutes after ingestion. Estimation of the presence of intact hormone was made on the basis of the elution profile of samples when run on gel chromatography as well as reversed-phase high-performance liquid chromatography. Isolated samples from portal blood also bound competitively to placental membranes bearing IGF receptors. In contrast, rh125I-IGF-II could not be demonstrated in receptor-active form in portal blood. Chromatography showed appropriate sized peaks with greater activity in portal than cardiac samples, but competitive binding was not appreciated. CONCLUSIONS: It is likely that at least milk-borne IGF-I is absorbed intact and may exert effects on liver and other peripheral tissues. In addition, this study lends further credence to the possibility of an enterohepatic circulation for IGF-I.

Clearance of IGFs and insulin from wounds: effect of IGF-binding protein interactions.

We have examined the role binding proteins have in regulating the clearance of exogenous growth factors from wounds. Hunt-Schilling chambers were subcutaneously implanted in rats, and the clearance of insulin-like growth factor (IGF) I from the chamber wound fluid was compared with IGF-II, LR3-IGF-I, which binds poorly to IGF-binding proteins (IGFBP), or insulin. Elimination rate constants of the slow phase of the decay curves did not differ between IGF-I and IGF-II. However, LR3-IGF-I and insulin were cleared more rapidly from wound fluid than IGF-I so that the half-lives for IGF-I, IGF-II, LR3-IGF-I, and insulin were 872, 861, 563, and 324 min, respectively. In wound fluid, minimal degradation of the IGFs occurred, whereas insulin was degraded considerably. The increased clearance of LR3-IGF-I and insulin equated with a reduced association with wound fluid IGFBPs, and increased amounts of radioactivity of these peptides were detected in the circulation and urine. These results show that this model of wound repair may be of use in examining the kinetics of growth factors and other bioactive molecules in extravascular spaces and support the hypothesis that IGFBPs can be significant regulators of IGF bioavailability in vivo.

Physiological inhibition of growth hormone secretion by both insulin-like growth factors-I and -II in chickens.

1. The role of both insulin-like growth factors (IGF)-I and -II in regulation of growth hormone (GH) secretion in chickens was examined. Seven-week-old male broiler chickens were injected intravenously (i.v.) with recombinant human IGF-I or IGF-II or specific anti-IGF-I or IGF-II immunoglobulins. Blood samples were taken before treatment and at 15 min intervals afterwards for 1 h. Controls received saline i.v. 2. Both IGF-I and IGF-II administration resulted in a rapid, significant decrease in plasma GH concentrations, but the concentrations of both triiodothyronine and thyroxine remained unchanged. 3. Immunisation against both IGF-I and IGF-II produced a significant elevation in plasma GH. 4. These data show that both IGFs can regulate GH concentrations in birds. Furthermore, the immunoneutralisation data suggest that these hormones have a physiological role in the regulation of GH secretion.

Serum insulin-like growth factors (IGFs) and IGF binding protein (IGFBP)-3 in patients with gastric cancer: IGFBP-3 protease activity induced by surgery.

Recent findings have indicated that insulin-like growth factors (IGF-I and IGF-II) may play a role in neoplasia. Alteration of serum IGFs or IGF Binding Proteins (IGFBPs) have been reported in some tumors. In this study, we measured serum IGF-I, IGF-II and IGFBPs profile in gastric cancer by radioimmunoassay and Western ligand blots. The serum IGF-I level in gastric cancer was significantly lower than in control subjects (65.2 +/- 26.5 vs 148.4 +/- 55.2 ng/ml, p < 0.01) and was further decreased to 45.5 +/- 20.9 ng/ml after surgery. The serum IGF-II level was slightly higher than that in control subjects (826.3 +/- 360.2 vs 735.7 +/- 154.6 ng/ml) but it was significantly decreased after surgery (525.7 +/- 220.1 ng/ml, p < 0.05). The serum IGFBP-3 level was not significantly different from those in control subjects. However, we observed a decreased level of serum IGFBP-3 after surgery, and incubation of postoperative serum with control serum resulted in a significant reduction of IGFBP-3 level. The reduction of IGFBP-3 in postoperative serum was mainly due to surgery associated IGFBP-3 protease activity. This protease activity was totally inhibited by aprotinin, EDTA and PMSF but not by pepstatin and leupeptin. This inhibition pattern is consistant with cation dependent serine protease. We speculate that proteolysis of IGFBP-3 may contribute to increase the bioavailability of IGFs.

Influence of insulin-like growth factor-binding protein-2 on plasma clearance and transfer of insulin-like growth factors-I and -II from plasma into mammary-derived lymph and milk of goats.

Plasma clearance of insulin-like growth factors-I and -II (IGF-I and -II) and insulin-like growth factor-binding protein-2 (IGFBP-2) from lactating goats (n = 4) was determined following a single intravenous injection of the corresponding 125I-labelled human protein. Transfer of these proteins out of the vascular space was monitored by their subsequent appearance in mammary-derived lymph and milk. Clearance of 125I-IGFBP-2 from circulation was 0.37 +/- 0.06 ml/min/kg, which is markedly greater than that of 125I-IGF-I or -II (0.11 +/- 0.01 and 0.12 +/- 0.01 ml/min/kg respectively). This was also reflected in longer elimination half-lives for IGF-I (353 +/- 6 min) and -II (254 +/- 8 min) compared with IGFBP-2 (110 +/- 9 min). Three hours after injection of the 125I-labelled protein, the plasma:lymph ratio of trichloroacetic acid-precipitable radioactivity was 1.54 +/- 0.04, 3.3 +/- 0.6 and 4.1 +/- 0.4 for IGFBP-2, IGF-I and -II respectively. The form of 125I-IGFBP-2 in lymph was not different from that of plasma. Elevation of plasma concentrations of IGFBP-2 by its intravenous infusion significantly decreased plasma half-life of both IGF-I and -II (251 +/- 8 and 198 +/- 7 min respectively). Although the amount and rate of transfer of IGF into mammary-derived lymph was decreased slightly by IGFBP-2, concentrations eventually obtained were not different from control. However, secretion of IGFs into milk was significantly reduced by IGFBP-2, particularly in the case of IGF-I. These results are consistent with the ability of all three compounds to cross the vascular endothelium intact and of IGFBP-2 to decrease the uptake of IGF by mammary epithelium and subsequent secretion into milk. IGFBP-2 may well have acted to target plasma IGF towards non-mammary tissues, thus explaining the more rapid plasma clearance of IGFs in the presence of elevated IGFBP-2.

Plasma clearance and tissue distribution of labelled chicken and human IGF-I and IGF-II in the chicken.

The metabolic clearance of chicken IGF-I (cIGF-I), cIGF-II, human IGF-I (hIGF-I), and hIGF-II was examined in the chicken using 125I-labelled growth factors. Superose-12 chromatography of plasma collected at 7.5 min post-infusion revealed peaks of radioactivity corresponding to 150 and 43 kDa and unbound tracer. Statistical analysis of trichloroacetic acid (TCA)-precipitable radioactivity in sequential plasma samples as well as following chromatography of the same samples revealed that clearance of the radiolabelled peptides followed an apparent triphasic pattern. The close similarity of the individual chromatographically defined pools in their clearance rate compared with the three components described by TCA precipitation strongly suggested their identity. Both free 125I-labelled cIGF-II (3.11 min) and hIGF-II (3.01 min) were cleared at a greater rate than their IGF-I counterparts. Unbound hIGF-I was cleared at a greater rate than cIGF-I (4.45 vs 5.66 min respectively). A similar pattern for clearance was evident in the radio-labelled growth factors associated with the 43 kDa component, although at a longer half-life. There was no difference in the apparent clearance of the radiolabelled growth factors associated with the 150 kDa component between IGF-I or -II or between species. Analysis of the chromatographic profiles of radioactive IGF-I peptides complexed to serum proteins versus those bound to labelled IGF-II peptides revealed the presence of a large molecular mass binding protein in vivo. Ligand blotting of chicken serum determined that a binding protein with a mass of 70 kDa was detectable with 125I-IGF-II probes only, and was not present in pig serum. In addition, tissue uptake of 125I-cIGF-I and -II was evaluated. Similar patterns of tissue distribution and uptake were observed for 125I-cIGF-I and -II, except that cIGF-II uptake by the liver exceeded that of 125I-cIGF-I at 15 min post-infusion. The rank order of tissue distribution was as follows: kidney > testis > heart > liver > pancreas > small intestine > cartilage > bursa > gizzard > leg muscle > breast muscle > brain. We conclude from these studies that the clearance of IGFs from the compartments identified in blood and the potential target tissues is dependent on their interactions with IGF-binding proteins and receptors.

The effects of recombinant human insulin-like growth factor-I and growth hormone on body composition in elderly women.

The purpose of this study was to determine the effects of recombinant human GH (rhGH; 0.025 mg/kg.day) and one of two doses of recombinant human insulin-like growth factor-I (rhIGF-I; 0.015 and 0.060 mg/kg, twice daily) on body composition in elderly women. Sixteen healthy elderly women (mean age +/- SEM, 71.9 +/- 1.3 yr) were randomly assigned to receive either rhGH (GH; n = 5), low dose rhIGF-I (n = 6), or high dose rhIGF-I (n = 5). A 2-week predrug baseline period was followed by 4 weeks of hormone treatment, with a standardized diet fed throughout. All groups experienced a significant increase in serum IGF-I and IGFBP-3 levels over the treatment period, accompanied by significant decreases in IGF-II (P < 0.05). Fat mass decreased in all groups, with significant increases in lean body mass and nitrogen retention occurring in the high dose IGF and GH groups. Total body water did not change, whereas increases observed in intracellular fluid approached significance (P = 0.06). These anabolic changes were accompanied by numerous negative side-effects in the GH and high dose IGF groups, including headaches, lethargy, joint swelling/pain, and bloatedness. The low IGF dose was well tolerated. These results demonstrate that the administration of rhGH and rhIGF-I for 4 weeks results in anabolic changes in body composition in elderly women.

Coordinated pattern of expression and localization of insulin-like growth factor-II (IGF-II) and IGF-binding protein-2 in the adult rat brain.

Insulin-like growth factors (IGFs) have numerous actions on neuronal and glial cell function in vitro, although their in vivo roles within the central nervous system (CNS) remain undefined. Levels of IGF-II are high in most rat tissues before the third postnatal week, but rapidly decrease thereafter, except in the brain and spinal cord, where elevated titers are present in the adult. This suggests a function of IGF-II within the CNS. IGF-binding proteins (IGFBPs) modify the type 1 IGF receptor-mediated activity of IGFs, thereby regulating the activities of IGF-II in the CNS. In this study, we use a ribonuclease protection assay, in situ hybridization, and immunohistochemistry to demonstrate that IGF-II and one of the major CNS binding proteins, IGFBP-2, show a striking congruency in their anatomical pattern of expression and localization throughout the adult rat brain. Both proteins are synthesized predominantly in the leptomeninges, choroid plexus, and parenchymal microvasculature, but become localized, remote from the site of synthesis, in the myelin sheaths of individual myelinated axons and in all of the myelinated nerve tracts in the brain, which presumably represents the site of IGF-II bioactivity. The spatial disparity between sites of synthesis and sites of bioactivity suggests a key role for IGFBP-2 in the regulation of IGF-II bioavailability within the brain.

Growth hormone insensitivity syndromes: a preliminary report on changes in insulin-like growth factors and their binding proteins during treatment with recombinant insulin-like growth factor I. Kabi Pharmacia Study Group on Insulin-like Growth Factor I Treatment in Growth Hormone Insensitivity Syndromes.

Serum levels of insulin-like growth factor (IGF) binding proteins (IGFBPs) 1, 2 and 3 were studied by radioimmunoassay in 29 patients with growth hormone (GH) insensitivity syndromes (GHIS) before and during treatment with IGF-I. As in normal subjects, there was a highly significant correlation between IGFs and IGFBP-3 but not between IGFs and the other binding proteins, though IGFBP-3 represented only about one-third of the total IGFBP concentration. In 6 patients with GH deficiency and in 5 patients with GHIS, the pharmacokinetic profile of IGF-I after a single injection was strongly dependent on the IGFBP-3 concentration. A slight but significant increase in IGFBP-3 was observed coincident with the IGF-I peak, whereas IGFBP-2 increased after a delay of about 10 hours. In the patients with GHIS, chronic IGF-I treatment, with twice-daily injections for 6 months, caused a significant steady decline of IGF-II and an increase in IGFBP-2, but had no effect on IGFBP-1 and IGFBP-3 levels. During IGF-I treatment, an inverse relationship between baseline IGF-I and GH levels was observed. The data suggest that total IGF-I and IGF-II serum levels are determined mainly by IGFBP-3, even in extreme situations such as GHIS, while other IGFBPs are less important. The IGFBP-3 concentration seems to be a major regulator of the pharmacokinetics of exogenous IGF-I, which, in turn, influences IGFBP-3 levels. This effect of IGF-I on IGFBP-3 is not through induction of IGFBP-3 synthesis, but possibly by reduction of IGFBP-3 clearance.(ABSTRACT TRUNCATED AT 250 WORDS)

Tumour suppression associated with expression of human insulin-like growth factor II.

Recent circumstantial evidence has implicated Insulin-like growth factor II in the genesis of several tumour types, notably developmental tumours (Scott et al., 1985; Schofield & Tate, 1987; Wilkins et al., 1989). This type of tumour, thought to originate during the defective differentiation of organ precursors (Miereau et al., 1987), often expresses greatly elevated levels of mRNA for IGF-II, a known mitogen for these cells and abundantly expressed in their presumed normal counterparts (Scott et al., 1985; Schofield & Tate, 1987; Gray et al., 1987). It has been proposed that continued, inappropriate expression of this gene drives tumour growth by an autocrine mechanism. In order to examine the potential role of IGF-II in the growth of tumour cells an IGF-II cDNA was introduced into a retroviral expression vector, and used to infect a cloned fibroblast cell line. Expression of IGF-II conferred a degree of serum independence of growth in cell culture, however, when cells were injected into nude mice as subcutaneous grafts, clones expressing IGF-II from the retrovirus were found to have a greatly increased (five fold) latency of sarcoma formation. After a prolonged lag all cell lines eventually gave rise to tumours in which the introduced IGF-II genes had either been lost or inactivated, suggesting that in this system IGF-II acts as a tumour suppressor gene.

Plasma clearance and tissue distribution of labelled insulin-like growth factor-I (IGF-I), IGF-II and des(1-3)IGF-I in rats.

Incubation of 125I-labelled insulin-like growth factor-I (IGF-I) with rat plasma at 4 degrees C led to the transfer of approximately half the radioactivity to 150 kDa and smaller complexes with IGF-binding proteins. The extent of association was greater with labelled IGF-II and essentially absent with the truncated IGF-I analogue, des(1-3)IGF-I. A greater degree of binding of IGF peptides with binding proteins occurred after i.v. injection of the tracers into rats, but most of the des(1-3)IGF-I radioactivity remained free. Measurement of the total plasma clearances showed the rapid removal of des(1-3)IGF-I compared with IGF-I and IGF-II; the mean clearances were 4.59, 1.20 and 1.34 ml/min per kg respectively. The mean steady-state volume of distribution was larger for des(1-3)IGF-I than for IGF-I and IGF-II (461, 167 and 181 ml/kg respectively), probably because of the differences in plasma protein binding. With all tracers, radioactivity appeared in the kidneys to a greater extent than in other organs. The amount of radioactivity found in the adrenals, brain, skin, stomach, duodenum, ileum plus jejunum and colon was in rank order, des(1-3)IGF-I greater than IGF-I greater than IGF-II. Since this ranking is the opposite of the abilities of the three IGF peptides to form complexes with plasma binding proteins, we propose that the plasma binding proteins inhibit the transfer of the growth factors to their tissue sites of action. Moreover, we suggest that IGF analogues that are cleared rapidly from blood may have greater biological potencies in vivo.

Disappearance of labelled IGF-2 from plasma of the ovine fetus in late gestation.

The role of IGF-2 in the fetus and its possible influence on fetal growth remains speculative. We investigated the size distribution of unsaturated binding sites for labelled oIGF-2 in ovine fetal plasma. In addition, the disappearance of each form of protein bound IGF-2 in the late gestation ovine fetus (125-135 days, n = 5) was estimated. One minute after injection into the fetal femoral vein, 125IoIGF- circulated in the fetal femoral artery bound primarily to a 50 kDa binding protein. Only a small amount of binding to a 150 kDa binding protein was seen with little to no free IGF-2 present. IGF-2 also circulated in association with a large molecular weight complex (ca. 250 kDa) presumed to be circulating receptor bound IGF-2. The half life of the 250 kDa form of IGF-2 was 385.9 +/- 65.4 min, for the 150 kDa form 308.0 +/- 65.0 min, for the 50 kDa form was 35.5 + 2.6 min and for the free form of IGF-2 was 1.6 +/- 0.6 min. There was no apparent movement of intact IGF-2 out of the fetal circulation into any of the fetal fluids or into the maternal circulation. Similarly there was no consistent placental uptake of IGF-2 from the fetal circulation.