Long-term effects of insulin-like growth factor (IGF)-I treatment on serum IGFs and IGF binding proteins in adolescent patients with growth hormone receptor deficiency

OBJECTIVE: The aim of this investigation was to study the effect of relatively high dose IGF-I therapy given for several months, on serum levels of IGF-I, IGF-II and IGFBP-3, and on IGF-I pharmacokinetics in patients with growth hormone insensitivity due to GH receptor dysfunction. DESIGN AND PATIENTS: Two adolescent subjects from Ecuador were treated with recombinant IGF-I at a dosage of 120 micrograms/kg s.c. twice daily, in combination with a GnRH analogue for 8 months. MEASUREMENTS: Serum was sampled at baseline and at 3-8 months, for determination of IGF-I, IGF-II and IGFBP-3 by radioimmunoassay, and for evaluation of IGFBPs and IGFBP-3 protease activity by Western ligand blot and protease assay, respectively. RESULTS: Peak serum IGF-I levels ranged from 272 to 492 micrograms/l. Mean serum IGF-II levels were decreased concurrently with the increase in IGF-I. Serum IGFBP-3 levels failed to rise with prolonged IGF-I treatment. There was no apparent change in the half-life of IGF-I during the treatment period. CONCLUSIONS: IGF-I administration does not increase serum levels of IGFBP-3 or significantly alter IGF-I pharmacokinetics.     

Insulin-like growth factor (IGF)-I but not IGF-II promotes lean growth and feed efficiency in broiler chickens

The efficacy of exogenous IGFs to stimulate growth and modulate protein and fat deposition was examined in a number of broiler chicken lines. From around 600 g body weight the chickens received a continuous infusion of vehicle (0.1 M acetic acid), human recombinant IGF-I or [Gly1]IGF-II at 300 microg/kg body weight per day, or a combined infusion of 150 microg/kg/day of each IGF for 2 weeks. Experiment 1 used commercial broiler female chickens and included measurements of nitrogen balance, Ntau-methylhistidine excretion and muscle protein synthesis rates. In Experiment 2 the same treatments were applied to three experimental lines of chickens selected for high food consumption (relatively fat), high food utilisation efficiency (relatively lean), or at random (control). IGF-I, but not IGF-II, significantly increased growth rate and food utilisation efficiency by around 10-15% in each experiment, an effect which was consistent across all genotypes. Nitrogen balance was significantly increased by IGF-I in Experiment 1 as was carcass nitrogen content in Experiment 2, indicating that the increased growth was in lean tissue. Carcass fat was consistently reduced in chickens receiving IGF-I and was related to the levels of circulating IGF-I (r2 = 0.30, P < 0.01) but not triiodothyronine. Protein synthesis rates were unaffected by treatment and could not account for increased growth rate. However, there was a significant reduction in Ntau-methylhistidine excretion indicating a reduced rate of muscle protein breakdown in IGF-I-treated chickens (1. 56%/day vs 2.05%/day for IGF-I-treated vs controls, P < 0.05). The efficiency of feed utilisation was inversely related to the rate of protein breakdown (r2 = 0.25, P < 0.01). In conclusion, these experiments are the first to report an enhancement of growth and food utilisation efficiency by broiler chickens receiving exogenous IGF-I. Our results show that IGF-I may be important in controlling the growth and efficiency of food utilisation of young chickens at least in part by modulating the rates of protein breakdown.     

Glutamate concentration in plasma, erythrocyte and muscle in relation to plasma levels of insulin-like growth factor (IGF)-I, IGF binding protein-1 and insulin in patients on haemodialysis

A simple, sensitive, and rapid method for simultaneous determination of residues of flumequine and its microbiologically active metabolite 7-hydroxyflumequine in 100 mg sheep edible tissues (muscle, liver, kidney, and fat) by liquid chromatography is reported. After liquid-liquid cleanup with ethyl acetate, tissue extracts were injected onto a Select B column. The 2 compounds were determined by ultraviolet and fluorimetric detection. The method was repeatable and reproducible for flumequine and 7-hydroxyflumequine in muscle, liver, kidney, and fat, with limits of detection below 2 and 3 micrograms/kg for flumequine and 7-hydroxyflumequine, respectively. Mean recoveries for flumequine were 90 +/- 7, 82 +/- 7, 89 +/- 5, and 82 +/- 6% in muscle, liver, kidney, and fat respectively. Mean recoveries for 7-hydroxyflumequine were 91 +/- 2, 90 +/- 4, 86 +/- 3, and 84 +/- 4% in muscle, liver, kidney, and fat, respectively.     

Proteolysis of insulin-like growth factors (IGF) and IGF binding proteins by cathepsin D

Various proteinases have been postulated to function in limited proteolysis of insulin-like growth factor binding proteins (IGFBPs) contributing to the regulation of IGF bioavailability. In this study, we report on the in vitro degradation of IGFs and IGFBPs by the purified acidic aspartylprotease cathepsin D that has been shown to proteolyze IGFBP-3. Recombinant human [125I] IGFBP-1 to -5 were processed by cathepsin D to fragments of defined sizes in a concentration dependent manner, whereas IGFBP-6 was not degraded. Ligand blotting revealed that none of the IGFBP-1 or -3 fragments formed by cathepsin D retain their ability to bind IGF. By N-terminal sequence analysis of nonglycosylated IGFBP-3 fragments produced by cathepsin D, at least four different cleavage sites were identified. Some of these cleavage sites were identical or differed by one amino acid from sites used by other IGFBP proteases described. The IGFBP-3 and -4 cleavage sites produced by cathepsin D are located in the nonconserved central region. IGF-I and -II, but not the unrelated platelet-derived growth factor BB, were degraded by cathepsin D in a time and concentration-dependent manner. We speculate that the major functional site of cathepsin D is intracellular and may be involved 1) in the selected clearance either of IGFBP or IGFs via different endocytic pathways or 2) in the general lysosomal inactivation of the IGF system.     

Growth factor regulation of insulin-like growth factor (IGF) binding proteins (IGFBP) and preadipocyte differentiation in porcine stromal-vascular cell cultures

The influence of anti-IGF-1 and anti-transforming growth factor beta (TGF-beta) neutralizing antibodies on preadipocyte differentiation and secretion of IGFBPs was examined in serum free porcine stromal-vascular cultures. Cultures were stained for morphological analysis and conditioned media were collected for: TGF-beta determination by ELISA, IGF-1 by RIA, and IGFBP analysis by ligand blotting. After 6 d of treatment, anti-TGF-beta increased fat proportions by 2.7 fold compared to controls. Anti-IGF-1 decreased fat cell proportions by 14-fold. Anti-TGF-beta increased concentrations of IGF-1 5.8-fold and IGFBP-2 and IGFBP-3 by 8- and 7-fold in conditioned media whereas IGFBP-4 decreased 5-fold. Anti-IGF-1 increased concentrations of IGFBP-2 and 3 by 9- and 35-fold, respectively. TGF-beta increased concentrations of IGFBP-1, 2 and 3 by 3-fold, 18-fold and 3-fold, respectively, after 9 d in culture (6 d of treatment). There was no change in TGF-beta levels in anti-IGF-1 treated cultures compared to controls. Control antibodies and negative controls had no effect. These results provide evidence that endogenously produced IGF-1 and TGF-beta has a major influence on preadipocyte differentiation in serum free media by modulating IGFBP production/secretion.     

Long-term effects of insulin-like growth factor (IGF)-I on serum IGF-I, IGF-binding protein-3 and acid labile subunit in Laron syndrome patients with normal growth hormone binding protein

A minority of patients with Laron syndrome have normal serum GH binding protein (GHBP), indicating that the defect is elsewhere than in the extracellular domain of the GH receptor. We have evaluated the effect of long-term IGF-I treatment on serum IGF-binding protein (IGFBP)-3 and the acid-labile subunit (ALS) in three sibling with Laron syndrome caused by a GH post-receptor defect and with normal GHBP. The children (a boy aged 3 years, a girl aged 4 years and a boy aged 10 years) were treated by daily s.c. injection of IGF-I in a dose of 150 micrograms/kg. IGFBP-3 was measured by RIA and Western ligand blotting, ALS by RIA. Based values of IGFBP-3 and ALS were low. During IGF-I treatment, the IGFBP-3 concentrations in the girl gradually increased, whereas in the boys there was a 60% decrease during the first week, followed by gradual increase towards baseline. The ALS concentrations followed a similar pattern. We conclude that IGF-I treatment induces and initial suppression and then an increase in the IGFBP-3 and ALS concentrations, confirming data from animal experiments that IGFBP-3 synthesis is not solely under GH control. The differences in responsiveness between the female and male siblings may reflect genetic differences, or lower circulating concentrations of IGF-I in the boys compared with the girl.     

Immunohistochemical pattern of insulin-like growth factor (IGF) I, IGF II and IGF binding proteins 1 to 6 in carcinoma in situ of the testis

AIM: To study the immunohistochemical localisation of insulin-like growth factor (IGF) I, IGF II, and IGF binding proteins 1-6 in intratubular germ cell neoplasia in the vicinity of solid germ cell tumours of the testis. METHODS: Testes were obtained from 13 patients (20-35 years old) who had undergone orchidectomy for treatment of a solid germ cell tumour. Tumour cells were verified histologically by their distinctive morphology and by visualisation of placental alkaline phosphatase immunoreactivity. RESULTS: The majority of carcinoma in situ (CIS) cells were immunopositive for IGF I, whereas no CIS cells stained for IGF II. Of all the IGF binding proteins investigated, CIS cells showed intense immunoreactivity for IGF binding protein 5 and lower expression of all other IGF binding proteins. CONCLUSIONS: These results suggest that the action of IGF binding protein 5 in CIS cells may modulate the activity of IGF I. This may be related to a proliferative advantage that could facilitate tumour development.     

Serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, sex steroids, osteocalcin and bone mineral density in male and female rats

Although it has been reported that the rate of weight gain and linear growth increases markedly during puberty in rats, little is known about the relationship between endocrine changes and bone mineral density (BMD) changes upon sexual maturation in these animals. The aim of this study was to examine the levels of serum insulin-like growth factor-I (IGF-I), IGF binding protein (IGFBP)-3, sex steroids and osteocalcin, and the changes in BMD in normal aging male and female rats. Male rats exhibited increases in serum IGF-I and IGFBP-3 concentrations before increases in serum testosterone levels. IGF-I and testosterone peaked at 9 weeks of age, and thereafter remained in a steady state, whereas IGFBP-3 reached a peak at 7 weeks of age, and then gradually declined. A strong correlation between serum IGF-I and IGFBP-3 levels was found in subjects 3-9 weeks old. A highly significant correlation between serum IGF-I and testosterone levels was also found. In females, serum 17 beta-estradiol, IGF-I and IGFBP-3 levels increased gradually from 3 to 5 weeks old, peaked at 9 weeks, and then decreased slowly thereafter. The correlation coefficient between serum IGF-I and IGFBP-3 was highly significant. The correlation coefficient between serum IGF-I and 17 beta-estradiol levels was weak, although it was strongest when the subjects were 3-9 weeks old. Serum osteocalcin is a marker of bone formation; its level remained relatively high from 3 to 9 and from 3 to 7 weeks of age in males and females, respectively, although osteocalcin in both sexes declined gradually with age. As for bone mass, sharp increases in BMD in the tibia, femur and lumbar vertebrae appeared earlier in female than in male rats, and the BMD in females tended to be higher than in males between 5 and 9 weeks old. After 9 weeks of age, BMD in males was higher than that in females, as BMD in males continued to increase whereas females tended to remain in a steady state after this stage. The correlation coefficients between tibial BMD and serum IGF-I or IGFBP-3 levels were highly significant when the subjects were from 3 to 9 weeks old. Taken together, these results suggest that BMD development occurs earlier in female than in male rats. This sex-related difference in changes in the BMD pattern may result from the earlier onset of puberty in females, and from sex-specific differences in concentrations of IGF-I, IGFBP-3 and sex steroids during maturation.     

Growth factors and intrauterine growth retardation. I. Serum growth hormone, insulin-like growth factor (IGF)-I, IGF-II, and IGF binding protein 3 levels in normally grown and growth-retarded human fetuses during the second half of gestation

The tumor necrosis factor receptor 2 (TNFR2) gene localizes to 1p36. 2, a genomic region characteristically deleted in neuroblastomas and other malignancies. In addition, TNFR2 is the principal mediator of the effects of TNF on cellular immunity, and it may cooperate with TNFR1 in the killing of nonlymphoid cells. Therefore, we undertook an analysis of the genomic structure and precise physical mapping of this gene. The TNFR2 gene is contained on 10 exons that span 26 kb. Most of the functional domains of TNFR2 are encoded by separate exons, and each of the repeats of the extracellular cysteine-rich domain is interrupted by an intron. The genomic structure reveals a close relationship to TNFR1, another member of the TNFR superfamily. Based on electrophoretic analysis of yeast artificial chromosomes, TNFR2 maps within 400 kb of the genetic marker D1S434. In addition, we have identified a new polymorphic dinucleotide repeat within intron 4 of TNFR2. The genetic sequence information and exon-intron boundaries we have determined will facilitate mutational analysis of this gene to determine its potential role in neuroblastoma, as well as in other cancers with characteristic deletions or rearrangements of 1p36.     

A protease-resistant form of insulin-like growth factor (IGF) binding protein 4 inhibits IGF-1 actions

Smooth muscle cells (SMC) secrete a serine protease that cleaves insulin-like growth factor (IGF) binding protein (IGFBP)-4 into fragments that have low affinity for IGF-1. When IGFBP-4 is added to monolayer cultures of cell types that do not secrete this protease, IGF-1 stimulation of DNA synthesis is significantly inhibited. In contrast, if cell types that secrete this protease are used, IGFBP-4 is a much less potent inhibitor. These studies were conducted to determine whether proteolysis of IGFBP-4 accounted for its reduced capacity to inhibit IGF-1-stimulated DNA synthesis. The cleavage site in IGFBP-4 that the SMC protease uses was determined to be lysine120, histidine121. A protease-resistant mutant form of IGFBP-4 was prepared, expressed, purified, and tested for biologic activity using porcine SMC cultures. Addition of the protease-resistant mutant resulted in inhibition of DNA and cell migration responses to IGF-1. The inhibition was concentration dependent and was maximal when 500 ng/ml (20 nM) of the mutant was added with 20 ng/ml (2.8 nM) of IGF-1. When the mutant was added in the absence of IGF-1, it had no activity. The results show that cleavage of IGFBP-4 at lysine120, histidine121 results in inactivation of the ability of IGFBP-4 to bind to IGF-1. Creation of a mutant form of IGFBP-4 that was not cleaved by the protease resulted in inhibition of IGF-1-stimulated actions. The results suggest that IGFBP-4 can act as a potent inhibitor of the anabolic effects of IGF-1 and that the variables that regulate protease activity may indirectly regulate IGF-1 actions.     

Analysis of insulin-like growth factors (IGF)-I, and -II, type II IGF receptor and IGF-binding protein-2 mRNA and peptide levels in normal and nephrectomized rat kidney

Immunocytochemistry, in situ hybridization, and radioimmunoassay were employed to examine the cellular distribution of mRNAs and proteins for IGF-I, II, IGF-II/M6P receptor, IGFBP2 as well as the levels of IGF-I and II in normal and unilaterally nephrectomized (Nx) adult rat kidneys. A similar distribution of immunoreactive IGF-I, and -II as well as IGF-II/M6P receptor was found in the principal cells of the cortical collecting duct and in all cells of the inner medullary collecting duct. In addition, immunostainable IGF-I and IGF-II/M6P receptor were noted in some inner medullary loops of Henle, while IGFBP2 was seen in the collecting ducts and loops of Henle of the inner medullar and the renal vasculature of all animals. By comparison, in situ hybridization revealed IGF-I mRNA only in the medullary thick ascending limbs while IGF-II mRNA was localized to the wall of the renal microvasculature in all kidneys. IGFBP2 mRNA was localized to the renal corpuscle and to inner medullary interstitial cells of all kidneys. These data suggest that renal IGF-I and IGFBP2 are synthesized at upstream sites along the nephron and then transported downstream for interaction with IGF receptors. Following nephrectomy, the renal levels of IGF-I peptide and mRNA were elevated at both 5 and 33 days post-nephrectomy, supporting a potential functional role for IGF-I in stimulating the structural and functional recovery in compensatory hypertrophy.     

Systemically but not orogastrically delivered insulin-like growth factor (IGF)-I and long [Arg3]IGF-I stimulates intestinal disaccharidase activity in two age groups of suckling rats

The growth mitogenic properties of IGF-I on tissues of the gastrointestinal tract are well established; however, IGF effects on enzyme maturation are less clear. To test whether IGF-I peptide administration stimulates disaccharidase activity, we administered IGF-I or the more potent analog, long [Arg3]IGF-I, at doses ranging between 2 and 12.5 micrograms g-1 d-1 to suckling Wistar rat pups by either continuous s.c. infusion or by three times daily orogastric gavage. Peptides were administered for approximately 6 d starting on d 6 or 12 postpartum with six to nine rats per group. The results of the study demonstrated that systemically but not orally administered IGF-I stimulated duodenal wet tissue weight (up to 85%) and length (up to 36%). Enzyme maturation was assessed by measuring disaccharidase biochemically in tissue homogenates. Enzyme activity was also localized histocytochemically in cryostat-sectioned duodenum. After systemic infusion of IGF-I, intestinal lactase activity increased proportional to mucosal mass in both age groups. Systemic infusion of the more potent analog, long [Arg3]IGF-I, precociously induced the decline in lactase activity and accelerated the appearance of sucrase activity in the rat pups infused during the later suckling period. These findings indicate that enzyme maturation can be accelerated by systemically derived IGF-I peptides. Orogastrically IGF-I peptides, delivered at pharmacologic doses, did not affect intestinal growth or digestive enzyme maturation in suckling rat pups treated between 6 and 18 d postpartum, indicating the efficacy of IGF-I peptides may depend on the route of delivery and postnatal age of the recipient.     

Structural changes in insulin-like growth factor (IGF) I mutant proteins affecting binding kinetic rates to IGF binding protein 1 and IGF-I receptor

Ligand binding properties of five single amino acid substituted variants (V11A, D12A, Q15A, Q15E, and F16A) of human insulin-like growth factor I (IGF-I) were analyzed with respect to their binding affinities and binding kinetics to recombinant IGF binding protein 1 (IGFBP-1) and a soluble form of the IGF type I receptor (sIGF-I(R)), respectively. Side chains of the substituted residues are all predicted to be the most surface exposed in the alpha-helical portion of the B-region of the IGF-I molecule. The IGF-I variants were produced as fusion proteins to a IgG(Fc) binding protein domain, Z. Ligand binding kinetic rates were determined using BIAcore biosensor interaction analysis technology. All IGF-I variants showed altered binding affinities to both IGFBP- I and sIGF-I(R). Secondary structure content of the IGF-I variants was estimated using far-UV circular dichroism spectroscopy, followed by variable selection secondary structure calculations. The amount of calculated alpha-helicity is reduced for all the mutants, most predominantly for IGF-I(V11A) and IGF-I(F16A) proteins. Surprisingly, most of the effects of reduced binding affinities to both target proteins are attributed to lowered on-rates of binding, and these are correlated with the amount of alpha-helicity in each IGF-I variant. In addition, in some of the IGF-I variants, lowered off-rates of binding are observed. From the results, we propose that IGF-I is unusually sensitive to structural changes by surface amino acid substitutions in the B-region of the molecule. Therefore, biochemical or biological properties of amino acid substituted variants of IGF-I cannot be used in a straightforward way to dissect the direct involvement in binding of individual amino acid residues since structural changes may be involved.     

Autocrine-paracrine regulation of human trophoblast invasiveness by insulin-like growth factor (IGF)-II and IGF-binding protein (IGFBP)-1

As a consequence of their mechanism of action, DNA (cytosine-5) methyltransferases from both prokaryotes and eukaryotes necessarily recognize mispaired bases in unusual DNA structures as catalytic transition-state analogs. A review of the available data suggests that the enzymes are designed to stall at these sites because they are unable to release substrates or products that are fixed in a conformation resembling the transition state. The enzymes can operate by a two-step process in which they first methylate extrahelical cytosines satisfying their recognition requirements and subsequently stall at the site of methylation. On RNA and DNA RNA hybrids they may operate by a similar one-step process in which they stall at transition-state analogs without methylating cytosine moieties. These natural capacities suggest that the enzymes may physically participate in stable nucleoprotein assemblies formed as components of normal chromatin structure or as intermediates in the repair of unusual structures. The methyltransferases, themselves, may physically participate in chromosome remodelling as part of a mechanism of inactivation or imprinting by stabilizing RNA DNA hybrids or RNA RNA secondary structure involving cis-acting untranslated RNAs like the product of the Xist gene. Methyl-transferase may physically participate in the repair of certain unusual structures by serving as a nucleation point. The affinity for secondary structure in nucleic acids may account for the spreading of DNA methylation patterns. Titration of host methyltransferase by RNA DNA hybrids and RNA secondary structure formed during retroviral replication in certain tumorigenic retroviruses, like MMTV, may account for global hypomethylation observed in retrovirally transformed cells. In a similar fashion, titration of methyltransferase by secondary structures associated with chromosome instability may account for global hypomethylation observed in association with local hypermethylation in tumorigenesis.     

Role of insulin-like growth factor binding proteins in the control of IGF actions

The insulin-like growth factor binding proteins have been shown to modify IGF actions. IGFBP-5 binds to extracellular matrix (ECM) and its ability to potentiate IGF activity is dependent upon the amount that is ECM associated. To determine the specific regions of IGFBP-5 that are required for ECM association, site directed mutagenesis has been used to prepare several forms of IGFBP-5. Mutants that have had the amino acids between positions 201 and 218 altered have been useful. Mutation of the lysine 211 resulted in no change in the affinity of IGFBP-5 for ECM or heparin Sepharose; however, it resulted in a major reduction in affinity for IGF-I following heparin binding. Other mutations which disrupted heparin binding also resulted in loss of this affinity shift. Most distruptive were mutations of amino acids 211, 214, 217 and 218 and 202, 206 and 207. Mutation of residues 201 plus 202 had some effect, but substitution for 207, 211, 217 and 218 had no effect. When binding to intact ECM was analyzed, similar results were obtained. This suggests that amino acids 202, 206 and 214 are definitely involved in heparin and ECM binding. When binding to proteoglycans such as tenascin and heparin sulfate proteoglycan was analyzed, similar results were obtained. IGFBP-5 also binds to other proteins in ECM, including type IV collagen and plasminogen activator inhibitor-I. Specific antisera for plasminogen activator inhibitor-1 can coprecipitate IGFBP-5. IGFBPs are degraded by specific proteases. Three proteases that degrade IGFBP-2, -4 and -5 have been characterized. They are serine proteases that cleave these proteins at basic residues. Although several well characterized serine proteases cleave IGFBP-4 or -5, the proteases in cell conditioned media appear to be distinct.     

Fetal programming of insulin-like growth factor (IGF)-I and IGF-binding protein-3: evidence for an altered response to undernutrition in late gestation following exposure to periconceptual undernutrition in the sheep

CD45-negative B-cell precursor acute lymphoblastic leukaemia (ALL) provides a unique model to study the stem cell compartment in ALL as leukaemic CD34-positive cells, unlike their normal counterparts, do not express CD45. By increasing the number of events analysed to 10(6), storing only the events in the region of interest (storage gate), using appropriate isotype controls and stringent washing procedures, a flow cytometric protocol was established to characterize rare CD34+ CD19- events. In eight of 12 patients (67%) with CD45-negative B-cell precursor ALL, a distinct CD34+ CD19- CD45+ candidate normal stem cell population could be detected. In one patient analysed by four-colour staining, the CD34+ CD19- CD45+ cells, unlike the CD45-negative leukaemic cells, expressed CD117 (c-kit), providing further evidence that these cells represent residual nonleukaemic normal cells. By multiparameter analysis, this population of candidate normal stem cells could be separated from contaminating leukaemic CD34+ CD19- CD45- cells, which were detected in 11 of the 12 patients within the CD34+ CD19- compartment.     

Ontogeny of insulin-like growth factors (IGF), IGF binding proteins, IGF receptors, and growth hormone receptor mRNA levels in porcine pancreas

We examined the ontogeny of mRNA levels of IGF-I and -II, IGF type 1 (IGFI-R) and type II receptors (IGFII-R), IGF binding protein-1 and -3 (IGFBP-1 and -3), GH receptor (GHR), and tissue concentrations of IGF and IGFBP in the pancreas of pigs. Tissues were collected from fetuses at 90 and 110 d of gestation and from pigs at 1, 21, 90 and 180 d of age. Northern blots were performed using total RNA hybridized with 32P-labeled cDNA probes (human IGF-I and human IGFI-R) and cRNA probes (rat IGF-II, human IGFII-R, human IGFBP-1, pig IGFBP-3, and pig GHR). There were two accelerated growth stages of the pancreas: the first one at 90 d of fetal life, which is characterized by cell hyperplasia (high ratio of DNA to body weight), and the second one at postnatal 90 d, which is attributed to cell hypertrophy (high ratios of pancreatic weight, RNA, and protein to DNA). The level of IGF-II mRNA and its tissue concentration were predominant during fetal life and low thereafter. The IGF-I mRNA level was high during fetal and early postnatal life and decreased thereafter. Messenger RNA levels of IGFI-R, IGFBP-3, and GHR and concentrations of IGFBP-1 and -2 were abundant during fetal and early postnatal life. In conclusion, IGF may be involved in various physiological periods of pancreatic development in pigs.     

Distinct sites of insulin-like growth factor (IGF)-II expression and localization in lesioned rat brain: possible roles of IGF binding proteins (IGFBPs) in the mediation of IGF-II activity

Although expression of the IGF-II has been demonstrated within the central nervous system (CNS), past studies have failed to reveal its precise roles or responses subsequent to a traumatic injury. To demonstrate that IGF-II, IGFBP, and IGF receptor (-R) expression alters in response to a penetrating CNS injury, we used the techniques of ribonuclease protection assay, in situ hybridization, immunohistochemistry, Western blotting, and RIA. Under normal physiology, IGF-II expression is restricted to the mesenchymal support structures of the brain, including the choroid plexus, where its expression is coincident with that of IGFBP-2. Between 1-7 days post lesion (dpl), in the acute phase following a penetrant wound to the CNS, IGF-II and IGF-IIR protein, but not messenger RNA, were colocalized, with IGF-I, IGF-IR, and IGFBP-1, -2, -3, and -6, to neurons, macrophages, astrocytes, and microglia within the damaged tissue. Within the cerebrospinal fluid (CSF), levels of IGF-II peptide increased to peak at 7 dpl. IGFBP-2, -3, and -6 were also observed within the CSF, with IGFBP-2 predominating and exhibiting an increase in binding efficiency from 7-10 dpl. In the chronic phase of injury (7-14 dpl), an increase in both IGF-II, IGF-IIR and IGFBP-5 messenger RNA and protein was observed specifically and focally in the marginal astrocytes forming the limiting glial membrane of the wound. Thus, our evidence suggests that there are two mechanisms of action for IGF-II within the injured rat brain. During the acute phase, the secretion of IGF-II from the choroid plexus into the CSF is up-regulated, resulting in increased transport of the peptide to the wound. In the CSF, transported IGF-II is complexed to IGFBP-2 and essentially demonstrates an endocrine mode of action with a balance of locally produced IGFBPs modulating its bioactivity in the wound. Later in the wounding response, levels of IGF-II decline in the CSF and the wound neuropil, possibly with the aid of increased IGFBP-5 levels that may help to locally sequester and down-regulate IGF-II activity. Hence, in the chronic phase of the injury response, IGF-II reasserts itself to a predominantly autocrine/paracrine role restricted to the mesenchymal support structures, including the glia limitans, which may help reestablish and maintain tissue homeostasis.