Differential dissociation kinetics explain the binding preference of insulin-like growth factor binding protein-6 for insulin-like growth factor-II over insulin-like growth factor-I

Insulin-like growth factor binding protein-6 binds insulin-like growth factor-II with a marked preferential affinity over insulin-like growth factor-I. The kinetic basis of this binding preference was studied using surface plasmon resonance. Binding of insulin-like growth factor-I and insulin-like growth factor-II to immobilized insulin-like growth factor binding protein-6 fitted a two-site binding kinetic model. Insulin-like growth factor-I and insulin-like growth factor-II association rates were similar whereas the dissociation rate was approximately 60-fold lower for insulin-like growth factor-II, resulting in a higher equilibrium binding affinity for insulin-like growth factor-II. The equilibrium binding affinities of a series of insulin-like growth factor-II mutants were also explained by differential dissociation kinetics. O-glycosylation had a small effect on the association kinetics of insulin-like growth factor binding protein-6. The insulin-like growth factor binding properties of insulin-like growth factor binding protein-6 are explained by differential dissociation kinetics.     

Matrix metalloproteinase-7 degrades all insulin-like growth factor binding proteins and facilitates insulin-like growth factor bioavailability

Proteolytic modification of insulin-like growth factor binding proteins (IGFBPs) plays an important physiological role in regulating insulin-like growth factor (IGF) bioavailability. Recently, we demonstrated that matrix metalloproteinase-7 (MMP-7)/Matrilysin produced by various cancer cells catalyzes the proteolysis of IGFBP-3 in vitro and regulates IGF bioavailability, resulting in an anti-apoptotic effect against anchorage-independent culture. In the present study, we investigated whether MMP-7 contributes to proteolysis of the other five IGFBPs, IGFBP-1, IGFBP-2, IGFBP-4, IGFBP-5, and IGFBP-6, and whether this results in phosphorylation of the IGF type 1 receptor (IGF-1R). MMP-7 cleaved all six IGFBPs, resulting in IGF-mediated IGF-1R phosphorylation, which was inhibited by EDTA treatment. These results suggest that MMP-7 derived from cancer cells can regulate IGF bioavailability in the microenvironment surrounding the tumor, where various kinds of IGF/IGFBP complexes are found, thereby favoring cancer cell growth and survival during the processes of invasion and metastasis.     

Decreased intracellular degradation of insulin-like growth factor binding protein-3 in cathepsin L-deficient fibroblasts

Proteolysis of insulin-like growth factor binding proteins (IGFBPs) is the major mechanism of releasing IGFs from their IGFBP complexes. Analysis of fibroblasts deficient for the lysosomal cysteine protease cathepsin L (CTSL) revealed an accumulation of IGFBP-3 in the medium which was due neither to alterations in IGFBP-3 mRNA expression nor to extracellular IGFBP-3 protease activity. Incubation of CTSL-deficient fibroblasts with radiolabeled IGFBP-3 followed by subcellular fractionation indicates that both intact and fragmented IGFBP-3 accumulate transiently in endosomal and lysosomal fractions of CTSL-deficient cells. This suggests the involvement of CTSL in the intracellular degradation of IGFBP-3 representing a new mechanism to regulate the extracellular concentration of IGFBP-3.     

Insulin-like growth factor binding protein-2: NMR analysis and structural characterization of the N-terminal domain

The insulin-like growth factor binding proteins are a family of six proteins (IGFBP-1 to -6) that bind insulin-like growth factors-I and -II (IGF-I/II) with high affinity. In addition to regulating IGF actions, IGFBPs have IGF-independent functions. IGFBP-2, the largest member of this family, is over-expressed in many cancers and has been proposed as a possible target for the development of novel anti-cancer therapeutics. The IGFBPs have a common architecture consisting of conserved N- and C-terminal domains joined by a variable linker domain. The solution structure and dynamics of the C-terminal domain of human IGFBP-2 have been reported (Kuang Z. et al. J. Mol. Biol. 364, 690-704, 2006) but neither the N-domain (N-BP-2) nor the linker domain have been characterised. Here we present NMR resonance assignments for human N-BP-2, achieved by recording spectra at low protein concentration using non-uniform sampling and maximum entropy reconstruction. Analysis of secondary chemical shifts shows that N-BP-2 possesses a secondary structure similar to that of other IGFBPs. Although aggregation hampered determination of the solution structure for N-BP-2, a homology model was generated based on the high degree of sequence and structure homology exhibited by the IGFBPs. This model was consistent with experimental NMR and SAXS data and displayed some unique features such as a Pro/Ala-rich non-polar insert, which formed a flexible solvent-exposed loop on the surface of the protein opposite to the IGF-binding interface. NMR data indicated that this loop could adopt either of two alternate conformations in solution - an entirely flexible conformation and one containing nascent helical structure. This loop and an adjacent poly-proline sequence may comprise a potential SH3 domain interaction site for binding to other proteins.     

Attenuation of insulin secretion by insulin-like growth factor binding protein-1 in pancreatic beta-cells

IGFBP-1 is involved in glucohomeostasis, but the direct action of IGFBP-1 on the beta-cell remains unclear. Incubation of dispersed mouse beta-cells with IGFBP-1 for 30min inhibited insulin secretion stimulated by glucose, glucagon-like peptide 1 (GLP-1) or tolbutamide without changes in basal release of insulin and in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and NAD(P)H evoked by glucose. In contrast, IGFBP-1 augmented glucose-stimulated insulin secretion in intact islets, associated with a reduced somatostatin secretion. These results suggest a suppressive action of IGFBP-1 on insulin secretion in isolated beta-cells through a mechanism distal to energy generating steps and not involving regulation of [Ca(2+)](i). In contrast, IGFBP-1 amplifies glucose-stimulated insulin secretion in intact islets, possibly by suppressing somatostatin secretion. These direct modulatory influences of IGFBP-1 on insulin secretion may imply an important regulatory role of IGFBP-1 in vivo and in the pathogenesis of type 2 diabetes, in which loss of insulin release is an early pathogenetic event.     

Identification of a novel insulin-like growth factor binding protein gene homologue with tumor suppressor like properties

Here we report the identification of a new insulin-like growth factor binding protein homologue, provisionally designated insulin-like growth factor binding related protein-4 (IGFBP-rP4). IGFBP-rP4 was found to be most closely related to IGFBP-7 with 52% amino acid homology and 43% amino acid identity, and shares a similar domain structure. Semi-quantitative RT-PCR expression analysis demonstrated a pattern of downregulation of this gene in multiple tumor samples including lung and colon cancer, compared to matched adjacent normal tissue. Western blotting revealed a protein of approximately 38kDa expressed in both the cell pellet and secreted into the supernatant of transiently transfected Cos-7 cells. Cos-7 supernatants containing IGFBP-RP4 protein were observed to suppress the growth of HeLa cells in culture compared to vector controls. IGFBP-RP4 directly transiently transfected into HeLa cells also further confirmed the growth suppressive properties of this protein. Together these data suggest that IGFBP-RP4 may be a novel putative tumor suppressor protein.     

ADAM28 is activated by MMP-7 (matrilysin-1) and cleaves insulin-like growth factor binding protein-3

ADAM28, a member of a disintegrin and metalloproteinase (ADAM) family, has two isoforms, membrane-type form (ADAM28m) and secreted form (ADAM28s). Although ADAM28 is expressed and synthesized in a precursor form (proADAM28) by lymphocytes and some cancer cells, its activation mechanism and substrates remain unclear. Here, we report that proADAM28s of 65kDa is processed with active matrix metalloproteinase-7 (MMP-7) to 42- and 40-kDa forms which corresponds to active ADAM28s without propeptide. Processed ADAM28s digested insulin-like growth factor binding protein-3 (IGFBP-3) in both free and complex forms with IGF-I or IGF-II, and the digestion was prevented with EDTA, 1,10-phenanthroline, KB-R7785, tissue inhibitor of metalloproteinases-3 (TIMP-3), and TIMP-4. These data provide the first evidence that proADAM28s is activated by MMP-7 and ADAM28 digests IGFBP-3.     

In vivo processed fragments of IGF binding protein-2 copurified with bioactive IGF-II

Proteolysis of insulin-like growth factor binding proteins (IGFBPs), the major carrier of insulin-like growth factors (IGFs) in the circulation, is an essential mechanism to regulate the bioavailability and half-live of IGFs. Screening for peptides in human hemofiltrate, stimulating the survival of PC-12 cells, resulted in the isolation of C-terminal IGFBP-2 fragments and intact IGF-II co-eluting during the chromatographic purification procedure. The IGFBP-2 fragments exhibited molecular masses of 12.7 and 12.9kDa and started with Gly169 and Gly167, respectively. The fragments were able to bind both IGFs. The stimulatory effect of the purified fraction on the survival of the PC-12 cells could be assigned exclusively to IGF-II, since it was abolished by the addition of neutralizing IGF-II antibodies. We suggest that in the circulation IGF-II is not only complexed with intact IGFBP but also with processed IGFBP-2 fragments not impairing the biological activity of IGF-II.     

Epigenetic regulation of insulin-like growth factor binding protein-3 (IGFBP-3) in cancer

Epigenetics refers to heritable changes in gene expression that are independent of alterations in DNA sequence. It is now accepted that disruption of epigenetic mechanisms plays a key role in the pathogenesis of cancer: culminating in altered gene function and malignant cellular transformation. DNA methylation and histone modifications are the most widely studied changes but non-coding RNAs such as miRNAs are also considered part of the epigenetic machinery. The insulin-like growth factor (IGF) axis is composed of two ligands, IGF-I and –II, their receptors and six high affinity IGF binding proteins (IGFBPs). The IGF axis plays a key role in cancer development and progression. As IGFBP genes have consistently been identified among the most common to be aberrantly altered in tumours, this review will focus on epigenetic regulation of IGFBP-3 in cancer for which the majority of evidence has been obtained.     

Cellular localisation of insulin-like growth factor binding protein-2 (IGFBP-2) during development of the marine fish, Sparus aurata

The spatial localisation of insulin-like growth-factor-binding protein-2 (IGFBP-2) and its mRNA was investigated during larval and post-larval developmental stages of the gilthead seabream (Sparus aurata) by immunohistochemistry and in situ hybridisation with specific antisera and riboprobes. During larval development, immunoreactivity was found in skin, muscle, gills, pharynx, intestine, liver and olfactory epithelium. After metamorphosis, immunoreactivity was found in the oesophageal epithelium (the strongest reaction) and in red skeletal muscle, heart muscle, the thymus and the epithelium of renal tubules. In the adult, immunostaining with IGFBP-2 antibody was also found in the saccus vasculosus, ovary and testis. IGFBP-2 mRNA was detected by in situ hybridisation mainly in the intestine, skeletal musculature and ovary. These results show that IGFBP-2 protein and mRNA are expressed in a variety of seabream tissues, suggesting that IGFBP-2 regulates the actions of IGFs on these tissues during development and growth.This work was supported by grants from the University of Padua (Progetto di Ateneo, 2001) and by the US–Israel Binational Agricultural Research and Development Fund (BARD, Project IS-2769-96R)     

PTEN-induction in U251 glioma cells decreases the expression of insulin-like growth factor binding protein-2

PTEN is a tumor suppressor gene whose loss of function is observed in approximately 40-50% of human cancers. Although insulin-like growth factor binding protein-2 (IGFBP-2) was classically described as a growth inhibitor, multiple recent reports have shown an association of overexpression and/or high serum levels of IGFBP-2 with poor prognosis of several malignancies, including gliomas. Using an inducible PTEN expression system in the PTEN-null glioma cell line U251, we demonstrate that PTEN-induction is associated with reduced proliferation, increased apoptosis, and a substantial reduction of the high levels of IGFBP-2 expression. The PTEN-induced decrease in IGFBP-2 expression could be mimicked with the PI3-kinase inhibitor LY294002, indicating that the lipid phosphatase activity of PTEN is responsible for the observed effect. However, the rapamycin analog CCI-779 did not affect IGFBP-2 expression, suggesting that the PTEN-induced decrease in IGFBP-2 expression is not attributable to decreased mTOR signalling. Recombinant human IGFBP-2 was unable to rescue U251-PTEN cells from the antiproliferative effects of PTEN, and IGFBP-2 siRNA did not affect the IGF-dependent or -independent growth of this cell line. These results suggest that the clinical data linking IGFBP-2 expression to poor prognosis may arise, at least in part, because high levels of IGFBP-2 expression correlate with loss of function of PTEN, which is well known to lead to aggressive behavior of gliomas. Our results motivate translational research regarding the relationship between IGFBP-2 expression and loss of function of PTEN.     

Characterization of insulin-like growth factor binding protein-1 kinases from human hepatoma cells

The phosphorylation of insulin-like growth factor binding protein-I (IGFBP-1) alters its binding affinity for insulin-like growth factor I (IGF-I) and thus regulates the bioavailability of IGF-I for binding to the IGF-I receptor. The kinase(s) responsible for the phosphorylation of IGFBP-1 has not been identified. This study was designed to characterize the IGFBP-1 kinase activity in HepG2 human hepatoma cells, a cell line that secretes IGFBP-1 primarily as phosphorylated isoforms. IGFBP-1 kinase activity was partially purified from detergent extracts of the cells by phosphocellulose chromatography and gel filtration. Two kinases of approximate M_r 150,000 (peak I kinase) and M_r 50,000 (peak II kinase) were identified. Each kinase phosphorylated IGFBP-1 at serine residues that were phosphorylated by intact HepG2 cells. The kinases were distinct based on their differential sensitivity to inhibition by heparin (IC₅₀ = 2.5 and 16.5 μg/ml, peak I and II kinase, respectively) and inhibition by the isoquinoline sulfonamide CKI-7 (IC₅₀ = 50 μM and 100 μM, peak I and II kinase, respectively). In addition, a tenfold molar excess of nonradioactive GTP relative to [gamma-³²P]ATP lowered the incorporation of ³²P into IGFBP-1 by 80% when the reaction was catalyzed by the peak I kinase, whereas GTP had no effect on the reaction catalyzed by the peak II kinase. In the presence of polylysine, IGFBP-1 was radiolabeled by the partially purified kinase activity when [gamma-³²P]GTP served as the phosphate donor indicating the presence of casein kinase II activity. Furthermore, IGFBP-1 was phosphorylated by purified casein kinase I and casein kinase II at sites phosphorylated by the peak I and peak II kinases. Our data suggest that at least two kinases could be responsible for the phosphorylation of IGFBP-1 in intact HepG2 cells and that the kinases are related to the casein kinase family of protein kinases. © 1996 Wiley-Liss, Inc.     

Cloning and sequence determination of bovine insulin-like growth factor binding protein-2 (IGFBP-2): comparison of its structural and functional properties with IGFBP-1.

Insulin-like growth factor binding proteins (IGFBPs) are secreted by several cell types and can modify IGF actions. Mandin-Darby Bovine Kidney (MDBK) cells have been shown to secrete a 34,000 Da form of IGF binding protein whose N-terminal sequence is similar to a form of IGFBP purified from rat BRL-3A cells that has recently been named IGFBP-2. These studies report the complete amino acid sequence of bovine IGFBP-2 and compare its functional properties with human IGFBP-1. The protein is 81% identical to rat IGFBP-2. When compared with both rat IGFBP-2 and human IGFBP-1, the positions of all 18 cysteine residues are conserved. Similarly an RGD sequence is present near the carboxyl terminus in both proteins. IGFBP-2 has a higher affinity for IGF-II than for IGF-I and its affinity for both forms of IGF is greater than for human IGFBP-1. Like IGFBP-1 the protein can enhance the DNA synthesis response of porcine aortic smooth muscle cells to IGF-I; however, IGFBP-2 was much less potent. The maximum potentiation of the IGF-mediated mitogenic response that could be achieved was approximately 42% that of IGFBP-1. This potentiation is dependent upon a factor contained in platelet poor plasma and if this factor is omitted from the incubation medium, IGFBP-2 inhibits DNA synthesis. The purification of IGFBP-2 will allow more detailed comparisons to be made between it and other forms of IGFBPs in physiologic test systems.     

Insulin-like growth factor-I and insulin-like growth factor binding protein-3 levels of children living in an iodine- and selenium-deficient endemic goiter area

Serum insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-3 (IGFBP-3) levels were investigated in 31 children living in an endemic goiter area and 33 healthy subjects living in an nonendemic area. Serum IGF-I and IGFBP-3 levels of iodine- and selenium-deficient children were found to be lower than those of control subjects (p<0.001). There was a positive correlation between the IGF-I with chronological age and body mass index. There was also positive correlation between the IGF-I and IGFBP-3. No significant difference was found between the goitrous and nongoitrous children. These results suggest that IGF-I and IGFBP-3 levels are affected by thyroid dysfunction as a result of iodine and selenium deficiency. However, IGF-I and IGFBP-3 levels are not associated with goiter.     

Influence of exercise duration on serum insulin-like growth factor and its binding proteins in athletes

The changes in circulating concentrations of insulin-like growth factors during exercise have to date remained incomplete in their documentation. Therefore, we examined in 25 healthy athletes the effects of three different durations of three types of exercise – incremental ergometer cycling exercise (ICE), long-distance Nordic ski race (NSR) and a treadmill-simulated soccer game (TSG) lasting 20 min, 3 h, and 2 × 45 min separated by a 15-min half-time rest respectively, on plasma concentrations of growth hormone ([GH]), insulin-like growth factor-1 ([IGF-I]) and its binding proteins 1 and 3 ([IGFBP-1], [IGFBP-3]). Compared to baseline, serum [GH] increased by 15.2-fold after ICE (P < 0.001), 2.9-fold after NSR (P < 0.01) and 4.6-fold after TSG. Serum [IGF-I] rose by 11.9% after ICE (P < 0.001), while it decreased by −14.6% after NSR (P < 0.001) and was unchanged after TSG. Serum [IGFBP-1] was slightly increased (1.7-fold) after ICE (P < 0.01), but increased markedly (11.8-fold) after NSR (P < 0.001) and by 6.3-fold after the second session of TSG (P < 0.01) (it remained unchanged at the end of the first period of TSG, i.e. after 45-min exercise). The [IGFBP-3] increased by 14.7% after ICE (P < 0.001) and by 6% after TSG (P < 0.05) while it did not change after NSR. From our results it would appear that [IGFBP-1] increase to bind free IGF and hinder their insulin-like action during long-term exercise (lasting beyond 45 min). It is suggested that IGFBP-1 might thus contribute both to preventing hypoglycaemic action of IGF and to facilitating glucose uptake by muscle cells when muscle glycogen stores become deplete. Accepted: 27 May 1998     

Inhibition of IGFBP-5 binding to extracellular matrix and IGF-I–stimulated DNA synthesis by a peptide fragment of IGFBP-5

Insulin-like growth factor binding protein-5 (IGFBP-5) is synthesized and secreted by smooth muscle cells (SMC). IGFBP-5 synthesis is stimulated five- to sixfold by IGF-I, and IGFBP-5 has been shown to augment IGF-I–stimulated DNA synthesis in this cell type. The ability of IGFBP-5 to augment the SMC response to IGF-I is dependent upon its binding to extracellular matrix. A highly charged region of IGFBP-5 that contains amino acids in positions 201–218 has been shown to mediate binding of IGFBP-5 to human fibroblast extracellular matrix (ECM), and a synthetic peptide containing this sequence inhibits IGFBP-5 binding to fibroblast ECM. In this study we show that exposure of SMC cultures that are constituitively synthesizing IGFBP-5 to a synthetic peptide (termed peptide A) containing this sequence has no effect on its synthesis but reduces its abundance within the ECM. The addition of increasing concentrations of the peptide to SMC cultures resulted in a concentration-dependent reduction in ECM-associated IGFBP-5. In contrast, a control peptide (peptide B), which contained the region of amino acids in positions 131–141 and had a similar charge-to-mass ratio, caused a minimal decrease in ECM binding. This effect was functionally significant since the addition of 10 μg/ ml of peptide A inhibited the cellular replication response to 10 ng/ ml IGF-I by 51%, and peptide B had no effect. The effects of peptide A were not due to nonspecific cytotoxicity since it had no inhibitory effect on the response of these cells to human serum and was associated with only minimal inhibition of the cellular response to platelet-derived growth factor. The findings suggest that inhibiting IGFBP-5 binding to porcine SMC ECM results in reduced cellular responses to IGF-I. J. Cell. Biochem. 71:375–381, 1998. © 1998 Wiley-Liss, Inc.     

Serum insulin-like growth factor-I, insulin-like growth factor binding protein-3, and the pubertal growth spurt in the female rhesus monkey

While there is good evidence suggesting IGF-I links to pubertal development and crown-rump length growth among rhesus monkeys, linkages between IGF-I and other measures of morphological growth have not been established. In this study, the pubertal growth spurt in a number of morphological characteristics of female rhesus monkeys is related to serum endocrine status of insulin-like growth factor-I (IGF-I) and its binding protein, insulin-like growth factor binding protein-3 (IGFBP-3), to test the hypothesis that elevations in IGF-I and IGFBP-3 coincide with the time of greatest growth rate of different morphological characteristics. A longitudinal study of pubertal growth among four female rhesus monkeys was carried out across a 3-year period. Morphometric measurements included weight, crown-rump length, foot-length, and skinfolds at five sites (biceps, triceps, abdominal, subscapular, and suprailiac). These measures were taken as being representative of total mass, skeletal growth of the trunk and head, limb length, and body fatness, respectively. Measurements were carried out as closely as possible to 3-monthly, with interpolations being performed to standardise the data to exactly 3-monthly intervals for all individuals. Blood samples were taken at time of morphometry. Elevations in serum IGF-I and IGFBP-3 took place in a manner similar to that of humans, and across the period associated with onset of puberty. Mean 3-monthly gain in crown-rump length and foot length showed significant peaks across the measurement period, while mean 3-monthly gains in weight and sum of five skinfolds did not. Greatest foot length gain occurred on average between 3-3.5 years of age, while crown-rump length gain was greatest between 3.75-4 years of age. Periods of greatest gain in crown-rump length and foot length took place across the period of elevated serum IGF-I levels, which was between 3-4.5 years of age. Significant elevations in IGF-I and IGFBP-3 were not coincident with greatest gains in foot length or crown-rump length. Thus the hypothesis does not hold true for the two measures showing significant peaks in 3-monthly gain across the measurement period. The nature of the endocrine impact on macaque morphology remains unclear, although this may be fundamental to the understanding of the variation in the pubertal growth spurt and its influence on morphology at maturity both within and across primate species.     

The heparin-binding domain of IGFBP-2 has insulin-like growth factor binding-independent biologic activity in the growing skeleton

Insulin-like growth factor-binding protein 2 (IGFBP-2) is a member of a family of six highly conserved IGFBPs that are carriers for the insulin-like growth factors (IGFs). IGFBP-2 levels rise during rapid neonatal growth and at the time of peak bone acquisition. In contrast, Igfbp2(-/-) mice have low bone mass accompanied by reduced osteoblast numbers, low bone formation rates, and increased PTEN expression. In the current study, we postulated that IGFBP-2 increased bone mass partly through the activity of its heparin-binding domain (HBD). We synthesized a HBD peptide specific for IGFBP-2 and demonstrated in vitro that it rescued the mineralization phenotype of Igfbp2(-/-) bone marrow stromal cells and calvarial osteoblasts. Consistent with its cellular actions, the HBD peptide ex vivo stimulated metacarpal periosteal expansion. Furthermore, administration of HBD peptide to Igfbp2(-/-) mice increased osteoblast number, suppressed marrow adipogenesis, restored trabecular bone mass, and reduced bone resorption. Skeletal rescue in the Igfbp2(-/-) mice was characterized by reduced PTEN expression followed by enhanced Akt phosphorylation in response to IGF-I and increased β-catenin signaling through two mechanisms: 1) stimulation of its cytosolic accumulation and 2) increased phosphorylation of serine 552. We conclude that the HBD peptide of IGFBP-2 has anabolic activity by activating IGF-I/Akt and β-catenin signaling pathways. These data support a growing body of evidence that IGFBP-2 is not just a transport protein but rather that it functions coordinately with IGF-I to stimulate growth and skeletal acquisition.     

Differential expression of insulin-like growth factor-I and insulin-like growth factor binding protein-1 in the diabetic rat

Summary Multiple factors contribute to the growth retardation which is a characteristic feature of uncontrolled diabetes. In this report we have examined the effects of streptozotocin-induced (STZ) diabetes on expression of insulin-like growth factor-I (IGF-I) and insulin-like growth factor binding protein-1 (IGFBP-1) in various tissues. As early as 7 days after STZ administration there was a modest reduction in IGF-I mRNA abundance. The reduction (10–30%) was of similar magnitude in each of the 7 tissues examined; liver, kidney, lung, diaphragm, quadraceps, heart and adipose tissue. However, the reduction achieved statistical significance only in the lung (p < 0.05) and diaphragm (p < 0.01). A further reduction in IGF-I mRNA abundance was seen in many tissues, 32 and 91 days after STZ administration. In contrast to the decrease in IGF-I mRNA, IGFBP-1 mRNA was significantly increased in the liver and kidney of diabetic rats. IGFBP-1 mRNA was detectable at only very low levels in other tissues but was increased in diabetic rats compared non-diabetic rats. In diabetic rats, a highly significant correlation (R = 0.75, p < 0.001) between hepatic IGFBP-1 mRNA and glucose was observed whereas there was no significant correlation between serum glucose and hepatic IGF-I mRNA abundance (R = 0.24, p = NS). Treatment of diabetic rats with insulin resulted in a small, non significant increase in hepatic and renal IGF-I mRNA and a significant decrease in renal IGFBP-1 mRNA abundance. The observations reported here are consistent with the hypothesis that diminished IGF-I expression and inhibition of available IGF-1 by increased levels of IGFBP-1 may explain the impaired growth seen in diabetic animals.