Insulin-like Growth factor-I Analogue Research Articles

Insights into pathological mutations in insulin-like growth factor I through in silico screening and molecular dynamics simulation.

Insulin-like growth factor I (IGF-I) is an anabolic growth hormone indispensable for cell growth, proliferation, differentiation, and other metabolic processes. Three missense mutations in IGF-I have been identified to be disease-related, while more mutations are waiting for phenotype annotation. However, there is no previous work regarding effective and accurate identification of pathological mutations of IGF-I, neither regarding the effects of mutations on the protein structure and dynamics. In this study, we first predicted potential deleterious mutations present in IGF-I using 16 in silico tools. Then, these mutations were further evaluated through multiple bioinformatics methods including conservation analysis, physicochemical characterization, and molecular dynamics simulation. After rigorous screening, five mutations (T4M, V17M, V44M, R50W, and M59R) were finally selected, of which two have been previously reported to be deleterious. These mutations locate at conserved regions and change the residue size locally. In the conventional simulations, the mutations destabilized the overall IGF-I structure by destroying two important hydrogen bonds within the key region of "C-neck." This finding was further confirmed by the thermal unfolding simulations and the free-energy calculations, where the mutants were associated with faster and greater loss of helix and lower energy barriers in comparison with the wild-type protein. The rigorous phenotype prediction and comprehensive structural analysis of missense mutations will not only pave the way of screening for harmful mutations in IGF-I but also provide new prospects for the rational design of IGF-I analogues and tailored medicine.

LongR3 enhances Fc-fusion protein N-linked glycosylation while improving protein productivity in an industrial CHO cell line

Abstract Glycosylation is a critical quality attribute for many therapeutic glycoproteins. Numerous studies in academia and industry have sought to understand and control protein glycosylation in order to obtain consistent glycoform profiles in production. In many cases, the resulting enhancement of protein glycosylation comes at the expense of protein productivity. Here we examine the impact of insulin-like growth factor-I analogue, LongR3 (LR3), on an Fc-fusion producing Chinese Hamster Ovary (CHO) cell process. We observe that LR3 improves protein productivity and significantly increases protein N-linked glycosylation. The beneficial effects can be seen at low concentrations (micrograms/L), which allows LR3 to be a cost-efficient additive for improving cell culture performances. The recombinant protein examined in this study has both higher sialic acid content and lower percent of asialylated species of N-linked glycans in the presence of LR3. Finally, CHO-specific glycosylation Real-Time Reverse Transcription (RT2) PCR Arrays combined with an enzymatic assay demonstrate that LR3 significantly down-regulates cytosolic sialidase gene Neu2 and hexosaminidase-D gene Hexdc, decreases extracellular sialidase activity, and therefore prevents recombinant glycoprotein desialylation.

Insulin-like growth factor-I (IGF-I) misuse in athletes and potential methods for detection

To athletes, insulin-like growth factor-I (IGF-I) is an attractive performance-enhancing drug, particularly as an alternative to growth hormone (GH) because IGF-I mediates many of the anabolic actions of GH. IGF-I has beneficial effects on muscle protein synthesis and glycogen storage that could enhance performance in several sporting disciplines. Recombinant human IGF-I (rhIGF-I) is used in clinical practice, but a variety of IGF-I compounds and IGF-I analogues are also advertised on the internet and many have been available on the black market for several years. Although methods for detecting GH misuse are now well established and there have been several cases in which athletes have tested positive for GH, no test is yet in place for detecting IGF-I misuse. The GH-2004 research group has been investigating methods for detection of IGF-I misuse and a test is being developed on the basis of the principles of the successful GH-2000 marker method, in which markers from the IGF axis and markers of collagen and bone turnover are used to detect GH misuse. Commercial immunoassays for these markers have been validated for anti-doping purposes but new methods, including IGF-I measurement by use of mass spectrometry, should improve the performance of the tests and help in the detection of athletes who are doping with these peptide hormones.

Therapeutic potential of PEGylated insulin-like growth factor I for skeletal muscle disease evaluated in two murine models of muscular dystrophy

ObjectiveDuchenne muscular dystrophy (DMD) is a fatal monogenetic disease with affected males displaying severe and progressive muscle wasting and weakness eventually leading to premature death. Possible therapeutic benefits of insulin-like growth factor I (IGF-I) have been studied extensively in various models of muscle disease and DMD with IGF-I as a mediator of improved skeletal muscle regeneration by enhancing myoblast proliferation and differentiation. DesignWe tested the efficacy of a novel IGF-I analogue, a polyethylene glycol modified IGF-I (PEG-IGF-I), to ameliorate the pathophysiology of muscular dystrophy in two mouse models of DMD. We used mdx mice which lack dystrophin (as in DMD) but exhibit only a relatively mild phenotype, and the dko mouse which is a transgenic model lacking utrophin in addition to dystrophin, and which exhibits a more severe, lethal phenotype like that in DMD. ResultsIn young mdx mice, twice-weekly PEG-IGF-I s.c. injections for 6weeks protected the diaphragm muscle against fatigue and the tibialis anterior (TA) muscle against contraction-induced injury. However, this beneficial effect of PEG-IGF-I was less pronounced in mdx mice when treatment was initiated later in adulthood. In severely affected dko mice PEG-IGF-I treatment did not affect pathophysiological parameters including animal survival. ConclusionsThese data suggest a therapeutic benefit with PEG-IGF-I treatment only in mild muscle pathologies, since its potential to ameliorate the pathophysiology in models of severe muscular dystrophies was limited. Treatment should be initiated only for mild muscle pathologies if functional benefits are to be realised and therefore may be relevant as a short-term therapy to hasten the functional repair of otherwise healthy muscles after injury.

Insulin analogues display IGF‐I‐like mitogenic and anti‐apoptotic activities in cultured cancer cells

Insulin analogues are widely used in the treatment of diabetes mellitus. Some insulin analogues were reported to display atypical activities in vitro that resemble those of insulin-like growth factor-I (IGF-I). The aim of this study was to investigate whether two long-acting insulin analogues [glargine (Lantus, Sanofi Aventis, Germany) and detemir (Levemir, Novo Nordisk, Denmark)] and two short-acting analogues [lispro (Humalog, Eli Lilly, Indianapolis, USA) and aspart (Novolog, Novo Nordisk, Denmark)] exhibit IGF-I-like activities on cultured cancer cells in comparison with IGF-I and regular human insulin. HCT-116 (colorectal cancer), PC-3 (prostate cancer) and MCF-7 (breast adenocarcinoma) cell lines were treated with insulin, IGF-I or insulin analogues, and proliferation and protection from apoptosis were measured by cell counting and fluorescent-activated cell sorter (FACS) analysis, respectively. In addition, Western blots were used to identify signalling molecules activated by the analogues. Glargine, detemir and lispro had proliferative effects that resemble IGF-I action. Insulin, however, did not stimulate cellular proliferation. In addition, glargine and detemir displayed an IGF-I-like anti-apoptotic activity. Glargine, like insulin and IGF-I, induced phosphorylation of both ERK and AKT, suggesting that the analogue is able to stimulate both the ras-raf-mitogen-activated protein kinase (MAPK) and PI3K-AKT pathways. Furthermore, glargine induced both insulin receptor (IR) and IGF-IR phosphorylation. Glargine, detemir and lispro, unlike regular insulin, exhibit in vitro proliferative and anti-apoptotic activities in a number of cancer cell lines. These actions resemble some of the effects of IGF-I, a growth factor involved in cancer initiation and progression. Insulin had no increased IGF-I activity. The specific receptor/s involved in mediating analogues actions remains to be identified.

Insulin‐like growth factor‐I analogue protects muscles of dystrophic mdx mice from contraction‐mediated damage

Contraction-mediated injury is a major contributing factor to the pathophysiology of muscular dystrophy and therefore therapies that can attenuate this type of injury have clinical relevance. Systemic administration of insulin-like growth factor-I (IGF-I) has been shown to improve muscle function in dystrophic mdx mice, an effect associated with a shift towards a more oxidative muscle phenotype and a reduced susceptibility to contraction-mediated damage. The actions of IGF-I in vivo are modulated by IGF binding proteins (IGFBPs), which generally act to inhibit IGF-I signalling. We tested the hypothesis that an analogue of IGF-I (LR IGF-I), which has significantly reduced binding affinity for IGFBPs, would improve the dystrophic pathology by reducing the susceptibility to muscle injury. Dystrophic mdx and wild-type (C57BL/10) mice were administered LR IGF-I continuously ( approximately 1.5 mg kg(-1) day(-1)) via osmotic mini-pump for 4 weeks. Administration of LR IGF-I reduced the susceptibility of extensor digitorum longus, soleus and diaphragm muscles to contraction damage, as evident from lower force deficits after a protocol of lengthening contractions. In contrast to the mechanism of protection conferred by administration of IGF-I, the protection conferred by LR IGF-I was independent of changes in muscle fatigue and oxidative metabolism. This study further indicates that modulation of IGF-I signalling has therapeutic potential for muscular diseases.

Alanine Scanning of a Putative Receptor Binding Surface of Insulin-like Growth Factor-I

Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor alpha subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe(23), Tyr(24), Tyr(31), Arg(36), Arg(37), Val(44), Tyr(60), and Ala(62)). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu(9) (His(B10)), Asp(12) (Glu(B13)), Phe(16) (Leu(B17)), Asp(53) (Ser(A12)), Leu(54) (Leu(A13)), and Glu(58) (Glu(A17)). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu(9), Asp(12), Phe(16), Leu(54), and Glu(58) results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33-100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.

Differential effects of insulin-like growth factor-I and gonadotropins on the proliferative activity of two subgroups of granulosa cells: cumulus oophorus and mural granulosa cells

Objective: To evaluate the physiological role of insulin-like growth factor-I (IGFI) and its interaction with gonadotropins in cell replication of two types of granulosa cells (cumulus oophorus, CC) and mural granulosa cells (MGC). Design: Controlled randomized study of the action and interaction of gonadotropins and IGFI on granulosa cell replication in the rat. Setting: A university reproductive biology laboratory. Intervention(s): The study examined the in vivo treatment with gonadotropins or an analogue of IGFI, long Arg 3-insulin-like growth factor (LR 3-IGFI), which does not bind to IGFI-binding proteins. Main Outcome Measure(s): Granulosa cell replication was evaluated by the use of 3H-thymidine incorporation. Result(s): It was noted that the CC replicate much faster than the MGC. These two types of granulosa cells have very different dose response curves to IGFI. Differential responses were seen in animal cells exposed to long Arg 3-insulin-like growth factor in vivo and then exposed to FSH and LH in vitro. Conclusion(s): Although murine granulosa cells show proliferative activity when they are exposed to IGFI, the two types, CC and MGC, respond differently. IGFI is not the sole mediator of the action of FSH, and these two chemicals may act independently or in concert.

Insulin-like growth factor-I (IGF-I) analogue, LR(3)IGF-I, ameliorates the loss of body weight but not of skeletal muscle during food restriction.

Insulin-like growth factor-I (IGF-I) is known to have anabolic effects in freely fed rats. We have investigated the ability of infused LR(3)IGF-I, an analogue of IGF-I, to attenuate the loss of lean tissue due to food restriction in young (5 weeks) and adult (12 weeks) rats. Groups of rats received food at 100%, 78%, 56% or 33% of ad libitum levels. Within each nutrition group the rats were continuously infused with LR(3)IGF-I at (98 nmol/day)/kg body weight or vehicle for 7 days. At each level of food intake, rats infused with LR(3)IGF-I maintained higher body weight (around 3-8%;P< 0.001) and nitrogen retention (P< 0.001) than those infused with vehicle alone but muscle protein was not conserved. LR(3)IGF-I infusion increased fat loss only in young rats (P< 0.05) despite a reduction in plasma insulin levels in both age groups (P< 0.01). Muscle protein turnover rates were unaffected by LR(3)IGF-I in young rats. In adult rats LR(3)IGF-I exacerbated the effects of food restriction through increased rates of protein breakdown, reduced RNA content and reduced rates of protein synthesis (P< 0.05) despite their larger fat reserves. Although young and adult rats show differing metabolic responses, we conclude that infusion of LR(3)IGF-I to either group during short-term food restriction does not ameliorate the loss of lean tissue by allowing more efficient utilization and/or partitioning of nutrients.

Differential Regulation of Phospholipase C-β Isozymes in Cardiomyocyte Hypertrophy

Cardiac hypertrophy is a major predictor of heart failure and of morbidity and mortality in developed countries. Many hormones and growth factors induce cardiac hypertrophy via activation of members of the phospholipase C (PLC) family. The expression pattern of the PLCβ isozyme subfamily was investigated in neonatal rat cardiomyocytes after stimulation with different hypertrophic stimuli. Under control conditions and after stimulation with norepinephrine, cardiomyocytes expressed similar amounts of PLCβ3 mRNA. In the presence of fetal calf serum (FCS), additional expression of PLCβ1 was induced. Growth hormone (GH) and insulin-like growth factor-I (IGF-I) both induced a substantial increase in PLCβ3 mRNA expression. The response to GH could not be abolished by the IGF-I receptor blocker IGF-I analogue indicating an IGF-I-independent action of GH. The upregulation of PLCβ3 by IGF-I was abolished by preincubation of cardiomyocytes with the IGF-I receptor antagonist IGF-I analogue, the tyrosine kinase inhibitor genistein, the extracellular signal-related kinase (ERK) inhibitor PD 98059, the phosphatidylinositol-3- (PI-3) kinase inhibitor wortmannin and the p70 S6 kinase inhibitor rapamycin. Induction of the immediate early genes c-myc, c-fos, and c-jun by IGF-I was abolished by preincubation with antisense oligos against PLCβ3. It is concluded that the expression of PLCβ isozymes in cardiomyocytes is differentially regulated by different hypertrophic stimuli. The upregulation of PLCβ3 by IGF-I is dependent on the activity of tyrosine kinase, ERK, PI3 kinase, and p70 S6 kinase and PLCβ3 expression seems to be required for the induction of immediate early genes by IGF-I. The involvement of the PLCβ subfamily in signal transduction of receptors other than G-protein-coupled receptors is suggested.

Insulin-like growth factor I production is essential for anabolic effects of thyroid hormone in osteoblasts.

Thyroid hormone (T3) and insulin-like growth factor I (IGF-I) are critical regulators of skeletal function. T3 increases IGF-I production in bone. To assess the potential role of IGF-I as a mediator of T3 actions, we characterized phenotypic markers of osteoblast activity in two osteoblast models, normal mouse osteoblasts and MC3T3-E1 cells, exposed to T3 alone or under conditions that interfere with IGF-I actions. T3 significantly increased osteoblast 3H-proline incorporation, alkaline phosphatase (ALP), and osteocalcin. Both alphaIR3, a neutralizing monoclonal antibody to the IGF-I receptor, and JB1, an IGF-I analogue antagonist, attenuated the stimulatory effects of T3. T3 effects also were decreased in cells transfected with antisense oligonucleotide (AS-ODN) to the IGF-I receptor gene. Both IGF-I and T3 had mitogenic effects that were inhibited by the antagonists. IGF-I by itself did not stimulate 3H-proline incorporation, ALP, and osteocalcin in the models used, revealing that although IGF-I is essential for the anabolic effects of T3, it acts in concert with other factors to elicit these phenotypic responses.

Novel Studies on Influence of Gonadotropins and Insulin-like Growth Factor-I on Growth of Cumulus Oophorus in the Rat

In the final developmental stage of a Graafian follicle, there are two functionally distinct types of granulosa cells: the cumulus cells (CCs) and the mural granulosa cells (MGCs). Previous studies focused on follicle-stimulating hormone (FSH) and insulin-like growth factor I (IGF-I) interactions in MGCs. Our goal was to study these interactions in CC proliferation. Immature rats received in vivo treatments of either saline, equine chorionic gonadotropin (eCG) with high FSH activity, an IGF-I analog (LR3-IGF-I) with poor binding to IGF-I binding proteins, or a combination of both hormones. CCs from each in vivo treatment were then cultured and treated in vitro with either saline, FSH, or IGF-I. CCs proliferation were assessed by measurement of 3H-thymidine incorporation. Prior in vivo treatment with eCG resulted in the highest proliferative activity of CCs when combined with FSH in vitro treatment. In vivo treatment with LR3-IGF-I had no effect on CC replication. CC replication was higher in FSH in vitro treatment than of IGF-I. The combination of eCG and LR3-IGF-I was the only in vivo treatment to stimulate higher CC proliferation with IGF-I in vitro treatment. This study suggests that FSH does not act through IGF-I, a mechanism previously proposed.

Protein kinases C-γ and -δ are involved in insulin-like growth factor I–induced migration of colonic epithelial cells

Background & Aims: The mechanisms by which epithelial cells migrate during the repair of damaged colonic mucosa are poorly understood. This study investigated the insulin-like growth factor I (IGF-I) signaling pathway leading to HT29-D4 human colonic epithelial cell line migration. Methods: IGF-stimulated cell migration was determined using a wound model in the presence or absence of kinase inhibitors. Activation of protein kinase C (PKC) was determined by immunodetection. Results: IGF-I and insulin induce cell migration without affecting cell proliferation through their cognate receptors. Des(1-3)-IGF-I, a truncated analogue of IGF-I, was more potent than IGF-I, suggesting that IGF-binding proteins secreted in the medium modulated IGF-I–induced cell migration. Inhibition of phosphatidylinositol 3-kinase, PKC, and mitogen-activated protein kinases eliminated cell restitution. Long-term exposure of cells to phorbol myristate acetate caused the depletion of PKC-δ and -γ and prevented also IGF-I–induced cell motility. IGF-I also induced activation of PKC-δ and -γ only. Conclusions: IGF-I stimulates colonic restitution through the activation of multiple signaling pathways including activation of phosphatidylinositol 3-kinase, PKC-δ and -γ, and mitogen-activated protein kinases. GASTROENTEROLOGY 1999;116:64-77

Preparation and characterization of the recombinant selenomethionine analogue of insulin-like growth factor-I.

Insulin-like growth factor-I (IGF-I), a single-chain polypeptide consisting of 70 amino acids and 3 disulfide bridges, is a member of a class of growth factors that are involved in many proliferative and metabolic processes. To assist in solving the crystallographic three-dimensional structure, we have expressed a recombinant fusion protein precursor of IGF-I in a methionine auxotrophic strain of Escherichia coli grown in the presence of selenomethionine. An homogeneous preparation of selenomethionyl-IGF-I was then obtained by chemical cleavage of the fusion protein. The selenomethionine analogue of IGF-I was characterized by electrospray mass spectrometry, peptide mapping, analytical chromatography, and electrophoresis as well as by biological assays. The final preparation of IGF-I was found to incorporate about 90% of selenium and fully retained the functional activity.

Insulin-like growth factor-I analogue prevents apoptosis mediated through an interleukin-I β converting enzyme (caspase-1)-like protease of cerebellar external granular layer neurons: developmental stage-specific mechanisms of neuronal cell death

Using an organotypic slice culture system of neonatal rat cerebellum, we examined developmental stage-specific mechanisms of cell death of granule neurons. This culture system allows a serial process of granule neuron development including their proliferation during the early culture period and the proceeding migration from the external granular layer to the internal granular layer in the presence of a supraphysiological concentration (5 μg/ml) of insulin. Insulin deprivation induced apoptosis of granule neurons in external granular layer but not in internal granular layer. A truncated analogue of insulin-like growth factor-I (des (1–3) insulin-like growth factor-I) prevented this apoptosis at a concentration of 65–650 ng/ml. Some apoptotic granule neurons expressed proliferating cell nuclear antigen but not TAG-1, a marker protein of the postmitotic and premigratory granule neurons. Thus, this apoptosis occurred at a specific stage in granule neuron development: at the stage before TAG-1 expression and at least partly at the proliferative state. Ac-YVAD-CHO, an inhibitor of interleukin-1 β converting enzyme (caspase-1)-like proteases, had a protective effect on this apoptosis. Interleukin-1 β converting enzyme (caspase-1)-like protease activity increased under the apoptosis-induced condition. High concentration of K +, which is known to prevent granule neuron apoptosis in dissociated cultures, had a partial protective effect on this apoptosis. These findings suggest that (i) cerebellar granule neurons fall into apoptosis at the specific developmental stage unless stimulated by insulin-like growth factor-I (analogue), (ii) this apoptosis is mediated through an interleukin-1 β converting enzyme-like protease, and (iii) this apoptosis consists of K +-sensitive and K +-insensitive components.

The regulation of type-I collagen synthesis by insulin-like growth factor-I in human osteoblast-like SaOS-2 cells.

The effect of insulin-like growth factor-I on collagen synthesis was studied using cultured human osteoblast-like SaOS-2 cells by measuring the incorporation of tritiated L-proline into immunoprecipitable type-I collagen. Tritiated L-proline incorporation into collagen was significantly stimulated by insulin-like growth factor-I in a time- and concentration-dependent manner. Unlabelled L-proline and alpha-(methylamino) isobutyric acid inhibited either the influx into cells, or the incorporation into collagen, of tritiated L-proline. The increase in incorporation of tritiated L-proline was significantly reduced by cycloheximide and actinomycin D. L-Proline incorporation into collagen was also stimulated by insulin-like growth factor-II, insulin-like growth factor-I analogues and insulin. The insulin-like growth factor-I-stimulated L-proline incorporation was inhibited by one of its binding proteins, insulin-like growth factor binding protein-4, in a concentration-dependent manner.

Effects of chronic renal failure on plasma clearance of insulin-like growth factor I, des-(1-3)IGF-I, and LR3IGF-I.

Using a rat model of chronic renal failure (CRF), we examined insulin-like growth factor I (IGF-I) clearance, degradation, organ distribution, and IGF binding profiles in plasma. The effects of IGF-binding proteins (IGFBP) on IGF clearance and degradation in CRF were studied using the IGF-I analogues des-(1-3)IGF-I and LR3IGF-I, which bind poorly to IGFBP. Although total clearance of IGF-I was not significantly altered in CRF, half-life and area under the curve were increased in the rapid distribution phase and were reduced in the slow elimination phase. Total clearance of LR3IGF-I was significantly increased. Reduced binding of IGF-I in the 150-kDa complex and increased binding to smaller-molecular-weight IGFBP were observed in CRF. Increased degradation of both IGF-I and LR3IGF-I was associated with reduced IGF binding in the 150-kDa complex. The results suggest that the accumulation of lower-molecular-weight IGFBP with reduced IGF binding in the 150-kDa complex, associated with increased degradation of peptide, may explain, at least in part, the reduced bioactivity of IGF-I observed in CRF.

The regulation of L-proline transport by insulin-like growth factor-I in human osteoblast-like SaOS-2 cells.

The effect of insulin-like growth factor-I on amino acid transport was studied by measuring the uptake of tritiated L-proline in the cultured human osteoblast-like SaOS-2 cells. The uptake of L-proline was supported by both transport system A, ASC and Gly and by Na+-dependent amino acid transport system A, and by Na+-independent system L. The initial rate of total L-proline uptake as a function of concentration showed saturation and obeyed Michaelis-Menten kinetics with Michaelis constant (Km) and maximum velocity (Vmax) values of 1.87 mM and 8.89 nmol x (mg protein)-1 x (3 min)-1, respectively. Na+-dependent L-proline uptake was significantly stimulated by insulin-like growth factor-I in a time- and concentration-dependent manner. Kinetic analysis showed that insulin-like growth factor-I enhanced transport activity by increasing the Vmax of transport without significant changes in the affinity (Km) of the carrier for the substrate. The increase in transport activity was significantly reduced by cycloheximide. The stimulated increment above basal L-proline uptake was completely inhibited by alpha-(methylamino) isobutyric acid, suggesting that only system A was affected by insulin-like growth factor-I. Na+-dependent L-proline uptake was also stimulated by insulin-like growth factor-II and insulin-like growth factor-I analogues. The insulin-like growth factor-I-stimulated L-proline uptake was inhibited by one of its binding protein, insulin-like growth factor binding protein-4, in a concentration-dependent manner.

The individual responsiveness to growth hormone (GH) treatment in GH-deficient adults is dependent on the level of GH-binding protein, body mass index, age, and gender.

The aim of the present trial was to study the individual responsiveness to GH treatment in terms of body composition and to search for possible predictors of the response in GH-deficient adults. Sixty-eight patients (44 men and 24 women) with a mean age of 44.3 (1.2) yr and verified GH deficiency participated in a 2-phase treatment trial with an initial randomized, double blind, placebo-controlled, 6-month period, followed by an open treatment period, thereby ensuring all patients 12 months of GH treatment. Recombinant human GH was administered sc daily at bedtime, with a target dose of 12 micrograms/kg x day. GHBP was measured by ligand-mediated immunofunctional assay, and serum insulin-like growth factor I (IGF-I) was determined by RIA after acid-ethanol extraction, using a truncated IGF-I analog as the radioligand. Lean body mass (LBM) and body fat (BF) were determined by dual energy x-ray absorptiometry, and total body water (TBW) was determined by bioelectrical impedance. During the placebo control period, serum IGF-I,LBM, and TBW increased (P < 0.001), whereas BF decreased (P < 0.001) and serum GHBP was unchanged in the group treated with GH compared with the patients treated with placebo. After 12 months of GH treatment, the individual changes in BF ranged from -12.5 to 4.3 kg and from -4.5 to 10.1 kg in LBM. Age (P < 0.05) and baseline GHBP level (P < 0.01) were inversely correlated with the increase in LBM. The GH-induced increment in IGF-I and TBW was greater in men than in women (P < 0.01), whereas the decreases in BF were similar in men and women. This trial demonstrates the variability in responsiveness to GH administration in GH-deficient adults. The best response to GH was obtained in younger patients with low GHBP levels. Furthermore, men responded better than women.

Insulin-like Growth Factor-I (IGF-I) Regulates IGF-binding Protein-5 Synthesis through Transcriptional Activation of the Gene in Aortic Smooth Muscle Cells

Previous studies have shown that porcine aortic smooth muscle cells (SMCs) secrete two insulin-like growth factor-binding proteins (IGFBP), IGFBP-2 and -4, and that these IGFBPs modulate IGF-I-stimulated SMC proliferation and migration. In this study we demonstrate that porcine SMCs express IGFBP-5 mRNA and synthesize and secrete the protein. In this cell type, the biosynthesis of IGFBP-5 is up-regulated by IGF-I. This increase in IGFBP-5 synthesis is accompanied by an increase in the steady-state mRNA levels. The induction of IGFBP-5 mRNA by IGF-I is time- and dose-dependent and requires de novo protein synthesis. IGF-II and insulin also increase IGFBP-5 mRNA levels at high doses. An IGF-I analog with normal affinity for the IGF-I receptor but reduced affinity for IGFBPs evokes a similar increase. Another analog that binds to IGFBPs but not to the receptor has no effect, indicating that this effect of IGF-I is mediated through the IGF-I receptor. The IGF-I-induced IGFBP-5 gene expression is cell type-specific because IGF-I had no such effect in other cell types examined. Nuclear run-on assays revealed that IGF-I increased transcription rate of the IGFBP-5 gene, while IGF-I did not change the IGFBP-5 mRNA stability. Furthermore, the IGFBP-5 promoter was 3.5-fold more active in directing expression of the luciferase reporter gene in IGF-I-treated aortic SMCs as compared to control cells, whereas the luciferase activity remained the same in control- and IGF-I-treated fibroblasts. These results suggest that IGF-I up-regulates IGFBP-5 synthesis by transcriptionally activating the IGFBP-5 gene in aortic SMCs.