Insulin-like Growth Factor I Release Research Articles

Insulin-like growth factor I releasing silk fibroin scaffolds induce chondrogenic differentiation of human mesenchymal stem cells

Growth factor releasing scaffolds are an emerging alternative to autologous or allogenous implants, providing a biologically active template for tissue (re)-generation. The goal of this study is to evaluate the feasibility of controlled insulin-like growth factor I (IGF-I) releasing silk fibroin (SF) scaffolds in the context of cartilage repair. The impact of manufacturing parameters (pH, methanol treatment and drug load) was correlated with IGF-I release kinetics using ELISA and potency tests. Methanol treatment induced water insolubility of SF scaffolds, allowed the control of bioactive IGF-I delivery and did not affect IGF-I potency. The cumulative drug release correlated linearly with the IGF-I load. To evaluate the chondrogenic potential of the scaffolds, hMSC were seeded on unloaded and IGF-I loaded scaffolds in TGF-β supplemented medium. Chondrogenic differentiation of hMSC was observed on IGF-I loaded scaffolds, starting after 2 weeks and more strongly after 3 weeks, whereas no chondrogenic responses were observed on unloaded control scaffolds. IGF-I loaded porous SF scaffolds have the potential to provide chondrogenic stimuli to hMSC. Evidence for in vivo cartilage (re)generation must be demonstrated by future, pre-clinical proof of concept studies.

Somatostatin inhibits basal and growth hormone-stimulated hepatic insulin-like growth factor-I production

Growth of vertebrates is controlled by the growth hormone (GH)-insulin-like growth factor-I (IGF-I) system, and somatostatins (SSs) have been shown to inhibit growth by reducing the release of growth hormone (GH) from the pituitary. In this study, we used rainbow trout to assess the effects of SSs on the production of IGF-I. Somatostatin-14 (SS-14-I) implantation for 15 days significantly reduced steady-state levels of IGF-I mRNA in liver and lowered IGF-I concentration in plasma compared to control animals. The direct effects of SSs were examined on hepatocytes incubated in vitro. SS-14-I inhibited basal and GH-stimulated IGF-I mRNA expression. SS-14-I inhibition of GH-stimulated IGF-I expression was concentration- and time-dependent; the ED 50 was ca. 40 ng/ml and the maximum response was observed after 6 h. All SS isofoms tested, including the N-terminally extended form of SS-14-I, SS-28-I, and the [Tyr 7, Gly 10]-substituted forms of SS, SS-14-II, SS-25-II and SS-28-II, inhibited GH-stimulated IGF-I mRNA expression. The inhibitory effects of SS-14-I on steady-state levels of IGF-I mRNA resulted from reduced IGF-I mRNA transcription and not from altered mRNA stability. SS-14-I also reduced basal and GH-stimulated release of IGF-I into culture medium. These results indicate that SSs regulate growth in an extrapituitary manner by reducing hepatic IGF-I biosynthesis and secretion.

Cathepsin K inhibitors prevent matrix-derived growth factor degradation by human osteoclasts

The coupling between bone formation and resorption creates a therapeutic impasse in osteoporosis: antiresorptive therapy halts bone loss, but also inhibits bone formation, and therefore does not cure the condition. Surprisingly, recent preliminary reports suggest that inhibition of resorption by cathepsin K (CathK) inhibitors augments bone formation. Uniquely amongst resorption-inhibitors, CathK-inhibitors suppress degradation of the organic matrix of bone while allowing demineralization. We hypothesized that these unique characteristics might explain a capacity of CathK inhibitors to enhance bone formation: the inhibitors might prevent degradation not only of collagen, but also other proteins, including growth factors embedded in matrix. We tested this hypothesis using osteocalcin and insulin-like growth factor I (IGF-I) as examples of matrix-embedded proteins, and found that CathK-inhibitors, unlike other resorption-inhibitors, dramatically increased the concentrations of these matrix-derived proteins in supernatants of osteoclasts on bone, most likely through protection against intracellular degradation. We found that protons are both necessary and sufficient for the release of IGF-I from bone matrix, and that recombinant CathK can degrade both marker proteins. In the presence of a CathK-inhibitor, the amount of IGF-I released from matrix substantially exceeded the amount secreted by osteoclasts. CathK-inhibition similarly augmented bone morphogenetic protein (BMP)-2 release. Lastly, MC3T3-E1 numbers were greater after co-culture with osteoclasts on bone with versus without CathK-inhibitor, showing that, in the presence of CathK-inhibitor, osteoclasts release biologically-significant quantities of biologically-active matrix-derived growth factors. These results support a model in which osteoclastic secretion of protons demineralizes bone, causing release of growth factors from bone matrix. Normally these are largely degraded, with collagen, in the resorptive hemivacuole and during transcytosis to the basal surface of the osteoclast, but in the presence of CathK inhibitor they are released intact, and so might augment bone formation.

Efeito da suplementação de L-arginina sobre a secreção de hormônio do crescimento e fator de crescimento semelhante à insulina em adultos

Based on presumptions that the infusion of amino acids can augment the release of human growth hormone (hGH) and that this metabolism is related with secretion of insulin-like growth factor I (IGF-I), the purpose of this study was to verify the effect of L-arginine supplementation on GH and IGF-I in adults. Seventeen male individuals participated on the study and were randomized to receive L-arginine (n= 10) or placebo (n= 7), seven grams per day for seven days. Before and after the supplementation period, the volunteers realized blood collection in fasting to verify both GH and IGF-I levels, as well as urine collection to verify urea excretion. At the end of the experimental period, it was verified that the group that received L-arginine augmented the urea in urine excretion (to 2684.1 +/- 475.2 mg/dl from 2967.2 +/- 409.7 mg/dl, p= 0.002), therefore it did not alter significantly the release of hormones evaluated. The group which received placebo did not alter significantly any evaluated parameters. The L-arginine supplementation during seven days was ineffective to augment both GH and IGF-I release in individual male adults.

Insulin-like growth factor I (IGF-I) in a growth-enhanced transgenic (GH-overexpressing) bony fish, the tilapia (Oreochromis niloticus): indication for a higher impact of autocrine/paracrine than of endocrine IGF-I

Several lines of growth hormone (GH)-overexpressing fish have been produced and analysed for growth and fertility parameters. However, only few data are available on the growth-promoting hormone insulin-like growth factor I (IGF-I) that mediates most effects of GH, and these are contradictory. Using quantitative real-time RT-PCR, radioimmunoassay, in situ hybridization, immunohistochemistry, and radiochromatography we investigated IGF-I and IGF binding proteins (IGFBPs) in an adult (17 months old) transgenic (GH-overexpressing) tilapia (Oreochromis niloticus). The transgenics showed an around 1.5-fold increase in length and an approximately 2.3-fold higher weight than the non-transgenics. Using radioimmunoassay, the serum IGF-I levels were lower (6.22 +/- 0.75 ng/ml) in transgenic than in wild-type (15.01 +/- 1.49 ng/ml) individuals (P = 0.0012). Radioimmunoassayable IGF-I in transgenic liver was 4.2-times higher than in wild-type (16.0 +/- 2.21 vs. 3.83 +/- 0.71 ng/g, P = 0.0017). No hepatocytes in wild-type but numerous hepatocytes in transgenic liver contained IGF-I-immunoreactivity. RT-PCR revealed a 1.4-times higher IGF-I mRNA expression in the liver of the transgenics (10.51 +/- 0.82 vs. 7.3 +/- 0.49 pg/microg total RNA, P = 0.0032). In correspondence, in situ hybridization showed more IGF-I mRNA containing hepatocytes in the transgenics. A twofold elevated IGF-I mRNA expression was determined in the skeletal muscle of transgenics (0.33 +/- 0.02 vs. 0.16 +/- 0.01 pg/microg total RNA, P < 0.0001). Both liver and serum of transgenics showed increased IGF-I binding. The increased IGFBP content in the liver may lead to retention of IGF-I, and/or the release of IGF-I into the circulation may be slower resulting in accumulation of IGF-I in the hepatocytes. Our results indicate that the enhanced growth of the transgenics likely is due to enhanced autocrine/paracrine action of IGF-I in extrahepatic sites, as shown here for skeletal muscle.

Recombinant Human Insulin-Like Growth Factor I (rhIGF-I) and rhIGF-I/rhIGF-Binding-Protein-3: New Growth Treatment Options?

INSULIN-LIKE GROWTH FACTOR I (IGF-I) AND GROWTH he growth effect that gives growth hormone (GH) its name is the result of GH stimulation of IGF-I production in the liver (endocrine IGF-I) and peripheral tissues, particularly bone and muscle (autocrine/paracrine IGF-I). Hepatic IGF-I circulates almost entirely ( 99%) bound to IGF-binding proteins (IGFBPs). The IGFBPs are a family of six tructurally related proteins with a high affinity for binding IGF. The principal BP, IGFBP-3, which binds 75% to 90% of irculating IGF-I, is a large ternary complex consisting of IGFBP-3, acid labile subunit (ALS), and IGF molecules. ALS and GFBP-3 are produced in the liver as a direct effect of GH. The ALS stabilizes the IGF–IGFBP-3 complex, reduces the passage f IGF-I to the extravascular compartment, and extends its half-life. The remainder of bound IGF-I is mostly with IGFBP-1 nd IGFBP-2. IGFBP-1 concentrations are controlled by nutritional status as reflected in insulin levels, with the highest GFBP-1 concentrations found in the fasting, hypoinsulinemic state. Similarly, circulating concentration of IGFBP-2 is under egative control by GH directly, independently of IGF-I, and is elevated with GH deficiency or resistance; the positive effect f IGF-I on IGFBP-2 results in further elevation when patients who are GH receptor deficient (GHRD, Laron syndrome) are reated with IGF-I. The IGFBPs modulate IGF action by controlling storage and release of IGF-I in the circulation and nfluencing its binding to its receptor, facilitating storage of IGF-I in extracellular matrices, and they also exert independent ctions. IGF-I binds to the type 1 IGF receptor with high affinity and to the structurally similar insulin receptor with an affinity hat is approximately 100-fold less that of insulin. Because IGF-I is present in the circulation at molar concentrations that are 000 times those of insulin, even a small insulin-like effect of IGF-I as a result of its binding to the insulin receptor could be ore important than that of insulin itself, were it not for the IGFBPs that control the availability and activity of IGF-I, and dapt to changing energy status. The importance of IGF-I in normal intrauterine growth in humans has been demonstrated in a patient with a homozygous artial deletion of the IGF-I gene, in a patient with mutation of the IGF-I gene resulting in high circulating levels of an neffective IGF-I, and in patients with mutations of the IGF-I receptor, who all had severe intrauterine growth retardation. ntrauterine IGF-I synthesis, however, does not appear to be GH dependent because most patients with genetically determined evere IGF-I deficiency have normal or only minimally reduced intrauterine growth. Standard deviation score (SDS) for length eclines rapidly after birth in these conditions, demonstrating the immediate need for GH-stimulated IGF-I synthesis for ostnatal growth. Growth velocity in the absence of GH is typically half normal, but it has been reported to be normal or upranormal despite absence of GH in some hypothalamic conditions characterized by hyperphagia, with obesity or rapid weight ain. A similar phenomenon was described in a placebo-controlled study of rhIGF-I treatment of patients with severe IGF-I eficiency from GHRD, in which 3 of the 9 placebo-treated subjects grew over the -month study period at an accelerated rate comparable with that of the rhIGF-I–treated ubjects, attributed to improved nutrition in the investigative milieu.

Stimulation of Steroid-Suppressed Cutaneous Healing by Repeated Topical Application of IGF-I: Different Mechanisms of Action Based Upon the Mode of IGF-I Delivery

Insulin-like growth factor-I (IGF-I) is accepted as a potent stimulus of wound healing when applied in combination with its binding proteins. However, there is only one study published that has investigated the effect of repeated topical application of unbound IGF-I on ischemic wound healing. The aim of this study was to show the effect of daily topical IGF-I therapy on cutaneous ulcer healing in a steroid-suppressed wound model. Full-thickness wounds were created on the back of 40 male Sprague-Dawley rats. Before surgery, animals received depot-steroids subcutaneously. Wounds were treated daily with either a standard hydrogel dressing (control), topical IGF-I dissolved in 0.2% methylcellulose gel (IGF-I gel), or a hydrogel dressing containing IGF-I (IGF-I dressing). After 7 days of treatment, wounds were excised and measured by photoplanimetry. SMA- and PCNA-expression as well as the formation of granulation tissue were assessed in tissue sections. Results are given as median(min-max). Differences between groups were calculated by the Mann-Whitney U test. Subcutaneous injection of depot-steroids induced a significant delay in healing, as shown by an enlarged wound size [44(33-65) versus 25(20-35)] mm(2); P = 0.001). In steroid-treated rats, both IGF-I gel and IGF-I dressing enhanced excisional healing, as shown by a significant reduction in wound size (P = 0.0001), with IGF-I released from the dressing being even more effective than IGF-I gel (P = 0.03). However, in these animals only IGF-I released from the hydrogel dressing stimulated SMA- (P = 0.03) as well as PCNA-expression (P = 0.001) and increased granulation tissue formation (P = 0.018). Our data indicate that a repeated application of topical IGF-I enhances cutaneous ulcer healing. In addition, only the controlled release of IGF-I from the hydrogel dressing is capable of reversing the steroid-induced delay of healing, suggesting different mechanisms of action with respect to the mode of IGF-I delivery.

Experimental ischemic wounds: Correlation of cell proliferation and insulin‐like growth factor I expression and its modification by different local IGF‐I release systems

We investigated cell proliferation and local insulin-like growth factor-I (IGF-I) expression in ischemic wounds after topical application of IGF-I through different delivery systems. IGF-I dressings were fabricated from an IGF-I containing polyvinyl alcohol film placed on a standard hydrogel dressing. In vitro, the release of IGF-I from this dressing was assessed by enzyme-linked immunosorbent assay. For animal experiments, a standardized ischemic skin flap containing a full-thickness wound was created on the back of male Sprague-Dawley rats. An identical wound outside the flap served as control. We initially investigated intracutaneous pO2 (p(ti)O2), cell proliferation, and local IGF-I expression. In a second setting, wounds were treated either with IGF-I dissolved in methylcellulose gel or with an IGF-I dressing, and ulcer size and cell proliferation were assessed. In vitro, approximately 60% of IGF-I was released from the IGF-I dressing, compared to a 97% release from methylcellulose gel. In vivo, ischemic wounds showed less cell proliferation and decreased IGF-I expression than nonischemic wounds. A lower local p(ti)O2 correlated with larger wound size, less cell proliferation, and decreased IGF-I expression. Ulcer size was reduced after treatment with either IGF-I dressing or methylcellulose gel. However, cell proliferation only increased after treatment with IGF-I dressing, but not after methylcellulose gel treatment. We conclude that IGF-I expression is decreased in ischemic wounds and correlates with low cell proliferation. This can be reversed by local IGF-I application, but the efficacy of treatment depends on the delivery system.

Insulin-like Growth Factor I Plasmid Therapy Promotes in Vivo Angiogenesis

Angiogenesis, the formation of neovessels from the endothelium of preexisting vessels, is stimulated by soluble angiogenic factors. Insulin-like growth factor I (IGF-I) stimulates myogenesis and induces nerve regeneration after injury, and it has been shown to stimulate angiogenesis. However, the in vivo angiogenic effects of IGF-I in regenerating and diabetic muscle have yet to be described. Therefore, we studied the effects of human IGF-I (hIGF-I) delivered by a plasmid-mediated therapy on angiogenesis in mouse models of these two conditions. Plasmid hIGF-I was delivered to the injured tibialis muscle by direct intramuscular injection followed by electroporation. Initial experiments compared two muscle-specific hIGF-I-expressing constructs containing either a skeletal actin 3′UTR (pAV2001) or a human growth hormone (GH) 3′UTR (pAV2002). Skeletal actin 3′UTR mediates sequestration of hIGF-I in the muscle and was more active, while the GH 3′UTR mediated release of IGF-I into the circulation. Treatment of regenerating muscle with pAV2001 and sequestration of IGF-I in muscle led to increased expression of vascular endothelial growth factor (VEGF) and VEGF receptors fetal liver kinase-1 and FmS-like tyrosine kinase receptor-1, as well as platelet endothelial cell adhesion molecule-1, on endothelial cells. These results indicate that IGF-I can amplify angiogenic responses in regenerating muscle. In a mouse diabetic model, plasmid-mediated IGF-I therapy reversed diabetic microangiopathy, as shown by increased angiogenesis and arterial flow as analyzed by Doppler imaging. These studies show that plasmid IGF-I delivery and sequestration in muscle can augment angiogenesis in regenerating muscle and increase blood flow and angiogenesis in the diabetic limb.

Action of protein kinase A regulators on secretory activity of porcine granulosa cells in vitro

To understand the role of protein kinase A (PKA) in the control of ovarian secretory activity, we examined effects of stimulators (db-cAMP, 6-Phe-cAMP, Sp-cDBIMPS) or inhibitors (Rp-cAMPS, KT5720) of PKA on the release of insulin-like growth factor I (IGF-I), progesterone (P) and estradiol (E) by cultured porcine granulosa cells using RIA. All the PKA stimulators db-cAMP (10–10,000 ng/ml), 6-Phe-cAMP (10–10,000 pmol) or Sp-cDBIMPS (1–10,000 pmol) increased IGF-I almost at all doses tested. P release was stimulated by db-cAMP (at doses 100–10,000 ng/ml), Sp-cDBIMPS (at 10–1000 pmol) and 6-Phe-cAMP (at 1000 and 10,000 pmol). The release of E was stimulated by Sp-cDBIMPS (1–100 pmol), db-cAMP (1000 and 10,000 ng/ml) and 6-Phe-cAMP (1000 and 10,000 pmol). Since Sp-cDBIMPS, which activates preferentially PKA isozyme type II, showed stimulating effects at doses lower than those of 6-Phe-cAMP, a preferential activator of both, type I and II of PKA, it is assumed that PKA type II is more important for the control of ovarian steroidogenesis than type I. A PKA inhibitor Rp-cAMPS inhibited release of IGF-I (10,000 pmol), P (1000 pmol) and E (1000 and 10,000 pmol), whereas Rp-cAMPS, at doses higher than 1000 pmol, tended to reverse this inhibitory effect. Other PKA inhibitor KT5720 suppressed P (at 10–1000 ng/ml), but not IGF-I or E release. The stimulation of growth factor and sex steroid release by PKA activators, and suppression of the secretion some of these substances by PKA inhibitors may indicate the implication of PKA (probably site B) in up- and down-regulation of ovarian IGF-I and steroid release.

GH/IGF-I axis in anorexia nervosa

Patients with anorexia nervosa (AN) may develop multiple endocrine abnormalities, including amenorrhea, hyperactivity of the hypothalamus-pituitary-adrenal axis, hypothyroidism and particular changes in the activity of the growth hormone (GH)/insulin-like growth factor I (IGF-I) axis. Exaggerated GH secretion and reduced IGF-I levels are usually found in AN, as well as in conditions of malnutrition and malabsorption, insulin-dependent diabetes mellitus, liver cirrhosis and catabolic states. In AN, GH hypersecretion at least partially reflects malnutrition-induced peripheral GH resistance, which leads to reduced IGF-I synthesis and release; this implies an impairment of the negative IGF-I feedback action on GH secretion. On the other hand, primary alterations in the neural control of GH secretion cannot be ruled out. The neuroendocrine alterations include enhanced somatotroph responsiveness to growth hormone releasing hormone (GHRH) and impaired GH response to most central nervous system-mediated stimuli. Particular resistance to cholinergic manipulation has also been demonstrated, thus suggesting a somewhat specific alteration in the somatostatin (SS)-mediated cholinergic influence on GH secretion. Moreover, paradoxical GH responses to glucose load, thyrotropin releasing hormone (TRH) and luteinizing hormone releasing hormone (LHRH) have also been reported. The effect of reduced leptin levels on GH hypersecretion in AN is still unclear, but ghrelin (the gastric hormone that is a natural ligand of the GH secretagogue receptor and strongly stimulates somatotroph secretion) is thought to play a major role. Regardless of the supposed central and peripheral alterations, it has to be emphasised that the activity of the GH/IGF-I axis in AN is generally restored by nutritional and stable weight gain. It therefore reflects an impaired nutritional state and cannot be considered a primary hallmark of the disease.

SOM230: a novel somatostatin peptidomimetic with broad somatotropin release inhibiting factor (SRIF) receptor binding and a unique antisecretory profile.

The aim of the present study was to identify a small, metabolically stable somatotropin release inhibiting factor (SRIF) analog with a more universal binding profile similar to that of natural somatostatin, resulting in improved pharmacological properties and hence new therapeutic uses. A rational drug design approach was followed by synthesizing alanine-substituted SRIF-14 analogs to determine the importance of single amino acids in SRIF-14 for SRIF receptor subtype binding. The incorporation of structural elements of SRIF-14 in a stable cyclohexapeptide template in the form of modified unnatural amino acids resulted in the identification of the novel cyclohexapeptide SOM230. SOM230 binds with high affinity to SRIF receptor subtypes sst1, sst2, sst3 and sst5 and displays a 30- to 40-fold higher affinity for sst1 and sst5 than Sandostatin (octreotide; SMS 201-995) or Somatuline (BIM 23014). In vitro, SOM230 effectively inhibited the growth hormone releasing hormone (GHRH)-induced growth hormone (GH) release in primary cultures of rat pituitary cells with an IC(50) of 0.4+/-0.1 nmol/l (n=5). In vivo, SOM230 also potently suppressed GH secretion in rats. The ED(50) values determined at 1 h and 6 h post injection of SOM230 indicated its very long duration of action in vivo. This property was also reflected in pharmacokinetic studies comparing plasma levels of SMS 201-995 and SOM230 after subcutaneous application. Whereas SMS 201-995 had a terminal elimination half life of 2 h, this was markedly prolonged in SOM230-treated animals (t(1/2)=23 h). Furthermore, in rats SOM230 demonstrated a much higher efficacy in lowering plasma insulin-like growth factor-I (IGF-I) levels compared with SMS 201-995. The infusion of 10 microg/kg/h of SOM230 using subcutaneously implanted minipumps decreased plasma IGF-I levels far more effectively than SMS 201-995. After 126 days of continuous infusion of SOM230 plasma IGF-I levels were decreased by 75% of placebo-treated control animals. For comparison SMS 201-995, when used under the same experimental conditions, resulted in only a 28% reduction of plasma IGF-I levels, indicating a much higher efficacy for SOM230 in this animal model. It is important to note that the inhibitory effect of SOM230 was relatively selective for GH and IGF-I in that insulin and glucagon secretion was inhibited only at higher doses of SOM230. This lack of potent inhibition of insulin and glucagon release was also reflected in the lack of effect on plasma glucose levels. Even after high dose treatment over 126 days no obvious adverse side effects were noticed, including changes in plasma glucose levels. We have identified a novel short synthetic SRIF peptidomimetic, which exhibits high affinity binding to four of the five human SRIF receptor subtypes and has potent, long lasting inhibitory effects on GH and IGF-I release. Therefore SOM230 is a promising development candidate for effective GH and IGF-I inhibition and is currently under evaluation in phase 1 clinical trials.

Stimulating Effect of Recombinant Human Erythropoietin (rhEPO) on Insulin-like Growth Factor-I (IGF-I) Production by Hepatocytes in Rats with or without Subtotal Nephrectomy

S We investigated the effect of recombinant human erythropoietin (rhEPO) on insulin-like growth factor-I (IGF-I) production in primary cultured rat hepatocytes. Hepatocytes were obtained by in situ collagenase perfusion from adult male Wistar rats with sham-operation (control rats) or 5/6 nephrectomy (chRF rats), in which renal function was impaired and associated with renal anemia 4 weeks after the surgery. The hepatocytes were cultured at a density of 5 X 10 5 cells/mL in Williams' medium E with different doses of rhEPO for 48 h. IGF-I concentrations were measured by specific radioimmunoassay after acid-ethanol extraction. Plasma EPO levels were lower in chRF rats than in control rats at 4 weeks after the surgery (1.8′0.1 vs 2.6′0.3 mU/mL, P<0.05), whereas plasma IGF-I levels were rather higher in chRF rats (778.3 ′53.5 vs 636.0′70.0, P<0.05). Medium IGF-I levels were not different between the two groups (control rats 9.8′1.7 ng/mL vs chRF rats 10.1′ 1.8 ng/mL) and were dose-dependently increased by rhEPO (10 - 1 1 -10 - 8 M) in both groups of animals. An increment of medium IGF-I after the incubation with 10 - 8 M rhEPO for 48 h was 5 fold greater in control rats than in chRF rats (Δ13.6′3.9 vs Δ2.7′1.5 ng/mL, P<0.05). These findings suggest that EPO directly stimulates IGF-I release from rat hepatocytes and that the stimulating effect of EPO is blunted in chRF rats.

Stabilizing insulin-like growth factor-I in poly( d,l-lactide-co-glycolide) microspheres

This study aimed at developing a controlled drug delivery system for recombinant human insulin-like growth factor-I (IGF-I) for localized delivery in bone healing. IGF-I was microencapsulated into an end-group uncapped 14 kDa poly( d,l-lactide-co-glycolide) 50:50 (PLGA 50:50) by solvent extraction from a W 1/O/W 2 dispersion. Prior to encapsulation, IGF-I was exposed to ultrasonication in a water/dichloromethane dispersion, and its stability tested in the presence and absence of various excipients in the W 1 phase. HPLC and RIA were used for the assessment of IGF-I stability. Microencapsulated IGF-I was tested again for its structural intactness and also for in vitro release from various formulations containing appropriate co-encapsulated excipients. A specific fat cell assay was used to determine the biological activity of released IGF-I. Moderate ultrasonic treatment of aqueous IGF-I/dichloromethane mixtures caused approx. 50% IGF-I degradation. However, IGF-I was fully protected when bovine serum albumin, succinylated gelatin or poly(ethyleneglycol) were added to the aqueous IGF-I. Co-encapsulation of these excipients protected efficiently the protein upon microencapsulation. IGF-I release from microsphere formulations was sustained for up to 13 days featuring a moderately pulsatile pattern, depending on the microsphere composition. Typically, the amounts of IGF-I released within the first 24 h (burst) and during the second release pulse were in the order of 20 and 40%, respectively, of the total dose. The biological activity of released IGF-I was confirmed at selected time-points by the fat cell assay. In conclusion, the developed microspheres proved to be suitable to release biologically intact IGF-I over up to 13 days, a time-period considered to be relevant to promote bone fracture healing.

Effect of cGMP analogues and protein kinase G blocker on secretory activity, apoptosis and the cAMP/protein kinase A system in porcine ovarian granulosa cells in vitro

The aim of the present study was to examine the role of cGMP-dependent intracellular mechanisms in control of ovarian functions. In the first series of experiments we studied the effects of the cGMP analogues 8-pCPT-cGMP (0.001–100 nM), Rp-8-pCPT-cGMPS (0.01–100 nM), Rp-8-Br-cGMPS (0.01–100 nM), and Rp-8-Br-PET-cGMPS (0.01–100 nM) on the release of progesterone, insulin-like growth factor I (IGF-I) and oxytocin by cultured porcine granulosa cells. In a second series of experiments, the effects of Rp-8-Br-PET-cGMPS (50 nM) and KT5822 (100 ng/ml), specific inhibitor of cGMP-dependent protein kinase (PKG), on cAMP, PKA, oxytocin and the occurrence of apoptosis in cultured cells were compared. The release of hormones and IGF-I into the culture medium was evaluated using a RIA, while the percentage of cells containing visible oxytocin, cAMP, as well as the regulatory and catalytic subunits of PKA was assessed using immunocytochemistry. Occurrence of apoptosis in these cells was detected using the TUNEL method. The stimulatory (8-pCPT-cGMP and Rp-8-pCPT-cGMPS), inhibitory (Rp-8-Br-cGMPS) and biphasic (Rp-8-Br-PET-cGMPS) effect of cGMP analogues on progesterone release was observed. All cGMP analogues used suppressed IGF-I release. All cGMP analogues decreased oxytocin release, but 8-pCPT-cGMP and Rp-8-Br-cGMPS, when given at low doses (0.01–0.1 and 1–10 nM, respectively) stimulated oxytocin output. Both, Rp-8-Br-PET-cGMPS and KT5822 increased the rate of incidence of apoptosis and percentage of cells containing immunoreactive cAMP. Both Rp-8-Br-PET-cGMPS and KT5822 decreased the proportion of cells containing immunoreactive oxytocin and regulatory subunit of PAK KT5822, but not Rp-8-Br-PET-cGMPS, increased the number of cells containing catalytic subunit of PKA. The present observations suggest the involvement of cGMP and PKG in control of the production of steroid, nonapeptide hormone, growth factor, cAMP and cAMP-dependent PKA, as well as the induction of apoptosis in porcine ovarian cells.

Growth hormone axis overview--somatomedin hypothesis.

The possibility that the action of growth hormone (GH) on cartilage is mediated by a separate hormonal agent found in serum was suggested by incubation with hypophysectomized rat costal cartilage. The stability of this tissue permitted long incubations and the measurement of the uptake of 35S-sulfate provided a convenient index of growth stimulation. Under the conditions arbitrarily selected, normal rat serum, but not serum from hypophysectomized rats, induced a great stimulation of 35S uptake. In contrast, GH added directly to cartilage in these incubations was virtually inactive. It was suggested that a serum sulfation factor, now known as insulin-like growth factor-I (IGF-I), was a mediator of GH action. Recently it has been observed that addition of 35S-sulfate after 24 h of preincubation with GH permitted the direct effect of GH to be recognized. Other observations in intact hypophysectomized rats have established that GH can induce the expression of IGF-I in cartilage that acts in an autocrine-paracrine manner. The relative importance of the endocrine and autocrine-paracrine routes of IGF-I action on the growth of cartilage is in dispute. It is clearly established that serum IGF-I exerts a negative feedback on GH secretion by action on the hypothalamus and pituitary. Serum IGF-I concentrations reflect GH action in postnatal life. Measurement of serum IGF-I is the most-valuable index of GH hypersecretion in acromegaly and in conditions of growth impairment. GH receptor deficiency leads to a marked decrease in circulating IGF-I. Hypernutrition and hyperinsulinism of obesity directly promote hepatic IGF-I release and inhibit GH secretion by the pituitary. Differences in hepatic IGF-I synthesis in response to GH may contribute to physiological differences in stature.

Decreased autocrine release of insulin-like growth factor-I (IGF-I) from immune cells is influenced by Aβ 1–42 fragment and is associated with reduced brain volume in subjects with Alzheimer's disease (AD)

Decreased autocrine release of insulin-like growth factor-I (IGF-I) from immune cells is influenced by Aβ 1–42 fragment and is associated with reduced brain volume in subjects with Alzheimer's disease (AD)

Insulin-like growth factor I in the anterior pituitary of the clawed frog Xenopus laevis: immunocytochemical and autoradiographic indication for a paracrine action and corelease with prolactin.

Insulin-like growth factor I (IGF-I) and its receptor are present in human and rat anterior pituitary. However, few data exist on the potential presence of IGF-I or its receptor in the non-mammalian pituitary and the cellular sites of IGF-I production have not been identified in any species. Thus, we investigated the anterior pituitary of the clawed frog Xenopus laevis which is widely used to study growth and differentiation. The study was performed with antisera against mammalian insulin-like growth factor I (IGF-I), prolactin (PRL) and growth hormone (GH) using immunohistochemical and immunocytochemical techniques. IGF-I binding was determined by in-vitro receptor autoradiography. The PRL-and GH-immunoreactive cells exhibited distinct distribution patterns. Neither at the light nor the electron microscopical level any colocalization of PRL-and GH-immunoreactivities was apparent. The PRL-immunoreactive cells exhibited round granules of medium electron density (mean diameter: 312 nm) and the GH-immunoreactive cells spherical granules of medium electron density (mean diameter: 165 nm). By the use of serial semithin sections IGF-I-immunoreactivity was exclusively located in PRL-immunoreactive cells. At the ultrastructural level, IGF-I-immunoreactivity was confined to the secretory granules in coexistence with PRL-immunoreactivity using the double labelling immunogold technique. Specific IGF-I binding sites were localized throughout the pituitary. The results provide evidence for a concomitant release of PRL and IGF-I and suggest autocrine/paracrine actions of IGF-I in the anterior pituitary.

Adsorption and release of insulin-like growth factor-I on porous tricalcium phosphate implant

In order to develop bone substitutes, the design of biomaterials like calcium phosphate ceramic loaded with bone growth factor are of great interest. However, it is necessary to control the amount of growth factor adsorbed onto ceramics and the kinetics of its release. Radiolabeling of insulin-like growth factor-I (IGF-I) with 125-iodine ([125I]-IGF-I) and its adsorption onto porous tricalcium phosphate (TCP) cylinders enabled us to establish the time-adsorption and time-release curves using various concentrations of IGF-I. The adsorption curve increased rapidly and then flattened out at 72 h; 90% of the maximum was already reached at 24 h; and 20% of the adsorbed IGF-I was released in water within 4 days. In human serum the release was faster at 82% within 4 days. In vivo evaluation on an animal model was then performed. Rabbits' bilateral femoral cylindrical bone defects were filled with the TCP cylinders, which were either carrying IGF-I or implanted alone as a control in each rabbit. Bone turnover and ceramic resorption were stimulated by IGF-I loaded TCP according to standard radiography, dual-energy X-ray absorptiometry, histology, and histomorphometry. © 2000 John Wiley & Sons, Inc. J Biomed Mater Res, 49, 415–421, 2000.

Adsorption and release of insulin-like growth factor-I on porous tricalcium phosphate implant.

In order to develop bone substitutes, the design of biomaterials like calcium phosphate ceramic loaded with bone growth factor are of great interest. However, it is necessary to control the amount of growth factor adsorbed onto ceramics and the kinetics of its release. Radiolabeling of insulin-like growth factor-I (IGF-I) with 125-iodine ([(125)I]-IGF-I) and its adsorption onto porous tricalcium phosphate (TCP) cylinders enabled us to establish the time-adsorption and time-release curves using various concentrations of IGF-I. The adsorption curve increased rapidly and then flattened out at 72 h; 90% of the maximum was already reached at 24 h; and 20% of the adsorbed IGF-I was released in water within 4 days. In human serum the release was faster at 82% within 4 days. In vivo evaluation on an animal model was then performed. Rabbits' bilateral femoral cylindrical bone defects were filled with the TCP cylinders, which were either carrying IGF-I or implanted alone as a control in each rabbit. Bone turnover and ceramic resorption were stimulated by IGF-I loaded TCP according to standard radiography, dual-energy X-ray absorptiometry, histology, and histomorphometry.