Increased Insulin-like Growth factor-I mRNA Research Articles

Cysteine induces longitudinal bone growth in mice by upregulating IGF-I.

Cysteine (Cys) is known to exert various effects, such as antioxidant, antipancreatitic and antidiabetic effects. However, the effects of Cys on longitudinal bone growth have not been elucidate to date. Thus, the aim of the present study was to evaluate the effects of Cys on bone growth. Growth-plate thickness and bone parameters, such as bone volume/tissue volume (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), connectivity density (Conn.D) and total porosity were analyzed by means of micro-computed tomography (μCT). The levels of serum insulin-like growth factor-I (IGF-I) were measured by enzyme-linked immunosorbent assay (ELISA). Hepatic IGF-I mRNA expression was analyzed by quantitative polymerase chain reaction (qPCR). The phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 5 (STAT5) was investigated by western blot analysis. Our results revealed that Cys increased IGF-I mRNA expression in HepG2 cells. The thickness of the growth plates was increased following treatment with Cys. Moreover, BV/TV, Tb.Th, TbN, Conn.D and total porosity were improved following treatment with Cys. Hepatic IGF-I mRNA expression and serum IGF-I levels were increased by Cys. The levels of phosphorylated JAK2 and STAT5 were elevated by Cys. The findings of our study indicate that Cys increases the thickness of growth plates through the upregulation of IGF-I, which results from the phosphorylation of JAK2-STAT5. Thus, our data suggest that Cys may have potential for use as a growth-promoting agent.

Acute heat stress and dietary methionine effects on IGF-I, GHR, and UCP mRNA expression in liver and muscle of quails

This study evaluated the expression of insulin-like growth factor I (IGF-I), growth hormone receptor (GHR), and uncoupling protein (UCP) mRNA in muscle and liver of quails that were in thermal comfort or exposed to heat stress and that were fed diets with or without methionine supplementation. Meat quails were fed a diet that either met the nutritional demands for methionine (MS) or did not meet this demand (methionine-deficient diet, MD). The animals were either kept at a thermal comfort temperature (25°C) or exposed to heat stress (38°C for 24 h starting on the 6th day). RNA was extracted from liver and breast muscle, and cDNA was synthesized and amplified using quantitative reverse transcription-polymerase chain reaction. Animals that were fed the MS diet and remained at the thermal comfort temperature exhibited increased IGF-I mRNA expression in the liver (0.56 AU). The GHR mRNA expression in the liver and muscle was influenced by both the study variables. Animals receiving the MS diet showed higher GHR expression, while increased expression was observed in animals at the thermal comfort temperature. The UCP mRNA expression in the muscle was influenced by both methionine supplementation and heat stress. Higher expression was observed in animals that received the MD diet (2.29 vs 3.77 AU) and in animals kept in thermal comfort. Our results suggest that heat stress negatively affects the expression of growth-related genes and that methionine supplementation is necessary to appropriately maintain the levels of IGF-I, GHR, and UCP transcripts for animal metabolism.

Effects of dietary arginine on endocrine growth factors of channel catfish, Ictalurus punctatus

Thyroid (TH) and growth (GH) hormones, and insulin-like growth factor I (IGF-I) are anabolic regulators in fish and responsive to nutrient intake. A study was conducted to determine if previously reported growth effects of dietary arginine (ARG) in channel catfish were related to the activation of endocrine axes. In a first experiment, catfish were fed incremental levels of ARG (0.5 – 4% of diet) for 6weeks and sampled at 2-week intervals. In a second experiment, fasted (48h) fish were fed a single ration of ARG (0.5 or 4% of diet) and sampled at various intervals (0 to 72h postprandial, PP). Experiment 1 did not reveal any influence of ARG on circulating TH, GH, or IGF-I despite the significantly increased growth of fish fed ARG-enriched diets. In experiment 2, feeding the 4% ARG diet significantly increased the amplitude of pulsatile plasma GH levels and also significantly increased IGF-I mRNA in liver and muscle, (at 2h PP) and plasma IGF-I levels (at 6h PP). Although relatively infrequent sampling failed to reveal alterations in TH or GH levels in response to ARG-induced growth activation, PP high frequency sampling unveiled high amplitude pulsatile GH secretions and may be important in activating IGF production in target tissues. Additionally, expressed and secreted IGF-I exhibited discernible patterns which closely correlate with ARG-induced growth effects in catfish.

Molecular cloning, expression analysis of insulin-like growth factor I (IGF-I) gene and IGF-I serum concentration in female and male Tongue sole (Cynoglossus semilaevis)

Insulin-like growth factor I (IGF-I) is a polypeptide hormone that regulates growth during all stages of development in vertebrates. To examine the mechanisms of the sexual growth dimorphism in the Tongue sole (Cynoglossus semilaevis), molecular cloning, expression analysis of IGF-I gene and IGF-I serum concentration analysis were performed. As a result, the IGF-I cDNA sequence is 911 bp, which contains an open reading frame (ORF) of 564 bp encoding a protein of 187 amino acids. The sex-specific tissue expression was analyzed by using 14 tissues from females, normal males and extra-large male adults. The IGF-I mRNA was predominantly expressed in liver, and the IGF-I expression levels in females and extra-large males were 1.9 and 10.2 times as much as those in normal males, respectively. Sex differences in IGF-I mRNA expressions at early life stages were also examined by using a full-sib family of C. semilaevis, and the IGF-I mRNA was detected at all of the 27 sampling points from 10 to 410 days old. An increase in IGF-I mRNA was detected after 190 day old fish. The significantly higher levels of IGF-I mRNA in females were observed after 190 days old in comparison with males (P<0.01). The IGF-I concentrations in serum of mature individuals were detected by ELISA. The IGF-I level in the serum of females was approximately two times as much as that of males. Consequently, IGF-I may play an important role in the endocrine regulation of the sexually dimorphic growth of C. semilaevis.

Resveratrol improves cognitive function in mice by increasing production of insulin-like growth factor-I in the hippocampus

We examined whether resveratrol increases insulin-like growth factor-I (IGF-I) production in the hippocampus by stimulating sensory neurons in the gastrointestinal tract, thereby improving cognitive function in mice. Resveratrol increased calcitonin gene-related peptide (CGRP) release from dorsal root ganglion (DRG) neurons isolated from wild-type (WT) mice. Increases in tissue levels of CGRP, IGF-I, and IGF-I mRNA and immunohistochemical expression of IGF-I were observed in the hippocampus at 3 weeks after oral administration of resveratrol in WT mice. Significant enhancement of angiogenesis and neurogenesis was observed in the dentate gyrus of the hippocampus in these animals ( P<.01). Improvement of spatial learning in the Morris water maze was observed in WT mice after administration of resveratrol. None of the effects of resveratrol observed in WT mice were seen after resveratrol administration in CGRP-knockout (CGRP −/−) mice. Although red wine containing 20 mg/L of resveratrol produced effects similar to those of resveratrol administrationl in WT mice, neither red wine containing 3.1 mg/L of resveratrol nor white wine exhibited such effects in WT mice. Resveratrol was undetectable in the hippocampus of WT mice administered resveratrol and red wine containing 20 mg/L of resveratrol. These observations strongly suggest that resveratrol increases hippocampal IGF-I production via sensory neuron stimulation in the gastrointestinal tract, thereby improving cognitive function in mice.

Impact of viral-mediated IGF-I gene transfer on skeletal muscle following cast immobilization.

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that plays a critical role in muscle regeneration and muscle hypertrophy. The purpose of this study was to evaluate the effect of IGF-I overexpression on the recovery of muscle size and function during reloading/reambulation after a period of cast immobilization in predominantly fast twitch muscles. In addition, we investigated concomitant molecular responses in IGF-I receptor and binding proteins (BPs). Recombinant adeno-associated virus vector for IGF-I (rAAV-IGF-IA) was injected into the anterior compartment of one of the hindlimbs of young (3 wk) C57BL6 female mice. At 20 wk of age, both hindlimbs were cast immobilized in a shortened position for 2 wk to unload the tibialis anterior (TA) and extensor longus digitorum (EDL) muscles. The TA and EDL muscles were removed bilaterally after 2 wk of cast immobilization and after 1 and 3 wk of free cage reambulation. Increases in IGF-I mRNA and protein levels with IGF-I overexpression were associated with significant increases in muscle wet weight, fiber size, and tetanic force, although overexpression did not protect against cast immobilization-induced muscle atrophy. After 1 wk of reambulation, evidence of enhanced muscle regeneration was noted in IGF-I-overexpressing muscles with an increased prevalence of central nuclei, embryonic myosin, and Pax7 positive fibers. We also observed larger relative gains in muscle size (wet weight and fiber area), but not force, during the 3-wk reambulation period in hindlimb muscles overexpressing IGF-I compared with contralateral control legs. Changes in IGFBP-5 mRNA expression during cast immobilization and reambulation paralleled those of IGF-I, whereas IGFBP-3 expression changed inversely to IGFBP-5.

Dietary isoflavone increases insulin-like growth factor-I production, thereby promoting hair growth in mice

Sensory neurons release calcitonin gene-related peptide (CGRP) upon activation. We previously demonstrated that CGRP increases insulin-like growth factor-I (IGF-I) production in various tissues of mice including the skin. We demonstrated that isoflavone increases the CGRP synthesis in the dorsal root ganglion (DRG) neurons in rats. Since IGF-I plays a critical role in hair growth, we hypothesized that isoflavones may promote hair growth by increasing the IGF-I production in hair follicles. We examined this hypothesis using wild-type (WT) and CGRP-knockout (CGRP −/−) mice. Isoflavone significantly increased the CGRP mRNA levels in DRG neurons isolated from WT mice ( P<.01). Administration of isoflavone for 3 weeks increased the dermal levels of CGRP, IGF-I and IGF-I mRNA in WT mice, but not in CGRP −/− mice. Isoflavone administration increased the immunohistochemical expression of IGF-I in hair follicle dermal papilla cells in WT mice. Significant enhancements of hair follicle morphogenesis, hair regrowth, and hair pigmentation were also observed in WT mice administered isoflavone. However, none of these effects in WT mice were observed in CGRP −/− mice. These observations strongly suggest that isoflavone might increase IGF-I production in the hair follicle dermal papilla cells in mice through increasing CGRP production in the sensory neurons, thereby promoting hair growth associated with melanogenesis in mice.

Serum IGF-I-Deficiency Does Not Prevent Compensatory Skeletal Muscle Hypertrophy in Resistance Exercise

The involvement of circulating insulin-like growth factor-I (IGF-I) in the skeletal muscle response to resistance exercise is currently unclear. To address this, we utilized the liver IGF-I-deficient (LID) mouse model, in which the igf1 gene has been disrupted in the hepatocytes, resulting in ~80% reduction in serum IGF-I. Twelve- to 13-month-old male LID and control (L/L) mice were subjected to 16 weeks of resistance training. Resistance exercise resulted in equal strength gains in both L/L and LID mice. Basal IGF-I mRNA levels were greater in LID muscles than in L/L, and exercise increased IGF-I mRNA in quadriceps, gastrocnemius, and plantaris muscles. LID mice had elevated tyrosine phosphorylation of IGF-IR and Stat5b, the latter possibly reflective of increased serum GH. Tyrosine phosphorylation of IGF-IR was increased, while phospho-Stat5b was reduced after resistance training of both wild-type and LID mice. These data suggest that: 1) performance and recovery in response to resistance training is normal even when there is severe deficiency of circulating IGF-I; and 2) upregulation of local IGF-I may be involved in the compensatory growth of muscle that occurs in response to resistance training. Decreased levels of p-Stat5b in exercised mice suggests that the upregulation of local IGF-I gene expression in response to exercise may be GH-independent.

RETRACTED: Stimulation of sensory neurons by capsaicin increases tissue levels of IGF-I, thereby reducing reperfusion-induced apoptosis in mice

Calcitonin gene-related peptide (CGRP) increases insulin-like growth factor-I (IGF-I) production in fetal rat osteoblasts in vitro, suggesting that stimulation of sensory neurons might increase IGF-I production, thereby preventing apoptosis. We examined whether stimulation of sensory neurons by capsaicin might reduce reperfusion-induced hepatic apoptosis by increasing IGF-I production. Administration of capsaicin increased tissue levels of IGF-I and IGF-I mRNA in various organs in wild-type (WT) mice, but not in CGRP-knock-out (CGRP-/-) mice. Administration of CGRP increased tissue levels of IGF-I and IGF-I mRNA in both WT and CGRP-/- mice. Increases in hepatic tissue levels of TNF, serum levels of transaminases, hepatic apoptosis and hepatic tissue levels of caspase-3 after hepatic ischemia/reperfusion (I/R) were more marked in CGRP-/- mice than in WT mice. Hepatic IGF-I levels were increased in WT mice after reperfusion, while they were not changed in CGRP-/- mice. Although administration of capsaicin enhanced increases in IGF-I levels and reduced reperfusion-induced events in WT mice, it had no effect in CGRP-/- mice. Administration of CGRP and IGF-I reduced reperfusion-induced effects in both strains of mice. These observations suggested that capsaicin-induced sensory neuron activation, which leads to release of CGRP, might increase IGF-I production, thereby reducing reperfusion-induced liver injury by reducing apoptosis.

IGF‐I response to tourniquet‐induced injury in young and old mice

The age-associated decline in skeletal muscle’s ability to regenerate is a concern among elderly patients of limb injury or orthopedic surgery. Changes in insulin-like growth factor-I (IGF-I) response have been implicated in this phenomenon. Previously, our laboratory has shown a drastic increase in IGF-I mRNA 7 days following tourniquet (TK) induced damage in adult rats, a response not seen in elderly rats. This study investigated the short-term time course of IGF-I expression in adult (6 month-old) and elderly (24 month-old) mice following 1, 3, 5, and 7-day recovery from 2 hour TK induced ischemia/reperfusion injury. RT-PCR analysis reveals an increase in IGF-I expression in both TK groups. Levels peaked ~20-fold 3 days post-injury in adult and ~8-fold 5 days post-injury in elderly subjects. Adult expression decreased sharply at day 5 and rose by day 7, suggesting the differential, temporal-specific expression of the IGF-I Ea and Eb isoforms in injury repair described by Goldspink et al. The early IGF-I response seen in adult subjects was not present in elderly subjects.

Metabolic hormones modulate the effect of growth hormone (GH) on insulin-like growth factor-I (IGF-I) mRNA level in primary culture of salmon hepatocytes

Liver production of insulin-like growth factor-I (IGF-I) is a major point of control in the growth hormone (GH)/IGF axis, the endocrine system regulating body growth in fishes and other vertebrates. Pituitary GH stimulates hepatocyte production of IGF-I; however, in catabolic states, hepatocyte GH resistance results in decreases in liver IGF-I production. To investigate endocrine mechanisms leading to the development of hepatocyte GH resistance, we examined the regulation of IGF-I mRNA level by GH and metabolic hormones in primary culture of salmon hepatocytes. Cells were cultured in RPMI medium, and exposed to insulin (Ins, 10(-6) M), glucagon (Glu, 10(-6) M), triiodothyronine (T3, 10(-7) M), dexamethasone (Dex, 10(-6) M) and glucagon-like peptide (GLP, 10(-6) M), in the presence and absence of GH (5 x 10(-9) M). GH always increased IGF-I mRNA. None of the other hormones tested alone affected IGF-I mRNA. However, Dex, Ins and Glu reduced the response to GH. The response to GH was inhibited by Dex at concentrations of 10(-12) M and above, by Ins at 10(-9) M and above, and by Glu only at 10(-6) M. Inhibition of GH response by glucocorticoids is found in other vertebrates. Salmon hepatocytes were very sensitive to Dex, suggesting that glucocorticoids may play an important role in salmon growth regulation even in unstressed conditions. Inhibition of GH response by Ins is the opposite of what is found in mammals and chickens, suggesting that the role of Ins in growth regulation may differ between fishes and tetrapods. To examine mechanisms for modulation of GH sensitivity, we measured hepatocyte GH receptor (GHR) mRNA levels. Ins inhibited and Dex stimulated GHR mRNA, suggesting that different mechanisms mediate the inhibition of GH response by these hormones. This study shows that glucocorticoids, Ins, and Glu induce GH resistance in cultured salmon hepatocytes.

Insulin-Like Growth Factor-I Stimulated DNA Replication in Mouse Endometrial Stromal Cells

Much evidence has suggested that sex steroid hormone-induced growth of uterine cells is mediated by polypeptide growth factors synthesized in uterine tissues. The present study aimed to clarify the effect of insulin-like growth factor-I (IGF-I) on the proliferation of mouse endometrial stromal cells obtained from immature mice. IGF-I and IGF-I receptor (type I) mRNAs were detected in the endometrial stromal cells. IGF-I increased bromodeoxyuridine (BrdU) uptake in the endometrial stromal cells, indicating an increase in DNA replication. E2 increased IGF-I mRNA levels in the endometrial stromal cells. IGF-I receptor is a tyrosine kinase receptor, and treatment with genistein, a tyrosine kinase inhibitor, reduced IGF-I-induced BrdU-uptake in the endometrial stromal cells. IGF-I signaling pathways involve mitogen-activated protein (MAP) kinase and phosphatidylinositol-3 kinase (PI-3 kinase). Treatment with 10(-7) M of the MAP kinase inhibitor PD098059 and 10(-5) M of the PI-3 kinase inhibitor LY294002 decreased IGF-I-induced BrdU-uptake in the endometrial stromal cells. However, LY294002 (10(-5) M) also decreased the BrdU-uptake in the absence of IGF-I treatment. These results suggest that endometrial IGF-I is involved in the proliferation of endometrial stromal cells in a paracrine or autocrine manner, and that the MAP kinase pathway is involved in DNA replication of endometrial stromal cells.

Uterine androgen receptors: roles in estrogen-mediated gene expressionand DNA synthesis.

The localization of androgen receptors (AR) and their ligands in the uterine microenvironment at early pregnancy suggest a role for AR in uterine physiology. We have evaluated AR expression in the pig uterine endometrium and examined whether AR ligands modulate peri-implantation uterine gene expression. Northern blot analysis demonstrated the approximately 10.5 kilobase AR transcript in endometrium. Endometrial levels of AR mRNA and protein were greater at early than at mid- or late pregnancy. Estrogen receptor-alpha mRNA levels showed similar maximal expression at early pregnancy. Immunocytochemical analysis of endometrium at early pregnancy localized AR to nuclei of glandular epithelial (GE) and stromal (ST) cells. To evaluate a role for AR in uterine gene regulation, the levels of mRNAs for insulin-like growth factor-I (IGF-I), proliferative cell nuclear antigen (PCNA), and AR itself were assessed in uterine endometrial explant cultures treated with estradiol-17beta (E), testosterone (T), and 19-nortestosterone (N). Induction by E of AR mRNA abundance occurred in endometrium from Day 10 but not from Day 12 pregnant animals and this was partially blocked by coaddition of N or T, although neither androgen alone had any effect. Abundance of IGF-I and PCNA mRNAs was increased by E and inhibited by coaddition of either T or N in Day 10 pregnant pig endometrium. In endometrium from Day 12 pregnant animals, addition of either N or T with E increased IGF-I mRNA levels over that of controls, although E alone was without effect. In contrast, PCNA mRNA abundance was suppressed by all steroid treatments in these explants. DNA synthesis in primary cultures of GE cells from endometrium at Days 10 and 12 of pregnancy was increased by E and was suppressed by T, the latter only at Day 12. E did not affect DNA synthesis in ST cells from endometrium at either pregnancy day, although T inhibited this process in an E-dependent manner in ST cells from pregnancy Day 12. Results identify AR in the pig endometrium during the window of maternal receptivity for implantation and demonstrate the functional, albeit complex, interactions of androgens and estrogens in the regulation of uterine endometrial gene expression and cell growth in vitro. Further elucidation of the role of androgens and their receptor in early pregnancy events may be relevant to an understanding of peri-implantation embryo loss.

IGF‐I and microglia/macrophage proliferation in the ischemic mouse brain

We have used a model of hypoxic-ischemic brain injury in adult male C57BL/6 mice to study insulin-like growth factor-I (IGF-I) and IGF-binding protein (IGFBP) expression in response to cerebral hypoxia-ischemia (H/I) in the adult mouse. A period of 20 min of H/I that resulted in histopathology in cortex, striatum, and thalamus was correlated with induction of mRNA for IGF-I, IGFBP-2, IGFBP-3, IGFBP-5, and glial fibrillary acidic protein (GFAP) by 4 days of recovery. Increased IGF-I mRNA was located within damaged regions and was surrounded by IGFBP-2 mRNA expression. The results of combined immunostaining/in situ hybridzation showed that the cells expressing IGFBP-2 mRNA were also GFAP-positive and comprised a subset of activated astrocytes immediately surrounding areas of damage. In contrast, staining within damaged regions showed high numbers of cells immunopositive for F4/80 and lectin B(4) indicative of microglia and macrophages but no cells immunopositive for the astrocytic proteins GFAP or S-100beta. Microglia/macrophages within the damaged areas expressed IGF-I mRNA and were also immunopositive for the proliferating cell nuclear antigen. To determine whether expression of IGF-I could contribute to proliferation of microglia, we treated purified cultures of adult brain microglia with IGF-I in the presence of (3)H-thymidine. IGF-I stimulated a twofold increase in DNA synthesis in cultures of adult brain microglia. Taken together with previous data demonstrating that IGF-I promotes proliferation of peripheral macrophages, these data support the hypothesis that IGF-I is an autocrine/paracrine mitogen for microglia/macrophages after H/I.

Hormonal Control of Insulin-like Growth Factor I Gene Transcription in Human Osteoblasts: DUAL ACTIONS OF cAMP-DEPENDENT PROTEIN KINASE ON CCAAT/ENHANCER-BINDING PROTEIN δ

Insulin-like growth factor-I (IGF-I) is essential for somatic growth and promotes bone cell replication and differentiation. IGF-I production by rat osteoblasts is stimulated by activation of cAMP-dependent protein kinase (PKA). In this report, we define two interacting PKA-regulated pathways that control IGF-I gene transcription in cultured human osteoblasts. Stimulation of cAMP led to a 12-fold increase in IGF-I mRNA and enhanced IGF-I promoter activity through a DNA response element termed HS3D and the transcription factor CCAAT/enhancer-binding protein delta (C/EBPdelta). Under basal conditions, C/EBPdelta was found in osteoblast nuclei but was transcriptionally silent. Treatment with the PKA inhibitor H-89 caused redistribution of C/EBPdelta to the cytoplasm. After hormone treatment, the catalytic subunit of PKA accumulated in osteoblast nuclei. Inhibition of active PKA with targeted nuclear expression of PKA inhibitor had no effect on the subcellular location of C/EBPdelta but prevented hormone-induced IGF-I gene activation, while cytoplasmic PKA inhibitor additionally caused the removal of C/EBPdelta from the nucleus. These results show that IGF-I gene expression is controlled in human osteoblasts by two PKA-dependent pathways. Cytoplasmic PKA mediates nuclear localization of C/EBPdelta under basal conditions, and nuclear PKA stimulates its transcriptional activity upon hormone treatment. Both mechanisms are indirect, since PKA failed to phosphorylate human C/EBPdelta in vitro.

Mammalian follicle-stimulating hormone and insulin-like growth factor I (IGF-I) up-regulate IGF-I gene expression in organ culture of newt testis.

We previously showed that porcine follicle-stimulating hormone (pFSH) and human recombinant insulin-like growth factor (rhIGF-I) promote the differentiation of secondary spermatogonia into primary spermatocytes in organ cultures of newt testes, respectively. To elucidate the molecular action of FSH and IGF-I, we cloned cDNAs for newt IGF-I and IGF-I receptor (IGF-IR), and examined their mRNA expression in organ culture during newt spermatogenesis. Northern blot and reverse transcription-polymerase chain reaction (RT-PCR) analyses revealed that IGF-I mRNA was highly expressed in somatic cells (mostly Sertoli cells) at the secondary spermatogonial stage but barely in germ cells, and that IGF-IR mRNA was expressed in both germ and somatic cells at all stages examined. The addition of pFSH to newt testis markedly increased IGF-I mRNA expression. Also, rhIGF-I increased IGF-I mRNA expression, whereas IGF-IR mRNA expression declined slightly. These results suggest that the ability of FSH to promote the differentiation of secondary spermatogonia is at least partly mediated by somatic cell-derived IGF-I, and that IGF-I mRNA expression in somatic cells is auto-upregulated.

Direct adenovirus-mediated insulin-like growth factor I gene transfer enhances transplant chondrocyte function.

Cell-based cartilage-resurfacing procedures may be enhanced by the addition of insulin-like growth factor I (IGF-I) to the transplant biomatrix. Given the relatively short half-life of IGF-I in biological systems, however, maintenance of effective concentrations of this peptide necessitates either high initial doses, or repeated treatment. This study investigated IGF-I delivery via adenoviral gene therapy, targeting graftable articular chondrocytes. Cultured articular chondrocytes were infected with an E1-deleted adenoviral vector containing IGF-I-coding sequence under CMV promoter control. Increased adenovirus-IGF-I concentrations resulted in coordinate increase in IGF-I mRNA and ligand expression; however, chondrocyte matrix synthesis was maximized by the lower adenovirus-IGF-I concentration (100 MOI) without additional increase at 200 or 500 MOI. Using 100 MOI, infected monolayers produced medium IGF-I content of at least 10 ng/ml in each 48-hr period for 28 days, reaching a day 4 peak concentration of 66 +/- 4.0 ng/ml. These concentrations were sufficient to produce significant stimulation of normal cartilage matrix gene expression. The concentration of secreted matrix products in medium from infected monolayers was increased up to 8-fold over uninfected control cultures. Moreover, compared with uninfected cultures, cells in infected cultures were more resistant to de-differentiation over time under serum-starved conditions, maintaining a normal chondrocyte molecular phenotype for at least 28 days. These data indicate that cultured chondrocytes are readily transduced by recombinant adenoviral vectors. The adenoviral-IGF transgene is abundantly expressed and its product secreted at therapeutic concentrations for at least 28 days, resulting in increased matrix biosynthesis and maintenance of the chondrocytic phenotype. Combined, this information suggests that there may be significant value in preimplantation adenoviral-IGF gene therapy for chondrocytes destined for cartilage resurfacing.

Effect of growth hormone and insulin-like growth factor I (IGF-I) on the expression of IGF-I messenger ribonucleic acid and peptide in rat tibial growth plate and articular chondrocytes in vivo.

Conflicting data exist as to whether insulin-like growth factor I (IGF-I) messenger RNA (mRNA) and peptide are expressed within chondrocytes. This question is pertinent to the mode of GH action on longitudinal bone growth. We have, therefore, investigated this issue in normal rats and in hypophysectomized rats treated for 24 h with GH or IGF-I using in situ hybridization and immunohistochemistry. Serum IGF-I, body weight, and tibial growth plate, but not articular cartilage, height increased with both treatments. Both IGF-I mRNA and IGF-I immunoreactivity occurred in all chondrocyte layers of growth plate and articular cartilage. The percentage of cells with IGF-I mRNA correlated well with IGF-I immunoreactivity under all experimental conditions. In normal rats, IGF-I expression was highest in the upper hypertrophic zone in growth plate (68-71%) and articular cartilage (32-34%). Hypophysectomy, GH, or IGF-I did not significantly affect this percentage. In the stem cell and proliferative and lower hypertrophic zones of growth plate, hypophysectomy dramatically reduced the percentage of labeled chondrocytes, and GH restored it. IGF-I increased IGF-I mRNA and immunoreactivity only in the proliferative zone. In articular cartilage, both remained unchanged under all experimental conditions. Together with our previous finding that GH infusion of hypophysectomized rats enhances chondrocyte maturation at all differentiation stages, the present results are compatible with the idea that IGF-I produced by all chondrocyte layers under the influence of GH mediates chondrocyte maturation and thus longitudinal bone growth in an autocrine/paracrine manner.

Glycosaminoglycans co-administration enhance insulin-like growth factor-I neuroprotective and neuroregenerative activity in traumatic and genetic models of motor neuron disease: a review

In this report it is shown how glycosaminoglycans and insulin-like growth factor-I (IGF-I) promote muscle reinnervation and prevent motor neuron death in experimental models of motor neuron disease. Such effect appears to be mediated by insulin-like growth factor-1. The glycosaminoglycan moiety of proteoglycans is a constituent of the basal lamina active on nerve regeneration by means of the interaction with laminin and with several growth factors. We have previously shown that supplementation by means of subcutaneous injections of glycosaminoglycans affects neuronal degeneration and regeneration. In this study we report that following neonatal lesion of the rat sciatic nerve, glycosaminoglycan treatment promoted extensor digitorum longus muscle reinnervation with consequent improvement of muscle morphology. In saline-treated rats, reinnervation was only partial and there was a marked muscle fibre atrophy, whereas, glycosaminoglycan treatment of lesioned rats increased IGF-I mRNA and protein in the reinnervated muscle, and IGF-I and insulin-like growth factor binding protein-3 plasma levels. Similarly, treatment of lesioned rats with IGF-I promoted muscle reinnervation, and prevented muscle fibre atrophy, higher levels of IGF-I in the reinnervated muscle, of IGF-I, and insulin-like growth factor binding proteins in plasma. In the wobbler mouse IGF-I and glycosaminoglycans alone promote only a partial motor neuron survival and the preservation of forelimb function decays after 3 weeks of treatment. However when glycosaminoglycans and insulin-like growth factor are administered together the motor neuron disease in the wobbler mouse is halted and there is no more loss of motor neurons.

Nandrolone decanoate does not enhance training effects but increases IGF-I mRNA in rat diaphragm.

To examine whether concomitant anabolic steroid treatment combined with training might enhance previously observed training effects (A. Bisschop, G. Gayan-Ramirez, H. Rollier, R. Gosselink, R. Dom, V. de Bock, and M. Decramer. Am. J. Respir. Crit. Care Med. 155: 1583-1589, 1997) and whether insulin-like growth factor I (IGF-I) was involved in these changes, male and female rats were submitted to inspiratory muscle training (IMT) for 8 wk (30 min/day, 5 times/wk) and were compared with untrained controls. During the last 5 wk of training, trained rats were divided to receive weekly either low-dose (LD; 1.5 mg/kg) or high-dose (HD; 7.5 mg/kg) nandrolone decanoate or saline for the IMT and control rats. In both sexes, diaphragm muscle mass and contractile properties were unchanged with treatment. In males, HD resulted in decreased diaphragm type I cross-sectional area (-15%; P < 0.05, HD vs. IMT), whereas no changes were observed in females. Finally, an increase in IGF-I mRNA levels was present in HD male (+73%; P < 0.05, HD vs. IMT) and female treated rats [LD (+58%) and HD (+96%) vs. IMT; P < 0.001]. We conclude that administration of nandrolone decanoate did not enhance the previously observed training effects in rat diaphragm, although it increased the IGF-I mRNA expression levels.