Insulin-like growth factor-1 contributes to neovascularization in age-related macular degeneration

Choroidal neovascularization (CNV) is a debilitating complication of age-related macular degeneration and a leading cause of vision loss. Along with other angiogenic factors like vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF)-1 and its receptor, IGF-1R, have been implicated in CNV. IGF-1 is produced in neurons and retinal pigment epithelium (RPE) but its targets and impact in CNV are not understood. IGF-1 immunoreactivity was abundant throughout surgically isolated human CNV tissues and RPE cells were immunopositive for IGF-1R. Cultured RPE cells obtained from CNV tissues expressed IGF-1R. IGF-1 stimulation of cultured cells from CNV tissues induced monophasic sustained rises in intracellular free Ca(2+). VEGF concentration in the medium of unstimulated RPE cell cultures from CNV tissues increased with time to a steady-state (8h) which was increased twofold by IGF-1 stimulation. Thus, in RPE cells IGF-1 stimulates the second messenger Ca(2+) and increases VEGF secretion which, in turn, induces neovascularization.     

Induction of autocrine epidermal growth factor receptor ligands in human keratinocytes by insulin/insulin-like growth factor-1

Autocrine activation of the epidermal growth factor (EGF) receptor on keratinocytes has been recognized as an important growth regulatory mechanism involved in epithelial homeostasis, and, possibly, hyperproliferative diseases. Insulin-like growth factor (IGF)-1 and insulin have been shown to be paracrine keratinocyte mitogens that bind to the type I IGF receptor which is expressed on actively proliferating keratinocytes in situ. In this report, we demonstrate that IGF-1/insulin induced production of keratinocyte-derived autocrine growth factors that bind to the EGF receptor. Increased steady-state mRNA levels for transforming growth factor alpha (TGF-α) and for amphiregulin (AR) were observed upon incubation of keratinocytes with mitogenic concentrations of IGF-1. IGF-1 also induced production and secretion of TGF-α and AR proteins as detected by immunoassays. An EGF receptor antagonistic monoclonal antibody abolished the mitogenic effect of IGF-1 on cultured keratinocytes. These results suggest that stimulation of keratinocyte growth by IGF-1 requires activation of an EGF receptor-mediated autocrine loop. © 1995 Wiley-Liss, Inc.     

Intraocular gutless adenoviral-vectored VEGF stimulates anterior segment but not retinal neovascularization

Vascular endothelial growth factor (VEGF) and insulin-like growth factor-1 (IGF-1) have been implicated as important stimulatory factors for retinal neovascularization. In this study, we used intraocular gene transfer with gutless adenoviral (AGV) vectors to determine the effect of increased intraocular expression of VEGF, IGF-1, or sphingosine kinase (SPK), which produces sphingosine-1-phosphate, another angiogenic factor. Retinal neovascularization did not occur from intravitreous AGV-vectored VEGF, IGF-1, SPK, or combined VEGF and IGF-1, except occasionally adjacent to the retinal penetration site from the injection. However, corneal and iris neovascularization occurred after 2 weeks in all eyes injected with AGV.VEGF, but not those injected with only AGV.IGF-1 or AGV.SPK. These data suggest that the superficial capillary bed of the retina is relatively insensitive to VEGF, IGF-1, or SPK in adult mice, except when combined with retinal trauma. However, AGV-vectored VEGF is sufficient to consistently cause severe corneal and iris neovascularization. This provides a model for anterior segment neovascularization, which unlike previous models is relatively inexpensive and is not plagued by spontaneous regression, and therefore, may be useful for identification of new treatments.     

Short-term insulin treatment and aortic expressions of IGF-1 receptor and VEGF mRNA in diabetic rats

We investigated the relationship between the changes in vascular responsiveness and growth factor mRNA expressions induced by 1-wk treatment with high-dose insulin in control and established streptozotocin (STZ)-induced diabetes. Aortas from diabetic rats, but not those from insulin-treated diabetic rats, showed impaired endothelium-dependent relaxation in response to ACh (vs. untreated controls). The ACh-induced nitrite plus nitrate (NOx) level showed no significant difference between controls and diabetics. Insulin treatment increased NOx only in diabetics. In diabetics, insulin treatment significantly increased the aortic expressions of endothelial nitric oxide synthase (eNOS) mRNA and VEGF mRNA. The expression of IGF-1 mRNA was unaffected by diabetes or by insulin treatment. In contrast, the mRNA for the aortic IGF-1 receptor was increased in diabetics and further increased in insulin-treated diabetics. In aortic strips from age-matched control rats, IGF-1 caused a concentration-dependent relaxation. This relaxation was significantly stronger in strips from STZ-induced diabetic rats. These results suggest that in STZ-diabetic rats, short-term insulin treatment can ameliorate endothelial dysfunction by inducing overexpression of eNOS and/or VEGF mRNAs possibly via IGF-1 receptors. These receptors were increased in diabetes, perhaps as result of insulin deficiency.     

AMPK activation inhibits the expression of HIF-1alpha induced by insulin and IGF-1

Insulin, insulin like growth factor (IGF)-1, and AMP-activated protein kinase (AMPK) signaling regulate independently angiogenesis through vascular endothelial growth factor (VEGF) expression. In the present study, we investigated a potential cross-talk between these signaling pathways on hypoxia-inducible factor (HIF)-1alpha and VEGF expression. Retinal epithelial ARPE-19 cells were treated with AICAR, an AMPK activator, alone or in combination with insulin and IGF-1. AICAR stimulated VEGF mRNA expression, but did not modify the insulin- and IGF-1-induced VEGF expression. We have investigated the effect of AICAR on insulin and IGF-1 signaling pathways. We observed that AICAR increased insulin- and IGF-1-induced phosphorylation of PKB, whereas phosphorylation of S6K-1 was decreased. Moreover, AICAR and metformin inhibited the ability of insulin and IGF-1 to induce HIF-1alpha expression. These results show that AICAR and insulin/IGF-1 regulate VEGF expression through different mechanisms.     

Insulin and β-adrenergic Receptors Inhibit Retinal Endothelial Cell Apoptosis Through Independent Pathways

Diabetic retinopathy results from altered insulin receptor signaling. Based on previous studies demonstrating an interaction between β-adrenergic receptors and insulin signaling in hyperglycemic conditions, we hypothesized that β-adrenergic receptor stimulation and insulin stimulation would act synergistically to inhibit one of the hallmarks of diabetic retinopathy, namely retinal endothelial cell apoptosis. To test this hypothesis, human retinal endothelial cells were grown in high glucose (25 mM) medium and treated with a β-1-adrenergic receptor agonist (xamoterol, 10 μM) alone, insulin alone (10 nM) or xamoterol + insulin. We then assessed changes in the levels of insulin receptor, insulin-like growth factor (IGF-1) receptor, and Akt phosphorylation, as well as cleaved caspase 3. Xamoterol alone significantly decreased insulin receptor, IGF-1 receptor and Akt phosphorylation, whereas insulin alone increased insulin receptor, IGF-1 receptor, and Akt phosphorylation. Xamoterol significantly decreased apoptosis of retinal endothelial cells. This data suggests that both β-adrenergic receptors and insulin can inhibit retinal endothelial cell apoptosis in hyperglycemic conditions, but inhibition occurs through independent pathways. These findings have implications for treatments of diabetic retinopathy.     

Inhibition of protein kinase C decreases prostaglandin-induced breakdown of the blood-retinal barrier

Breakdown of the blood-retinal barrier (BRB) occurs in several retinal diseases and is a major cause of visual loss. Vascular endothelial growth factor (VEGF) has been implicated as a cause of BRB breakdown in diabetic retinopathy and other ischemic retinopathies, and there is evidence to suggest that other vasopermeability factors may act indirectly through VEGF. In this study, we investigated the effect of several receptor kinase inhibitors on BRB breakdown resulting from VEGF, tumor necrosis factor-alpha (TNF-alpha), interleukin-1beta (IL-1beta), insulin-like growth factor-1 (IGF-1), prostaglandin E1 (PGE(1)), or PGE(2). Inhibitors of VEGF receptor kinase, including PKC412, PTK787, and SU1498, decreased VEGF-induced breakdown of the BRB. None of the inhibitors blocked leakage caused by TNF-alpha, IL-1beta, or IGF-1 and only PKC412, an inhibitor of protein kinase C (PKC) as well as VEGF and platelet-derived growth factor (PDGF) receptor kinases, decreased leakage caused by prostaglandins. Since the other inhibitors of VEGF and/or PDGF receptor kinases that do not also inhibit PKC had no effect on prostaglandin-induced breakdown of the BRB, these data implicate PKC in retinal vascular leakage caused by prostaglandins. PKC412 may be useful for treatment of post-operative and inflammatory macular edema, in which prostaglandins play a role, as well as macular edema associated with ischemic retinopathies.     

Free-ligand accelerated dissociation of insulin-like growth factor 1 (IGF-1) from the type I IGF receptor is reduced by insulin-like growth factor binding protein 3

Insulin-like growth factor binding proteins (IGFBP) can inhibit or accentuate the mitogenic activities of insulin-like growth factor 1 (IGF-1) depending upon the experimental model employed. Inhibitory effects may be attributed to sequestration of IGF-1 onto IGFBP rather than the type I IGF receptor. We have demonstrated that the presence of IGFBP in a simple equilibrium binding assay significantly reduces the total amount of IGF-1 bound to the type I IGF receptor and increases the IC₅₀ for IGF-1 binding. On the basis of such an experiment, performed at equilibrium, IGFBP should reduce the mitogenic activity of IGF-1. Recent work has demonstrated an inverse correlation between the dissociation rate of insulin-like molecules from their receptors and their mitogenic activity. It has also been suggested that the increased rate of dissociation of insulin and IGF-1 from their receptors at increased ligand concentrations serves as a ‘dampening’ mechanism to decrease mitogenic signalling. We have demonstrated increased rates of dissociation of IGF-1 from the type I IGF receptor with increasing concentrations of IGF-1. Furthermore, IGFBP-3 inhibits the acceleration of dissociation rates due to increased IGF-1 levels. Thus, under receptor saturating conditions IGFBP-3 may act to increase mitogenesis by increasing the residence time of individual molecules of IGF-1 upon the type I IGF receptor.     

Insulin, insulin analogues and diabetic retinopathy

Insulin is absolutely vital for living beings. It is not only involved in metabolism, but also in the regulation of growth factors, e.g. IGF-1. In this review we address the role insulin has in the natural evolution of diabetic retinopathy. On the one hand, chronic deficiency of insulin and IGF-1 at the retina is thought to cause capillary degeneration, with subsequent ischaemia. On the other hand, acute abundance of (exogenously administered) insulin and IGF-1 enhances ischaemia-induced VEGF expression. A critical ratio of tissue VEGF-susceptibility: VEGF-availability triggers vascular proliferation (i.e. of micro-aneurysms and/or abnormal vessels). The patent-protected insulin analogues Lispro, Glulisine, Aspart, Glargine and Detemir are artificial insulin derivatives with altered biological responses compared to natural insulin (e.g. divergent insulin and /or IGF-1 receptor-binding characteristics, signalling patterns, and mitogenicity). Their safety profiles concerning diabetic retinopathy remain to be established by randomised controlled trials. Anecdotal reports and circumstantial evidence suggest that Lispro and Glargine might worsen diabetic retinopathy.     

Inhibition of vascular endothelial growth factor-induced retinal neovascularization by retinoic acid in experimental retinopathy of prematurity

Vascular endothelial growth factor (VEGF) has an important role in the pathogenesis of retinopathy of prematurity (ROP) and inhibition of VEGF expression in the neovascular phase might prevent destructive neovascularization in ROP. It is suggested that retinoids exert a highly potent antiangiogenic activity by inhibiting VEGF expression. The aim of this study was to demonstrate the preventive effect of retinoic acid (RA) on the VEGF-induced retinal neovascularization in a rat model of ROP. Wistar albino rats were placed into incubators at birth and exposed to an atmosphere alternating between 50 % and 10 % O(2) every 24 hours. After 14 days, the animals were removed to room air and received either an intraperitoneal injection of RA (5 mg/kg/day) (n=9) or saline (n=4) daily for six days, and sacrificed at 21 days. Other rats (n=4) were raised in room air and served as age-matched controls. The globe of each eye was cut through the cornea and embedded in paraffin. Serial sections were stained with hematoxylin-eosin for quantification of neovascular nuclei. The avidin-biotin peroxidase method was performed for evaluation of VEGF expression. The average number of neovascular nuclei was significantly lower in the control group compared to that in the ROP groups. In addition, it significantly decreased in the RA-treated ROP group compared to that of the saline-administrated ROP group. VEGF immunostaining was overall negative in room air-exposed rats. The VEGF immunostaining score significantly decreased in the RA-treated ROP group compared to that in the saline-administered ROP group. RA treatment might be beneficial in preventing neovascularization resulting from oxygen-induced retinopathy by downregulation of VEGF expression.     

Coupling of insulin-responsive glucose transport to receptors for insulin-like growth factor 1 in primary human fibroblasts

We have recently described an insulin-resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine----proline mutation at amino acid position 233 in the alpha-chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2-deoxyglucose uptake by insulin and insulin-like growth factor 1 (IGF-1). Insulin hardly stimulates basal 2-deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2-deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF-1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF-1. With both types of fibroblasts, maximal IGF-1 response is reached at about 10 nM IGF-1, the ED50 being approximately 4 nM. The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF-1. Insulin binds with an approximately 200-fold lower affinity to IGF-1 receptors, compared to homologous IGF-1 binding. As an insulin concentration of 10 microM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF-1, it seems that occupation of IGF-1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.     

Disruption of the insulin-like growth factor type 1 receptor in osteoblasts enhances insulin signaling and action

Defective bone formation is common in patients with diabetes, suggesting that insulin normally exerts anabolic actions in bone. However, because insulin can cross-activate the insulin-like growth factor type 1 receptor (IGF-1R), which also functions in bone, it has been difficult to establish the direct (IGF-1-independent) actions of insulin in osteoblasts. To overcome this problem, we examined insulin signaling and action in primary osteoblasts engineered for conditional disruption of the IGF-1 receptor (DeltaIGF-1R). Calvarial osteoblasts from mice carrying floxed IGF-1R alleles were infected with adenoviral vectors expressing the Cre recombinase (Ad-Cre) or green fluorescent protein (Ad-GFP) as control. Disruption of IGF-1R mRNA (>90%) eliminated IGF-1R without affecting insulin receptor (IR) mRNA and protein expression and eliminated IGF-1R/IR hybrids. In DeltaIGF-1R osteoblasts, insulin signaling was markedly increased as evidenced by increased phosphorylation of insulin receptor substrate 1/2 and enhanced ERK/Akt activation. Microarray analysis of RNA samples from insulin-treated, DeltaIGF-1R osteoblasts revealed striking changes in several genes known to be downstream of ERK including Glut-1 and c-fos. Treatment of osteoblasts with insulin induced Glut-1 mRNA, increased 2-[1,2-(3)H]-deoxy-d-glucose uptake, and enhanced proliferation. Moreover, insulin treatment rescued the defective differentiation and mineralization of DeltaIGF-1R osteoblasts, suggesting that IR signaling can compensate, at least in part, for loss of IGF-1R signaling. We conclude that insulin exerts direct anabolic actions in osteoblasts by activation of its cognate receptor and that the strength of insulin-generated signals is tempered through interactions with the IGF-1R.     

MiR-18b suppresses high-glucose-induced proliferation in HRECs by targeting IGF-1/IGF1R signaling pathways

MicroRNAs (miRNAs) are important for the proliferation of endothelial cells and have been shown to be involved in diabetic retinopathy (DR). In previous study, we found that miRNAs might play a critical role in hyperglycemia-induced endothelial cell proliferation based on miRNA expression profiling. Here, the roles of microRNA-18b (miR-18b) in the proliferation of human retinal endothelial cells (HRECs) were investigated in an in vitro model of HRECs grown in high glucose. We identified that levels of miR-18b were decreased in high-glucose-induced HRECs, compared with those in cells incubated in normal glucose. However, the reduction of miR-18b up-regulated vascular endothelial growth factor (VEGF) secretion and promoted effects on in vitro proliferation of HRECs. Mechanistically, insulin growth factor-1 (IGF-1) was identified as a target of miR-18b. IGF-1 simulation could antagonize the effect induced by miR-18b up-regulation, promoting cell proliferation and increasing VEGF production. In contrast, the opposite results were observed with silencing IGF-1, which was consistent with the effects of miR-18b overexpression. MiR-18b exerted its function on VEGF synthesis and cell proliferation by suppressing the IGF-1/insulin growth factor-1 receptor (IGF1R) pathway, consequently inhibiting the downstream phosphorylation of Akt, MEK, and ERK. Hence, this may provide a new insight into understanding the mechanism of DR pathogenesis, as well as a potential therapeutic target for proliferative DR.     

Angiogenesis is not impaired in connective tissue growth factor (CTGF) knock-out mice.

Connective tissue growth factor (CTGF) is a member of the CCN family of growth factors. CTGF is important in scarring, wound healing, and fibrosis. It has also been implicated to play a role in angiogenesis, in addition to vascular endothelial growth factor (VEGF). In the eye, angiogenesis and subsequent fibrosis are the main causes of blindness in conditions such as diabetic retinopathy. We have applied three different models of angiogenesis to homozygous CTGF(-/-) and heterozygous CTGF(+/-) mice to establish involvement of CTGF in neovascularization. CTGF(-/-) mice die around birth. Therefore, embryonic CTGF(-/-), CTGF(+/-), and CTGF(+/+) bone explants were used to study in vitro angiogenesis, and neonatal and mature CTGF(+/-) and CTGF(+/+) mice were used in models of oxygen-induced retinopathy and laser-induced choroidal neovascularization. Angiogenesis in vitro was independent of the CTGF genotype in both the presence and the absence of VEGF. Oxygen-induced vascular pathology in the retina, as determined semi-quantitatively, and laser-induced choroidal neovascularization, as determined quantitatively, were also not affected by the CTGF genotype. Our data show that downregulation of CTGF levels does not affect neovascularization, indicating distinct roles of VEGF and CTGF in angiogenesis and fibrosis in eye conditions.     

Secretion of Annexin II via activation of insulin receptor and insulin-like growth factor receptor

Annexin II is secreted into the extracellular environment, where, via interactions with specific proteases and extracellular matrix proteins, it participates in plasminogen activation, cell adhesion, and tumor metastasis and invasion. However, mechanisms regulating annexin II transport across the cellular membrane are unknown. In this study, we used coimmunoprecipitation to show that Annexin-II was bound to insulin and insulin-like growth factor-1 (IGF-1) receptors in PC12 cells and NIH-3T3 cells overexpressing insulin (NIH-3T3(IR)) or IGF-1 receptor (NIH-3T3(IGF-1R)). Stimulation of insulin and IGF-1 receptors by insulin caused a temporary dissociation of annexin II from these receptors, which was accompanied by an increased amount of extracellular annexin II detected in the media of PC12, NIH-3T3(IR), and NIH-3T3(IGF-1R) cells but not in that of untransfected NIH-3T3 cells. Activation of a different growth factor receptor, the platelet-derived growth factor receptor, did not produce such results. Tyrphostin AG1024, a tyrosine kinase inhibitor of insulin and IGF-1 receptor, was shown to inhibit annexin II secretion along with reduced receptor phosphorylation. Inhibitors of a few downstream signaling enzymes including phosphatidylinositol 3-kinase, pp60c-Src, and protein kinase C had no effect on insulin-induced annexin II secretion, suggesting a possible direct link between receptor activation and annexin II secretion. Immunocytochemistry revealed that insulin also induced transport of the membrane-bound form of annexin II to the outside layer of the cell membrane and appeared to promote cell aggregation. These results suggest that the insulin receptor and its signaling pathways may participate in molecular mechanisms mediating annexin II secretion.     

Roles of insulinlike growth factor 1 (IGF-1) and the IGF-1 receptor in epidermal growth factor-stimulated growth of 3T3 cells.

BALB/c3T3 cells are exquisitely growth regulated and require platelet-derived growth factor, epidermal growth factor (EGF), and insulinlike growth factor 1 (IGF-1) for growth. When BALB/c3T3 cells are transfected with plasmids constitutively expressing both EGF and the human IGF-1 receptor mRNAs, the cells are capable of growing in serum-free medium without the addition of any exogenous growth factor. These cells, called p5 cells, can grow for prolonged periods in serum-free medium. BALB/c3T3 cells transfected with only the IGF-1 receptor expression plasmid (p6 cells) do not grow in serum-free medium but do grow if IGF-1 (or insulin in supraphysiological concentrations) is added. p6 cells also grow in response to EGF, confirming that the combination of EGF and an overexpressed IGF-1 receptor is sufficient for the growth of 3T3 cells. We have found that in EGF-stimulated p6 cells there is an increase in the expression of IGF-1 mRNA, that IGF-1 is secreted into the medium, and that the growth of p5 cells and EGF-stimulated p6 cells is inhibited by exposure to antisense oligodeoxynucleotides to IGF-1 receptor RNA. Finally, while cells constitutively expressing both EGF and EGF receptor RNAs grow, albeit modestly, in serum-free medium, their growth is also inhibited by an antisense oligodeoxynucleotide to IGF-1 receptor RNA. In contrast, in cells overexpressing the IGF-1 receptor, IGF-1-mediated cell growth occurs independently of the platelet-derived growth factor and EGF receptors (Z. Pietrzkowski, R. Lammers, G. Carpenter, A. M. Soderquist, M. Limardo, P. D. Phillips, A. Ullrich, and R. Baserga, Cell Growth Differ. 3:199-205, 1992, and this paper). These data indicate that an important role for EGF is participation in the activation of an autocrine loop based on the IGF-1-IGF-1 receptor interaction, which is obligatory for the proliferation of 3T3 cells.     

Constitutive expression of insulin-like growth factor 1 and insulin-like growth factor 1 receptor abrogates all requirements for exogenous growth factors.

BALB/c3T3 cells are exquisitely growth regulated and require both platelet-derived growth factor and insulin-like growth factor-1 (IGF-1) for optimal proliferation. BALB/c3T3 cells that constitutively express IGF-1 and elevated levels of IGF-1 receptor (IGF-1R) are capable of growth in serum-free medium without the addition of any exogenous growth factors. BALB/c3T3 cells overexpressing only the IGF-1R plasmid required IGF-1 or insulin for serum-free growth. Antisense oligodeoxynucleotides complementary to IGF-1R mRNA inhibited IGF-1-mediated cell growth. Under these conditions, neither the epidermal growth factor receptor nor phospholipase C gamma 1 was autophosphorylated. These findings indicate that constitutive expression of IGF-1 and IGF-1R allows 3T3 cells to grow in serum-free medium without addition of those exogenous growth factors that are required by the parent cell line.     

Effects of insulin and insulin-like growth factor 1 on osteoblast proliferation and differentiation: differential signalling via Akt and ERK

Insulin and insulin-like growth factor 1 (IGF-1) are evolutionarily conserved hormonal signalling molecules, which influence a wide array of physiological functions including metabolism, growth and development. Using genetic mouse studies, both insulin and IGF-1 have been shown to be anabolic agents in osteoblasts and bone development primarily through the activation of Akt and ERK signalling pathways. In this study, we examined the temporal signalling actions of insulin and IGF-1 on primary calvarial osteoblast growth and differentiation. First, we observed that the IGF-1 receptor expression decreases whereas insulin receptor expression increases during osteoblast differentiation. Subsequently, we show that although both insulin and IGF-1 promote osteoblast differentiation and mineralization in vitro, IGF-1, but not insulin, can induce osteoblast proliferation. The IGF-1-induced osteoblast proliferation was mediated via both MAPK and Akt pathways because the IGF-1-mediated cell proliferation was blocked by U0126, an MEK/MAPK inhibitor, or LY294002, a PI3-kinase inhibitor. Osteocalcin, an osteoblast-specific protein whose expression corresponds with osteoblast differentiation, was increased in a dose- and time-dependent manner after insulin treatment, whereas it was decreased with IGF-1 treatment. Moreover, insulin treatment dramatically induced osteocalcin promoter activity, whereas IGF-1 treatment significantly inhibited it, indicating direct effect of insulin on osteocalcin synthesis.