Insulin-like Growth factor-I Treatment Research Articles

Upregulation of IGF-I in the goldfish retinal ganglion cells during the early stage of optic nerve regeneration

Goldfish retinal ganglion cells (RGCs) can regrow their axons after optic nerve injury. However, the reason why goldfish RGCs can regenerate after nerve injury is largely unknown at the molecular level. To investigate regenerative properties of goldfish RGCs, we divided the RGC regeneration process into two components: (1) RGC survival, and (2) axonal elongation processes. To characterize the RGC survival signaling pathway after optic nerve injury, we investigated cell survival/death signals such as Bcl-2 family members in the goldfish retina. Amounts of phospho-Akt (p-Akt) and phospho-Bad (p-Bad) in the goldfish retina rapidly increased four- to five-fold at the protein level by 3–5 days after nerve injury. Subsequently, Bcl-2 levels increased 1.7-fold, accompanied by a slight reduction in caspase-3 activity 10–20 days after injury. Furthermore, level of insulin-like growth factor-I (IGF-I), which activates the phosphatidyl inositol-3-kinase (PI3K)/Akt system, increased 2–3 days earlier than that of p-Akt in the goldfish retina. The cellular localization of these molecular changes was limited to RGCs. IGF-I treatment significantly induced phosphorylation of Akt, and strikingly induced neurite outgrowth in the goldfish retina in vitro. On the contrary, addition of the PI3K inhibitor wortmannin, and IGF-I antibody inhibited Akt phosphorylation and neurite outgrowth in an explant culture. Thus, we demonstrated, for the first time, the signal cascade for early upregulation of IGF-I, leading to RGC survival and axonal regeneration in adult goldfish retinas through PI3K/Akt system after optic nerve injury. The present data strongly indicate that IGF-I is one of the most important molecules for controlling regeneration of RGCs after optic nerve injury.

Disturbed Cross Talk between Insulin-Like Growth Factor I and AMP-Activated Protein Kinase as a Possible Cause of Vascular Dysfunction in the Amyloid Precursor Protein/Presenilin 2 Mouse Model of Alzheimer's Disease

Cerebrovascular dysfunction appears to be involved in Alzheimer's disease (AD). In double mutant amyloid precursor protein/presenilin 2 (APP/PS2) mice, a transgenic model of AD, vessel homeostasis is disturbed. These mice have elevated levels of vascular endothelial growth factor (VEGF) and increased brain endothelial cell division but abnormally low brain vessel density. Examination of the potential involvement of insulin-like growth factor I (IGF-I) in these alterations revealed that treatment with IGF-I, a potent vessel growth promoter in the brain that ameliorates cognitive dysfunction in APP/PS2 mice, counteracted vascular dysfunction as follows: VEGF levels and endothelial cell proliferation were reduced, whereas vascular density was normalized. Notably, abnormally elevated brain IGF-I receptor levels in APP/PS2 mice were also normalized by IGF-I treatment. Analysis of possible processes involved in these alterations indicated that AMP-activated protein kinase (AMPK), a cell energy sensor that intervenes in angiogenic signaling and interacts with IGF-I, was also abnormally activated in APP/PS2 brains. Examination of the consequences of AMPK activation on cultured brain endothelial cells revealed increased VEGF levels together with enhanced endothelial cell proliferation and metabolism. Although these effects were also independently elicited by IGF-I, when both IGF-I and AMPK pathways were simultaneously activated on brain endothelial cells, VEGF production and endothelial cell proliferation ceased while cells remained metabolically activated (glucose use, peroxide production, and mitochondrial activity were elevated) and became more resistant to oxidative stress. Therefore, high IGF-I receptor and phosphoAMPK levels in APP/PS2 brains may reflect imbalanced IGF-I and AMPK angiogenic cross talk that could underlie vascular dysfunction in this model of AD.

IGF‐I‐induced oligodendrocyte progenitor proliferation requires PI3K/Akt, MEK/ERK, and Src‐like tyrosine kinases

Insulin-like growth factor-I (IGF-I) is required for the growth of oligodendrocytes, although the underlying mechanisms are not fully understood. Our aim was to investigate the role of phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase kinase (MEK1), and Src family tyrosine kinases in IGF-I-stimulated proliferation of oligodendrocyte progenitors. IGF-I treatment increased the proliferation of cultured oligodendrocyte progenitors as determined by measuring incorporation of [(3)H]-thymidine and bromodeoxy-uridine (BrdU). IGF-I stimulated a transient phosphorylation of 3-phosphoinositide-dependent kinase-1 (PDK1) and extracellular signal-regulated kinases (ERK1/2) (targets of MEK1), as well as a rapid and sustained activation of Akt (a target of PI3K). Furthermore, inhibitors of PI3K (LY294002 and Wortmannin), MEK1 (PD98059 and U0126), and Src family tyrosine kinases (PP2) decreased IGF-I-induced proliferation, and blocked ERK1/2 activation. LY294002, Wortmannin and PP2 also blocked Akt activation. To further determine whether Akt is required for IGF-I stimulated oligodendrocyte progenitor proliferation, cultures were infected with adenovirus vectors expressing dominant-negative mutants of Akt or treated with pharmacological inhibitors of Akt. All treatments reduced IGF-I-induced oligodendrocyte progenitor proliferation. Our data indicate that stimulation of oligodendrocyte progenitor proliferation by IGF-I requires Src-like tyrosine kinases as well as the PI3K/Akt and MEK1/ERK signaling pathways.

Signaling through the small G‐protein Cdc42 is involved in insulin‐like growth factor‐I resistance in aging articular chondrocytes

During aging, chondrocytes become unresponsive to insulin-like growth factor-I (IGF-I). This study examined the role of Cdc42 (cell-division-cycle 42) in IGF-I signaling during aging. Experiments were performed using cartilage and chondrocytes isolated from horses ages 1 day-25 years. Northern analysis was used to examine expression of the small GTPases Cdc42, Rac, and RhoA. Western analysis was utilized to assess total Cdc42 (GTP + GDP-bound); active, GTP-Cdc42 was assessed using a pulldown assay with Western analysis. GTP-Cdc42 was also measured following IGF-I treatment. Gene expression for Cdc42 and Rac were decreased in mature samples, but there was no difference in total Cdc42 (GTP + GDP-bound) protein expression due to age. GTP-Cdc42 was significantly greater in prepubescent samples compared to other age groups. IGF-I diminished the GTP-bound state of Cdc42 in prepubescent chondrocytes; however, this effect was lost during aging. No differences in results were observed due to sample type; that is, cartilage tissues versus isolated chondrocytes. These studies suggest that loss of IGF-I-mediated regulation of Cdc42 activation may be a mechanism for the chondrocyte unresponsive state during aging. Further, the activation state of Cdc42, measured in native and IGF-I-treated cartilage tissue for the first time, is similar to that of isolated chondrocytes, indicating that the activation state of small G-proteins is not affected by isolation of chondrocytes from the extracellular matrix. Continued studies will identify the upstream regulators of Cdc42, which will further elucidate the molecular mechanism of IGF-I resistance during aging thereby providing insight into targeted strategies for age-related osteoarthritis.

Regulatory role of c-Met in insulin-like growth factor-I receptor–mediated migration and invasion of human pancreatic carcinoma cells

Pancreatic carcinoma cells overexpress the insulin-like growth factor-I (IGF-I) receptor (IGF-IR) and the hepatocyte growth factor (HGF) receptor, c-Met, which are both known to mediate tumor cell migration and invasion. We hypothesized that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells and that IGF-I-mediated migration and invasion depend on c-Met. Migration and invasion assays were done with the human pancreatic cancer cell line L3.6pl treated with PBS, IGF-I, HGF, or IGF-I plus HGF. To determine if c-Met is necessary for IGF-IR-mediated migration and invasion, c-Met was down-regulated in L3.6pl cells via adenoviral infection with a c-Met ribozyme before IGF-I treatment. IGF-I and HGF increased cell migration and invasion. Furthermore, IGF-I plus HGF had a greater than additive effect on cell migration and invasion compared with either growth factor alone. Down-regulation of c-Met nearly completely inhibited IGF-I-mediated migration and invasion. Our findings suggest that IGF-IR and c-Met cooperate to induce migration and invasion of human pancreatic carcinoma cells. Furthermore, c-Met is required for both HGF- and IGF-I-mediated migration and invasion. Elucidation of the signaling pathways that contribute to tumor progression and metastasis should provide a foundation for the development of targeted therapies for pancreatic carcinoma.

Intranasal administration of IGF-I improves behavior and Purkinje cell pathology in SCA1 mice

Spinocerebellar ataxia type 1 (SCA1) is a neurodegenerative disease caused by the expansion of polyglutamine repeat within ataxin-1 protein. Cerebellar Purkinje cells are the primary targets of SCA1 pathology. These cells synthesize insulin-like growth factor-I (IGF-I) and express its receptors during their entire life. The aim of present study was to determine if intranasally administered IGF-I to SCA1 transgenic mice suppresses toxic effects of ataxin-1. Two-week old SCA1 heterozygous animals were randomly divided into two treatment groups of IGF-I (30 and 60 μg IGF-I/animal) and a vehicle-treated control group. The wildtype animals served as normal controls. IGF-I or vehicle was administered at 48 h intervals for the total of 10 doses. Animals were then subjected to rotarod test, sacrificed, brains removed and processed for immunohistochemical and Western blot analysis. Radiolabeled IGF-I and bioactive TAT peptide accumulated in the brains of SCA1 mice following intranasal administration validating the use of intranasal route. SCA1 mice showed SCA1 pathology with impaired motor function and downregulation of calcium binding proteins as compared to wildtype mice. However, 30 and 60 μg IGF-I-treated animals showed improved performance on the rotarod as compared to vehicle-treated SCA1 mice with significant improvement ( p < 0.05) on day 3 in 60 μg IGF-I group. The immunohistochemical data further showed partial recovery in the expression of calbindin D28k and protein kinase C-γ in Purkinje cells in IGF-I-treated SCA1 animals. Our results indicate that suppression of ataxin-1-mediated adverse effects by intranasal IGF-I treatment may be of a therapeutic value to treat SCA1.

Long-term IGF-I treatment of children with Laron syndrome increases adiposity

Laron syndrome (LS) is an autosomal recessive disease caused by deletions or mutations in the GH receptor gene leading to an inability of insulin-like growth factor I (IGF-I) generation. Among the major resulting body changes are dwarfism and obesity. The only effective treatment is daily administration of biosynthetic IGF-I. Body composition determination by DEXA (dual energy X-ray absorptiometry) of three girls with LS treated by IGF-I for 1, 3 and 11 1/2 years, respectively, revealed that concomitantly with the increase in growth there was a significant increase in body adipose tissue to double or triple the normal values. Due to the underdevelopment of the muscular and skeletal systems body mass index (BMI) did not accurately reflect the degree of obesity. In conclusion, IGF-I similar to insulin, exerts an adipogenic effect.

IGF-I-induced VEGF expression in HUVEC involves phosphorylation and inhibition of poly(ADP-ribose)polymerase

Insulin-like growth factor-I (IGF-I) has been shown to promote angiogenesis by enhancing vascular endothelial growth factor (VEGF) expression. However, how IGF-I-induces VEGF expression is not yet fully understood. With this investigation, we propose a new possible mechanism involving downregulation of poly(ADP-ribosyl)ation (pADPR). We first demonstrated that IGF-I increased VEGF protein expression in endothelial cells. Inhibitors of mitogen activated kinase (PD 98059), phosphatidyl-3-inositol-kinase (LY 294002), and protein kinase C (staurosporine) diminished the IGF-I effect suggesting the involvement of signal transduction. Since there is an established link between pADPR and transcriptional activity, we focused on a possible role of poly(ADP-ribose)polymerase (PARP). The inhibition of PARP by 3-aminobenzamide or nicotinamide enhanced VEGF expression. Additionally, IGF-I markedly decreased PARP activity. Furthermore, the IGF-I-mediated inhibition of PARP could be demonstrated as a result of protein phosphorylation since phosphorylation of PARP decreased its activity in vitro and IGF-I treatment of endothelial cells induced PARP phosphorylation. The IGF-I-mediated phosphorylation and inhibition of PARP represent a novel mechanism of VEGF protein expression.

Individualized therapy for growth hormone deficiency

The use of human growth hormone to treat children with short stature resulting from growth hormone deficiency or insufficiency has now accrued over 40 years of clinical experience with a satisfactory safety and efficacy record. Growth hormone deficiency is the primary indication for growth hormone treatment in childhood. It is basically a clinical diagnosis, based upon auxologic features, and confirmed by biochemical testing. For assurance of compliance, dosing and, perhaps, safety considerations, a dosing algorithm based upon insulin-like growth factor-I response seems to be appropriate. Current data suggest that such algorithms reflect the true growth hormone needs of a patient, and allow optimization of growth hormone treatment. For patients who display a suboptimal growth response or in whom the insulin growth factor levels remain low with assurance of adherence to the injection schedule, it is reasonable to increase the growth hormone dose. The availability of recombinant human insulin-like growth factor-I treatment may provide an alternative for massively increasing the dose of growth hormone. Dose reductions should be considered for patients with serum insulin-like growth factor-I levels substantially above the normal range.

Multiple Signaling Pathways are Activated During Insulin-like Growth Factor-I (IGF-I) Stimulated Breast Cancer Cell Migration

In order to display the full metastatic phenotype, the cancer cell must acquire the ability to migrate. In breast cancer, we have previously shown that insulin-like growth factor I (IGF-I) enhances cell motility in the highly metastatic MDA-231BO cell line by activating the type I IGF receptor (IGF1R). This motility response requires activation of IRS-2 and integrin ligation. In order to identify the key molecules downstream of IRS-2, we examined several signaling pathways known to be involved in cell motility. Focal adhesion kinase (FAK) was not activated by IGF-I, but IGF-I caused redistribution of FAK away from focal adhesion plaques. IGF-I treatment of MDA-231BO cells activated RhoA and inhibition of Rho-kinase (ROCK) inhibited the IGF-mediated motility response. The mitogen activated protein kinase (MAPK), p38, was also activated by IGF-I and inhibition of p38 by SB203580 blocked IGF-I induced cell motility. ROCK inhibition with Y-27632 also inhibited p38 phosphorylation suggesting that p38 lies downstream of ROCK. Both Erk1,2 and phosphatidyl-3 kinase (PI3K) were required for IGF-I stimulated cell motility, but only PI3K appeared to be directly downstream of IGF-I. Thus, IGF-I activation of its receptor coordinates multiple signaling pathways required for cell motility. Defining the key molecules downstream of the type I IGF receptor may provide a basis for optimizing therapies directed at this target.

Changes in insulin sensitivity induced by short-term growth hormone (GH) and insulin-like growth factor I (IGF-I) treatment in GH-deficient adults are not associated with changes in adiponectin levels

To test potential effects of altered insulin sensitivity on circulating adiponectin levels, we studied patients with GH deficiency (GHD) undergoing high dose GH and IGF-I treatment in a well-defined short-term setting. This design allowed to exclude confounding effects of GH and IGF-I treatment on body composition. Six patients (three women and three men) with acquired GHD were treated in a randomised cross-over trial with GH (subcutaneous injections of GH 0.03 IU/kg/daily) or IGF-I (continuous subcutaneous infusion of 5 μg/kg/h recombinant IGF-I) for 5 days. Median age of patients was 60 (range 25–72) years, BMI 24.7 (20.7–30.7) kg/m 2, and baseline IGF-I concentration 60 (30–83) μg/L. HOMA scores (to assess insulin sensitivity) at baseline were 0.8 (0.3–11.7) and adiponectin concentrations were 7.0 (2.5–14.8) mg/L. HOMA scores increased rapidly and significantly up to a maximum of 2.7-fold (from baseline) at day 5 of GH treatment and returned promptly to baseline when GH was stopped, while adiponectin serum levels remained within the baseline range throughout the study period. HOMA scores dropped to a nadir of 0.66 from baseline at day 2 of IGF-I treatment whereas adiponectin levels remained within the baseline range. These data suggest that changes in insulin sensitivity (induced by high dose and short-term GH and IGF-I treatment) in patients with GHD are not associated with changes in adiponectin levels.

Insulin-like growth factor I (IGF-I) replacement therapy increases albumin concentration in liver cirrhosis: Results of a pilot randomized controlled clinical trial

Insulin-like growth factor I (IGF-I) is an anabolic hormone synthesized in the liver whose levels decrease sharply in liver cirrhosis. We conducted a randomized double-blind placebo-controlled clinical trial to evaluate the effect of subcutaneous administration of IGF-I (20 microg/kg/day with dose escalation to 50-100 microg/kg/day) for 4 months in patients with alcoholic or primary biliary cirrhosis (PBC) and subnormal IGF-I levels. Eight alcoholics and one PBC entered the placebo group and seven alcoholics and two PBC the treatment group. Biochemistry, body composition, muscle mass and strength, and resting energy expenditure (REE) were evaluated. Total serum IGF-I and IGF-I/IGFBP-3 ratio (a surrogate marker of IGF-I biovailability) increased in the treatment group but IGF-I values still remained below normal limits in the treated patients. No differences were observed in body composition, muscle strength or muscle mass between groups. However, IGF-I therapy increased significantly serum albumin (P = 0.038) and this improvement correlated positively with variation of IGF-I/IGFBP-3 ratio. IGF-I treatment also tended to increase REE (P = 0.085); this difference was significant (P = 0.049) in the subgroup of alcoholic patients. A short course of IGF-I increased albumin levels and tended to improve energy metabolism in liver cirrhosis. These findings warrant larger clinical trials to assess the clinical benefit of IGF-I in cirrhotic patients.

Intrapericardial IGF-I Improves Cardiac Function in an Ovine Model of Chronic Heart Failure

Background. Following myocardial infarction, progressive deterioration of left ventricular function often follows, leading eventually to overt heart failure. In the myocardium, there is increased expression of insulin-like growth factor I (IGF-I) mRNA, protein and receptor levels, particularly in the peri-infarct zone, suggesting that IGF-I has a role to play in post-infarct cardiac structure and function. In this study, we examine the effects of exogenous IGF-I on cardiac function. Methods. Intrapericardial IGF-I (150 μg/kg/d, n = 3) or vehicle (sterile saline, n = 3) was administered to sheep in chronic heart failure and the results of intrapericardial delivery compared with those of subcutaneous delivery. Left ventricular ejection fraction (EF) was measured to assess cardiac performance. Concentrations of plasma IGF-I were quantified by radioimmunoassay. Results. Intrapericardial delivery of IGF-I resulted in a rapid and sustained increase ( P < 0.001) in EF, which remained elevated 14 days after cessation of treatment. Subcutaneous IGF-I treatment did not affect EF. Both subcutaneous and intrapericardial IGF-I administration increased concentrations of plasma IGF-I, although concentrations declined prior to the cessation of treatment. Conclusions. We conclude that the higher concentration of IGF-I in the myocardium, which results from intrapericardial delivery significantly increases EF in chronic heart failure but that subcutaneous delivery of IGF-I does not.

Fluid Shear Stress Synergizes with Insulin-like Growth Factor-I (IGF-I) on Osteoblast Proliferation through Integrin-dependent Activation of IGF-I Mitogenic Signaling Pathway

This study tested the hypothesis that shear stress interacts with the insulin-like growth factor-I (IGF-I) pathway to stimulate osteoblast proliferation. Human TE85 osteosarcoma cells were subjected to a steady shear stress of 20 dynes/cm(2) for 30 min followed by 24-h incubation with IGF-I (0-50 ng/ml). IGF-I increased proliferation dose-dependently (1.5-2.5-fold). Shear stress alone increased proliferation by 70%. The combination of shear stress and IGF-I stimulated proliferation (3.5- to 5.5-fold) much greater than the additive effects of each treatment alone, indicating a synergistic interaction. IGF-I dose-dependently increased the phosphorylation level of Erk1/2 by 1.2-5.3-fold and that of IGF-I receptor (IGF-IR) by 2-4-fold. Shear stress alone increased Erk1/2 and IGF-IR phosphorylation by 2-fold each. The combination treatment also resulted in synergistic enhancements in both Erk1/2 and IGF-IR phosphorylation (up to 12- and 8-fold, respectively). Shear stress altered IGF-IR binding only slightly, suggesting that the synergy occurred primarily at the post-ligand binding level. Recent studies have implicated a role for integrin in the regulation of IGF-IR phosphorylation and IGF-I signaling. To test whether the synergy involves integrin-dependent mechanisms, the effect of echistatin (a disintegrin) on proliferation in response to shear stress +/- IGF-I was measured. Echistatin reduced basal proliferation by approximately 60% and the shear stress-induced mitogenic response by approximately 20%. It completely abolished the mitogenic effect of IGF-I and that of the combination treatment. Shear stress also significantly reduced the amounts of co-immunoprecipitated SHP-2 and -1 with IGF-IR, suggesting that the synergy between shear stress and IGF-I in osteoblast proliferation involves integrin-dependent recruitment of SHP-2 and -1 away from IGF-IR.

IGF‐I neuroprotection in the immature brain after hypoxia‐ischemia, involvement of Akt and GSK3β?

Insulin-like growth factor I (IGF-I) is a neurotrophic factor that promotes neuronal growth, differentiation and survival. Neuroprotective effects of IGF-I have previously been shown in adult and juvenile rat models of brain injury. We wanted to investigate the neuroprotective effect of IGF-I after hypoxia-ischemia (HI) in 7-day-old neonatal rats and the mechanisms of IGF-I actions in vivo. We also wanted to study effects of HI and/or IGF-I on the serine/threonine kinases Akt and glycogen synthase kinase 3beta (GSK3beta) in the phophatidylinositol-3 kinase (PI3K) pathway. Immediately after HI, phosphorylated Akt (pAkt) and phosphorylated GSK3beta (pGSK3beta) immunoreactivity was lost in the ipsilateral and reduced in the contralateral hemisphere. After 45 min, pAkt levels were restored to control values, whereas pGSK3beta remained low 4 h after HI. Administration of IGF-I (50 microg i.c.v.) after HI resulted in a 40% reduction in brain damage (loss of microtubule-associated protein) compared with vehicle-treated animals. IGF-I treatment without HI was shown to increase pAkt whereas pGSK3beta decreased in the cytosol, but increased in the nuclear fraction. IGF-I treatment after HI increased pAkt in the cytosol and pGSK3beta in both the cytosol and the nuclear fraction in the ipsilateral hemisphere compared with vehicle-treated rats, concomitant with a reduced caspase-3- and caspase-9-like activity. In conclusion, IGF-I induces activation of Akt during recovery after HI which, in combination with inactivation of GSK3beta, may explain the attenuated activation of caspases and reduction of injury in the immature brain.

Inhibition of Src-like Kinases Reveals Akt-dependent and -independent Pathways in Insulin-like Growth Factor I-mediated Oligodendrocyte Progenitor Survival

Insulin-like growth factor I (IGF-I) has been previously shown to promote survival of oligodendrocyte progenitors; however, the underlying mechanisms are not fully understood. Our aim was to investigate the involvement of phosphatidylinositol 3-kinase (PI3K), MEK1, and Src family tyrosine kinases in IGF-I-mediated oligodendrocyte progenitor survival. In agreement with previous studies, IGF-I promoted cell survival. We show that IGF-I prevented apoptosis induced by growth factor deprivation in a PI3K-dependent and MEK/ERK-independent manner. In addition, IGF-I activated Akt while inhibiting caspase-3 activation, and these effects were reversed by the PI3K inhibitors LY 294002 and wortmannin, but not by the MEK1 inhibitor PD 98059. Interestingly, PP2, a specific Src-like kinase inhibitor, blocked the tyrosine phosphorylation of Src, Fyn, and Lyn and IGF-I-stimulated Akt activation, yet had no significant effects on caspase-3 activation or progenitor survival. To further determine whether Akt is required for IGF-I-mediated survival, oligodendrocyte progenitors were transduced with defective Akt mutants or treated with an Akt inhibitor. Although the Akt mutants and inhibitor decreased Akt activity and reduced basal cell survival, IGF-I could partially rescue oligodendrocyte progenitors by decreasing caspase-3 activation. These results suggest that 1) PI3K is essential for IGF-I-promoted cell survival, 2) downstream activation of Akt-dependent and -independent pathways is involved, and 3) Src-like tyrosine kinases participate in IGF-I-induced Akt activation. Therefore, an unidentified effector(s) of PI3K appears to be involved in conferring complete IGF-I-mediated protection of oligodendrocyte progenitors.

129 INSULIN-LIKE GROWTH FACTOR-I PROMOTES BLASTOCYST DEVELOPMENT OF HEAT-SHOCKED BOVINE EMBRYOS INDEPENDENT OF ITS ANTI-APOPTOTIC EFFECTS REQUIRING P13K SIGNALING

Insulin-like growth factor-I (IGF-I) reduces effects of heat shock on blastocyst development and induction of apoptosis. The present objective was to test whether IGF-I would allow for blastocyst development following heat shock because of its anti-apoptotic effects. Because anti-apoptotic actions of IGF-I require signaling through the phosphatidylinositol 3-kinase (PI3K) pathway, an inhibitor of PI3K (LY 294002) was used in Exp. 1 to determine whether it would prevent the thermoprotective effects of IGF-I on development. Embryos were produced in vitro in KSOM-BE2 medium �100 ng mL-1 of IGF-I. Embryos e16 cells at 5 d post-insemination (dpi) were placed in fresh drops containing the same IGF-I treatment as well as LY 294002 (100 �m) or vehicle (0.1% DMSO) and cultured at either 38.5�C or 41�C for 15 h. All groups were then cultured at 38.5�C until 8 dpi when blastocyst development was assessed (10 replicates; 112 to 142 embryos per treatment). For embryos in DMSO, IGF-I did not increase the percentage that became blastocysts when cultured at 38.5�C (59.1 and 57.1% for control and IGF-I embryos, respectively; SEM = 3.6%). Heat shock reduced blastocyst development (P &lt; 0.01), and IGF-I blocked this decrease (percent blastocyst = 42.0 and 54.7% for control and IGF-I embryos, respectively; SEM = 3.6%). Similar results were obtained for embryos cultured with LY 294002. The percentage of embryos becoming blastocysts for control embryos was 53.5 and 35.5%, respectively, at 38.5 and 41�C, development for IGF-I-treated embryos was, respectively, 55.8 and 49.4% at 38.5 and 41�C (SEM = 4.8%). Analysis of the entire data set revealed an IGF-I � temperature interaction (P &lt; 0.05) but no interactions with inhibitor treatment. Thus, IGF-I protected embryos from heat shock in the presence and absence of LY 294002. In Exp. 2, procedures were similar, except that embryos were cultured with a caspase-3 inhibitor (z-DEVD-fmk; 100 �m) instead of LY 294002 (12 replicates; 114 to 137 embryos per treatment). For embryos in DMSO, blastocyst development was reduced by heat shock (P &lt; 0.06) and increased by IGF-I (P &lt; 0.06). The percentage of embryos becoming blastocysts was 67.0 and 55.8% for control embryos at 38.5 and 41�C, respectively, vs. 74.7 and 70.8% for IGF-I embryos at 38.5 and 41�C, respectively (SEM = 5.2%). For embryos cultured with z-DEVD-fmk, heat shock reduced blastocyst development (P &lt; 0.01), and IGF-I was no longer effective in blocking the reduction in blastocyst development caused by heat shock (IGF-I � inhibitor treatment; P &lt; 0.01). Blastocyst development was 66.4 and 58.7% for control embryos at 38.5 and 41�C, respectively, vs. 61.8 and 48.6% for IGF-I embryos at 38.5 and 41�C, respectively (SEM = 4.6%). Note that z-DEVD-fmk did not protect embryos from the anti-developmental effects of heat shock but exacerbated heat shock effects for IGF-I-treated embryos. In conclusion, the ability of IGF-I to allow heat-shocked embryos to continue development to the blastocyst stage is independent of its anti-apoptotic effects involving the PI3K pathway but may depend on active caspase-3. This work was supported by USDA NRICGP 2002-35203-12664, BARD US-3551-04, and USDA TSTAR 2004-34135-14715.

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.

Insulin-like Growth Factor I Controls Adhesion Strength Mediated by α5β1Integrins in Motile Carcinoma Cells

One of the intriguing questions regarding cell motility concerns the mechanism that makes stationary cells move. Here, we provide the first physical evidence that the onset of breast cancer cell motility in response to insulin-like growth factor I (IGF-I) correlates with lowering of adhesion strength from 2.52 +/- 0.20 to 1.52 +/- 0.13 microdynes/microm2 in cells attached to fibronectin via alpha5beta1 integrin. The adhesion strength depends on the dose of IGF-I and time of IGF-I treatment. Weakening of cell-matrix adhesion is blocked significantly (p < 0.01) by the catalytically inactive IGF-I receptor (IGF-IR) and the phosphoinositide 3-kinase (PI-3 kinase) inhibitor LY-294002, but it is unaffected by mitogen-activated protein kinase kinase inhibitor UO-126 and Src kinase inhibitor PP2. Sustained blockade of Rho-associated kinase (ROCK) with Y-27632 down-regulates adhesion strength in stationary, but not in IGF-I-treated, cells. Jasplakinolide, a drug that prevents actin filament disassembly, counteracts the effect of IGF-I on integrin-mediated cell adhesion. In the absence of growth factor signaling, ROCK supports a strong adhesion via alpha5beta1 integrin, whereas activation of the IGF-IR kinase reduces cell-matrix adhesion through a PI-3K-dependent, but ROCK-independent, mechanism. We propose that disassembly of the actin filaments via PI-3 kinase pathway contributes to weakening of adhesion strength and induction of cell movement. Understanding how cell adhesion and migration are coordinated has an important application in cancer research, developmental biology, and tissue bioengineering.

Mammary tumorigenesis in growth hormone deficient spontaneous dwarf rats; effects of hormonal treatments

This study was carried out to investigate mammary tumorigenesis in growth hormone (GH) deficient spontaneous dwarf rats (SDR). At 50-60 days of age, the rats were divided into five groups. Group 1 received bovine (b) GH (prolonged release formulation) administered at a dose of 40-50 mg/kg body wt. in 50 microl weekly injections; group 2 received recombinant human insulin-like growth factor-I (IGF-I) at a dose of 1 mg/kg body wt./day administered via osmotic pumps; animals in group 3 were fitted with subcutaneous silastic capsule containing 30 microg 17 beta-estradiol (E2) plus 30 mg progesterone (P4), replaced every 2 months; group 4 received both bGH and E2 plus P4 treatments at the same doses as above, and control animals (group 5) received sham treatments (vegetable oil injection, silastic capsules containing cellulose). After 1 week of treatment, all animals were injected intraperitoneally with the carcinogen N-methyl-N-nitrosourea (MNU) at a dose of 50 mg/kg body wt. Other groups of animals, receiving identical hormonal treatment to those exposed to MNU, were treated for 10 days only and then sacrificed for assessment of circulating concentrations of hormones and mammary gland characteristics at the time of carcinogen exposure. The hormonal treatments of the animals exposed to the MNU were continued for an additional 20 weeks and mammary tumor development monitored by weekly palpation and tumors collected as necessary. The rats were weighed weekly. At the end of the treatment period, all animals were sacrificed and remaining tumors were collected. Rats in all groups continued to gain weight throughout the experimental period, but the largest weight gain was see in animals receiving GH either alone or with E2 and P4. Animals treated with IGF-I also gained weight compared to controls, but this weight gain was less than that seen in GH-treated rats. GH treatment alone increased mammary tumor incidence from 4.8% in controls to 100%. Average tumor load and latency in the GH-treated rats were 7.0 +/- 0.8 tumors/tumor-bearing rat (mean +/- SEM) and 57.3 +/- 2.7 days (mean +/- SEM), respectively. As in intact Sprague-Dawley rats, approximately 90% of the tumors that developed in the GH-treated rats were ovarian dependent for growth. IGF-I treatment also increased mammary tumor development to 62.5%. Average tumor load and latency in the IGF-I-treated rats were 1.6 +/- 0.4 tumors/tumor-bearing rat (mean +/- SEM) and 96.2 +/- 14.5 days (mean +/- SEM), respectively. However E2 + P4 treatments did not significantly alter tumorigenesis and, surprisingly, simultaneous treatment with E2 + P4 and GH obliterated the GH-stimulated increase in tumor development. Prolactin (PRL) did not appear to influence mammary tumorigenesis in the SDRs, as untreated SDRs had significantly elevated serum concentration of PRL as compared with normal Sprague-Dawley (SD) rats, whereas GH-treated SDRs had PRL levels similar to that of normal SD rats. No obvious structural characteristics were associated with high or low susceptibility to mammary tumorigenesis, as assessed by mammary gland whole mounts from the different animal groups sacrificed at the time of carcinogen administration. Enhanced expression of the extracellular signal-regulated kinase 1/2 (ERK1/2), and activation (phosphorylation) of ERK1/2 were associated with an increase in mammary tumorigenesis. Similarly, the expression of the estrogen receptor-alpha (ER alpha) was significantly elevated in animal groups with the highest susceptibility to tumorigenesis, whereas the levels of cyclin D1 expression were not related to mammary tumorigenesis.