Insulin-like Growth factor-I Stimulation Research Articles

Oxytocin Mediates Some Effects of Insulin-Like Growth Factor-I on Porcine Ovarian Follicles

The aims of the present study were (1) to investigate the influence of insulin-like growth factor-I (IGF-I) on follicular size, on the secretion of oxytocin (OT), progesterone (P), estradiol (E), IGF binding protein-3 (IGFBP-3), inhibin A, inhibin B and cAMP and on the expression of proliferation-associated peptide PCNA, ERK-related mitogen activated protein kinase (MAPK/ERK1, 2) and protein kinase A (PKA) in cultured porcine ovarian follicles; (2) to examine the effects of OT on IGF-I and on these functions; and (3) to determine whether the effects of IGF-I can be mediated by OT. To define the involvement of OT in mediating IGF-I action, we compared responses of porcine ovarian follicles to IGF-I and OT and examined whether blockade of endogenous OT by specific antiserum can affect IGF-I action. It was observed that IGF-I (1, 10 or 100 ng/ml) was able to prevent a decrease in the size of ovarian follicles during culture and caused an increase in the diameter of some follicles. It also stimulated the secretion of OT, P, IGFBP-3, inhibin A and cAMP, decreased the secretion of E and inhibin B (RIA/EIA/ELISA), and induced the expression of PCNA, PKA, MAPK/ERK1, but not MAPK/ERK2 (Western blotting). Like IGF-1, OT (100 ng/ml) prevented decrease in follicular size and increased the diameter of some follicles. It also stimulated the secretion of P and IGF-I, but not E. Antiserum against OT (1%), when given alone, did not affect the reduction of follicular size but slightly increased the percentage of follicles increasing their diameter during culture. The antiserum also inhibited secretion of OT and cAMP but not the secretion of P, E, IGFBP-3 or the expression of PKA, MAPK/ERK1 or 2. When given together with IGF-I, the antiserum prevented the stimulatory action of IGF-I on the proportion of enlarged follicles and on OT, IGFBP-3 and MAPK/ERK1. It augmented the effect of IGF-I on P, but not the effect on E, cAMP, PKA or MAPK/ERK2. These observations demonstrate the involvement of IGF-I and OT in the control of ovarian follicular size and follicular cell proliferation, progestagen, estrogen, IGFBP-3, inhibin A and B secretion and in cAMP/PKA- and MAPK/ERK1-dependent intracellular mechanisms. Furthermore, the reciprocal stimulation of IGF-I and OT and the similarity of some their effects, together with the prevention or augmentation of some IGF-I effects after OT blockade, suggest that IGF-I action can be mediated by OT.

The Effects of Insulin-like Growth Factor-I (IGF-I) in Mouse Lung Cancer Cells.

Insulin-like growth factor-I (IGF-I) is an important mitogen in many types of malignancies. The purpose of this study was to evaluate the role of the IGF system on cell proliferation and cell death in mouse lung cancer cell lines (3LL). Northern analysis was performed in 3LL cells. We evaluated the phosphorylation of IGF-I receptor (IGF-IR) with IGF-I stimulation. MTT assay was performed after treating 3LL cells with IGF-I and the treatment effect on cell death in the presence of anticancer drug was investigated. Northern analysis revealed the presence of IGF-I and IGF-IR mRNA expression in 3LL cells. IGF-I increased cellular proliferation in serum free media. IGF-I also stimulated the tyrosine phosphorylation of two proteins: one, with a molecular mass of 95 kDa, was the beta-subunit of IGF-IR; the other, with an approximate molecular mass of 185 kDa, was originally identified as the insulin receptor substrate-I (IRS-I). IGF-I at a low concentration inhibited the cell death induced by adriamycin. IGF-I, a mitogen through the phosphorylation of the IGF-IR beta-subunit, acts as a survival factor to inhibit cell death. Therefore, these findings suggest that IGF-I and IGF-IR are involved in both the cell proliferation and cell death associated with cancer cell growth.

Deficient expression of insulin receptor substrate-1 (IRS-1) fails to block insulin-like growth factor-I (IGF-I) stimulation of brain growth and myelination

To determine whether insulin receptor substrate-1 (IRS-1) is essential in mediating insulin-like growth factor-I (IGF-I) stimulation of brain growth and myelination in vivo, we cross-bred IGF-I transgenic (Tg) mice with IRS-1 null mutant (IRS-1 −/−) mice and examined brain growth and expression of myelin-specific proteins in mice that overexpress IGF-I with or without IRS-1 expression. We found that while IGF-I overexpression stimulates a dramatic increase in brain weight (43%) by 7–8 weeks of age in the absence of IRS-1, it stimulates a greater increase (50%) with intact IRS-1 expression. To evaluate myelination we investigated IGF-I-stimulated expression of myelin basic protein (MBP) and proteolipid protein (PLP) in the cerebral cortex CTX and brainstem, and found similar increases in each region in IRS-1 −/− and wild type mice. In studies using mixed glial cultures derived from IRS-1 −/− mice, IGF-I also increased the abundance of MBP and PLP mRNA. To assess possible alternate mediators of IGF-I actions, we examined IRS-2 and IRS-4 and found that the abundance of each is increased in the CTX of IRS-1 −/− mice and IGF-I Tg mice. Our results suggest that IRS-1 is not essential in IGF-I promotion of oligodendrocyte development and myelination, and that IRS-2 and IRS-4 may compensate for the loss of IRS-1 expression and function in the cells of oligodendrocyte lineage. Nonetheless, the finding that IGF-I stimulates brain growth less well in the absence of IRS-1 suggests that IRS-1-mediated signaling may be more central to IGF-I action in cells other than glia and oligodendrocytes.

RACK1, an insulin-like growth factor I (IGF-I) receptor-interacting protein, modulates IGF-I-dependent integrin signaling and promotes cell spreading and contact with extracellular matrix.

The insulin-like growth factor I (IGF-I) receptor (IGF-IR) is known to regulate a variety of cellular processes including cell proliferation, cell survival, cell differentiation, and cell transformation. IRS-1 and Shc, substrates of the IGF-IR, are known to mediate IGF-IR signaling pathways such as those of mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K), which are believed to play important roles in some of the IGF-IR-dependent biological functions. We used the cytoplasmic domain of IGF-IR in a yeast two-hybrid interaction trap to identify IGF-IR-interacting molecules that may potentially mediate IGF-IR-regulated functions. We identified RACK1, a WD repeat family member and a Gbeta homologue, and demonstrated that RACK1 interacts with the IGF-IR but not with the closely related insulin receptor (IR). In several types of mammalian cells, RACK1 interacted with IGF-IR, protein kinase C, and beta1 integrin in response to IGF-I and phorbol 12-myristate 13-acetate stimulation. Whereas most of RACK1 resides in the cytoskeletal compartment of the cytoplasm, transformation of fibroblasts and epithelial cells by v-Src, oncogenic IR or oncogenic IGF-IR, but not by Ros or Ras, resulted in a significantly increased association of RACK1 with the membrane. We examined the role of RACK1 in IGF-IR-mediated functions by stably overexpressing RACK1 in NIH 3T3 cells that expressed an elevated level of IGF-IR. RACK1 overexpression resulted in reduced IGF-I-induced cell growth in both anchorage-dependent and anchorage-independent conditions. Overexpression of RACK1 also led to enhanced cell spreading, increased stress fibers, and increased focal adhesions, which were accompanied by increased tyrosine phosphorylation of focal adhesion kinase and paxillin. While IGF-I-induced activation of IRS-1, Shc, PI3K, and MAPK pathways was unaffected, IGF-I-inducible beta1 integrin-associated kinase activity and association of Crk with p130(CAS) were significantly inhibited by RACK1 overexpression. In RACK1-overexpressing cells, delayed cell cycle progression in G(1) or G(1)/S was correlated with retinoblastoma protein hypophophorylation, increased levels of p21(Cip1/WAF1) and p27(Kip1), and reduced IGF-I-inducible Cdk2 activity. Reduction of RACK1 protein expression by antisense oligonucleotides prevented cell spreading and suppressed IGF-I-dependent monolayer growth. Our data suggest that RACK1 is a novel IGF-IR signaling molecule that functions as a positive mediator of cell spreading and contact with extracellular matrix, possibly through a novel IGF-IR signaling pathway involving integrin and focal adhesion signaling molecules.

Four PSM/SH2-B alternative splice variants and their differential roles in mitogenesis.

An SH2 domain originally termed SH2-B had been identified as a direct cellular binding target of a number of mostly mitogenic receptors. The complete cellular protein, termed PSM, and respective sequence variants share additional Pro-rich and PH regions, as well as similarities with APS and Lnk. A role of these mediators has been implicated in signaling pathways found downstream of growth hormone receptor and receptor tyrosine kinases, including the insulin, insulin-like growth factor-I (IGF-I), platelet-derived growth factor (PDGF), nerve growth factor, hepatocyte growth factor, and fibroblast growth factor receptors. As a result of this report a total of four PSM/SH2-B sequence variants termed alpha, beta, gamma, and delta have now been identified in the mouse and have been compared with the available rat and human sequences. Variant differences are based on alternative splicing and define distinct last exons 7, 8, and 9 that result in reading frameshifts and unique carboxyl-terminal amino acid sequences. Variant sequences have been identified from cDNA libraries and directly by reverse transcription-polymerase chain reaction. Sequence analysis predicts four distinctly sized protein products that have been demonstrated after cDNA expression. All were found phosphorylated on tyrosine specifically in response to IGF-I and PDGF stimulation. cDNA expression of the four variants caused variant-dependent levels of stimulation of IGF-I- and PDGF-induced mitogenesis. The most pronounced increase in mitogenesis was consistently observed for the gamma variant followed by delta, alpha, and beta with decreasing responses. In contrast, the mitogenic response to epidermal growth factor consistently remained unaffected. The variants are expressed in most mouse tissues, typically, most strongly in pairs of alpha and delta or beta and gamma. Our findings implicate differential roles of the PSM/SH2-B splice variants in specific mitogenic signaling pathways.

The proliferative and antiapoptotic actions of growth hormone and insulin-like growth factor-1 are mediated through distinct signaling pathways in the Pro-B Ba/F3 cell line.

Biological actions of GH can be direct or mediated through insulin-like growth factor I (IGF-I). In the interleukin-3 (IL-3)-dependent Ba/F3 cell line, IGF-I induces cell cycle entry and proliferation. Ba/F3 cells expressing the rat GH receptor (Ba/F3 GHR cells) have been shown to escape from apoptosis and to proliferate under GH stimulation. Using the Ba/F3 GHR cell model, we sought to dissect the signals elicited specifically by IGF-I or GH. In contrast to IGF-I or IL-3, GH is able to maintain cell cycle entry of Ba/F3 GHR cells cultured for 7 days in the absence of serum. The presence of IGF-I messenger RNA was not detected by RT-PCR, and by RIA, IGF-I was not found in culture medium of Ba/F3 GHR cells, unstimulated or stimulated by GH. Moreover, the addition of an anti-IGF-I antibody that blocks IGF-I effects suggests that the actions of GH are not mediated by IGF-I, but appear to be direct. GH or IGF-I stimulation increased expression of cyclins A and D(1) with comparable kinetics, whereas expression of p21(waf1/cip1) seemed delayed in IGF-I-stimulated cells compared with that in GH-stimulated cells. Contrary to GH or IL-3, IGF-I did not induce nuclear factor-kappaB DNA-binding activity in Ba/F3 cells. Inhibition of nuclear factor-kappaB through expression of the mutant IkappaBalpha (A32/36) abrogated the GH-mediated survival signal, but did not result in alterations of the cell cycle in Ba/F3 GHR cells treated with IGF-I. Phosphatidylinositol 3-kinase was required for both survival and proliferative responses to IGF-I. Transfection of a dominant negative form of AKT (AH-AKT) resulted in suppression of IGF-I-mediated cell survival, but not of the antiapoptotic effect of GH in Ba/F3 GHR cells. Thus, GH and IGF-I are able to promote cell survival and proliferation through independent and different pathways in Ba/F3 cells.

S-myotrophin promotes the hypertrophy of myotube as insulin-like growth factor-I does

We have reported previously that a novel muscle cell growth factor, having a structure of a peptide with sugar chains, was successfully purified from porcine skeletal muscle. It was named s-myotrophin. To determine the role of s-myotrophin in skeletal muscle growth, the effect of s-myotrophin on primary cultured chick skeletal muscle cells (composed almost totally of multinucleated myotubes) was investigated by comparing s-myotrophin with Insulin-like growth factor-I (IGF-I). Both s-myotrophin and IGF-I significantly increased creatine kinase activity of the cultures; both substances gave similar responses. Intracellar protein content was also increased by the addition of these factors. The content of myosin and actin in s-myotrophin treated culture in the differentiation medium was significantly higher than that of the control (unstimulated). The content of those proteins in IGF-I treated culture was also higher than that of control, but the differences were not statistically significant. Immunoblot analysis confirmed that the amounts of myosin and actin in the myocytes were greatly increased by s-myotrophin stimulation and also by IGF-I stimulation. Morphological observations using an anti-desmin antibody staining procedure demonstrated that the size of both s-myotrophin and IGF-I treated myotubes was appreciably larger than that of control myotubes. These results suggest that s-myotrophin is a potent mediator of skeletal muscle cell hypertrophy thorough the accumulations of muscle structural proteins.

Insulin resistance and cardiovascular disease.

I RESISTANCE describes an impaired biological response to insulin (1–4). In the early states of insulin resistance there is a compensatory increase in insulin concentrations. Although hyperinsulinemia may compensate for resistance to some biological actions of insulin, it may result in overexpression of actions in tissues that retain normal or minimally impaired sensitivity to insulin. Also, high concentrations of insulin can act through receptors for insulin-like growth factor I (IGF-I) (5–8). Thus, accentuation of some actions of insulin with concurrent resistance to other actions gives rise to diverse clinical manifestations and sequelae of the insulin resistance syndrome. In general, insulin resistance can be due to a prereceptor, receptor, or postreceptor abnormality (1). One signaling pathway for insulin and IGF-I is the phosphatidylinositide 3-kinase (PI3 -kinase) system. Upon binding to their receptors, there is autophosphorylation of the b-subunit, which mediates noncovalent but stable interactions between the receptor and cellular proteins (1). Several proteins are then rapidly phosphorylated on tyrosine residues by ligandbound insulin receptors, including insulin receptor substrate-1 (IRS-1) (1). IRS docking proteins bind strongly to the enzyme PI3 -kinase (1), a heterodimer consisting of a p85 regulatory subunit and a p110 catalytic subunit, via SH-2 domain interaction with the p85 subunit (1). Insulin and IGF-I stimulation increases the amount of PI3 -kinase associated with IRS, and the binding process is associated with increased activity of the enzyme. Activation of the enzyme is crucial for transducing the actions of these peptides in cardiovascular (CV) tissue (9–14) as well as conventional insulin-sensitive tissues (1). The interruption of this pathway creates a resistance to the actions of insulin/IGF-I in stimulating vascular nitric oxide (NO) production (9, 10), CV cation transport mechanisms (11–15), as well as glucose transport (1, 6) (Fig. 1) in classically sensitive tissues such as muscle and adipose tissue. PI3-kinase mediates the increases in NO, Na pump, K channel, and calcium (Ca) myofilament sensitivity by increasing the trafficking and translocation of NO synthase and cation pump units as well as glucose transporters (1, 9, 16) (Fig. 2). Therefore, resistance to the actions of insulin and IGF-I in these tissues occurs whenever there is reduced PI3 -kinase activation (Fig. 2).

Involvement of PI 3-kinase in IGF-I stimulation of jejunal Na+-K+-ATPase activity and nutrient absorption.

Mechanisms responsible for increased jejunal transport rates observed in tissues treated with orally administered insulin-like growth factor-I (IGF-I) were studied in 5-day-old colostrum-deprived piglets. Human recombinant IGF-I (3.5 mg. kg(-1). day(-1)) or control vehicle was given orogastrically for 4 days. Disaccharidase activity, fructose uptake, and Na+-glucose cotransporter SGLT-1 protein abundance were similar between groups. Oral IGF-I produced greater rates of enterocyte Na+-K+-ATPase activity with no significant differences in Na+-K+-ATPase abundance. Cellular mechanisms responsible for transport changes were studied in Ussing chambers. In control tissues, the presence of IGF-I in mucosal solutions increased basal short-circuit current (I(sc)), potential difference, D-glucose-stimulated I(sc), and Na+-K+-ATPase activity; these changes were abolished by preincubation of tissues with wortmannin, a phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor. The results suggest that the effect of IGF-I on jejunal ion and nutrient transport involves activation of PI 3-kinase and stimulation of Na+-K+-ATPase activity in enterocytes.

IGF-I stimulation of extracellular acidification is not linked to cell proliferation for autocrine cells.

Insulin-like growth factor-I (IGF-I) increases extracellular acidification rate (ECAR), a measure correlated with proliferation for nonautocrine cells. To evaluate the effect of autocrine IGF-I secretion on cell responsiveness, a cell line that secretes IGF-I was tested. SV40-lGF-I cells also registered concentration-dependent increases in ECAR; however, unlike the parental cell line, signal attenuation upon repeat challenges was not evident. Furthermore, SV40-IGF-I cells did not proliferate in response to IGF-I. We investigated if lack of proliferation was due to differences in the protocols of the assays ([3H]thymidine incorporation and microphysiometry). We identified three key differences in the protocols: surface substrate, cell density, and fluid residence time. We found no increase in [3H]thymidine incorporation for cells on either tissue-culture plastic or polycarbonate transwells. Control levels of [3H]thymidine incorporation were cell-density-dependent, but IGF-I did not increase proliferation at any density studied. Finally, we investigated IGF-I stimulation for cells under microphysiometer flow conditions and found no proliferative response to IGF-I. We found that the cells do respond to IGF-I with increased amino acid uptake. These data suggest that IGF-I signaling is operational in the SV40-IGF-I cells, but the transduction pathway for IGF-I-induced proliferation is compromised, despite the fact that these cells respond to fetal bovine serum with increased growth. Ongoing studies are focused on identifying which elements in the signaling cascade are altered by autocrine secretion of IGF-I.

Protein Kinase C α-mediated Negative Feedback Regulation Is Responsible for the Termination of Insulin-like Growth Factor I-induced Activation of Nuclear Phospholipase C β1 in Swiss 3T3 Cells

Previous studies from several independent laboratories have demonstrated the existence of an autonomous phosphoinositide (PI) cycle within the nucleus, where it is involved in both cell proliferation and differentiation. Stimulation of Swiss 3T3 cells with insulin-like growth factor-I (IGF-I) has been shown to induce a transient and rapid increase in the activity of nuclear-localized phospholipase C (PLC) beta1, which in turn leads to the production of inositol trisphosphate and diacylglycerol in the nucleus. Nuclear diacylglycerol provides the driving force for the nuclear translocation of protein kinase C (PKC) alpha. Here, we report that treatment of Swiss 3T3 cells with Go6976, a selective inhibitor of PKC alpha, caused a sustained elevation of IGF-I-stimulated nuclear PLC activity. A time course study revealed an inverse relationship between nuclear PKC activity and the activity of nuclear PLC in IGF-I-treated cells. A time-dependent association between PKC alpha and PLC beta1 in the nucleus was also observed following IGF-I treatment. Two-dimensional phosphopeptide mapping and site-directed mutagenesis demonstrated that PKC promoted phosphorylation of PLC beta1 at serine 887 in the nucleus of IGF-I-treated cells. Overexpression of either a PLC beta1 mutant in which the PKC phosphorylation site Ser(887) was replaced by alanine, or a dominant-negative PKC alpha, resulted in a sustained activation of nuclear PLC following IGF-I stimulation. These results indicate that a negative feedback regulation of PLC beta1 by PKC alpha plays a critical role in the termination of the IGF-I-dependent signal that activates the nuclear PI cycle.

Nitric oxide inhibits chondrocyte response to IGF-I: inhibition of IGF-IRbeta tyrosine phosphorylation.

Chondrocytes in arthritic cartilage respond poorly to insulin-like growth factor I (IGF-I). Studies with inducible nitric oxide synthase (iNOS) knockout mice suggest that NO is responsible for part of the cartilage insensitivity to IGF-I. These studies characterize the relationship between NO and chondrocyte responses to IGF-I in vitro, and define a mechanism by which NO decreases IGF-I stimulation of chondrocyte proteoglycan synthesis. Lapine cartilage slices, chondrocytes, and cartilage from osteoarthritic (OA) human knees were exposed to NO from the donors S-nitroso-N-acetylpenicillamine (SNAP) or (Z)-1-[2-(2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1- ium-1, 2-diolate] (DETA NONOate), by transduction with adenoviral transfer of iNOS (Ad-iNOS), or by activation with interleukin-1 (IL-1). NO synthesis was estimated from medium nitrite, and proteoglycan synthesis was measured as incorporation of (35)SO(4). IGF-I receptor phosphorylation was evaluated with Western analysis. SNAP, DETA NONOate, endogenously synthesized NO in Ad-iNOS-transduced cells, or IL-1 activation decreased IGF-I-stimulated proteoglycan synthesis in cartilage and monolayer cultures of chondrocytes. OA cartilage responded poorly to IGF-I; however, the response to IGF-I was restored by culture with N(G)-monomethyl-L-arginine (L-NMA). IGF-I receptor phosphotyrosine was diminished in chondrocytes exposed to NO. These studies show that NO is responsible for part of arthritic cartilage/chondrocyte insensitivity to anabolic actions of IGF-I; inhibition of receptor autophosphorylation is potentially responsible for this effect.

IGF-I stimulation of luteinizing hormone secretion, IGF-binding proteins (IGFBPs) and expression of mRNAs for IGFs, IGF receptors and IGFBPs in the ovine pituitary gland.

Circulating concentrations of insulin-like growth factor-I (IGF-I) are reduced in juvenile sheep during nutritional growth restriction and the associated delay in puberty. Since exogenous IGF-I has been shown to stimulate luteinizing hormone (LH) secretion, it is postulated that endogenous IGF-I may act as a stimulatory metabolic signal to the pubertal ovine hypothalamo-pituitary axis, yet its site of action is unknown. Using coronal hypothalamic and pituitary sections from pubertal ewe lambs, in vitro autoradiography was used to localise 125I-labelled IGF-I binding, and gene expression for components of the IGF system was localised by in situ hybridisation using oligonucleotide probes. High concentrations of 125I-IGF-I binding were seen in the pars tuberalis (PT) and pars distalis (PD) of the pituitary, and relatively little in the hypothalamus; binding in the PT but not the PD was displaced by excess unlabelled IGF-I. Large amounts of mRNA were detected for the type-1 receptor (IGF-1R) and for IGF-binding protein (IGFBP)-5, localised to the PT and PD, and less intense specific hybridisation signals were obtained with mRNAs for IGF-II, type-2 receptor (IGF-2R) and IGFBP-3. There was some evidence for specific hybridisation to IGFBP-4 mRNA in the PT. IGF-I, IGFBP-1 and IGFBP-2 mRNAs were not detected in PT and PD. None of the genes were expressed in hypothalamic tissue. Western-ligand binding on PD extracts from male castrates revealed by their molecular weights the likely presence of IGFBPs-2, -3, and -5. Finally, cultured PD cells from abattoir-killed sheep were challenged with IGF-I (0.1, 1, 10 or 30 nM) or luteinizing hormone-releasing hormone (LHRH, 10 nM) alone, or both together. Basal LH output was stimulated by 10 nM IGF-I (120+/-11.2%, P>0.05), 30 nM IGF-I (148+/-12.8%, P<0.01), and LHRH alone (200+/-16.1%, P<0.001); there was no additive or subtractive effect of LHRH and IGF-I given together. Thus, an intrapituitary IGF system exists in sheep and the present results are consistent with an endocrine role for IGF-I in nutritional modulation of LH secretion at the level of the pituitary gland.

Mechanical and Physicochemical Regulation of the Action of Insulin-Like Growth Factor-I on Articular Cartilage

The development and maintenance of healthy joints is a complex process involving many physical and biological stimuli. This study investigates the interaction between insulin-like growth factor-I (IGF-I) and static mechanical compression in the regulation of articular cartilage metabolism. Bovine cartilage explants were treated with concentrations of IGF-I from 0 to 300 ng/ml in the presence or absence of 0–50% static compression, and the transient and steady-state incorporation of [3H]proline and [35S]sulfate into matrix components were measured. In parallel studies, cartilage explants were treated with 0–300 ng/ml IGF-I at media pH ranging from 6.4 to 7.2 and the steady-state incorporation of [3H]proline and [35S]sulfate was measured. The effect of 50% static compression on IGF-I transport was determined by measuring the uptake of 125I-labeled IGF-I into cartilage explants. Static compression decreased both [3H]proline and [35S]sulfate incorporation in a dose-dependent manner in the presence or absence of IGF-I. IGF-I increased [3H]proline and [35S]sulfate incorporation in a dose-dependent manner in the presence or absence of compression, but the anabolic effect of the growth factor was lessened when the tissue was compressed by 50%. The response of cartilage explants to IGF-I was similarly lessened in unstrained tissue cultured in media at pH 6.4, a condition which results in a similar intratissue pH to that when cartilage is compressed by 50%. The characteristic time constant (τ) for IGF-I stimulation of cartilage explants was approximately 24 h, while τ for inhibition of biosynthesis by static compression was approximately 2 h. Samples which were both compressed and treated with IGF-I demonstrated an initial decrease in biosynthetic activity at 2 h, followed by an increase at 24 h. Static compression did not alter τ for 125I-labeled IGF-I transport into cartilage but decreased the concentration of 125I-labeled IGF-I in the tissue at equilibrium.

Effects of age on serum dehydroepiandrosterone sulfate, IGF-I, and IL-6 levels in women.

Data from animal and in vitro studies suggest that the growth-promoting effects of the adrenal androgen dehydroepiandrosterone sulfate (DHEAS) may be mediated by stimulation of insulin-like growth factor-I (IGF-I) and/or inhibition of interleukin 6 (IL-6), a cytokine mediator of bone resorption. This study tests the hypotheses that there are effects of age on serum DHEAS, IGF-I, and IL-6 levels, and that levels of IGF-I and IL-6 are related to DHEAS levels. The study included 102 women: 27 premenopausal and 75 postmenopausal, including 35 postmenopausal women with osteoporosis, as defined by bone mineral density scores by dual X-ray energy absorptiometry. DHEAS levels decreased significantly with age (r = -0.52, P < 0.0001) and IGF-I levels decreased significantly with age (r = -0.49, P < 0.0001). IL-6 levels increased significantly with age (r = 0.36, P = 0.008). IGF-I was positively correlated to DHEAS levels (r = 0.43, P < 0. 0001, n = 102) and IL-6 levels were negatively correlated to DHEAS levels (r = -0.32, P = 0.021, n = 54). Levels of DHEAS and IGF-I were correlated with T scores of the spine and some hip sites. In a multiple variable model to predict DHEAS, age was an important predictor (P < 0.001), but osteoporosis status, IGF-I, and IL-6 were not. The median DHEAS level was lower in the postmenopausal osteoporotic women (67 microg/dl, n = 35) than in the nonosteoporotic postmenopausal women (106.3 microg/dl, n = 40, P = 0. 03), but this was not significant after correction for age. Age accounted for 32% of the variance in DHEAS levels. In summary, DHEAS levels decreased with age and had a positive association with IGF-I levels and a negative association with IL-6 levels. DHEA deficiency may contribute to age-related bone loss through anabolic (IGF-I) and anti-osteolytic (IL-6) mechanisms.

The critical role of Shc in insulin-like growth factor-I-mediated mitogenesis and differentiation in 3T3-L1 preadipocytes.

Insulin-like growth factor-I (IGF-I) stimulates mitogenesis in proliferating preadipocytes, but when cells reach confluence and become growth arrested, IGF-I stimulates differentiation into adipocytes. IGF-I induces signaling pathways that involve IGF-I receptor-mediated tyrosine phosphorylation of Shc and insulin receptor substrate 1 (IRS-1). Either of these adaptor proteins can lead to activation of the three-kinase cascade ending in activation of the extracellular signal-regulated kinase 1 and -2 (ERK-1 and -2) mitogen-activated protein kinases (MAPKs). Several lines of evidence suggest that activation of MAPK inhibits 3T3-L1 preadipocyte differentiation. We have shown that IGF-I stimulation of MAPK activity is lost as 3T3-L1 preadipocytes begin to differentiate. This change in MAPK signaling coincides with loss of IGF-I-mediated Shc, but not IRS-1, tyrosine phosphorylation. We hypothesized that down-regulation of MAPK via loss of proximal signaling through Shc is an early component in the IGF-I switch from mitogenesis to differentiation in 3T3-L1 preadipocytes. Treatment of subconfluent cells with the MEK inhibitor PD098059 inhibited both IGF-I-activation of MAPK as well as 3H-thymidine incorporation. PD098059, in the presence of differentiation-inducing media, accelerated differentiation in subconfluent cells as measured by expression of adipocyte protein-2 (aP-2), peroxisome proliferator-activated receptor gamma (PPARgamma) and lipoprotein lipase (LPL). Transient transfection of subconfluent cells with Shc-Y317F, a dominant-negative mutant, attenuated IGF-I-mediated MAPK activation, inhibited DNA synthesis, and accelerated expression of differentiation markers aP-2, PPARgamma, and LPL. We conclude that signaling through Shc to MAPK plays a critical role in mediating IGF-I-stimulated 3T3-L1 mitogenesis. Our results suggest that loss of the ability of IGF-I to activate Shc signaling to MAPK may be an early component of adipogenesis in 3T3-L1 cells.

An Optimal Condition of Bronchial Cell Proliferation Stimulated by Insulin-Like Growth Factor-I

Background: It is known that growth hormones such as insulin-like growth factor-I (IGF-I) and several kinds of cytokines are involved in the regeneration process of injured epithelial cells in chronic respiratory inflammatory diseases such as bronchial asthma. Repetitive degeneration/regeneration processes of the airway epithelial layer is supposed to be responsible for the remodeling and irreversible organic changes of the airway in bronchial asthma. The purpose of this study is to establish a simple and reliable in vitro method for studying airway epithelial cell growth and proliferation using IGF-I. Method: By altering the number of cultured epithelial cells (strain NCI-H₂₉₂), culture duration before stimulation with IGF-I, concentration of IGF-I, and duration of IGF-I stimulation, the optimum conditions for epithelial cell growth was determined. The epithelial cell growth was evaluated using [methyl-³H]thymidine uptake. Result: Among various culture conditions, the epithelial cells cultured at 1 × 10³ cells/well for 24 h followed by 24 h of stimulation by 10^–8 M of IGF-I showed the highest growth. Conclusion: The method for the evaluation of epithelial cell growth established in this study requires a small number of cells and has no complicated procedure. This simple model enables us to investigate the effect of various substances on bronchial epithelial cell growth in the presence of IGF-I.

Role of nitric oxide in the vasodilator effect of recombinant human growth hormone in patients with dilated cardiomyopathy

Dilated cardiomyopathy is characterized by elevated arterial vascular resistance and impaired nitric oxide (NO)-dependent vasodilation. Insulin-like growth factor-I (IGF-I) has been shown to stimulate endothelial NO-synthase resulting in endothelium-dependent vasodilation. Growth hormone (GH) substitution therapy leads in GH-deficient patients to significant increases of IGF-I which may alter systemic vascular resistance by stimulating NO production. This study was designed to evaluate the effects of treatment with recombinant human growth hormone (GH) on NO production and NO-dependent vascular effects in patients with dilated cardiomyopathy. 50 patients with dilated cardiomyopathy were randomly assigned to double-blind treatment with 2 I.U. of GH or placebo for 3 months. Central hemodynamics were determined by Swan-Ganz catheter and cardiac output was obtained by the thermodilution method. Serum GH and IGF-I levels were measured and systemic NO production was determined from urinary nitrate and cyclic GMP excretion rates in 42 patients. GH treatment caused in comparison to the placebo group a significant increase of IGF-I by 91 ng/ml (P = 0.0001). Urinary excretion rates of nitrate and cyclic GMP increased also significantly by 38 mumol/mmol creatinine (P = 0.027) and 65 nmol/mmol creatinine (P = 0.003), respectively. The parallel increase of both marker molecules indicates increased systemic NO production during GH treatment. GH treatment induces a significant, but moderate increase of systemic NO production in patients with dilated cardiomyopathy. This effect may be mediated by IGF-I stimulating endothelial NO synthase.

Phosphatidylinositol 3-kinase is required for insulin-like growth factor-I-induced vascular smooth muscle cell proliferation and migration.

Insulin-like growth factor-I (IGF-I) plays an important role in regulating vascular smooth muscle cell (VSMC) proliferation and directed migration. The mitogenic and chemotactic actions of IGF-I are mediated through the IGF-I receptor, but how the activation of the IGF-I receptor leads to these biological responses is poorly understood. In this study, we examined the role of phosphatidylinositol 3-kinase (PI3 kinase) in mediating the mitogenic and chemotactic signals of IGF-I. IGF-I treatment resulted in a significant increase in phosphotyrosine-associated PI3 kinase activity in cultured primary VSMCs. To determine whether insulin receptor substrate (IRS)-1, -2, or both are involved in IGF-I signaling in VSMCs, cell lysates were immunoprecipitated with either an anti-IRS-1 or an anti-IRS-2 antibody, and the associated PI3 kinase activity was determined. IGF-I stimulation resulted in a significant increase in IRS-1- but not IRS-2-associated PI3 kinase activity, suggesting that IGF-I primarily utilizes IRS-1 to transmit its signal in VSMCs. The IGF-I-induced increase in IRS-I-associated PI3 kinase activity was concentration dependent. At the maximum concentration (50 ng/mL), IGF-I induced a 60-fold increase. This activation occurred within 5 minutes and was sustained at high levels for at least 6 hours. IGF-I also caused a concentration-dependent and long-lasting activation of protein kinase B (PKB/Akt). Inhibition of PI3 kinase activation by LY294002 or wortmannin abolished IGF-I-stimulated VSMC proliferation and reduced IGF-I-directed VSMC migration by approximately 60%. These results indicate that activation of PI3 kinase is required for both IGF-I-induced VSMC proliferation and migration.

Lycopene Interferes With Cell Cycle Progression and Insulin-Like Growth Factor I Signaling in Mammary Cancer Cells

Recent studies have shown that high insulin-like growth factor I (IGF-I) blood level is a risk factor in breast and prostate cancer. The aim of this study was to determine whether the mitogenic activity of IGF-I in mammary cancer cells can be reduced by the dietary carotenoid lycopene. The anticancer activity of lycopene, the major tomato carotenoid, has been suggested by in vitro, in vivo, and epidemiological studies. Growth stimulation of MCF7 mammary cancer cells by IGF-I was markedly reduced by physiological concentrations of lycopene. The inhibitory effects of lycopene on MCF7 cell growth were not accompanied by apoptotic or necrotic cell death, as determined by annexin V binding to plasma membrane and propidium iodide staining of nuclei in unfixed cells. Lycopene treatment markedly reduced the IGF-I stimulation of tyrosine phosphorylation of insulin receptor substrate 1 and binding capacity of the AP-1 transcription complex. These effects were not associated with changes in the number or affinity of IGF-I receptors, but with an increase in membrane-associated IGF-binding proteins, which were previously shown in different cancer cells to negatively regulate IGF-I receptor activation. The inhibitory effect of lycopene on IGF signaling was associated with suppression of IGF-stimulated cell cycle progression of serum-starved, synchronized cells. Moreover, in cells synchronized by mimosine treatment, lycopene delayed cell cycle progression after release from the mimosine block. Collectively, the above data suggest that the inhibitory effects of lycopene on MCF7 cell growth are not due to the toxicity of the carotenoid but, rather, to interference in IGF-I receptor signaling and cell cycle progression.