Human Insulin-like Growth factor-I Receptor Research Articles

Lessons Learned from Targeting IGF-I Receptor in Thyroid-Associated Ophthalmopathy.

Complex immunological mechanisms underlie the pathogenesis of thyroid-associated ophthalmopathy (TAO). Historical models of Graves’ disease and TAO have focused almost entirely on autoimmune reactivity directed against the thyrotropin receptor (TSHR). The insulin-like growth factor-I receptor (IGF-IR) has been proposed as a second participating antigen in TAO by virtue of its interactions with IGFs and anti-IGF-IR antibodies generated in Graves’ disease. Furthermore, the IGF-IR forms with TSHR a physical and functional complex which is involved in signaling downstream from both receptors. Inhibition of IGF-IR activity results in attenuation of signaling initiated at either receptor. Based on the aggregate of findings implicating IGF-IR in TAO, the receptor has become an attractive therapeutic target. Recently, teprotumumab, a human monoclonal antibody IGF-IR inhibitor was evaluated in two clinical trials of patients with moderate to severe, active TAO. Those studies revealed that teprotumumab was safe and highly effective in reducing disease activity and severity. Targeting IGF-IR with specific biologic agents may result in a paradigm shift in the therapy of TAO.

Teprotumumab for the treatment of thyroid eye disease

ABSTRACT Introduction Thyroid eye disease is a debilitating, disfiguring, and potentially blinding periocular condition. Teprotumumab is a human insulin-like growth factor-I receptor monoclonal inhibitor antibody which indicated for treating thyroid eye disease. Areas covered The authors performed a systematic review of the literature using the PubMed database, and the following keywords were used: ‘teprotumumab,’ ‘thyroid eye disease,’ and ‘insulin-like growth factor I receptor.’ The chemical property, mechanism of action, pharmacokinetics, clinical efficacy, and safety of teprotumumab were introduced in this paper. Expert opinion Teprotumumab is a human monoclonal antibody targeting insulin-like growth factor-I receptor. Clinical trials indicated that proptosis response of teprotumumab was 83%, and clinical activity score, diplopia, and quality of life were also better than placebo. Teprotumumab was well tolerated, common adverse reactions included muscle spasm, nausea, alopecia, diarrhea, fatigue, hyperglycemia, hearing impairment, dysgeusia, headache, and dry skin.

Potency of Full-Length MGF to Induce Maximal Activation of the IGF-I R Is Similar to Recombinant Human IGF-I at High Equimolar Concentrations.

AimsTo compare full-length mechano growth factor (full-length MGF) with human recombinant insulin-like growth factor-I (IGF-I) and human recombinant insulin (HI) in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor (IR-A) and the human insulin receptor-B (IR-B), respectively. In addition, we tested the stimulatory activity of human MGF and its stabilized analog Goldspink-MGF on the IGF-IR.MethodsThe effects of full-length MGF, IGF-I, human mechano growth factor (MGF), Goldspink-MGF and HI were compared using kinase specific receptor activation (KIRA) bioassays specific for IGF-I, IR-A or IR-B, respectively. These assays quantify activity by measuring auto-phosphorylation of the receptor upon ligand binding.ResultsIGF-IR: At high equimolar concentrations maximal IGF-IR stimulating effects generated by full-length MGF were similar to that of IGF-I (89-fold vs. 77-fold, respectively). However, EC50 values of IGF-I and full-length MGF for the IGF-I receptor were 0.86 nmol/L (95% CI 0.69–1.07) and 7.83 nmol/L (95% CI: 4.87–12.58), respectively. No IGF-IR activation was observed by human MGF and Goldspink-MGF, respectively. IR-A/IR-B: At high equimolar concentrations similar maximal IR-A stimulating effects were observed for full -length MGF and HI, but maximal IR-B stimulation achieved by full -length MGF was stronger than that by HI (292-fold vs. 98-fold). EC50 values of HI and full-length MGF for the IR-A were 1.13 nmol/L (95% CI 0.69–1.84) and 73.11 nmol/L (42.87–124.69), respectively; for IR-B these values were 1.28 nmol/L (95% CI 0.64–2.57) and 35.10 nmol/L (95% 17.52–70.33), respectively.ConclusionsFull-length MGF directly stimulates the IGF-IR. Despite a higher EC50 concentration, at high equimolar concentrations full-length MGF showed a similar maximal potency to activate the IGF-IR as compared to IGF-I. Further research is needed to understand the actions of full-length MGF in vivo and to define the physiological relevance of our in vitro findings.

Novel cross talk between IGF-IR and DDR1 regulates IGF-IR trafficking, signaling and biological responses.

The insulin-like growth factor-I receptor (IGF-IR), plays a key role in regulating mammalian development and growth, and is frequently deregulated in cancer contributing to tumor initiation and progression. Discoidin domain receptor 1 (DDR1), a collagen receptor tyrosine-kinase, is as well frequently overexpressed in cancer and implicated in cancer progression. Thus, we investigated whether a functional cross-talk between the IGF-IR and DDR1 exists and plays any role in cancer progression.Using human breast cancer cells we found that DDR1 constitutively associated with the IGF-IR. However, this interaction was enhanced by IGF-I stimulation, which promoted rapid DDR1 tyrosine-phosphorylation and co-internalization with the IGF-IR. Significantly, DDR1 was critical for IGF-IR endocytosis and trafficking into early endosomes, IGF-IR protein expression and IGF-I intracellular signaling and biological effects, including cell proliferation, migration and colony formation. These biological responses were inhibited by DDR1 silencing and enhanced by DDR1 overexpression.Experiments in mouse fibroblasts co-transfected with the human IGF-IR and DDR1 gave similar results and indicated that, in the absence of IGF-IR, collagen-dependent phosphorylation of DDR1 is impaired.These results demonstrate a critical role of DDR1 in the regulation of IGF-IR action, and identify DDR1 as a novel important target for breast cancers that overexpress IGF-IR.

Phosphatidylinositol 3-Kinase (PI3K) Activity Bound to Insulin-like Growth Factor-I (IGF-I) Receptor, which Is Continuously Sustained by IGF-I Stimulation, Is Required for IGF-I-induced Cell Proliferation

Continuous stimulation of cells with insulin-like growth factors (IGFs) in G(1) phase is a well established requirement for IGF-induced cell proliferation; however, the molecular components of this prolonged signaling pathway that is essential for cell cycle progression from G(1) to S phase are unclear. IGF-I activates IGF-I receptor (IGF-IR) tyrosine kinase, followed by phosphorylation of substrates such as insulin receptor substrates (IRS) leading to binding of signaling molecules containing SH2 domains, including phosphatidylinositol 3-kinase (PI3K) to IRS and activation of the downstream signaling pathways. In this study, we found prolonged (>9 h) association of PI3K with IGF-IR induced by IGF-I stimulation. PI3K activity was present in this complex in thyrocytes and fibroblasts, although tyrosine phosphorylation of IRS was not yet evident after 9 h of IGF-I stimulation. IGF-I withdrawal in mid-G(1) phase impaired the association of PI3K with IGF-IR and suppressed DNA synthesis the same as when PI3K inhibitor was added. Furthermore, we demonstrated that Tyr(1316)-X-X-Met of IGF-IR functioned as a PI3K binding sequence when this tyrosine is phosphorylated. We then analyzed IGF signaling and proliferation of IGF-IR(-/-) fibroblasts expressing exogenous mutant IGF-IR in which Tyr(1316) was substituted with Phe (Y1316F). In these cells, IGF-I stimulation induced tyrosine phosphorylation of IGF-IR and IRS-1/2, but mutated IGF-IR failed to bind PI3K and to induce maximal phosphorylation of GSK3β and cell proliferation in response to IGF-I. Based on these results, we concluded that PI3K activity bound to IGF-IR, which is continuously sustained by IGF-I stimulation, is required for IGF-I-induced cell proliferation.

727 ANTISENSE OLIGONUCLEOTIDE TARGETING INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR ENHANCES PACLITAXEL SENSITIVITY IN A PACLITAXEL- RESISTANT PROSTATE CANCER MODEL

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011727 ANTISENSE OLIGONUCLEOTIDE TARGETING INSULIN-LIKE GROWTH FACTOR-1 RECEPTOR ENHANCES PACLITAXEL SENSITIVITY IN A PACLITAXEL- RESISTANT PROSTATE CANCER MODEL Junya Furukawa, Hideaki Miyake, Masato Fujisawa, Christopher Wraight, Brett Monia, Martin Gleave, and Michael Cox Junya FurukawaJunya Furukawa Kobe, Japan More articles by this author , Hideaki MiyakeHideaki Miyake Kobe, Japan More articles by this author , Masato FujisawaMasato Fujisawa Kobe, Japan More articles by this author , Christopher WraightChristopher Wraight Victoria, Australia More articles by this author , Brett MoniaBrett Monia Carlsbad, CA More articles by this author , Martin GleaveMartin Gleave Vancouver, Canada More articles by this author , and Michael CoxMichael Cox Vancouver, Canada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.1696AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Altered expression of insulin-like growth factor (IGF) axis components have been consistently found in prostate cancer (PCa) and associated with adaptive growth and survival signaling implicated in castrate-resistant disease (CRPC) progression. These activities are mediated by activation of the IGF-I receptor (IGF-IR). While CRPC patients treated with taxanes alone or in combination with estramustine phosphate or predonisone show a survival benefit, development of chemotherapeutic resistance eventually occurs. Therefore novel strategies targeting the molecular basis of androgen deprivation therapy and chemotherapy resistance are required. In this study, we establish paclitaxel-resistant androgen-independent PCa cells (PtxR PC3) and assess the potency and anti-cancer activity of a 2 EMOE-modified antisense oligonucleotide (ASO) targeting human IGF-IR, ATL1101, on PtxR PC3 cells in vitro and in vivo. METHODS PtxR PC3 cells were established by culturing in step-wise increased drug concentrations. IGF-IR mRNA and protein expression in ATL1101- and control oligonucleotides (ODN)-treated PtxR PC3 cells were measured by QT-PCR and immunoblotting. The effect of IGF-1R ASO and paclitaxel combination therapy on PtxR PC3 cell growth and apoptosis in vitro was examined by crystal violet assay and flow cytometry. In vitro combination index (CI) was calculated by Calcusyn® softoware. For in vivo xenograft studies, PtxR PC-3 cells were inoculated s.c. in the flanks of athymic nude mice and tumor volume kinetics were compared for mice treated with paclitaxel injected i.v. and either ATL1101 or control ODN injected i.p. RESULTS The IC50 for paclitaxel in PtxR PC3 was 15-fold higher than that in parental cells. PtxR PC3 also shows cross-resistance to docetaxel and mitoxantrone. We observed dose- and sequence-specific suppression of IGF-IR mRNA and protein expression in ATL1101-treated PtxR PC3 cells in vitro which correlated with decreased proliferation and increased apoptosis of PtxR PC3 cells. Combination of ATL1101 with paclitaxel showed CI values below 1 at the IC50, IC75, suggesting the drug interactions resulted in synergy. Compared to control ODN, ATL1101 significantly suppressed PtxR PC3 tumor growth as a combination therapy with paclitaxel in murine xenografts. CONCLUSIONS This study reports the first preclinical proof-of-principle data that this novel IGF-IR ASO suppresses growth of paclitaxel resistant PC tumors, synergistically enhancing paclitaxel sensitivity in vitro and in vivo. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e292 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Junya Furukawa Kobe, Japan More articles by this author Hideaki Miyake Kobe, Japan More articles by this author Masato Fujisawa Kobe, Japan More articles by this author Christopher Wraight Victoria, Australia More articles by this author Brett Monia Carlsbad, CA More articles by this author Martin Gleave Vancouver, Canada More articles by this author Michael Cox Vancouver, Canada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

The Role of GH and IGF-I in Mediating Anabolic Effects of Testosterone on Androgen-Responsive Muscle

Testosterone (T) supplementation increases skeletal muscle mass, circulating GH, IGF-I, and im IGF-I expression, but the role of GH and IGF-I in mediating T's effects on the skeletal muscle remains poorly understood. Here, we show that T administration increased body weight and the mass of the androgen-dependent levator ani muscle in hypophysectomized as well as castrated plus hypophysectomized adult male rats. T stimulated the proliferation of primary human skeletal muscle cells (hSKMCs) in vitro, an effect blocked by transfecting hSKMCs with small interference RNA targeting human IGF-I receptor (IGF-IR). In differentiation conditions, T promoted the fusion of hSKMCs into larger myotubes, an effect attenuated by small interference RNA targeting human IGF-IR. Notably, MKR mice, which express a dominant negative form of the IGF-IR in skeletal muscle fibers, treated with a GnRH antagonist (acyline) to suppress endogenous T, responded to T administration by an attenuated increase in the levator ani muscle mass. In conclusion, circulating GH and IGF-I are not essential for mediating T's effects on an androgen-responsive skeletal muscle. IGF-I signaling plays an important role in mediating T's effects on skeletal muscle progenitor cell growth and differentiation in vitro. However, IGF-IR signaling in skeletal muscle fibers does not appear to be obligatory for mediating the anabolic effects of T on the mass of androgen-responsive skeletal muscles in mice.

Insulin glargine is more potent in activating the human IGF-I receptor than human insulin and insulin detemir

Objective To investigate whether human insulin (HI) and insulin analogues differ in their ability to activate the human IGF-I receptor (IGF-IR), the human insulin receptor A (IR-A) and the human insulin receptor B (IR-B) in vitro. Methods HI, short-acting insulin analogues (insulin aspart; insulin lispro) and long-acting insulin analogues (insulin glargine; insulin detemir) were compared by using kinase receptor activation (KIRA) bioassays specific for IGF-IR, IR-A or IR-B, respectively. These assays quantify ligand activity by measuring receptor auto-phosphorylation upon ligand binding. HI and insulin analogues were tested in a range from 0.1 to 100 nM. Results Short-acting analogues: Overall, short-acting insulin analogues did not differ substantially from HI, nor from each other. Insulin lispro was slightly more potent than HI and insulin aspart in activating the IGF-IR, only reaching statistical significance at 100 nM (p < 0.01). Long-acting analogues: At < 10 nM insulin glargine was as potent as HI in activating the IRs and IGF-IR. At 10–100 nM insulin glargine was significantly more potent than HI in activating the IR-B (p < 0.05) and IGF-IR (p < 0.001). Insulin glargine was more potent than insulin detemir in activating all three receptors (p < 0.001). Insulin detemir was less potent than HI in activating the IRs at 1–10 nM (p < 0.01) and IGF-IR at > 1 nM (p < 0.05). Conclusions Insulin glargine was more potent in activating the IGF-IR than HI and insulin detemir. Since KIRA bioassays do not mimic the exact in vivo situation, further research is needed to find out whether our data have implications for clinical use of insulin glargine.

Abstract 5376: Antisense oligonucleotide targeting insulin-like growth factor-1 receptor (IGF-1R) enhances paclitaxel sensitivity in a castrate-resistant and paclitaxel-resistant prostate cancer model

Abstract While prostate cancer (PCa) initially responds to androgen deprivation therapy (ADT), castrate-resistant disease (CRPC) relapse is inevitable. Altered expression of insulin-like growth factor (IGF) axis components have been consistently found in PCa and associated with adaptive growth and survival signaling implicated in CRPC progression. These activities are mediated by activation of the IGF-I receptor (IGF-IR). While CRPC patients treated with taxanes (paclitaxel or docetaxel) alone or in combination with estramustine phosphate or predonisone show a survival benefit, development of chemotherapeutic resistance eventually occurs. Therefore novel strategies targeting the molecular basis of ADT and chemotherapy resistance are required. In this study, we assess the potency and anti-cancer activity of a 2′-MOE-modified antisense oligonucleotide (ASO) targeting human IGF-IR, ATL1101, on the CRPC cell line, PC3 and a paclitaxel-resistant androgen-independent PC3-derived cell line, PtxR PC3, in vitro and in vivo. PtxR PC3 cells were established by culturing in step-wise increased drug concentrations. IGF-IR mRNA and protein expression in ATL1101- and control oligonucleotides (ODN)-treated PtxR PC3 cells were measured by QT-PCR and immunoblotting. The effect of IGF-1R ASO and paclitaxel combination therapy on parental and PtxR PC3 cell growth and apoptosis in vitro was examined by crystal violet assay and flow cytometry. In vitro combination index (CI) was calculated by Calcusyn® softoware. For xenograft studies, parental and PtxR PC-3 cells were inoculated s.c. in the flanks of athymic male nude mice and tumor volume kinetics were compared for mice treated with paclitaxel injected i.v. and either ATL1101 or control ODN injected i.p. In vitro, the IC50 for paclitaxel in PtxR PC3 was 20-fold higher than that in parental cells. PtxR PC3 also show significant cross-resistance to docetaxel and mitoxantrone. We observed equivalent dose- and sequence-specific suppression of IGF-IR mRNA and protein expression, and comparable decreased proliferation and increased apoptosis in ATL1101-treated parental and PtxR PC3 cells in vitro. Combination of ATL1101 with paclitaxel showed CI values below 1 at the IC50, IC75, IC90, suggesting the drug interactions resulted in synergy. In vivo, ATL1101 significantly increased sensitivity of PtxR PC3 tumors to combination therapy with paclitaxel as compared to control ODN in murine xenografts. This study reports the first preclinical proof-of-principle data that this novel IGF-IR ASO suppresses growth of paclitaxel resistant PC tumors, synergistically enhancing paclitaxel sensitivity in vitro and in vivo. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 5376.

A Phase I Study of Weekly R1507, A Human Monoclonal Antibody Insulin-like Growth Factor-I Receptor Antagonist, in Patients with Advanced Solid Tumors

A phase I study was conducted to evaluate the pharmacokinetics, pharmacodynamics, safety, and tolerability of R1507-a fully human IgG1 type monoclonal antibody directed against the human insulin-like growth factor-I receptor. Patients with advanced solid tumors were assigned to receive i.v. R1507 weekly (qW), starting with 1 mg/kg. Subsequent cohorts were dosed at 3 and then 9 mg/kg. An additional 12 patients received 9 mg/kg R1507 qW. Patients remained on the study until the development of a dose-limiting toxicity or progressive disease. In total, 37 patients were treated with R1507 qW. No dose-limiting toxicities were identified and the maximum tolerated dose was not reached. The pharmacokinetics of R1507 were characterized by a slow clearance and limited volume of distribution, with an estimated elimination half-life justifying weekly administration. Serum IGF-I ligand levels increased proportionally to dose during the first 72 hours in all cohorts. R1507 was well tolerated. Two patients diagnosed with Ewing's sarcoma had partial responses of 11.5 and >26 months (ongoing at time of submission); 13 patients had stable disease; and 16 had progressive disease as best response by the Response Evaluation Criteria in Solid Tumors. R1507 is well tolerated and shows antitumor activity in patients with solid neoplasms, in particular Ewing's sarcoma. The recommended dose for the weekly schedule is 9 mg/kg qW.

A Fully Human Insulin-Like Growth Factor-I Receptor Antibody SCH 717454 (Robatumumab) Has Antitumor Activity as a Single Agent and in Combination with Cytotoxics in Pediatric Tumor Xenografts

The insulin-like growth factor-I receptor (IGF-IR) and its ligands (IGF-I and IGF-II) have been implicated in the growth, survival, and metastasis of a broad range of malignancies including pediatric tumors. Blocking the IGF-IR action is a potential cancer treatment. A fully human neutralizing monoclonal antibody, SCH 717454 (19D12, robatumumab), specific to IGF-IR, has shown potent antitumor effects in ovarian cancer in vitro and in vivo. In this study, SCH 717454 was evaluated in several pediatric solid tumors including neuroblastoma, osteosarcoma, and rhabdomyosarcoma. SCH 717454 is shown here to downregulate IGF-IR as well as inhibit IGF-IR and insulin receptor substrate-1 phosphorylation in pediatric tumor cells. IGF-IR and insulin receptor substrate-1 phosphorylation in the tumor cells. In vivo, SCH 717454 exhibits activity as a single agent and significantly inhibited growth of neuroblastoma, osteosarcoma, and rhabdomyosarcoma tumor xenografts. Combination of SCH 717454 with cisplatin or cyclophosphamide enhanced both the degree and the duration of the in vivo antitumor activity compared with single-agent treatments. Furthermore, SCH 717454 treatment markedly reduced Ki-67 expression and blood vessel formation in tumor xenografts, showing that the in vivo activity is derived from its inhibition of tumor cell proliferation and angiogenesis activity.

Insulin-Mediated Acceleration of Breast Cancer Development and Progression in a Nonobese Model of Type 2 Diabetes

Epidemiologic studies suggest that type 2 diabetes (T2D) increases breast cancer risk and mortality, but there is limited experimental evidence supporting this association. Moreover, there has not been any definition of a pathophysiological pathway that diabetes may use to promote tumorigenesis. In the present study, we used the MKR mouse model of T2D to investigate molecular mechanisms that link T2D to breast cancer development and progression. MKR mice harbor a transgene encoding a dominant-negative, kinase-dead human insulin-like growth factor-I receptor (IGF-IR) that is expressed exclusively in skeletal muscle, where it acts to inactivate endogenous insulin receptor (IR) and IGF-IR. Although lean female MKR mice are insulin resistant and glucose intolerant, displaying accelerated mammary gland development and enhanced phosphorylation of IR/IGF-IR and Akt in mammary tissue, in the context of three different mouse models of breast cancer, these metabolic abnormalities were found to accelerate the development of hyperplastic precancerous lesions. Normal or malignant mammary tissue isolated from these mice exhibited increased phosphorylation of IR/IGF-IR and Akt, whereas extracellular signal-regulated kinase 1/2 phosphorylation was largely unaffected. Tumor-promoting effects of T2D in the models were reversed by pharmacological blockade of IR/IGF-IR signaling by the small-molecule tyrosine kinase inhibitor BMS-536924. Our findings offer compelling experimental evidence that T2D accelerates mammary gland development and carcinogenesis,and that the IR and/or the IGF-IR are major mediators of these effects.

Transforming Growth Factor-β–Stimulated Clone-22 Is an Androgen-Regulated Gene That Enhances Apoptosis in Prostate Cancer following Insulin-Like Growth Factor-I Receptor Inhibition

PURPOSE: Inhibition of insulin-like growth factor (IGF) signaling using the human IGF-I receptor monoclonal antibody A12 is most effective at inducing apoptosis in prostate cancer xenografts in the presence of androgen. We undertook this study to determine mechanisms for increased apoptosis by A12 in the presence of androgens. Experimental Methods: The castrate-resistant human xenograft LuCaP 35 V was implanted into intact or castrate severe combined immunodeficient mice and treated with A12 weekly. After 6 weeks of tumor growth, animals were sacrificed and tumors were removed and analyzed for cell cycle distribution/apoptosis and cDNA arrays were done. RESULTS: In castrate mice, the tumors were delayed in G(2) with no apoptosis; in contrast, tumors from intact mice underwent apoptosis with either G(1) or G(2) delay. Transforming growth factor-beta-stimulated clone-22 (TSC-22) was significantly elevated in tumors from the intact mice compared with castrate mice, especially in those tumors with the highest levels of apoptosis. To further determine the function of TSC-22, we transfected various human prostate cancer cell lines with a plasmid expressing TSC-22. Cell lines overexpressing TSC-22 showed an increase in apoptosis and a delay in G(1). When these cell lines were placed subcutaneously in athymic nude mice, a decreased number of animals formed tumors and the rate of tumor growth was decreased compared with control tumors. CONCLUSIONS: These data indicate that IGF-I receptor inhibition in the presence of androgen has an enhanced effect on decreasing tumor growth, in part, through increased expression of the tumor suppressor gene TSC-22. (Clin Cancer Res 2009;15(24):7634-41).

Alanine Scanning of a Putative Receptor Binding Surface of Insulin-like Growth Factor-I

Current evidence supports a binding model in which the insulin molecule contains two binding surfaces, site 1 and site 2, which contact the two halves of the insulin receptor. The interaction of these two surfaces with the insulin receptor results in a high affinity cross-linking of the two receptor alpha subunits and leads to receptor activation. Evidence suggests that insulin-like growth factor-I (IGF-I) may activate the IGF-I receptor in a similar mode. So far IGF-I residues structurally corresponding to the residues of the insulin site 1 together with residues in the C-domain of IGF-I have been found to be important for binding of IGF-I to the IGF-I receptor (e.g. Phe(23), Tyr(24), Tyr(31), Arg(36), Arg(37), Val(44), Tyr(60), and Ala(62)). However, an IGF-I second binding surface similar to site 2 of insulin has not been identified yet. In this study, we have analyzed whether IGF-I residues corresponding to the six residues of the insulin site 2 have a role in high affinity binding of IGF-I to the IGF-I receptor. Six single-substituted IGF-I analogues were produced, each containing an alanine substitution in one of the following positions (corresponding insulin residues in parentheses): Glu(9) (His(B10)), Asp(12) (Glu(B13)), Phe(16) (Leu(B17)), Asp(53) (Ser(A12)), Leu(54) (Leu(A13)), and Glu(58) (Glu(A17)). In addition, two analogues with 2 and 3 combined alanine substitutions were also produced (E9A,D12A IGF-I and E9A,D12A,E58A IGF-I). The results show that introducing alanine in positions Glu(9), Asp(12), Phe(16), Leu(54), and Glu(58) results in a significant reduction in IGF-I receptor binding affinity, whereas alanine substitution at position 53 had no effect on IGF-I receptor binding. The multiple substitutions resulted in a 33-100-fold reduction in IGF-I receptor binding affinity. These data suggest that IGF-I, in addition to the C-domain, uses surfaces similar to those of insulin in contacting its cognate receptor, although the relative contribution of the side chains of homologous residues varies.

Lectin-induced alterations of the interaction of insulin and insulin-like growth factor 1 receptors with their ligands

In order to study whether the carbohydrate moieties of the human placental IGF-I receptor (IGF1R), IGF-II receptor (IGF2R) and insulin receptors (IRs) play a role in ligand binding, solubilised cell membrane preparations were incubated with 125I-labelled IGF-I, IGF-II and insulin in the presence of lectins with different sugar specificities. Three incubation procedures were tested: ligand-first, co-incubation and lectin-first incubation. Wheat germ agglutinin (WGA), concanavalin A (Con A) and phytohaemagglutinin (PHA) altered the binding of IGF-I and insulin to their high-affinity receptors in a lectin specific and dose-dependent manner, whereas these lectins did not affect the interaction of IGF-II with its receptor(s). Moreover, the same lectins either inhibited or enhanced IGF-I and insulin binding, depending on the incubation scheme. These results also suggest that IR-A and IR-B from human placenta might be differently glycosylated.

Recombinant Human Insulin-Like Growth Factor-I Treatment Inhibits Gluconeogenesis in a Transgenic Mouse Model of Type 2 Diabetes Mellitus

IGF-I and insulin are structurally related polypeptides that mediate a similar pattern of biological effects via receptors that display considerably homology. Administration of recombinant human IGF-I (rhIGF-I) has been proven to improve glucose control and liver and muscle insulin sensitivity in patients with type 2 diabetes mellitus (DM). The effect of rhIGF-I treatment was evaluated in a mouse model of type 2 DM (MKR mouse), which expresses a dominant-negative form of the human IGF-I receptor under the control of the muscle creatine kinase promoter specifically in skeletal muscle. MKR mice have impaired IGF-I and insulin signaling in skeletal muscle, leading to severe insulin resistance in muscle, liver, and fat, developing type 2 DM at 5 wk of age. Six-week-old MKR mice were treated with either saline or rhIGF-I for 3 wk. Blood glucose levels were decreased in response to rhIGF-I treatment in MKR mice. rhIGF-I treatment also increased body weight in MKR with concomitant changes in body composition such as a decrease in fat mass and an increase in lean body mass. Insulin, fatty acid, and triglyceride levels were not affected by rhIGF-I, nor were insulin or glucose tolerance in MKR mice. Hyperinsulinemic-euglycemic clamp analysis demonstrated no improvement in overall insulin sensitivity. Pyruvate and glutamine tolerance tests proved that there was a decrease in the rate of glucose appearance in MKR mice treated with rhIGF-I, suggesting a reduction in the gluconeogenic capacity of liver, kidney, and small intestine. Taken together these results demonstrate that the improvement of the hyperglycemia was achieved by inhibition of gluconeogenesis rather than an improvement in insulin sensitivity. Also, these results suggest that a functional IGF-I receptor in skeletal muscle is required for IGF-I to improve insulin sensitivity in this mouse model of type 2 DM.

Ectodomain shedding-dependent transactivation of epidermal growth factor receptors in response to insulin-like growth factor type I.

Diverse extracellular stimuli activate the ERK1/2 MAPK cascade by transactivating epidermal growth factor (EGF) receptors. Here, we have examined the role of EGF receptors in IGF-I-stimulated ERK1/2 activation in several cultured cell lines. In human embryonic kidney 293 cells, IGF-I triggered proteolysis of heparin binding (HB)-EGF, increased tyrosine autophosphorylation of EGF receptors, stimulated EGF receptor inhibitor (AG1478)-sensitive ERK1/2 phosphorylation, and promoted EGF receptor endocytosis. In a mixed culture system that employed IGF-I receptor null murine embryo fibroblasts (MEFs) (R(-) cells) to detect paracrine signals produced by MEFs expressing the human IGF-I receptor (R(+) cells), stimulation of R(+) cells provoked rapid activation of green fluorescent protein-tagged ERK2 in cocultured R(-) cells. The R(-) cell response was abolished by either the broad-spectrum matrix metalloprotease inhibitor batimastat or by AG1478, indicating that it resulted from the proteolytic generation of an EGF receptor ligand from adjacent R(+) cells. These data suggest that the paracrine production of EGF receptor ligands leading to EGF receptor transactivation is a general property of IGF-I receptor signaling. In contrast, the contribution of transactivated EGF receptors to IGF-I-stimulated downstream events, such as ERK1/2 activation, varies in a cell type-dependent manner.

TDAG51 Mediates the Effects of Insulin-like Growth Factor I (IGF-I) on Cell Survival

Insulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I.

Activation of the phosphatidylinositol-3' kinase pathway and DNA synthesis by a mutant insulin-like growth factor I receptor lacking the NPXY motif.

We have investigated the role of the NPXY motif in the insulin-like growth factor I receptor (IGF-IR) by focusing on the activation of the phosphatidylinositol-3' kinase (PI3-K) pathway and DNA synthesis following IGF-I stimulation. For this purpose, we established stable R-cell lines, which are deficient in endogenous IGF-IR, and express human IGF-IR lacking the whole NPEY(950) sequence (DeltaNPEY). The DeltaNPEY cells showed an apparent autophosphorylation of IGF-IR, albeit with reduced sensitivity to stimulation compared with cells expressing similar levels of wild-type IGF-IR. Activation of insulin receptor substrate (IRS)-1 and IRS-2 was severely impaired in DeltaNPEY cells even at high concentrations of IGF-I. However, recruitment of p85, a regulatory subunit of PI3-K, to activated IRS-2 was similar between the cell lines, but recruitment of p85 to IRS-1 was reduced in DeltaNPEY cells. Essentially similar levels of p85- or phosphotyrosine-associated PI3-K and Akt activities were observed between the cell lines, although the sensitivity to stimulation was reduced in DeltaNPEY cells. Activation of extracellular signal-regulated kinase and DNA synthesis were virtually unaffected by the mutation, in terms of both sensitivity to stimulation and responsiveness. DNA synthesis was completely inhibited by the PI3-K inhibitor, LY294002. These results indicate that the IGF-IR is able to activate the PI3-K pathway and induce DNA synthesis in a normal fashion without the NPXY motif when the receptor is fully activated.

Insulin-like growth factor I receptor does not contribute to heat shock-induced Activation of Akt and extracellular signal-regulated kinase (ERK) in mouse embryo fibroblasts.

We have investigated the role of insulin-like growth factor I receptor (IGF-IR) in heat shock-induced activation of the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3 ' kinase (PI3-K) pathways. We utilized mouse embryo fibroblasts (MEFs) devoid of endogenous IGF-IR (R-) and MEFs overexpressing human IGF-IR (WT) and examined the activation kinetics of extracellular signal-regulated kinase (ERK) and Akt following heat shock treatment. There were no differences in the kinetics or temperature dependence of activation of either ERK or Akt between the cell lines. As expected, heat shock failed to induce autophosphorylation of IGF-IR overexpressed in WT cells. Surprisingly, the autophosphorylation of endogenous epidermal growth factor receptor (EGFR), which is thought to play an important role in heat shock-induced activation of the MAPK and PI3-K pathways, was not observed in either WT or R-cells. These results suggest that neither IGF-IR nor EGFR contributes to the heat shock -induced activation of ERK and Akt in these cell lines.