The Insulin-Like Growth Factor System and Neurological Complications in Diabetes

The IGF system plays vital roles in neuronal development, metabolism, regeneration and survival. It consists of IGF-I, IGF-II, insulin, IGF-I-receptor, and those of IGF-II and insulin as well as IGF-binding proteins. In the last decades it has become clear that perturbations of the IGF system play important roles in the pathogenesis of diabetic neurological complications. In the peripheral nervous system IGF-I, insulin, and C-peptide particularly in type 1 diabetes participate in the development of axonal degenerative changes and contributes to impaired regenerative capacities. These abnormalities of the IGF system appear to be less pronounced in type 2 diabetes, which may in part account for the relatively milder neurological complications in this type of diabetes. The members of the IGF system also provide anti-apoptotic effects on both peripheral and central nervous system neurons. Furthermore, both insulin and C-peptide and probably IGF-I possess gene regulatory capacities on myelin constituents and axonal cytoskeletal proteins. Therefore, replenishment of various members of the IGF system provides a reasonable rational for prevention and treatment of diabetic neurological complications.     

Insulin-Like Growth Factor I/Somatomedin-C: A Rapid Isolation Procedure with FPLC

Insulin-like growth factor I (IGF l)/somatomedin-C (SM-C) was purified from lyophilized human serum by acid-ethanol extraction. The extract was precipitated with acetone-ethanol. The precipitate was purified by Sephadex G-50 chromatography. The protein peak within a molecular weight range of 5000 – 10 000 was further purified with FPLC-reversed phase chromatography using a Pep RPC HR 5/5 column (Pharmacia) with a solvent system of acetonitrile (CH₃ CN) and 0.1% trifluoroacetic acid (TFA) in water. The purification of IGF I was monitored by radioimmunoassay for SM-C. Purity was established by analytical isoelectric focusing and by SDS poly-acrylamide gel electrophoresis. Analytical isoelectric focusing showed one single protein band with an apparent pi of 8.3 0.1. SDS polyacryl-amide gel electrophoresis showed also one single protein band with an apparent molecular weight of 7000. Biological activity was demonstrated by measureing the (³H)thymidine incorporation into DNA of cultured arterial smooth muscle cells.     

Demonstration of Type I Insulin-Like Growth Factor Receptors on Human Platelets

Abstract

Human platelets, freshly isolated from healthy human adults, express receptors for insulin-like growth factor I. The IC₅₀ for displacement of ¹²⁵I-IGF-I binding by unlabeled IGF-I was 0.2 nM, by IGF-II 32 nM and by insulin 160 nM. Scatchard analysis of IGF-I binding demonstrates dissociation constants of 0.14 ± 0.08 nM for high affinity binding site and 54 ± 18 nM for low affinty binding site. The presence of the α-subunit of type I IGF receptor, as high affinity binding site, was verified by affinity crosslinking of ¹²⁵I-IGF-I to platelet surface membranes. Under reducing con-conditions a M_r= 135,000 band was preferentially labeled. The complete type I IGF receptor complex, which revealed under nonreducing conditions, has an approximately molecular mass of M_r > 400,000. The immunoprecipitation of the ¹²⁵I-IGF-I cross-linked type I receptor with αIR-3 confirmed the results achieved by affinity crosslinking.     

Modeling the Insulin-Like Growth Factor System in Articular Cartilage

IGF signaling is involved in cell proliferation, differentiation and apoptosis in a wide range of tissues, both normal and diseased, and so IGF-IR has been the focus of intense interest as a promising drug target. In this computational study on cartilage, we focus on two questions: (i) what are the key factors influencing IGF-IR complex formation, and (ii) how might cells regulate IGF-IR complex formation? We develop a reaction-diffusion computational model of the IGF system involving twenty three parameters. A series of parametric and sensitivity studies are used to identify the key factors influencing IGF signaling. From the model we predict the free IGF and IGF-IR complex concentrations throughout the tissue. We estimate the degradation half-lives of free IGF-I and IGFBPs in normal cartilage to be 20 and 100 mins respectively, and conclude that regulation of the IGF half-life, either directly or indirectly via extracellular matrix IGF-BP protease concentrations, are two critical factors governing the IGF-IR complex formation in the cartilage. Further we find that cellular regulation of IGF-II production, the IGF-IIR concentration and its clearance rate, all significantly influence IGF signaling. It is likely that negative feedback processes via regulation of these factors tune IGF signaling within a tissue, which may help explain the recent failures of single target drug therapies aimed at modifying IGF signaling.     

胰岛素样生长因子IGF系统与鱼类性腺的研究进展

IGFs系统包含3个配体(IGF-1、IGF-2、IGF-3)、2个受体(IGF-1R、IGF-2R)和6个IGF结合蛋白(IGFBP).生殖和生长是生物体最基本的特征,两者既密切相关又相互区别,胰岛素样生长因子(IGFs)是生长轴和生殖轴相交联的关键因子.最近研究表明:鱼类性腺的发育及成熟伴随着细胞分化和组织生长,传统的生长因子IGF-1、IGF-2和最近发现的IGF-3,对鱼类性腺发挥着重要作用.本文重点介绍鱼类特有的配体IGF-3的结构,鱼类IGFs系统的信号通路及其与鱼类性腺的相关性研究进展.     

Effect of Dominant-Negative Epidermal Growth Factor Receptors on Cardiomyocyte Hypertrophy

Angiotensin II (AngII) induces heart growth via cardiomyocyte hypertrophy, and central to this is the capacity of the type 1 AngII receptor (AT₁R) to “transactivate” epidermal growth factor receptors (EGFRs)—a family with four main subtypes (HER1-4)—although the exact molecular mechanism remains unresolved. In this study, the pharmacological inhibition of AngII-stimulated ERK1/2 activation and cardiomyocyte hypertrophy by increasing concentrations of an EGFR inhibitor, AG1478, indicated that other EGFR subtypes, in addition to HER1, may be involved. We constructed expression vectors and adenoviruses expressing truncated mutant versions of HER1, HER2, and HER4 and determined their capacity to act as dominant-negative inhibitors when co-transfected with full-length EGFRs. It is surprising that adenoviral-mediated expression of these truncated EGFRs in cardiomyocytes led to paradoxical, ligand-independent increases in cardiomyocyte hypertrophy and unusual morphological changes. These results challenge our perception of AT₁R-mediated EGFR transactivation and imply that truncated EGFRs may affect cell function through unconventional mechanisms.     

Myostatin and Insulin-Like Growth Factor I: Potential Therapeutic Biomarkers for Pompe Disease

Objective

Myostatin and insulin-like growth factor 1 (IGF-1) are serum markers for muscle growth and regeneration. However, their value in the clinical monitoring of Pompe disease – a muscle glycogen storage disease – is not known. In order to evaluate their possible utility for disease monitoring, we assessed the levels of these serum markers in Pompe disease patients receiving enzyme replacement therapy (ERT).

Design

A case-control study that included 10 patients with Pompe disease and 10 gender- and age-matched non-Pompe disease control subjects was performed in a referral medical center. Average follow-up duration after ERT for Pompe disease patients was 11.7 months (range: 6–23 months). Measurements of serum myostatin, IGF-1, and creatine kinase levels were obtained, and examinations of muscle pathology were undertaken before and after ERT in the patient group.

Results

Compared with control subjects, Pompe disease patients prior to undergoing ERT had significantly lower serum IGF-1 levels (98.6 ng/ml vs. 307.9 ng/ml, p = 0.010) and lower myostatin levels that bordered on significance (1.38 ng/ml vs. 3.32 ng/ml, p = 0.075). After ERT, respective myostatin and IGF-1 levels in Pompe disease patients increased significantly by 129% (from 1.38 ng/ml to 3.16 ng/ml, p = 0.047) and 74% (from 98.6 ng/ml to 171.1 ng/ml, p = 0.013); these values fall within age-matched normal ranges. In contrast, myostatin and IGF-1 serum markers did not increase in age-matched controls. Follistatin, a control marker unrelated to muscle, increased in both Pompe disease patients and control subjects. At the same time, the percentage of muscle fibers containing intracytoplasmic vacuoles decreased from 80.0±26.4% to 31.6±45.3%.

Conclusion

The increase in myostatin and IGF-1 levels in Pompe disease patients may reflect muscle regeneration after ERT. The role of these molecules as potential therapeutic biomarkers in Pompe disease and other neuromuscular diseases warrants further study.     

Insulin-Like Growth Factor I Receptor Expression and Function in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Receptors for insulin-like growth factor I (IGF-I) were studied on PC12EY cells, a subclone of PC12. Differentiation of PC12EY cells with nerve growth factor (NGF) did not alter either the number of IGF-I receptors nor their affinity for IGF-I. IGF-I receptors remained fully functional during differentiation, promoting increases in thymidine incorporation, glucose uptake, amino acid uptake, and the phosphorylation of the S6 protein of the ribosomes. IGF-I also increased the proportion of differentiated cells found in S-phase. But although the addition of IGF-I to naive cells caused an increase in cell number, there was no comparable increase when IGF-I was added to differentiated cells. Thus, although the receptor for IGF-I continues to be present and functional, IGF-I fails to induce cell proliferation in differentiated PC12 cells.     

Insulin-Like Growth Factor I Receptor Binding in Brains of Alzheimer''s and Alcoholic Patients

Patients with chronic alcoholism and/or Alzheimer's disease show degenerative changes in the cerebral cortex and hippocampus. To investigate possible changes in insulin-like growth factor I receptor binding sites in brain tissue of patients with these pathological conditions, the number of 125I-insulin-like growth factor I binding sites was determined in tissues obtained from control patients and those with Alzheimer's and/or with a history of alcoholism. The four experimental groups examined consisted of patients from similar age groups. Postmortem histology and a clinical history were used for the diagnosis of Alzheimer's disease and alcoholism, respectively. Careful clinical records were kept concerning other variables such as immediate cause of death and medications administered before death. Specific binding of 125I-insulin-like growth factor I to homogenates prepared from cerebral cortex of Alzheimer's, alcoholic, alcoholic Alzheimer's, and age-matched control patients was similar, although Alzheimer's patients tended to have slightly higher binding values. No significant differences in insulin-like growth factor I binding in cerebral cortex were found with regard to age of patients, the interval between death and autopsy, and CNS-active medications. No statistical differences in 125I-insulin-like growth factor I binding were noted in hippocampal tissue from the four patient groups. Thus, human insulin-like growth factor I binding sites in cerebral cortex and hippocampus appear unaffected by several variables.     

Protein Phosphotyrosine in Mouse Brain: Developmental Changes and Regulation by Epidermal Growth Factor, Type I Insulin-Like Growth Factor, and Insulin

Using antiphosphotyrosine antibodies, we have investigated protein phosphorylation in mouse brain during development in intact animals and in reaggregated cerebral cultures. Under basal conditions, in vivo and in vitro, the levels of two main phosphoproteins, of Mr 120,000 and 180,000 (pp180), increased with development, reaching a maximum in the early postnatal period and decreasing thereafter. In adult forebrain, pp180 was still highly phosphorylated, but it was not detected in cerebellum or in peripheral tissues. In reaggregated cortical cultures, epidermal growth factor (EGF), type I insulin-like growth factor (IGF-I), and insulin enhanced protein tyrosine phosphorylation of several proteins, which were specific for EGF or IGF-I/insulin. In highly enriched neuronal or astrocytic monolayer cultures, some proteins phosphorylated in basal conditions, or in response to EGF and IGF-I, were found in both types of culture, whereas others appeared cell type specific. In addition, in each cell type, some proteins were phosphorylated under the action of both growth factors. These results indicate that tyrosine protein phosphorylation is maximal in mouse brain during development and is regulated by growth factors in neurons as well as in astrocytes.     

Osteocyte-Derived Insulin-Like Growth Factor I Is Not Essential for the Bone Repletion Response in Mice

The present study sought to evaluate the functional role of osteocyte-derived IGF-I in the bone repletion process by determining whether deficient expression of Igf1 in osteocytes would impair the bone repletion response to one week of dietary calcium repletion after two weeks of dietary calcium deprivation. As expected, the two-week dietary calcium depletion led to hypocalcemia, secondary hyperparathyroidism, and increases in bone resorption and bone loss in both Igf1 osteocyte conditional knockout (cKO) mutants and WT control mice. Thus, conditional disruption of Igf1 in osteocytes did not impair the calcium depletion-induced bone resorption. After one week of calcium repletion, both cKO mutants and WT littermates showed an increase in endosteal bone formation attended by the reduction in osteoclast number, indicating that deficient Igf1 expression in osteocytes also did not have deleterious effects on the bone repletion response. The lack of an effect of deficient osteocyte-derived IGF-I expression on bone repletion is unexpected since previous studies show that these Igf1 osteocyte cKO mice exhibited impaired developmental growth and displayed complete resistance to bone anabolic effects of loading. These studies suggest that there is a dichotomy between the mechanisms necessary for anabolic responses to mechanical loading and the regulatory hormonal and anabolic skeletal repletion following low dietary calcium challenge. In conclusion, to our knowledge this study has demonstrated for the first time that osteocyte-derived IGF-I, which is essential for anabolic bone response to mechanical loading, is not a key regulatory factor for bone repletion after a low calcium challenge.     

Amelioration of Diabetic Mouse Nephropathy by Catalpol Correlates with Down-Regulation of Grb10 Expression and Activation of Insulin-Like Growth Factor 1 / Insulin-Like Growth Factor 1 Receptor Signaling

Growth factor receptor-bound protein 10 (Grb10) is an adaptor protein that can negatively regulate the insulin-like growth factor 1 receptor (IGF-1R). The IGF1-1R pathway is critical for cell growth and apoptosis and has been implicated in kidney diseases; however, it is still unknown whether Grb10 expression is up-regulated and plays a role in diabetic nephropathy. Catalpol, a major active ingredient of a traditional Chinese medicine, Rehmannia, has been reported to possess anti-inflammatory and anti-aging activities and then used to treat diabetes. Herein, we aimed to assess the therapeutic effect of catalpol on a mouse model diabetic nephropathy and the potential role of Grb10 in the pathogenesis of this diabetes-associated complication. Our results showed that catalpol treatment improved diabetes-associated impaired renal functions and ameliorated pathological changes in kidneys of diabetic mice. We also found that Grb10 expression was significantly elevated in kidneys of diabetic mice as compared with that in non-diabetic mice, while treatment with catalpol significantly abrogated the elevated Grb10 expression in diabetic kidneys. On the contrary, IGF-1 mRNA levels and IGF-1R phosphorylation were significantly higher in kidneys of catalpol-treated diabetic mice than those in non-treated diabetic mice. Our results suggest that elevated Grb10 expression may play an important role in the pathogenesis of diabetic nephropathy through suppressing IGF-1/IGF-1R signaling pathway, which might be a potential molecular target of catalpol for the treatment of this diabetic complication.     

Chicken Insulin Discriminates Between Receptors for Insulin and Insulin-Like Growth Factor I on Centrally-and Peripherally-Derived Glial Cells

Abstract

Insulin and IGF-I receptors in G26–20 cells, derived from a mouse oligodendroglioma, and in RN-2 cells, derived from a rat Schwannoma, were characterized by specific binding to [¹²⁵I]insulin and [¹²⁵I]IGF-I respectively. In both cell lines, the Kd for insulin was 1.5 nM. Insulin receptor number was 33,000/cell for RN-2 cells and 17,000 receptors/ cell for G26–20 cells. RN-2 cells have 700,000 IGF-I receptors/cell with a Kd of 2 nM while G26–20 cells have 60,000 receptors/cell with an affinity of 4.9 nM. However, the independence of these two receptor populations in each cell type was equivocal since the subunit structure of these receptors appears identical by electrophoresis. In both cell lines, competition with insulin analogs for [¹²⁵I]insulin binding demonstrated chicken insulin>insulin>IGF-I. Competition for [¹²⁵I]IGF-I binding showed that IGF-I was approximately 85-fold more potent than insulin. Chicken insulin was ineffective at all concentrations. Thus, chicken insulin can be used as a specific ligand to unequivocally discriminate between IGF-I and insulin receptors and effects.     

Evidence for an Insulin-Like Growth Factor Autocrine-Paracrine System in the Retinal Photoreceptor-Pigment Epithelial Cell Complex

The interphotoreceptor matrix (IPM), lying between retinal photoreceptor and pigment epithelial (RPE) cells, contains insulin-like growth factor I (IGF-I) immunoreactivity that co-elutes with authentic human IGF-I in HPLC analyses. Cultured human RPE cells synthesize and release IGF-I, raising the possibility that the RPE serves as a source of IPM IGF-I in vivo. Photoreceptor rod outer segments and cultured monkey RPE cells express specific IGF-I receptors with alpha-subunits of 120 and 138 kDa, respectively. They thus appear to be of the "brain" (in photoreceptors) and "peripheral" (in RPE cells) receptor subtypes. Additionally, the IPM contains high levels of an IGF binding protein (IGF-BP) that specifically binds IGF-I and IGF-II. The IPM-BP is visualized as a single radiographic band by both ligand blot and affinity cross-linking procedures. With enzymes specific for removing N- and O-linked oligosaccharides, the IPM-BP was found to contain O- but not N-linked glycosylated side chains. The distinctive size and glycosylation pattern of the IPM-BP indicate that it is not derived from the vitreous or serum but instead is synthesized locally. The presence of IGF-I and IGF-BP in the IPM, together with the presence of IGF-I receptors on both photoreceptor and RPE cells, suggests the presence of an outer retina autocrine-paracrine system.     

Primary Culture of Canine Tracheal Smooth Muscle Cells in Serum-Free Medium: Effects of Insulin-Like Growth Factor I and Insulin

Abstract

The effects of growth factors on cell growth and muscarinic receptor (mAChR) expression of canine tracheal smooth muscle cells (TSMCs) were observed under serum-free medium supplemented with 0.1% BSA. In the presence of 0.1% BSA, TSMCs withdraw from cell cycle as compared with 10% FBS and allow to determine the effects of growth factors on mAChR expression. The individual components of growth factors (IGF-I, insulin, and aFGF) at the concentration used are not sufficient to stimulate growth of TSMCs in the primary culture with 0.1% BSA. IGF-I (10 ng/ml) and insulin (1 μg/m1), alone or in combination, could stimulate the expression of mAChRs of cultured TSMCs. Heparin could inhibit these stimulatory effects of mAChR expression. The stimulatory effects of IGF-I and insulin on mAChR expression were mediated through their own receptors since these effects were reversed by pretreatment of TSMCs with antibodies of the respective growth factor receptors. The pharmacological response of functional mAChRs, determined as accumulation of inositol phosphates induced by carbachol, is greater in the medium containing IGF-I and insulin than that cultured in 0.1% BSA. These results firmly establish that IGF-I and insulin could stimulate the expression of mAChRs in TSMCs under serum-free culture condition.     

Regulation of Human Trophoblast GLUT1 Glucose Transporter by Insulin-Like Growth Factor I (IGF-I)

Glucose transport to the fetus across the placenta takes place via glucose transporters in the opposing faces of the barrier layer, the microvillous and basal membranes of the syncytiotrophoblast. While basal membrane content of the GLUT1 glucose transporter appears to be the rate-limiting step in transplacental transport, the factors regulating transporter expression and activity are largely unknown. In view of the many studies showing an association between IGF-I and fetal growth, we investigated the effects of IGF-I on placental glucose transport and GLUT1 transporter expression. Treatment of BeWo choriocarcinoma cells with IGF-I increased cellular GLUT1 protein. There was increased basolateral (but not microvillous) uptake of glucose and increased transepithelial transport of glucose across the BeWo monolayer. Primary syncytial cells treated with IGF-I also demonstrated an increase in GLUT1 protein. Term placental explants treated with IGF-I showed an increase in syncytial basal membrane GLUT1 but microvillous membrane GLUT1 was not affected. The placental dual perfusion model was used to assess the effects of fetally perfused IGF-I on transplacental glucose transport and syncytial GLUT1 content. In control perfusions there was a decrease in transplacental glucose transport over the course of the perfusion, whereas in tissues perfused with IGF-I through the fetal circulation there was no change. Syncytial basal membranes from IGF-I perfused tissues showed an increase in GLUT1 content. These results demonstrate that IGF-I, whether acting via microvillous or basal membrane receptors, increases the basal membrane content of GLUT1 and up-regulates basal membrane transport of glucose, leading to increased transepithelial glucose transport. These observations provide a partial explanation for the mechanism by which IGF-I controls nutrient supply in the regulation of fetal growth.     

Insulin-Like Growth Factors,Insulin, and Growth Hormone Signaling in Breast Cancer: Implications for Targeted Therapy

ObjectiveIn recent decades, multiple therapeutics targeting the estrogen and human epidermal growth factor-2 (HER2) receptors have been approved for the treatment of breast cancer.MethodsThis review discusses a number of growth factor pathways that have been implicated in resistance to both anti-estrogen and HER2-targeted therapies. The association between growth factors and breast cancer is well established. Over decades, numerous laboratories have studied the link between insulin-like growth factor (IGF), insulin, and growth hormone (GH) to the development and progression of breast cancer.ResultsAlthough preclinical data demonstrates that blockade of these receptors inhibits breast cancer growth, progression, and drug resistance, therapies targeting the IGF, insulin, and GH receptors (GHRs) have not been successful in producing significant increases in progression- free, disease-free, or overall survival for patients with breast cancer. The failure to demonstrate a benefit of growth factor blockade in clinical trials can be attributed to redundancy in IGF, insulin, and GHR signaling pathways. All 3 receptors are able to activate oncogenic phosphoinositide- 3 kinase (PI3K) and mitogen-activated protein kinase (MAPK) pathways.ConclusionConsequently, multitargeted blockade of growth factor receptors and their common downstream kinases will be necessary for the successful treatment of breast cancer. (Endocr Pract. 2014;20:1214-1221)