Accumulated evidence suggests that the single transmembrane domain insulin-like growth factor-II/mannose 6-phosphate receptor (IGF-II/M6P or IGF-II receptor) plays an important role in the intracellular trafficking of lysosomal enzymes and endocytosis-mediated degradation of insulin like growth factor (IGF-II). However, the role of this receptor in signal transduction following IGF-II binding remains controversial. In the present study, we revealed that Leu(27)IGF-II, an analog which binds preferentially to the IGF-II receptor, can attenuate K(+)-as well as veratridine-evoked GABA release from the adult rat hippocampal formation. Tetrodotoxin failed to alter the effects of Leu(27)IGF-II on GABA release, thus suggesting the lack of involvement of voltage-dependent Na(+) channels. Interestingly, the effect is found to be sensitive to pertussis toxin (PTX), indicating the possible involvement of a Gi/o protein-dependent pathway in mediating the release of GABA from the hippocampal slices. Additionally, Leu(27)IGF-II was found to attenuate GABA release from frontal cortex but not from striatum. These results, together with the evidence that IGF-II receptors are localized on GABAergic neurons, raised the possibility that this receptor, apart from mediating intracellular trafficking, may also be involved in the regulation of endogenous GABA release by acting directly on GABAergic terminals.

Insulin and insulin-like growth factors (IGFs) act on tetrameric tyrosine kinase receptors controlling essential functions including growth, metabolism, reproduction and longevity. The insulin receptor (IR) binds insulin and IGFs with different affinities triggering different cell responses.

Type 2 insulin-like growth factor (IGF-II) levels are increased in fibrosing lung diseases such as idiopathic pulmonary fibrosis (IPF) and scleroderma/systemic sclerosis-associated pulmonary fibrosis (SSc). Our goal was to investigate the contribution of IGF receptors to IGF-II-mediated fibrosis in these diseases and identify other potential mechanisms key to the fibrotic process. Cognate receptor gene and protein expression were analyzed with qRT-PCR and immunoblot in primary fibroblasts derived from lung tissues of normal donors (NL) and patients with IPF or SSc. Compared to NL, steady-state receptor gene expression was decreased in SSc but not in IPF. IGF-II stimulation differentially decreased receptor mRNA and protein levels in NL, IPF, and SSc fibroblasts. Neutralizing antibody, siRNA, and receptor inhibition targeting endogenous IGF-II and its primary receptors, type 1 IGF receptor (IGF1R), IGF2R, and insulin receptor (IR) resulted in loss of the IGF-II response. IGF-II tipped the TIMP:MMP balance, promoting a fibrotic environment both intracellularly and extracellularly. Differentiation of fibroblasts into myofibroblasts by IGF-II was blocked with a TGFβ1 receptor inhibitor. IGF-II also increased TGFβ2 and TGFβ3 expression, with subsequent activation of canonical SMAD2/3 signaling. Therefore, IGF-II promoted fibrosis through IGF1R, IR, and IGF1R/IR, differentiated fibroblasts into myofibroblasts, decreased protease production and extracellular matrix degradation, and stimulated expression of two TGFβ isoforms, suggesting that IGF-II exerts pro-fibrotic effects via multiple mechanisms.

Insulin-like growth factor II (IGF2) enhances memory in rodents via the mannose-6-phosphate receptor (M6PR), but the underlying mechanisms remain poorly understood. We found that human IGF2 produces an enhancement of both synaptic transmission and neurite outgrowth in the marine mollusk Aplysia californica. These findings were unexpected since Aplysia lack the mammal-specific affinity between insulin-like ligands and M6PR. Surprisingly, this effect was observed in parallel with a suppression of neuronal excitability in a well-understood circuit that supports several temporally and mechanistically distinct forms of memory in the defensive withdrawal reflex, suggesting functional coordination between excitability and memory formation. We hypothesize that these effects represent behavioral adaptations to feeding that are mediated by the endogenous Aplysia insulin-like system. Indeed, the exogenous application of a single recombinant insulin-like peptide cloned from the Aplysia CNS cDNA replicated both the enhancement of synaptic transmission, the reduction of excitability, and promoted clearance of glucose from the hemolymph, a hallmark of bona fide insulin action.

Maternal ethanol (ETOH) exposure is associated with impaired fetal growth. Because insulin-like growth factors (IGFs) are thought to be important in the regulation of fetal somatic growth, we examined the influence of maternal ETOH exposure on fetal growth and plasma levels of IGF-I, IGF-II, and IGF binding proteins (IGFBPs) in the rat model. Control (A) dams were fed a standard rat chow ad libitum. ETOH (E) consuming dams were fed a 36% ETOH diet, and pair-fed (P) dams were fed isocaloric amounts of a control liquid diet. All animals were killed on day 20 of gestation. Plasma concentrations of IGF-I and -II were determined by radioimmunoassay after formic acid-acetone extraction and heat inactivation of IGFBPs. Levels of IGFBPs in fetal plasma were estimated by Western ligand blotting after protein separation by SDS-PAGE and electrotransfer to nitrocellulose. Membranes were probed with [125I]IGF-I, and IGFBPs were identified by autoradiography, quantified by scanning densitometry and results expressed relative to corresponding IGFBPs in control fetal plasma. Maternal weight gain from conception to 20 days of pregnancy was reduced for E compared to P and A dams (p < 0.05 E vs. P or A). The same pattern was reflected in fetal weight that tended to be lower in P compared with A pups, and was significantly reduced in E pups compared with both groups (p < 0.0001 E vs. P or A). Thus, fetal growth was more retarded in E animals despite equal caloric and protein intake by E and P dams.(ABSTRACT TRUNCATED AT 250 WORDS)

Chronic exposure to ethanol can induce widespread cell loss in the brain, in some cases even causing dementia. Although the underlying mechanism associated with ethanol toxicity has not yet been established, it is suggested that one of the ways in which ethanol disrupts neuronal functioning/survival is by targeting the actions of mitogenic growth factors. Insulin-like growth factors-I and -II and insulin are structurally related polypeptides with potent mitogenic and metabolic effects on the central and peripheral nervous systems. These growth factors and their respective receptors are widely distributed throughout the brain, including the hippocampus and cerebellum. Evidence indicates that ethanol can decrease plasma levels of insulin-like growth factors and can also inhibit the growth-promoting and cell survival effects of these growth factors under in vitro conditions. The present study was designed to determine if voluntary ethanol consumption over a 21-day period could alter [125I]insulin-like growth factor-I, [125I]insulin-like growth factor-II and [125I]insulin receptor-binding sites in the hippocampus and cerebellum-areas known to be severely affected following chronic exposure to ethanol. C57BL/6 mice were presented with either water only or a choice of water and a 10% v/v ethanol solution. Mice with access to the ethanol solution drank an average of 5.35+/-0.77 g of ethanol/kg body weight per day. [125I]Insulin-like growth factor-I receptor-binding sites were found to be significantly increased in all subfields of the hippocampal formation, but not in the cerebellum, of ethanol-treated mice compared to controls. [125I]Insulin-like growth factor-II and [125I]insulin receptor-binding sites, on the other hand, did not exhibit any alterations either in the hippocampus or cerebellum following chronic exposure to ethanol. These results, in keeping with earlier reports, suggest that hippocampal insulin-like growth factor-I is more sensitive to ethanol treatment than either insulin-like growth factor-II or insulin, and the observed increase in the [125I]insulin-like growth factor-I receptor levels possibly reflects an activity-dependent response to prevent/slow down neuronal degeneration and/or to regulate subtle functional alterations that follow chronic exposure to ethanol.

Serum IGF-I and IGF-II levels decline with age. IGF-I replacement therapy reduces the impact of age in rats. We have recently reported that IGF-II is able to act, in part, as an analogous of IGF-I in aging rats reducing oxidative damage in brain and liver associated with a normalization of antioxidant enzyme activities. Since mitochondria seem to be the most important cellular target of IGF-I, the aim of this work was to investigate whether the cytoprotective actions of IGF-II therapy are mediated by mitochondrial protection.

Metals pollution plays an important role in the regulation of gene expression through interference with signal transduction pathways which are important for cell bioactivity.

Autism spectrum disorder (ASD) is a developmental disability characterized by impairments in social interaction and repetitive behavior, and is also associated with cognitive deficits. There is no current treatment that can ameliorate most of the ASD symptomatology; thus, identifying novel therapies is urgently needed. We used male BTBR T+Itpr3tf /J (BTBR) mice, a model that reproduces most of the core behavioral phenotypes of ASD, to test the effects of systemic administration of insulin-like growth factor II (IGF-II), a polypeptide that crosses the blood-brain barrier and acts as a cognitive enhancer. We show that systemic IGF-II treatments reverse the typical defects in social interaction, cognitive/executive functions, and repetitive behaviors reflective of ASD-like phenotypes. In BTBR mice, IGF-II, via IGF-II receptor, but not via IGF-I receptor, reverses the abnormal levels of the AMPK-mTOR-S6K pathway and of active translation at synapses. Thus, IGF-II may represent a novel potential therapy for ASD.SIGNIFICANCE STATEMENT Currently, there is no effective treatment for autism spectrum disorder (ASD), a developmental disability affecting a high number of children. Using a mouse model that expresses most of the key core as well as associated behavioral deficits of ASD, that are, social, cognitive, and repetitive behaviors, we report that a systemic administration of the polypeptide insulin-like growth factor II (IGF-II) reverses all these deficits. The effects of IGF-II occur via IGF-II receptors, and not IGF-I receptors, and target both basal and learning-dependent molecular abnormalities found in several ASD mice models, including those of identified genetic mutations. We suggest that IGF-II represents a potential novel therapeutic target for ASD.

Monomers of the insulin receptor and type 1 insulin-like growth factor receptor (IGF-1R) can combine stochastically to form heterodimeric hybrid receptors. These hybrid receptors display ligand binding and signaling properties that differ from those of the homodimeric receptors. Here, we describe the cryoelectron microscopy structure of such a hybrid receptor in complex with insulin-like growth factor I (IGF-I). The structure (ca. 3.7 Å resolution) displays a single IGF-I ligand, bound in a similar fashion to that seen for IGFs in complex with IGF-1R. The IGF-I ligand engages the first leucine-rich-repeat domain and cysteine-rich region of the IGF-1R monomer (rather than those of the insulin receptor monomer), consistent with the determinants for IGF binding residing in the IGF-1R cysteine-rich region. The structure broadens our understanding of this receptor family and assists in delineating the key structural motifs involved in binding their respective ligands.

Human type 1 insulin-like growth factor receptor (IGF-1R) signals chiefly in response to the binding of insulin-like growth factor I. Relatively little is known about the role of insulin-like growth factor II signaling via IGF-1R, despite the affinity of insulin-like growth factor II for IGF-1R being within an order of magnitude of that of insulin-like growth factor I. Here, we describe the cryoelectron microscopy structure of insulin-like growth factor II bound to a leucine-zipper-stabilized IGF-1R ectodomain, determined in two conformations to a maximum average resolution of 3.2 Å. The two conformations differ in the relative separation of their respective points of membrane entry, and comparison with the structure of insulin-like growth factor I bound to IGF-1R reveals long-suspected differences in the way in which the critical C domain of the respective growth factors interact with IGF-1R.

Tumor-produced high molecular weight insulin-like growth factor-II (big insulin-like growth factor-II) is considered to cause non-islet cell tumor hypoglycemia. This paper presents a case of surgically resected retroperitoneal liposarcoma that produced big insulin-like growth factor-II.

The effects of recombinant basic fibroblast growth factor (bFGF), insulin-like growth factor (IGF)-II and transforming growth factor (TGF)-beta 1 on dental pulp cells were investigated by light and transmission electron microscopy after their implantation for 1 and 3 weeks at central sites of mechanically exposed pulps in dog molar and canine teeth. The implants were Millipore filters that have been soaked with solutions containing 100 or 500 ng/ml of bFGF or IGF-II or 100 ng/ml of TGF-beta 1. Control filters were soaked with dog albumin. No changes in cell organization or matrix synthesis were seen after implantation of control filters. Groups of columnar, polarized cells with numerous mitochondria and Golgi elements or elongated cells unassociated with any matrix deposition were demonstrated after 1 or 3 weeks, respectively, in close proximity to the filters that had been soaked with bFGF solution; at a distance from these implants enhanced formation of an osteotypic matrix was seen beneath the exposure site. No particular response was found in close proximity to the filters that had been soaked with IGF-II solution after 1 or 3 weeks implantation but thick zones of osteodentine were found beneath the exposure site and at adjacent circumferential dentine sites. Numerous elongated, polarized cells with long cytoplasmic extensions invading the filter pores were consistently seen after 1 week in close proximity to the filters that had been soaked with TGF-beta 1 solution. After 3 weeks implantation of these filters, deposition of a tubular matrix surrounding the implants was seen in association with the highly elongated odontoblast-like cells, while enhancement of circumferential dentine formation was also found at adjacent peripheral sites. These experiments demonstrate that TGF-beta 1 when implanted for short term periods at central pulp sites exerted dentine-specific effects, inducing differentiation of odontoblast-like cells and stimulating primary odontoblasts. Implantation of bFGF and IGF-II did not result in reparative dentine formation, but did stimulate osteotypical matrix deposition at a distance from the implants.

Insulin-like growth factor-1 (IGF-1) promotes the survival of cardiomyocytes by activating type 1 IGF receptor (IGF-1R). Within the myocardium, IGF-1 action is modulated by IGF binding protein-3 (IGFBP-3), which sequesters IGF-1 away from IGF-1R. Since cardiomyocyte apoptosis is implicated in anthracycline cardiotoxicity, we investigated the effects of the anthracycline, doxorubicin, on the IGF-1 system in H9c2 cardiomyocytes.

Administration of kainic acid evokes acute seizure in hippocampal pathways that results in a complex sequence of functional and structural alterations resembling human temporal lobe epilepsy. The structural alterations induced by kainic acid include selective loss of neurones in CA1-CA3 subfields and the hilar region of the dentate gyrus followed by sprouting and permanent reorganization of the synaptic connections of the mossy fibre pathways. Although the neuronal degeneration and process of reactive synaptogenesis have been extensively studied, at present little is known about means to prevent pathological conditions leading to kainate-induced cell death. In the present study, to address the role of insulin-like growth factors I and II, and insulin in neuronal survival as well as synaptic reorganization following kainate-induced seizure, the time course alterations of the corresponding receptors were evaluated. Additionally, using histological preparations, the temporal profile of neuronal degeneration and hypertrophy of resident astroglial cells were also studied. [125I]Insulin-like growth factor I binding was found to be decreased transiently in almost all regions of the hippocampal formation at 12 h following treatment with kainic acid. The dentate hilar region however, exhibited protracted decreases in [125I]insulin-like growth factor I receptor sites throughout (i.e. 30 days) the study. [125I]Insulin-like growth factor II receptor binding sites in the hippocampal formation were found to be differentially altered following systemic administration of kainic acid. A significant decrease in [125I]insulin-like growth factor II receptor sites was observed in CA1 subfield and the pyramidal cell layer of the Ammon's horn at all time points studied whereas the hilar region and the stratum radiatum did not exhibit alteration at any time. A kainate-induced decrease in [125I]insulin receptor binding was noted at all time points in the molecular layer of the dentate gyrus whereas binding in CA1-CA3 subfields and discrete layers of the Ammon's horn was found to be affected only after 12 h of treatment. These results, when analysed with reference to the observed histological changes and established neurotrophic/protective roles of insulin-like growth factors and insulin, suggest possible involvement of these growth factors in the cascade of neurotrophic events that is associated with the reorganization of the hippocampal formation observed following kainate-induced seizures.

The rapid rise in obesity, metabolic syndrome and type 2 diabetes is one of the major healthcare problems of the Western world. Affected individuals are often treated with statins (3-hydroxy-3-methylglutaryl co-enzyme A [HMG CoA] reductase inhibitors) to reduce circulating cholesterol levels and the risk of developing cardiovascular disease; given the evolving demographic profile of these conditions, such drugs are increasingly prescribed to women of reproductive age. We have previously shown that exposure of placental tissue to statins inhibits the action of insulin-like growth factors (IGF)-I and -II which are key regulators of trophoblast proliferation and placental development. N-linked glycans in the IGF receptor, IGF1R, influence its presentation at the cell surface. This study aimed to determine whether statins, which are known to affect N-glycosylation, modulate IGF1R function in placenta. Treatment of first trimester villous tissue explants with statins (pravastatin or cerivastatin) or inhibitors of N-glycosylation (tunicamycin, deoxymannojirimycin or castanospermine) altered receptor distribution in trophoblast and attenuated proliferation induced by IGF-I or IGF-II (Ki67; P < 0.05, n = 5). Decreased binding of Phaseolus vulgaris lectin and phytohaemagglutinin to IGF1R immunoprecipitated from treated explants demonstrated reduced levels of complex N-linked glycans. Co-incubation of tissue explants with statins and farnesyl pyrophosphate (which increases the supply of dolichol intermediates), prevented statin-mediated disruption of IGF1R localization and reversed the negative effect on IGF-mediated trophoblast proliferation. These data suggest that statins attenuate IGF actions in the placenta by inhibiting N-linked glycosylation and subsequent expression of mature IGF1R at the placental cell surface.

Tissue-specific stem cells have unique properties and growth requirements, but a small set of juxtacrine and paracrine signals have been identified that are required across multiple niches. Whereas insulin-like growth factor II (IGF-II) is necessary for prenatal growth, its role in adult stem cell physiology is largely unknown. We show that loss of Igf2 in adult mice resulted in a ∼50% reduction in slowly dividing, label-retaining cells in the two regions of the brain that harbor neural stem cells. Concordantly, induced Igf2 deletion increased newly generated neurons in the olfactory bulb accompanied by hyposmia, and caused impairments in learning and memory and increased anxiety. Induced Igf2 deletion also resulted in rapid loss of stem and progenitor cells in the crypts of Lieberkühn, leading to body-weight loss and lethality and the inability to produce organoids in vitro. These data demonstrate that IGF-II is critical for multiple adult stem cell niches.

We compared IGF responses of fetal and adult intestinal fibroblasts to identify a developmental difference in the IGF-axis. Intestinal fibroblasts were isolated from maternal and fetal jejunum. Media was conditioned at confluence and one week afterwards. The proliferative response at confluence to 5 nM IGF-I or -II was compared.

Stress seems to contribute to Parkinson's disease (PD) neuropathology, probably by dysregulation of the hypothalamic-pituitary-adrenal axis. Key factors in this pathophysiology are oxidative stress and mitochondrial dysfunction and neuronal glucocorticoid-induced toxicity. The insulin-like growth factor II (IGF-II), a pleiotropic hormone, has shown antioxidant and neuroprotective effects in some neurodegenerative disorders. Our aim was to examine the protective effect of IGF-II on a dopaminergic cellular combined model of PD and mild to moderate stress measuring oxidative stress parameters, mitochondrial and neuronal markers, and signalling pathways. IGF-II counteracts the mitochondrial-oxidative damage produced by the toxic synergistic effect of corticosterone and 1-methyl-4-phenylpyridinium, protecting dopaminergic neurons from death and neurodegeneration. IGF-II promotes PKC activation and nuclear factor (erythroid-derived 2)-like 2 antioxidant response in a glucocorticoid receptor-dependent pathway, preventing oxidative cell damage and maintaining mitochondrial function. Thus, IGF-II is a potential therapeutic tool for treatment and prevention of disease progression in PD patients suffering mild to moderate emotional stress.

Insulin and insulin-like growth factors I and II are closely related protein hormones. Their distinct evolution has resulted in different yet overlapping biological functions with insulin becoming a key regulator of metabolism, whereas insulin-like growth factors (IGF)-I/II are major growth factors. Insulin and IGFs cross-bind with different affinities to closely related insulin receptor isoforms A and B (IR-A and IR-B) and insulin-like growth factor type I receptor (IGF-1R). Identification of structural determinants in IGFs and insulin that trigger their specific signaling pathways is of increasing importance in designing receptor-specific analogs with potential therapeutic applications. Here, we developed a straightforward protocol for production of recombinant IGF-II and prepared six IGF-II analogs with IGF-I-like mutations. All modified molecules exhibit significantly reduced affinity toward IR-A, particularly the analogs with a Pro-Gln insertion in the C-domain. Moreover, one of the analogs has enhanced binding affinity for IGF-1R due to a synergistic effect of the Pro-Gln insertion and S29N point mutation. Consequently, this analog has almost a 10-fold higher IGF-1R/IR-A binding specificity in comparison with native IGF-II. The established IGF-II purification protocol allowed for cost-effective isotope labeling required for a detailed NMR structural characterization of IGF-II analogs that revealed a link between the altered binding behavior of selected analogs and conformational rearrangement of their C-domains.