Mannose 6-phosphate potentiates insulin-like growth factor II effects in cultured human neuroblastoma cells

Insulin-like growth factor II (IGF-II) and mannose 6-phosphate (man-6-P) bind to distinct sites on the same receptor. In the present study, we examined the effects of man-6-P on the growth promoting effects of IGF-II on SH-SY5Y cultured human neuroblastoma cells. Man-6-P alone increased cell number and neurite outgrowth by approximately 50%; as previously observed, IGF-II increased cell number and neurite outgrowth by approximately 110 and 30%, respectively. However, when cells were grown in the presence of both ligands, cell number increased by 330% and neurite outgrowth by 130%. These results suggest that man-6-P can potentiate the known growth promoting effects of IGF-II on human neuroblastoma cells. Furthermore, they indicate that the IGF-II/man-6-P receptor may serve as a means of integrating distinct growth promoting signals in neuronal cells.     

Insulin-like growth factor-II/mannose-6-phosphate receptor: widespread distribution in neurons of the central nervous system including those expressing cholinergic phenotype

The insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptor is single transmembrane glycoprotein that plays a critical role in the trafficking of lysosomal enzymes and the internalization of circulating IGF-II. At present, there is little information regarding the cellular distribution of the IGF-II/M6P receptor within the adult rat brain. With the use of immunoblotting and immunocytochemical methods, we found that the IGF-II/M6P receptor is widely but selectively expressed in all major brain areas, including the olfactory bulb, striatum, cortex, hippocampus, thalamus, hypothalamus, cerebellum, brainstem, and spinal cord. Intense IGF-II/M6P receptor immunoreactivity was apparent on neuronal cell bodies within the striatum, deeper layers (layers IV and V) of the cortex, pyramidal and granule cell layers of the hippocampal formation, selected thalamic nuclei, Purkinje cells of the cerebellum, pontine nucleus and motoneurons of the brainstem as well as in the spinal cord. Moderate neuronal labeling was evident in the olfactory bulb, basal forebrain areas, hypothalamus, superior colliculus, midbrain areas, granule cells of the cerebellum and in the intermediate regions of the spinal gray matter. We also observed dense neuropil labeling in many regions, suggesting that this receptor is localized in dendrites and/or axon terminals. Double-labeling studies further indicated that a subset of IGF-II/M6P receptor colocalizes with cholinergic cell bodies and fibers in the septum, striatum, diagonal band complex, nucleus basalis, cortex, hippocampus, and motoneurons of the brainstem and spinal cord. The observed widespread distribution and colocalization of IGF-II/M6P receptor in the adult rat brain provide an anatomic basis to suggest a multifunctional role for the receptor in a wide-spectrum of central nervous system neurons, including those expressing a cholinergic phenotype.     

The therapeutic potential of insulin-like growth factor-1 in central nervous system disorders

Central nervous system (CNS) development is a finely tuned process that relies on multiple factors and intricate pathways to ensure proper neuronal differentiation, maturation, and connectivity. Disruption of this process can cause significant impairments in CNS functioning and lead to debilitating disorders that impact motor and language skills, behavior, and cognitive functioning. Recent studies focused on understanding the underlying cellular mechanisms of neurodevelopmental disorders have identified a crucial role for insulin-like growth factor-1 (IGF-1) in normal CNS development. Work in model systems has demonstrated rescue of pathophysiological and behavioral abnormalities when IGF-1 is administered, and several clinical studies have shown promise of efficacy in disorders of the CNS, including autism spectrum disorder (ASD). In this review, we explore the molecular pathways and downstream effects of IGF-1 and summarize the results of completed and ongoing pre-clinical and clinical trials using IGF-1 as a pharmacologic intervention in various CNS disorders. This aim of this review is to provide evidence for the potential of IGF-1 as a treatment for neurodevelopmental disorders and ASD.     

Immunohistochemical study on the distribution of insulin-like growth factor I (IGF-I) receptor in the central nervous system of SOD1(G93A) mutant transgenic mice

In the present study, we used the SOD1(G93A) mutant transgenic mice as an in vivo model of ALS and performed immunohistochemical studies to investigate the changes of insulin-like growth factor I (IGF-I) receptor in the central nervous system. IGF-I receptor-immunoreactive astrocytes were detected in the spinal cord, brainstem, central gray and cerebellar nuclei of SOD1(G93A) transgenic mice. In contrast to transgenic mice, no IGF-I receptor-immunoreactive astrocytes were observed in any brain region of wtSOD1 transgenic mice although a few moderately stained neurons were observed. In the hippocampal formation of SOD1(G93A) transgenic mice, IGF-I receptor immunoreactivity was increased in the pyramidal cells of the CA1-3 regions and granule cells of the dentate gyrus. The present study provides the first evidence that IGF-I receptor immunoreactivity was increased in reactive astrocytes in the central nervous system of SOD(G93A) transgenic mice, suggesting that reactive astrocytes may play an important role in the pathogenesis and progress of ALS. The mechanisms underlying the increased immunoreactivity for IGF-I receptor, and the functional implications of these increases, require elucidation.     

RNA interference affects tumorigenicity and expression of insulin-like growth factor-1, insulin-like growth factor-1 receptor, and basic fibroblast growth factor-2 in rat C6 glioma cells

BACKGROUND： Human gliomas are more likely to express basic fibroblast growth factor-2 （FGF-2） insulin-like growth factor-1（IGF-1）, and IGF-1 receptor （IGF-1R） than normal brain tissue. These factors activate signal transduction systems of Ras/MAPK and PI3K/Akl, which promote glioma growth. OBJECTIVE： To utilize RNA interference （RNAi） technique to down-regulate FGF-2, IGF-1, and IGF-1R gene expression, and to investigate the effects of these genes on rat C6 glioma cells, as well as the feasibility of RNAi for treating glioma. DESIGN, TIME AND SETTING： This neurooncological, randomized, controlled, in vivo and in vitro experiment, which used RNAi methodology, was performed at the Laboratory of Molecular Biology, Institute of Biochemistry, Chinese Academy of Sciences between August 2005 and February 2008. MATERIALS： Rat C6 cell lines were purchased from Shanghai Institute of Cellular Biology Affiliated to Chinese Academy of Sciences. Small interfering RNA （siRNA） was synthesized by Shanghai GenePharma. Anti-IGF-1, anti-IGF-1R, anti-FGF-2, anti-mouse and anti-rabbit IgG G1-HRP antibodies were provided by Santa Cruz Biotechnology, USA. Four to six week-old BALB/c nude mice were purchased from the Laboratory Animal Center, Chinese Academy of Sciences. METHODS： C6 glioma cells were transfected with siRNA, which was chemically synthesized in vitro to correspond to endogenous FGF-2, IGF-1, and IGF-1R genes. The inhibition ratio of targeting mRNA expression was detected by semiquantitative RT-PCR, and protein expression was determined by Western blot analysis. C6 glioma cell proliferation was observed using a growth curve C6 glioma cell apoptosis rate and cell cycle were detected by flow cytometry. C6 glioma cell growth regression was observed by transwell migration assay. In addition, nude mouse subcutaneous tumor models were used in this study. For studying the anti-tumor effects of IGF-1 and IGF-1R siRNA, two blank control groups, with six mice each, were set up： A （2.5 μg siRNA was injected one week after C6 cells were inoculated, Le., when tumor volume reached 8 mm × 8 mm） and B （siRNA was injected at the same time with C6 cells were inoculated. To study the effects of FGF-2 siRNA, the groups consisted of a blank control group, negative control group, 2.6 μg siRNA group, 4 μg siRNA group, and 5.3 μg siRNA group, with six mice each. MAIN OUTCOME MEASURES： mRNA and protein inhibition ratio of FGF-2, IGF-1, and IGF-1 R; C6 glioma cell proliferation, apoptosis, and cycle growth arrest; C6 glioma cell growth regression and subcutaneous tumorigenicity rates. RESULTS： All siRNA constructs proved to be effective. After 48 hours, transfection of 200 nmol/L siRNA resulted in a FGF-2 or IGF-1R gene inhibition ratio 〉 80% and an IGF-1 gene inhibition ratio of approximately 70%. Protein expression levels for FGF-2, IGF-1, and IGF-1R decreased in a dose-dependent manner following siRNA transfection, with an inhibition rate 〉 85%, 60%, and 50%, respectively. C6 glioma cell proliferation and apoptosis rates increased in proportion to siRNA. The apoptosis rate of C6 glioma cells induced by FGF-2, IGF-1, and IGF-1R siRNA was 39.96%, 15.07% and 22.47%, respectively （P 〈 0.01）. Transfection of 200 nmol/L IGF or IGF-1R siRNA for 48 hours suppressed C6 glioma cell migration. At 30 days after intratumoral injection of 2.6, 4, and 5.3 tJg FGF-2 siRNA, tumor growth regression rate of FGF-2 siRNA was 56%, 67%, and 86%, respectively. The tumor growth regression rate was 71.88% and 45.71%, respectively, when IGF-1 or IGF-1R siRNA was intratumorally injected 1 week after C6 glioma cell transplantation. When IGF-1 or IGF-1 R siRNA was intratumorally injected during C6 glioma cell transplantation, the tumor growth regression rate was 78.13% and 74.29%, respectively. CONCLUSION： siRNA transfection downregulated gene expression of FGF-2, IGF-1, and IGF-1R In addition, siRNA treatment markedly suppressed glioma cell proliferation, growth, and migration, and concomitantly reduced subcutaneous tumorigenicity.     

Role of insulin-like growth factor binding proteins in limitation of IGF-I degradation into the N-methyl- -aspartate receptor antagonist GPE: evidence from gonadotrophin-releasing hormone secretion in vitro at two developmental stages

We showed previously that insulin-like growth factor-I (IGF-I) could inhibit the secretion of gonadotrophin-releasing hormone (GnRH) evoked in vitro by N-methyl- -aspartate (NMDA) or veratridine depolarization. Such an IGF-I effect appeared to be mediated by its physiological breakdown product, the N-terminal tripeptide GPE. That effect was developmentally regulated since IGF-I could inhibit GnRH secretion from hypothalamic explants of 50-day-old adult rats but not from immature 15-day-old explants. We hypothesized that the IGF-binding proteins (BPs) could limit the peptide availability to endopeptidases and account for the absent IGF-I effects at 15 days. In this paper, we show that the inhibition of GnRH secretion by 10⁻¹⁰ M of IGF-I at 50 days is prevented in a dose-dependent manner by 0.3 to 3 nM of IGF-BP2 as well as IGF-BP3. The inhibition caused by 10⁻¹⁰ M of GPE is not affected under similar conditions. Using explants obtained at 15 days, a significant inhibition of GnRH secretion can be obtained by 10⁻¹⁰ M of IGF-I in the presence of an anti IGF-BP2 antiserum used at 1:3000 and 1:1000 concentrations. These data indicate that in the immature rat brain, the IGF-BPs could act as modulators of IGF-I degradation into its subproduct GPE, a possible endogenous antagonist at NMDA receptors.     

Cloning, sequencing, chromosomal location, and function of cDNAs encoding an opioid growth factor receptor (OGFr) in humans

The native opioid growth factor (OGF), [Met(5)]-enkephalin, is a tonic inhibitory peptide that modulates cell proliferation and tissue organization during development, cancer, cellular renewal, wound healing, and angiogenesis. OGF action is mediated by a receptor mechanism. We have cloned and sequenced cDNAs encoding multiple spliced forms of a human OGF receptor. The open reading frame in the longest cDNA was found to encode a protein of 697 amino acids, and 8 imperfect repeats of 20 amino acids each were a prominent feature. Altogether, five alternatively spliced forms were observed. The cDNA hybridized to mRNA from a variety of normal and neoplastic cells and tissues. Functional studies using antisense oligonucleotides to OGFr demonstrated an enhancement in cell growth. Fluorescent in situ hybridization (FISH) experiments showed the chromosomal location to be 20q13.3. This OGF receptor has no homology to classical opioid receptors. These results provide molecular validity for the interaction of OGF and OGF receptor in the regulation of growth processes in humans.     

The insulin-like growth factor I system in the rat cerebellum: Developmental regulation and role in neuronal survival and differentiation

The developmental regulation of insulin-like growth factor I (IGF-I), its receptor, and its binding proteins (IGFBPs) was studied in the rat cerebellum. All the components of the IGF-I system were detectable in the cerebellum at least by embryonic day 19. Levels of IGF-I receptor and its mRNA were highest at perinatal ages and steadily decrease thereafter, although a partial recovery in IGF-I receptor mRNA was found in adults. Levels of IGF-I and its mRNA also peaked at early ages, although immunoreactive IGF-I showed a second peak during adulthood. Finally, levels of IGFBPs were also highest at early postnatal ages and abruptly decreased thereafter to reach lower adult levels. Since highest levels of the different components of the IGF-I system were found at periods of active cellular growth and differentiation we also examined possible trophic effects of IGF-I on developing cerebellar cells in vitro. We found a dose-dependent effect of IGF-I on neuron survival together with a specific increase of the two main neurotransmitters used by cerebellar neurons, GABA and glutamate. Analysis of cerebellar cultures by combined in vitro autoradiography and immunocytochemistry with cell-specific markers indicated that both Purkinje cells (calbindin-positive) and other neurons (neurofilament-positive) contain IGF-I binding sites. These results extend previous observations on a developmental regulation of the IGF-I system in the cerebellum and reinforce the notion of a physiologically relevant trophic role of IGF-I in cerebellar development. © 1994 Wiley-Liss, Inc.     

The distribution of the mRNA and protein products of the melanin-concentrating hormone (MCH) receptor gene, slc-1, in the central nervous system of the rat

Melanin-concentrating hormone (MCH), a 19 amino acid cyclic peptide, is largely expressed in the hypothalamus. It is implicated in the control of general arousal and goal-orientated behaviours in mammals, and appears to be a key messenger in the regulation of food intake. An understanding of the biological actions of MCH has been so far hampered by the lack of information about its receptor(s) and their location in the brain. We recently identified the orphan G-protein-coupled receptor SLC-1 as a receptor for the neuropeptide MCH. We used in situ hybridization histochemistry and immunohistochemistry to determine the distribution of SLC-1 mRNA and its protein product in the rat brain and spinal cord. SLC-1 mRNA and protein were found to be widely and strongly expressed throughout the brain. Immunoreactivity was observed in areas that largely overlapped with regions mapping positive for mRNA. SLC-1 signals were observed in the cerebral cortex, caudate-putamen, hippocampal formation, amygdala, hypothalamus and thalamus, as well as in various nuclei of the mesencephalon and rhombencephalon. The distribution of the receptor mRNA and immunolabelling was in good general agreement with the previously reported distribution of MCH itself. Our data are consistent with the known biological effects of MCH in the brain, e.g. modulation of the stress response, sexual behaviour, anxiety, learning, seizure production, grooming and sensory gating, and with a role for SLC-1 in mediating these physiological actions.     

Autoradiographic analysis of mu1, mu2, and delta opioid binding in the central nervous system of C57BL/6BY and CXBK (opioid receptor-deficient) mice

The recent development of in vitro autoradiography techniques has enabled investigators to determine the distribution and relative levels of multiple ligand binding sites in discrete anatomical areas. In this study we used semi-quantitative in vitro autoradiography to compare the levels of binding to central mu₁, mu₂, and delta opioids sites in two strains of mice, C57BL/6BY and CXBK. The CXBK strain is known to be deficient in whole brain opioid binding sites and to be less sensitive than the C57 strain to the analgesic and locomotor stimulatory effects of opiates and opioids. Delta sites were visualized using [³H][d-Ala²-d-Leu⁵]-enkephalin (DADL) plus a low concentration of morphine, total mu sites (mu₁ and mu₂) were visualized using [³H]dihydromorphine (DHM), and mu₂ sites were visualized using [³H]DHM plus a low concentration of DADL. Binding to mu₁ sites was determined by subtracting mu₂ binding from total mu binding. We found that the two strains did not consistently differ in the levels of delta sites; in some areas the CXBKs had lower levels but in many areas they had levels equal to or greater than those for the C57s. The CXBK strain, however, either had less or the same amount of mu binding as the C57 strain in all areas studied. The CXBK strain was especially deficient in mu₁ binding, particularly in areas involved in pain processing.     

The central fibroblast growth factor receptor/beta klotho system: Comprehensive mapping in Mus musculus and comparisons to nonhuman primate and human samples using an automated in situ hybridization platform

Central activation of fibroblast growth factor (FGF) receptors regulates peripheral glucose homeostasis and reduces food intake in preclinical models of obesity and diabetes. The current work was undertaken to advance our understanding of the receptor expression, as sites of ligand action by FGF19, FGF21, and FGF1 in the mammalian brain remains unresolved. Recent advances in automated RNAscope in situ hybridization and droplet digital PCR (ddPCR) technology allowed us to interrogate central FGFR/beta klotho (Klb) system at the cellular level in the mouse, with relevant comparisons to nonhuman primate and human brain. FGFR1-3 gene expression was broadly distributed throughout the CNS in Mus musculus, with FGFR1 exhibiting the greatest heterogeneity. FGFR4 expression localized only in the medial habenula and subcommissural organ of mice. Likewise, Klb mRNA was restricted to the suprachiasmatic nucleus (SCh) and select midbrain and hindbrain nuclei. ddPCR in the rodent hypothalamus confirmed that, although expression levels are indeed low for Klb, there is nonetheless a bonafide subpopulation of Klb+ cells in the hypothalamus. In NHP and human midbrain and hindbrain, Klb + cells are quite rare, as is expression of FGFR4. Collectively, these data provide the most robust central map of the FGFR/Klb system to date and highlight central regions that may be of critical importance to assess central ligand effects with pharmacological dosing, such as the putative interactions between the endocrine FGFs and FGFR1/Klb, or FGF19 with FGFR4.     

Tumor necrosis factor and interleukin-1β: suppression of food intake by direct action in the central nervous system

Intracerebroventricular microinfusion of recombinant human tumor necrosis factor (rhTNF) and recombinant human interleukin-1β (rhIL-1β) suppressed food intake in rats. Central infusion of heat-inactivated rhTNF and rhIL-1β, bovine serum albumin, heparin or transforming growth factor-β had no such effect. Central infusion of rhIL-1β did not affect the dipsogenic response to central administration of angiotensin II. Peripheral administration of rhTNF and rhIL-1β in doses equivalent to or higher than those administered centrally had no effect. Electrophoretically applied rhTNF and rhIL-1β specifically suppressed the activity of glucose-sensitive neurons in the lateral hypothalamic area. Glucose-insensitive neurons were little affected. The results suggest that TNF and IL-1β act directly in the central nervous system to suppress feeding, and this effect may be operative during acute and chronic disease.     

The distribution and function of DL-[H]2-Amino-4-Phosphonobutyrate binding sites in the rat striatum

The binding of the glutate-like radioligand,DL-[³H]2-amino-4-phosphhonobutyrate (DL-[³H]APB), to L-glutamate-sensitive sites in the rat striatum was investigated. A single, saturable population of binding sites, indistinguishable from that characterized previously on rat whole brain synaptic membranes, was identified. The effects of specific lesions of the striatum: (a) decortication; (b) striatal injection of kainic acid; and (c) 6-hydroxydopamine injections into the substantia nigra, were also examined. SpecificDL-[³H]APB binding in the striatum was elevated significantly following decortication. An increase in the number of binding sites was found to be responsible for this enhancement in binding. Lesions of the postsynaptic tartets of corticostriatal fibres reduced the number of DL-[³H]APB binding sites in the striatum without affecting binding site affinity. This finding suggests thatL-APB sensitive excitatory amino acid receptors are located predominantly on membranes derived from structures postsynaptic with regard to the glutamatergic innervation. The possible physiological role of these receptors was examined using an in vitro release technique. BothL-glutamate and L-APB were found to facilitate potassium evoked [³H]dopamine release from striatal slices. This finding supports the proposed existence of functional acidic amino acid receptors on dopaminergic terminals in the striatum. These receptors may play an important role in the control of motor function.     

Distribution of α2 agonist binding sites in the rat and human central nervous system: Analysis of some functional, anatomic correlates of the pharmacologic effects of clonidine and related adrenergic agents

Using [³H]para-aminoclonidine, α₂ adrenergic binding sites have been mapped in the rat and human CNS using in vitro labeling autoradiographic techniques. In both the rat and human thoracic spinal cord, high densities of α₂ binding sites were associated with the substantia gelatinosa and the intermediolateral cell column. In the rat medulla, high binding site density was observed in the medial nucleus of the solitary tract, dorsal motor nucleus of the vagus, raphe pallidus and the substantia gelatinosa of the trigeminal nucleus, while lower levels of specific binding were found in the lateral and ventrolateral medulla. In the human, a similar distribution was observed. However, significantly lower levels of specific binding were seen in the medial nts as opposed to the dmv. In the rat, high levels of specific binding were seen at pontine and midbrain levels in the locus coeruleus, parabrachial nucleus and periaqueductal gray. In the forebrain, several hypothalmic and limbic regions, including the paraventricular and arcuate nuclei of the hypothalamus, the central, medial and basal nuclei of the amygdala, lateral septum and bed nucleus of the stria terminalis and pyriform, entorhinal and insular cortex were labeled. Each of these regions are involved in either modulating autonomic functions directly or integrating somatosensory and/or affective function with autonomic mechanisms. Further, these regions are interrelated by reciprocal connections, and neurons that utilize nor-adrenaline or adrenaline as their neurotransmitter form a vital part of these connections. Thus, these functional, anatomical and neurochemical correlates of the α₂ binding site distribution establish a neurological basis for the complex pharmacological effects of centrally acting α₂ agonists.