The role of growth hormone and insulin-like growth factors in the immune system

Growth hormone (GH) and insulin-like growth factor I (IGF-I) can modulate the development and function of the immune system. In this chapter, we present data on the expression of receptors for GH and IGFs and the in vitro and in vivo effects of these proteins. We show that expression of GH and IGFs in the immune system opens up the possibility that these proteins are not only involved in endocrine control of the immune system but can also play a role as local growth and differentiation factors (cytokines). Endocrine control of GH could be direct or mediated via endocrine or autocrine/paracrine IGF-I. In addition, GH can act as an autocrine or paracrine factor itself. Furthermore, IGF-I in the immune system has been shown to be regulated by cytokines, such as interleukin-1 and interferon-γ, alluding to a cytokine-like function of IGF-I. In addition to data on the function of GH and IGF-I in the immune system, we present new findings which imply a possible function of IGF-II and IGF-binding proteins.     

New concepts in regulation and function of the insulin-like growth factors: implications for understanding normal growth and neoplasia

The insulin-like growth factors (IGFs) are a ubiquitous family of growth factors, binding proteins and receptors that are involved in normal growth and development. They are also implicated in numerous pathological states, including malignancy. IGF-II is a commonly expressed growth factor in many tumors and may enhance tumor growth, acting via the overexpressed IGF-I receptor, a cell-surface tyrosine kinase receptor. The IGF-I receptor may be overexpressed due to mutations in tumor suppression gene products such as p53 and WT-1 or growth factors such as bFGF and PDGF. Thus, this family of growth factors, especially the IGF-I receptor, may present an excellent target for new therapeutic agents in the treatment of cancer and other disorders of excessive cellular proliferation.     

Ethanol inhibits insulin expression and actions in the developing brain

Ethanol-induced cerebellar hypoplasia is associated with inhibition of insulin-stimulated survival signaling. The present work explores the mechanisms of impaired insulin signaling in a rat model of fetal alcohol syndrome. Real-time quantitative RT-PCR demonstrated reduced expression of the insulin gene in cerebella of ethanol-exposed pups. Although receptor expression was unaffected, insulin and insulin-like growth factor (IGF-I) receptor tyrosine kinase (RTK) activities were reduced by ethanol exposure, and these abnormalities were associated with increased PTP1b activity. In addition, glucose transporter molecule expression and steady-state levels of ATP were reduced in ethanol-exposed cerebellar tissue. Cultured cerebellar granule neurons from ethanol-exposed pups had reduced expression of genes encoding insulin, IGF-II, and the IGF-I and IGF-II receptors, and impaired insulin- and IGF-I-stimulated glucose uptake and ATP production. The results demonstrate that ethanol inhibits insulin-mediated actions in the developing brain by reducing local insulin production and insulin RTK activation, leading to inhibition of glucose transport and ATP production.Received 30 December 2004; received after revision 1 March 2005; accepted 10 March 2005     

Structure and function of the type 1 insulin-like growth factor receptor

The type 1 insulin-like growth factor receptor (IGF-1R), a transmembrane tyrosine kinase, is widely expressed across many cell types in foetal and postnatal tissues. Activation of the receptor following binding of the secreted growth factor ligands IGF-1 and IGF-2 elicits a repertoire of cellular responses including proliferation, and the protection of cells from programmed cell death or apoptosis. As a result, signalling through the IGF-1R is the principal pathway responsible for somatic growth in foetal mammals, whereas somatic growth in postnatal animals is achieved through the synergistic interaction of growth hormone and the IGFs. Forced overexpression of the IGF-1R results in the malignant transformation of cultured cells: conversely, downregulation of IGF-1R levels can reverse the transformed phenotype of tumour cells, and may render them sensitive to apoptosis in vivo. Elevated levels of IGF-IR are observed in a variety of human tumour types, whereas epidemiological studies implicate the IGF-1 axis as a predisposing factor in the pathogenesis of human breast and prostate cancer. The IGF-1R has thus emerged as a therapeutic target for the development of antitumour agents. Recent progress towards the elucidation of the three-dimensional structure of the extracellular domain of the IGF-1R represents an opportunity for the rational assembly of small molecule antagonists of receptor function for clinical use.     

Production of insulin-like growth factor I (IGF-I) and IGF-binding proteins by rat intestinal stromal cells in vitro

To determine if intestinal stromal cells secrete diffusible factors such as insulin-like growth factors (IGFs) capable of regulating epithelial cell growth in vitro, stromal cells were isolated by enzymatic digestion of rat intestine. Incorporation of [³H]thymidine into DNA and [¹⁴C]leucine into protein of IEC-6 cells, a model intestinal epithelial cell line, was significantly increased (two- to threefold) when the IEC-6 cells were co-cultured with stromal cells, relative to IEC-6 cells grown alone. Medium conditioned by stromal cells stimulated DNA synthesis of IEC-6 cells in a dose-dependent manner. Analysis of the conditioned medium revealed that intestinal stromal cells secreted IGF-I, but little IGF-II, in addition to an M _r 32,000 IGF-binding protein (IGFBP-2) and an IGFBP having M _r∼ 24,000. We conclude that rat intestinal stromal cells secrete one or more diffusible factors, which may include IGF-I and IGFBPs, capable of stimulating proliferation of IEC-6 cells in vitro. Received 25 August 1997; received after revision 7 November 1997; accepted 20 November 1997     

Chronic gestational exposure to ethanol causes insulin and IGF resistance and impairs acetylcholine homeostasis in the brain

In fetal alcohol syndrome (FAS), cerebellar hypoplasia is associated with impaired insulin-stimulated survival signaling. This study characterizes ethanol dose-effects on cerebellar development, expression of genes required for insulin and insulin-like growth factor (IGF) signaling, and the upstream mechanisms and downstream consequences of impaired signaling in relation to acetylcholine (ACh) homeostasis. Pregnant Long Evans rats were fed isocaloric liquid diets containing 0%, 2%, 4.5%, 6.5%, or 9.25% ethanol from gestation day 6. Ethanol caused dose-dependent increases in severity of cerebellar hypoplasia, neuronal loss, proliferation of astrocytes and microglia, and DNA damage. Ethanol also reduced insulin, IGF-I, and IGF-II receptor binding, insulin and IGF-I receptor tyrosine kinase activities, ATP, membrane cholesterol, and choline acetyltransferase (ChAT) expression. In vitro studies linked membrane cholesterol depletion to impaired insulin receptor binding and insulin-stimulated ChAT. In conclusion, cerebellar hypoplasia in FAS is mediated by insulin/IGF resistance with attendant impairments in energy production and ACh homeostasis. Received 4 May 2006; received after revision 13 June 2006; accepted 20 June 2006     

Specific cleavage of insulin-like growth factor-binding protein-1 by a novel protease activity

Insulin-like growth factor-binding protein-1 (IGFBP-1) is secreted in a highly phosphorylated form that binds IGF-I with high affinity and is resistant to proteolysis. We have purified IGFBP-1-specific protease activity from the urine of an individual with multiple myeloma. This protease efficiently cleaves both phosphorylated and non-phosphorylated IGFBP-1 at Ile130-Ser131, generating fragments that together have higher association and dissociation rates for IGFs compared with intact IGFBP-1. The proteolytic fraction contained azurocidin, a protease homologue hitherto considered inactive. After cleavage of IGFBP-1, there was a lower affinity, but higher capacity for IGF-I binding, suggesting both N- and C-terminal fragments may interact with ligand independently. There was decreased inhibition of IGF-II-stimulated cell growth and glucose uptake. Alone, proteolysed IGFBP-1 stimulated glucose uptake in muscle. We conclude that specific cleavage of IGFBP-1 at target tissues is important in cellular growth and metabolism and opens novel strategies for targeting IGFBP-1 in treatment of disease.     

A 30-kDa fragment of insulin-like growth factor (IGF) binding protein-3 in human pregnancy serum with strongly reduced IGF-I binding

Proteolytic cleavage of insulin-like growth factor (IGF) binding protein (IGFBP)-3 during pregnancy is likely to have both IGF-dependent and -independent effects on maternal, placental and fetal growth and metabolism. A 30-kDa proteolytic IGFBP-3 fragment was isolated from third trimester pregnancy human serum and identified by N- and C-terminal amino acid sequence analysis and mass spectrometry to correspond to residues 1–212 of the parent protein. This fragment is the dominating IGFBP-3 immunoreactive species in pregnancy serum. The 30-kDa fragment was also detected in serum of non-pregnant women where it coexists with intact IGFBP-3. Using biosensor technology, (1–212)IGFBP-3 was found to have 11-fold lower affinity for IGF-I compared to intact IGFBP-3, while a 4-fold decrease in affinity was found for IGF-II. Tests with des(1–3)IGF-I suggest fast binding of IGF-I to the N-terminal region of IGFBP-3 and similar affinity to a slow binding site in the C-terminal region. Received 24 April 2007; received after revision 11 June 2007; accepted 13 June 2007     

Effect of various stressors on the levels of lipid peroxide,antioxidants and Na+, K+-ATPase actrivity in rat brain

The level of malondialdehyde (MDA), an index of lipid peroxidation, and the antioxidants superoxide dismutase (SOD) and glutathione (GSH), as well as the activity of Na⁺, K⁺-ATPase, were assessed in whole rat brain after immobilization, anemic hypoxia (NaNO₂) and 72 h starvation. The effect of these stressors on plasma glucose and corticosterone levels was also observed. Hypoxia and starvation stimulated the lipidj peroxide formation in braini as indicated by an increase in the level of MDA, being higher after starvation than hypoxia. Brain SOD activity was also increased in response to hypoxia and starvation while GSH content was only diminished ini hypoxia. However, neither MDA nor antioxidants were affected by immobilization. On the other hand, the activity of brain Na⁺, K⁺-ATPase was significantly increased by immobilization and hypoxia but decreased in starvation. A similar pattern of change was also observed in plasma glucose and corticosterone levels in response to these stressors. These results elucidate differences in the biochemical response of animals towards various types of stress, with increased lipid peroxide formation in hypoxia and starvation.     

Differential expression of genes for uncoupling proteins 1, 2 and 3 in brown and white adipose tissue depots during rat development

The different expression patterns of genes for uncoupling proteins (UCPs) 1, 2 and 3 (ucp1, ucp2 and ucp3) were studied in interscapular brown adipose tissue (BAT) and in four white adipose tissue (WAT) depots (epididymal, inguinal, mesenteric and retroperitoneal) in male rats of different ages (18 days-12 months). UCP mRNA expression levels were determined by Northern blotting. In BAT, there were high levels of expression of UCP1 and UCP3 mRNA, but no detectable levels of UCP2 mRNA. Both ucp1 and ucp3 followed a similar expression pattern with age, with high levels in suckling rats which decreased to 50% or less in rats just under 2 months old, declining thereafter until 5 months and then recovering with age. However, an additional peak of expression was observed for ucp3 at the age of 3 months. In WAT, ucp1 expression was rare: occasional expression was found for UCP1 mRNA in the retroperitoneal depot in suckling rats and in the epididymal and inguinal depots in suckling and mature adult rats. ucp2 and ucp3 had different developmental expression patterns, but these were similar for each gene in the different depots studied. UCP3 mRNA was highly expressed in rats soon after birth, it decreased until 3 months, and increased thereafter, except for the mesenteric WAT where ucp3 expression decreased until 7 months before recovering. The fact that changes with age of both ucp1 and ucp3 expression have a similar profile in BAT, which is also similar to the ucp3 and also ucp1 profiles in some WAT depots, might reflect a common regulatory pattern for the expression of these genes, and also a common function. In contrast to ucp1 and ucp3, ucp2 had a peak of expression at about 2 months, and lower expression at 3 months, suggesting different regulation and probably a different role for this UCP.     

Microarray-based identification of differentially expressed growth- and metastasis-associated genes in pancreatic cancer

Pancreatic ductal adenocarcinoma (PDAC) has an extremely poor prognosis. To improve diagnosis and treatment, key mechanisms of deregulated molecular functions have to be identified. Using microarray analysis, the expression patterns of 5600 human genes were assessed in PDAC by comparison with the normal pancreas and chronic pancreatitis (CP). The expression of 467 of 5600 genes was increased in PDAC in comparison to the normal pancreas, and the expression of 120 of these genes was not increased in CP. In addition, 341 of 5600 genes were expressed at decreased levels in PDAC tissues, of which 96 were decreased in comparison to both normal and CP tissues. Thus, a total of 808 of 5600 human genes were differentially expressed in pancreatic cancer. The identification of a large panel of altered genes in PDAC will stimulate additional studies that will lead to improved understanding of the molecular mechanisms underlying pancreatic malignant growth.     

An isoform of the vacuolar (H+)-ATPase accessory subunit Ac45

The vacuolar (H⁺)-ATPase (V-ATPase) is the main regulator of intraorganellar pH and in neuroendocrine cells is controlled by its accessory subunit, Ac45. Here, we report the discovery of the first isoform of a V-ATPase accessory subunit, namely an Ac45-like protein, denoted Ac45LP. Phylogenetic analysis revealed a lineage-dependent evolutionary history: Ac45 is absent in birds, and Ac45LP is absent in placental mammals, whereas all other tetrapod species contain both genes. In contrast to Ac45, Ac45LP is not proteolytically cleaved, a prerequisite for proper Ac45 routing. Intriguingly, Xenopus Ac45LP mRNA was expressed in developing neural tissue and in neural crest cells. In adult Xenopus, Ac45 mRNA is widely expressed mostly in neuroendocrine tissues, while Ac45LP mRNA expression was found to be restricted to the kidney and the lung. This novel Ac45LP may provide additional possibilities for V-ATPase regulation during neurodevelopment as well as in kidney and lung cells.     

Molecular basis of cardiotoxicity upon cobra envenomation

Various clinical manifestations leading to death have been documented in most cases of bites caused by venomous snakes. Cobra envenomation is an extremely variable process and known to cause profound neurological abnormalities. The complexity of cobra venom can induce multiple-organ failure, leading to death in case of severe envenomation. Intramuscular administration of Malayan spitting cobra (Naja sputatrix) crude venom at 1 g/g dose caused death in mice in approximately 3 h. Analysis of gene expression profiles in the heart, brain, kidney, liver and lung revealed 203 genes whose expression was altered by at least 3-fold in response to venom treatment. Of these, 50% were differentially expressed in the heart and included genes involved in inflammation, apoptosis, ion transport and energy metabolism. Electrocardiogram recordings and serum troponin T measurements indicated declining cardiac function and myocardial damage. This not only sheds light on the cardiotoxicity of cobra venom but also reveals the molecular networks affected during envenomation.Received 7 August 2004; received after revision 11 October 2004; accepted 4 November 2004     

Differential removal of insulin-like growth factor binding proteins in rat serum by solvent extraction procedures

Solvent extraction of serum and other biological fluids at an acidic pH is a convenient method to remove the insulin-like growth factor binding proteins (IGFBPs); however, an incomplete removal of IGFBPs can occur and this can potentially interfere with the radioimmunoassay of insulin-like growth factors (IGFs). This study compared the removal of IGFBPs from normal adult rat serum and 5-day old neonatal rat serum by acid-gel filtration, and three solvent extraction methods, i.e., acid-ethanol (AE), acid-cryo-ethanol (ACE) and formic acid-acetone (FAA) treatments by western ligand blotting and slot-blotting analysis. In adult rat serum all three extraction methods removed nearly 75% of total IGFBPs present. For the neonatal serum, AE and FAA were very inefficient in eliminating the IGFBPs, while ACE was somewhat better, as it removed nearly 30% of IGFBPs. Ligand blots of extracted samples showed that IGFBPs of lower size range, 24 to 32 kDa (IGFBP-4, IGFBPs-1 and-2), were resistant to solvent extraction. Acid-gel filtration, in contrast, eliminated >95% of IGF-binding components in both sera. Determination of IGF-I concentrations in samples after gel filtration and extraction methods revealed lower IGF-I values in neonatal serum in acid extracted samples. These data caution against using solvent extractions for IGFBP removal in fetal/neonatal serum.     

CD24 controls Src/STAT3 activity in human tumors

CD24 is a glycosyl-phosphatidylinositol-anchored membrane protein that is frequently over-expressed in a variety of human carcinomas and is correlated with poor prognosis. In cancer cell lines, changes of CD24 expression can alter several cellular properties in vitro and tumor growth in vivo. However, little is known about how CD24 mediates these effects. Here we have analyzed the functional consequences of CD24 knock-down or over-expression in human cancer cell lines. Depletion of CD24 reduced cell proliferation and adhesion, enhanced apoptosis, and regulated the expression of various genes some of which were identified as STAT3 target genes. Loss of CD24 reduced STAT3 and FAK phosphorylation. Diminished STAT3 activity was confirmed by specific reporter assays. We found that reduced STAT3 activity after CD24 knock-down was accompanied by altered Src phosphorylation. Silencing of Src, similar to CD24, targeted the expression of prototype STAT3-regulated genes. Likewise, the over-expression of CD24 augmented Src-Y416 phosphorylation, the recruitment of Src into lipid rafts and the expression of STAT3-dependent target genes. An antibody to CD24 was effective in reducing tumor growth of A549 lung cancer and BxPC3 pancreatic cancer xenografts in mice. Antibody treatment affected the level of Src-phosphorylation in the tumor and altered the expression of STAT3 target genes. Our results provide evidence that CD24 regulates STAT3 and FAK activity and suggest an important role of Src in this process. Finally, the targeting of CD24 by antibodies could represent a novel route for tumor therapy.     

Role of hepcidin in murine brain iron metabolism

Brain iron homeostasis is maintained by a balance of both iron uptake and release, and accumulating evidence has revealed that brain iron concentrations increase with aging. Hepcidin, an iron regulatory hormone produced by hepatocytes in response to inflammatory stimuli, iron, and hypoxia, has been shown to be the long-sought hormone responsible for the regulation of body iron balance and recycling in mammals. In this study, we report that hepcidin is widely expressed in the murine brain. In cerebral cortex, hippocampus and striatum, hepcidin mRNA levels increased with aging. Injection of hepcidin into the lateral cerebral ventricle resulted in decreased Fpn1 protein levels in cerebral cortex, hippocampus, and striatum. Additionally, treatment of primary cultured neurons with hepcidin caused decreased neuronal iron release and Fpn1 protein levels. Together, our data provide further evidence that hepcidin may be involved in the regulation of brain iron metabolism.