Lifespan extension by increased expression of the Drosophila homologue of the IGFBP7 tumour suppressor

Mammals possess multiple insulin-like growth factor (IGF) binding proteins (IGFBPs), and related proteins, that modulate the activity of insulin/IGF signalling (IIS), a conserved neuroendocrine signalling pathway that affects animal lifespan. Here, we examine if increased levels of an IGFBP-like protein can extend lifespan, using Drosophila as the model organism. We demonstrate that Imaginal morphogenesis protein-Late 2 (IMP-L2), a secreted protein and the fly homologue of the human IGFBP7 tumour suppressor, is capable of binding at least two of the seven Drosophila insulin-like peptides (DILPs), namely native DILP2 and DILP5 as present in the adult fly. Increased expression of Imp-L2 results in phenotypic changes in the adult consistent with down-regulation of IIS, including accumulation of eIF-4E binding protein mRNA, increase in storage lipids, reduced fecundity and enhanced oxidative stress resistance. Increased Imp-L2 results in up-regulation of dilp2, dilp3 and dilp5 mRNA, revealing a feedback circuit that is mediated via the fly gut and/or fat body. Importantly, over-expression of Imp-L2, ubiquitous or restricted to DILP-producing cells or gut and fat body, extends lifespan. This enhanced longevity can also be observed upon adult-onset induction of Imp-L2, indicating it is not attributable to developmental changes. Our findings point to the possibility that an IGFBP or a related protein, such as IGFBP7, plays a role in mammalian aging.     

Attenuation of insulin signalling contributes to FSN‐1‐mediated regulation of synapse development

A neuronal F‐box protein FSN‐1 regulates Caenorhabditis elegans neuromuscular junction development by negatively regulating DLK‐mediated MAPK signalling. In the present study, we show that attenuation of insulin/IGF signalling also contributes to FSN‐1‐dependent synaptic development and function. The aberrant synapse morphology and synaptic transmission in fsn‐1 mutants are partially and specifically rescued by reducing insulin/IGF‐signalling activity in postsynaptic muscles, as well as by reducing the activity of EGL‐3, a prohormone convertase that processes agonistic insulin/IGF ligands INS‐4 and INS‐6, in neurons. FSN‐1 interacts with, and potentiates the ubiquitination of EGL‐3 in vitro, and reduces the EGL‐3 level in vivo. We propose that FSN‐1 may negatively regulate insulin/IGF signalling, in part, through EGL‐3‐dependent insulin‐like ligand processing.     

Insulin‐like receptor substrate gene chico regulates octopamine metabolism in Drosophila melanogaster

Octopamine, one of the main insect biogenic amines, plays an important role in the control of fitness in Drosophila melanogaster Meigen. The present study examines the effects of a null mutation of the gene of the insulin‐like receptor substrate (chico), in the heterozygous state, on octopamine metabolism, heat stress resistance and fecundity of D. melanogaster. A rise in the activity of one of the key enzymes of octopamine synthesis, tyrosine decarboxylase, as well as that of an enzyme of its degradation, octopamine‐dependent N‐acetyl transferase, is observed in chico^1/+ females. It is also found that the resistance to heat stress is decreased and fecundity is reduced dramatically in chico^1/+ flies. Such changes in these parameters in D. melanogaster females result from a rise in octopamine titre, which suggests that chico affects the octopamine level by regulating the activity of tyrosine decarboxylase.     

Serum concentrations of insulin‐like growth factor‐i and insulin‐like growth factor binding protein‐2 and ‐3 in eight hoofstock species

The somatotropic axis, which includes growth hormone, insulin‐like growth factor (IGF)‐I, and IGF binding proteins (IGFBP), is involved in the regulation of growth and metabolism. Measures of the somatotropic axis can be predictive of nutritional status and growth rate that can be utilized to identify nutritional status of individual animals. Before the somatotropic axis can be a predictive tool, concentrations of hormones of the somatotropic axis need to be established in healthy individuals. To begin to establish these data, we quantified IGF‐I, IGFBP‐2, and IGFBP‐3 in males and females of eight threatened hoofstock species at various ages. Opportunistic blood samples were collected from Bos javanicus (Java banteng), Tragelaphus eurycerus isaaci (bongo), Gazella dama ruficollis (addra gazelle), Taurotragus derbianus gigas (giant eland), Kobus megaceros (Nile lechwe), Hippotragus equines cottoni (roan antelope), Ceratotherium simum simum (white rhinoceros), and Elephas maximus (Asian elephant). Serum IGF‐I and IGFBPs were determined by radioimmunoassay and ligand blot, respectively. Generally, IGF‐I and IGFBP‐3 were greater in males, and IGFBP‐2 was greater in females. In banteng (P = 0.08) and male Nile lechwe (P<0.05), IGF‐I increased with age, but decreased in rhinoceros (P = 0.07) and female Nile lechwe (P<0.05). In banteng, IGFBP‐3 was greater (P<0.01) in males. In elephants (P<0.05) and antelope (P = 0.08), IGFBP‐2 were greater in females. Determination of concentrations of hormones in the somatotropic axis in healthy animals makes it possible to develop models that can identify the nutritional status of these threatened hoofstock species. Zoo Biol 30:275–284, 2011. © 2010 Wiley‐Liss, Inc.     

DILP‐producing median neurosecretory cells in the Drosophila brain mediate the response of lifespan to nutrition

Dietary restriction extends lifespan in diverse organisms, but the gene regulatory mechanisms and tissues mediating the increased survival are still unclear. Studies in worms and flies have revealed a number of candidate mechanisms, including the target of rapamycin and insulin/IGF‐like signalling (IIS) pathways and suggested a specific role for the nervous system in mediating the response. A pair of sensory neurons in Caenorhabditis elegans has been found to specifically mediate DR lifespan extension, but a neuronal focus in the Drosophila nervous system has not yet been identified. We have previously shown that reducing IIS via the partial ablation of median neurosecretory cells in the Drosophila adult brain, which produce three of the seven fly insulin‐like peptides, extends lifespan. Here, we show that these cells are required to mediate the response of lifespan to full feeding in a yeast dilution DR regime and that they appear to do so by mechanisms that involve both altered IIS and other endocrine effects. We also present evidence of an interaction between these mNSCs, nutrition and sleep, further emphasising the functional homology between the DILP‐producing neurosecretory cells in the Drosophila brain and the hypothalamus of mammals in their roles as integration sites of many inputs for the control of lifespan and behaviour.     

Impaired IGF-I signalling of hypertrophic hearts in the developmental phase of hypertension in genetically hypertensive rats

Insulin-like growth factor-I (IGF-I) signalling is reported to contribute to the modulation of blood pressure and set survival and hypertrophic responses in cardiac tissue. However, whether IGF-I signalling normally acts in cardiac tissues of hypertensive rats is unknown. In this study, using spontaneously hypertensive rats (SHR) and stroke-prone spontaneously hypertensive rats (SPSHR), both with early blood pressure increases, and Wistar-Kyoto (WKY) rats as controls, we measured the hypertrophic and IGF-I signalling activity changes in rat hearts at 4, 6 and 12 weeks of age. Both SHR and SPSHR were found to have significantly increased blood pressures and ratios of heart- and left ventricle- to body weight at 12 weeks of age. However, IGF-IR and its downstream signalling, including the protein levels of PI3K and phosphorylated Akt, known to maintain physiological cardiac hypertrophy and cardiomyocyte survival, were downregulated. The results of dot blotting showed that cardiac mRNA levels of IGF-I in hypertensive rats were higher than those in controls starting from the age of 4 weeks. This difference suggests the increased ligand IGF-I mRNA levels may be a compensatory response caused by the impaired IGF-I signalling. Moreover, enhanced cardiac cytosolic cytochrome-c, a mitochondria-dependent apoptotic pathway component, tended to occur in both hypertensive rats, although it did not reach a significant level. These findings indicate that impaired IGF-IR signalling occurs at early stages, and it may contribute, at least partially, to the development of hypertension and pathological cardiac hypertrophy and to cardiomyocyte apoptosis at later stages in SHR and SPSHR.     

Central insulin‐like growth factor‐1 (IGF‐1) restores whole‐body insulin action in a model of age‐related insulin resistance and IGF‐1 decline

Low insulin‐like growth factor‐1 (IGF‐1) signaling is associated with improved longevity, but is paradoxically linked with several age‐related diseases in humans. Insulin‐like growth factor‐1 has proven to be particularly beneficial to the brain, where it confers protection against features of neuronal and cognitive decline. While aging is characterized by central insulin resistance in the face of hyperinsulinemia, the somatotropic axis markedly declines in older humans. Thus, we hypothesized that increasing IGF‐1 in the brain may prove to be a novel therapeutic alternative to overcome central insulin resistance and restore whole‐body insulin action in aging. Utilizing hyperinsulinemic‐euglycemic clamps, we show that old insulin‐resistant rats with age‐related declines in IGF‐1 level demonstrate markedly improved whole‐body insulin action, when treated with central IGF‐1, as compared to central vehicle or insulin (P < 0.05). Furthermore, central IGF‐1, but not insulin, suppressed hepatic glucose production and increased glucose disposal rates in aging rats (P < 0.05). Taken together, IGF‐1 action in the brain and periphery provides a ‘balance’ between its beneficial and detrimental actions. Therefore, we propose that strategies aimed at ‘tipping the balance’ of IGF‐1 action centrally are the optimal approach to achieve healthy aging and longevity in humans.     

Expressions of Leptin and Insulin‐Like Growth Factor‐I Are Highly Correlated and Region‐Specific in Adipose Tissue of Growing Rats

Objective: Anatomically distinct adipose tissue regions differ in their predominant modality of growth (i.e., cellular hypertrophy vs. hyperplasia). We examined site‐specific patterns of expression of two genes whose products, leptin and insulin‐like growth factor‐I (IGF‐I), could be involved in mediating differential growth and metabolism of white adipose tissue. We also related these patterns of expression to measures of adipose depot cellularity. Research Methods and Procedures: Male Wistar rats were fed ad libitum and studied from ages 7 weeks to ～12 months. Terminal measures of body weights; weights, composition, and cellularity of four white adipose depots; circulating leptin and IGF‐I; and adipose depot‐specific expression levels of leptin and IGF‐I were measured in subsets of rats at 7, 12, 22, 42, and 46 weeks of age. Results: Both leptin and IGF‐I mRNAs are quantitatively expressed in a depot‐specific manner, in the following order: retroperitoneal ? epididymal > mesenteric > subcutaneous inguinal. Furthermore, there is a marked correlation between the expressions of these hormones in the various regions of adipose tissue of rats during the first year of life. The mechanisms that underlie the parallel expressions of leptin and IGF‐I appear to be related to fat‐cell volume. Discussion: Because both leptin and IGF‐I have been implicated in the regulation of energy homeostasis and are both expressed in adipose tissue, the depot‐specific linkage between the two genes suggests interaction at the autocrine level. This interaction may have an important role in determining functional properties particular to individual adipose depots.     

Expression of insulin‐like growth factor‐1 receptor in metastatic uveal melanoma and implications for potential autocrine and paracrine tumor cell growth

We investigated the importance of the insulin‐like growth factor‐1 receptor (IGF‐1R) in hepatic metastases of uveal melanoma. The expression pattern of IGF‐1R in archival tissue samples of hepatic metastasis from 24 patients was analyzed by immunohistochemistry. All the samples of hepatic metastases stained positive for IGF‐1R. To investigate the biological role of IGF‐1R on the growth of metastatic uveal melanoma, a long‐term cell line obtained from a hepatic metastasis (TJU‐UM001) was evaluated. TJU‐UM001 expressed cell surface IGF‐1R (>90%) and proliferated in response to exogenous and endogenous insulin‐like growth factor‐1 (IGF‐1). Correlatively, anti‐IGF‐1R antibody completely blocked IGF‐1‐induced growth of TJU‐UM001 cells. IGF‐1 preferentially induced phosphorylation of Akt (S473) in quiescent TJU‐UM001 cells, and this was blocked by anti‐IGF‐1R antibody. This study suggests that autocrine and paracrine mechanisms underlie IGF‐1‐induced growth of metastatic uveal melanoma and underscore the potential benefit of IGF‐1 or IGF‐1R antagonism in treatment for metastatic uveal melanoma.     

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA‐122‐5p (miR‐122) regulates the hepatic IR in vitro. We first found that the miR‐122 level was upregulated in the liver of rats fed with a high‐fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin‐like growth factor 1 receptor (IGF‐1R), a potential target of miR‐122, was downregulated in the diabetic liver. In vitro, glucosamine‐induced IR was introduced in HepG2 hepatic cells, and the levels of miR‐122 and IGF‐1R were further assessed. An increase of miR‐122 level and a decrease of IGF‐IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF‐1R as a direct target of miR‐122. Moreover, in IR HepG2 cells, antagonizing miR‐122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6‐phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR‐122 inhibitor in IR hepatic cells were impaired when IGF‐1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR‐122 inhibitor transfection. In conclusion, our study demonstrates that miR‐122 is able to regulate IR in hepatic cells by targeting IGF‐1R.     

Decreased caveolin‐1 levels contribute to fibrosis and deposition of extracellular IGFBP‐5

Our previous studies have demonstrated increased expression of insulin‐like growth factor binding protein‐5 (IGFBP‐5) in fibrotic tissues and IGFBP‐5 induction of extracellular matrix (ECM) components. The mechanism resulting in increased IGFBP‐5 in the extracellular milieu of fibrotic fibroblasts is unknown. Since Caveolin‐1 (Cav‐1) has been implicated to play a role in membrane trafficking and signal transduction in tissue fibrosis, we examined the effect of Cav‐1 on IGFBP‐5 internalization, trafficking and secretion. We demonstrated that IGFBP‐5 localized to lipid rafts in human lung fibroblasts and bound Cav‐1. Cav‐1 was detected in the nucleus in IGFBP‐5‐expressing fibroblasts, within aggregates enriched with IGFBP‐5, suggesting a coordinate trafficking of IGFBP‐5 and Cav‐1 from the plasma membrane to the nucleus. This trafficking was dependent on Cav‐1 as fibroblasts from Cav‐1 null mice had increased extracellular IGFBP‐5, and as fibroblasts in which Cav‐1 was silenced or lipid raft structure was disrupted through cholesterol depletion also had defective IGFBP‐5 internalization. Restoration of Cav‐1 function through administration of Cav‐1 scaffolding peptide dramatically increased IGFBP‐5 uptake. Finally, we demonstrated that IGFBP‐5 in the ECM protects fibronectin from proteolytic degradation. Taken together, our findings identify a novel role for Cav‐1 in the internalization and nuclear trafficking of IGFBP‐5. Decreased Cav‐1 expression in fibrotic diseases likely leads to increased deposition of IGFBP‐5 in the ECM with subsequent reduction in ECM degradation, thus identifying a mechanism by which reduced Cav‐1 and increased IGFBP‐5 concomitantly contribute to the perpetuation of fibrosis.     

Drosophila poly suggests a novel role for the Elongator complex in insulin receptor-target of rapamycin signalling

Multi-cellular organisms need to successfully link cell growth and metabolism to environmental cues during development. Insulin receptor-target of rapamycin (InR-TOR) signalling is a highly conserved pathway that mediates this link. Herein, we describe poly, an essential gene in Drosophila that mediates InR-TOR signalling. Loss of poly results in lethality at the third instar larval stage, but only after a stage of extreme larval longevity. Analysis in Drosophila demonstrates that Poly and InR interact and that poly mutants show an overall decrease in InR-TOR signalling, as evidenced by decreased phosphorylation of Akt, S6K and 4E-BP. Metabolism is altered in poly mutants, as revealed by microarray expression analysis and a decreased triglyceride : protein ratio in mutant animals. Intriguingly, the cellular distribution of Poly is dependent on insulin stimulation in both Drosophila and human cells, moving to the nucleus with insulin treatment, consistent with a role in InR-TOR signalling. Together, these data reveal that Poly is a novel, conserved (from flies to humans) mediator of InR signalling that promotes an increase in cell growth and metabolism. Furthermore, homology to small subunits of Elongator demonstrates a novel, unexpected role for this complex in insulin signalling.