Mutations in insulin/IGF-1 signaling pathway have been shown to lead to increased longevity in various invertebrate models. Therefore, the effect of the haplo- insufficiency of the IGF-1 receptor (Igf1r+/−) on longevity/aging was evaluated in C57Bl/6 mice using rigorous criteria where lifespan and end-of-life pathology were measured under optimal husbandry conditions using large sample sizes. Igf1r+/− mice exhibited reductions in IGF-1 receptor levels and the activation of Akt by IGF-1, with no compensatory increases in serum IGF-1 or tissue IGF-1 mRNA levels, indicating that the Igf1r+/− mice show reduced IGF-1 signaling. Aged male, but not female Igf1r+/− mice were glucose intolerant, and both genders developed insulin resistance as they aged. Female, but not male Igf1r+/− mice survived longer than wild type mice after lethal paraquat and diquat exposure, and female Igf1r+/− mice also exhibited less diquat-induced liver damage. However, no significant difference between the lifespans of the male Igf1r+/− and wild type mice was observed; and the mean lifespan of the Igf1r+/− females was increased only slightly (less than 5%) compared to wild type mice. A comprehensive pathological analysis showed no significant difference in end-of-life pathological lesions between the Igf1r+/− and wild type mice. These data show that the Igf1r+/− mouse is not a model of increased longevity and delayed aging as predicted by invertebrate models with mutations in the insulin/IGF-1 signaling pathway.
Dampening of insulin/insulin like growth factor-1 (IGF1) signaling results in extension of lifespan in invertebrate as well as murine models. The impact of this evolutionarily conserved pathway on modulation of human lifespan remains unclear. We previously identified two IGF1R mutations (Ala-37-Thr and Arg-407-His) that are enriched in Ashkenazi Jewish centenarians as compared to younger controls and are associated with reduced activity of the IGF1 receptor as measured in immortalized lymphocytes. To determine whether these human longevity-associated IGF1R mutations affect IGF1 signaling, we engineered mouse embryonic fibroblasts (MEFs) expressing the different human IGF1R variants in a mouse Igf1r null background. The results indicate that MEFs expressing the human longevity-associated IGF1R mutations attenuated IGF1 signaling, as demonstrated by significant reduction in phosphorylation of both IGF1R and AKT after IGF1 treatment, in comparisons to MEFs expressing the wild type IGF1R. The impaired IGF1 signaling caused by the IGF1R mutations resulted in reduced induction of the major IGF1-activated genes in MEFs, including EGR1, mCSF, IL3Rα, and TDAG51. Furthermore, the IGF1R mutations caused a delay in cell cycle progression after IGF1 treatment, indicating a dysfunctional physiological response to a cell proliferation signal. These results demonstrate that the human longevity-associated IGF1R variants are reduced-function mutations, implying that dampening of IGF1 signaling may be a longevity mechanism in humans.
human longevity; IGF1 signaling; genetic variation; gene expression
Insulin-like growth factor II (IGF-II) plays a pivotal role in fetal and cancer development by signaling through the IGF-I and insulin receptors and activating the estrogen signaling cascade. We previously showed that precursor IGF-II (proIGF-II, the predominant form expressed in cancer) and not mature IGF-II (mIGF-II) blocks resveratrol (RSV) (a phytoalexin/anticancer agent)-induced cell death in MCF-7 cells. We hypothesize that proIGF-II regulates antiapoptotic proteins and/or the mitochondria to inhibit RSV actions and promote cell survival. This study examines the effect of mIGF-II and proIGF-II on survivin expression and mitochondrial polarization in response to RSV. RSV inhibits survivin expression and stimulates mitochondrial depolarization, caspase 7 activation and cell death. These effects were completely blocked by the addition of proIGF-II. RSV treatment had no effect on transfected MCF-7 cells constitutively expressing proIGF-II, while IGF-II siRNA transfection decreased survivin levels. Our results provide new insights for the potential use of proIGF-II as target for new anticancer therapies.
Insulin-like growth factor II; survivin; resveratrol; MCF-7; breast cancer; mitochondria
It has been demonstrated in invertebrate species that the evolutionarily conserved insulin and insulin-like growth factor (IGF) signaling (IIS) pathway plays a major role in the control of longevity. In the roundworm Caenorhabditis elegans, single mutations that diminish insulin/IGF-1 signaling can increase lifespan more than twofold and cause the animal to remain active and youthful much longer than normal. Likewise, substantial increases in lifespan are associated with mutations that reduce insulin/IGF-1 signaling in the fruit fly Drosophila melanogaster. In invertebrates, multiple insulin-like ligands exist that bind to a common single insulin/IGF-1 like receptor. In contrast, in mammals, different receptors exist that bind insulin, IGF-1 and IGF-2 with different affinities. In several mouse models, mutations that are associated with decreased GH/IGF-1 signaling or decreased insulin signaling have been associated with enhanced lifespan. However, the increased complexity of the mammalian insulin/IGF-1 system has made it difficult to separate the roles of insulin, GH and IGF-1 in mammalian longevity. Likewise, the relevance of reduced insulin and IGF-1 signaling in human longevity remains controversial. However, studies on the genetic and metabolic characteristics that are associated with healthy longevity and old age survival suggest that the conserved ancient IIS pathway may also play a role in human longevity.
Insulin; IGF-1; longevity; signaling
IGF-1 receptor (IGF-1R) signaling regulates cell growth, transformation and survival. Haploinsufficiency of the IGF-1R is reported to paradoxically confer resistance to oxidative stress in vivo and in cells cultured from Igf1r+/− mice. In order to determine whether IGF-1R deficiency directly confers resistance to oxidative stress in specific cell types, an siRNA-mediated approach was applied to reduce IGF-1R in C2C12 myoblasts, NIH3T3 fibroblasts and MC3T3-E1 osteoblasts. Treating the IGF-1R deficient myoblasts with H2O2 resulted in significantly higher phosphorylation of Akt as compared to cells having normal expression of IGF-1R. Similar results were obtained with UV treatment, another inducer of oxidative stress. This enhanced activation of Akt was associated with reduced level of cleaved caspase-3 and PARP. Moreover, in the IGF-1R knockdown myoblasts, phosphorylation of the Akt substrate Bad was enhanced after peroxide treatment. However, in NIH-3T3 fibroblasts and MC3T3-E1 osteoblasts, the loss of IGF-1R by siRNA directed knockdown was associated with reduced levels of phosphorylated Akt on treatment with H2O2 or UV as compared to control cells and these cells showed more apoptosis. These results suggest a novel mechanism of cell type specific differential regulation of resistance to oxidative stress induced apoptosis by reduced levels of IGF-1R.
Luteolin is a 3',4',5,7-tetrahydroxyflavone found in various fruits and vegetables. We have shown previously that luteolin reduces HT-29 cell growth by inducing apoptosis and cell cycle arrest. The objective of this study was to examine whether luteolin downregulates the insulin-like growth factor-I receptor (IGF-IR) signaling pathway in HT-29 cells.
In order to assess the effects of luteolin and/or IGF-I on the IGF-IR signaling pathway, cells were cultured with or without 60 μmol/L luteolin and/or 10 nmol/L IGF-I. Cell proliferation, DNA synthesis, and IGF-IR mRNA levels were evaluated by a cell viability assay, [3H]thymidine incorporation assays, and real-time polymerase chain reaction, respectively. Western blot analyses, immunoprecipitation, and in vitro kinase assays were conducted to evaluate the secretion of IGF-II, the protein expression and activation of IGF-IR, and the association of the p85 subunit of phophatidylinositol-3 kinase (PI3K) with IGF-IR, the phosphorylation of Akt and extracellular signal-regulated kinase (ERK)1/2, and cell division cycle 25c (CDC25c), and PI3K activity.
Luteolin (0 - 60 μmol/L) dose-dependently reduced the IGF-II secretion of HT-29 cells. IGF-I stimulated HT-29 cell growth but did not abrogate luteolin-induced growth inhibition. Luteolin reduced the levels of the IGF-IR precursor protein and IGF-IR transcripts. Luteolin reduced the IGF-I-induced tyrosine phosphorylation of IGF-IR and the association of p85 with IGF-IR. Additionally, luteolin inhibited the activity of PI3K activity as well as the phosphorylation of Akt, ERK1/2, and CDC25c in the presence and absence of IGF-I stimulation.
The present results demonstrate that luteolin downregulates the activation of the PI3K/Akt and ERK1/2 pathways via a reduction in IGF-IR signaling in HT-29 cells; this may be one of the mechanisms responsible for the observed luteolin-induced apoptosis and cell cycle arrest.
In recent years, the activation of the insulin-like growth factor (IGF) system in cancer has emerged as a key factor for tumour progression and resistance to apoptosis. Therefore, a variety of strategies have been developed to block the type I IGF receptor (IGF-I-R), which is thought to mediate the biological effects of both IGF-I and IGF-II. However, recent data suggest that the IGF signalling system is complex and that other receptors are involved. To unravel the complexity of the IGF system in thyroid cancer, IGF-I and IGF-II production, and the expression and function of their cognate receptors were studied. Both IGFs were found to be locally produced in thyroid cancer: IGF-I by stromal cells and IGF-II by malignant thyrocytes. Values were significantly higher in malignant tissue than in normal tissue. IGF-I-Rs were overexpressed in differentiated papillary carcinomas but not in poorly differentiated or undifferentiated tumours, whereas insulin receptors (IRs) were greatly overexpressed in all tumour hystotypes, with a trend for higher values in dedifferentiated tumours. As a consequence of IR overexpression, high amounts of IR/IGF-I-R hybrids (which bind IGF-I with high affinity) were present in all thyroid cancer histotypes. Because of recent evidence that isoform A of IR (IR-A) is a physiological receptor for IGF-II in fetal life, the relative abundance of IR-A in thyroid cancer was measured. Preliminary data indicate that overexpressed IRs mainly occur as IR-A in thyroid cancer. These data indicate that both IR/IGF-I-R hybrids and IR-A play an important role in the overactivation of the IGF system in thyroid cancer and in IGF-I mitogenic signalling in these tumours. J Clin Pathol: Mol Pathol
insulin-like growth factor system; insulin receptor; insulin-like growth factor I receptor
Reduced insulin/IGF signaling increases lifespan in many animals. To understand how insulin/IGF mediates lifespan in Drosophila, we performed chromatin immunoprecipitation-sequencing analysis with the insulin/IGF regulated transcription factor dFOXO in long-lived insulin/IGF signaling genotypes. Dawdle, an Activin ligand, is bound and repressed by dFOXO when reduced insulin/IGF extends lifespan. Reduced Activin signaling improves performance and protein homeostasis in muscles of aged flies. Activin signaling through the Smad binding element inhibits the transcription of Autophagy-specific gene 8a (Atg8a) within muscle, a factor controlling the rate of autophagy. Expression of Atg8a within muscle is sufficient to increase lifespan. These data reveal how insulin signaling can regulate aging through control of Activin signaling that in turn controls autophagy, representing a potentially conserved molecular basis for longevity assurance. While reduced Activin within muscle autonomously retards functional aging of this tissue, these effects in muscle also reduce secretion of insulin-like peptides at a distance from the brain. Reduced insulin secretion from the brain may subsequently reinforce longevity assurance through decreased systemic insulin/IGF signaling.
It is widely known that reduced insulin/IGF signaling slows aging in many contexts. This process requires the forkhead transcription factor (FOXO). FOXO modulates the expression of many genes, and the list of those associated with slow aging is impressive. But there are few data indicating the mechanisms or genes through which FOXO actually slows aging. Here, we identify a novel FOXO target, dawdle, the Activin-like ligand in fruit flies. We show that down-regulation of Activin signaling in muscle, but not in adipose tissue, leads to extended lifespan. In part it does so when it alleviates the negative transcriptional repression of its Smox transcription factor (a Smad transcription factor) upon a keystone autophagy gene, Atg8a. This double signaling cascade autonomously improves muscle performance (measured at cellular and functional levels) and nonautonomously extends lifespan as it reduces the secretion of insulin peptides from the brain. The work develops the emerging model for interacting autonomous-nonautonomous roles of insulin/IGF signaling as a systems integrative mechanism of aging control.
Reduced function mutations in the insulin/IGF-I signaling pathway increase maximal lifespan and health span in many species. Calorie restriction (CR) decreases serum IGF-1 concentration by ~40%, protects against cancer and slows aging in rodents. However, the long-term effects of CR with adequate nutrition on circulating IGF-1 levels in humans are unknown. Here we report data from two long-term CR studies (1 and 6 years) showing that severe CR without malnutrition did not change IGF-1 and IGF-1 : IGFBP-3 ratio levels in humans. In contrast, total and free IGF-1 concentrations were significantly lower in moderately protein-restricted individuals. Reducing protein intake from an average of 1.67 g kg −1 of body weight per day to 0.95 g kg −1 of body weight per day for 3 weeks in six volunteers practicing CR resulted in a reduction in serum IGF-1 from 194 ng mL −1 to 152 ng mL −1 . These findings demonstrate that, unlike in rodents, long-term severe CR does not reduce serum IGF-1 concentration and IGF-1 : IGFBP-3 ratio in humans. In addition, our data provide evidence that protein intake is a key determinant of circulating IGF-1 levels in humans, and suggest that reduced protein intake may become an important component of anticancer and anti-aging dietary interventions.
aging; calorie restriction; IGF-1; metabolism; protein restriction
Although the literature suggests a protective (anabolic) effect of insulin-like growth factor-1 (IGF-1) on the musculoskeletal system during growth and aging, there is evidence that reductions in IGF-1 signaling are advantageous for promoting an increase in lifespan through reduction in oxidative stress-induced tissue damage. To better understand this paradox, we utilized the hepatocyte-specific IGF-1 transgenic (HIT) mice, which exhibit 3-fold increases in serum IGF-1, with normal IGF-1 expression in other tissues; and mice with an IGF-1 null background that exclusively express IGF-1 in the liver, which thereby delivers IGF-1 by the endocrine route only (KO-HIT mice). We found that in the total absence of tissue igf1 gene expression (KO-HIT), increases in serum IGF-1 levels were associated with increased levels of lipid peroxidation products in serum, increased mortality rate at 18 months, of age in both genders. Surprisingly, however, we found that in female mice, tissue IGF-1 plays an important role in preserving trabecular bone architecture as KO-HIT mice show bone loss in the femoral distal metaphysis. Additionally, in male KO-HIT mice increases in serum IGF-1 levels were insufficient to protect against age-related muscle loss. Overall, we conclude that elevations in serum IGF-1 have beneficial role in the aging musculoskeletal system but may have deleterious effects that lead to earlier mortality.
IGF-1; Bone; aging; oxidative stress
Deregulation of insulin-like growth factor (IGF)-I/IGF-IR signaling has been implicated in the development and progression of prostate cancer. Agents that can suppress the mitogenic activity of the IGF/IGF-IR growth axis may be of preventive or therapeutic value. We have previously demonstrated that apigenin, a plant flavone, modulates IGF signaling through upregulation of IGFBP-3. In this study, we investigated the mechanism(s) of apigenin action on the IGF/IGF-IR signaling pathway. Exposure of human prostate cancer DU145 cells to apigenin markedly reduced IGF-I-stimulated cell proliferation and induced apoptosis. Apigenin inhibited IGF-I-induced activation of IGF-IR and Akt in DU145 cells. Similar growth inhibitory and apoptotic responses were observed in PC-3 cells, which constitutively over-express this pathway. This effect of apigenin appears to be due partially to reduced autophosphorylation of IGF-IR. Inhibition of p-Akt by apigenin resulted in decreased phosphorylation of GSK-3β along with decreased expression of cyclin D1 and increased expression of p27/kip1. In vivo administration of apigenin to PC-3 tumor xenografts inhibited tumor growth, resulted in IGF-IR inactivation and dephosphorylation of Akt and its downstream signaling. These results suggest that inhibition of cell proliferation and induction of apoptosis by apigenin are mediated, at least in part, by its ability to inhibit IGF/IGF-IR signaling and the PI3K/Akt pathway.
prostate cancer; apigenin; IGF-I; IGF-IR; PI3K-Akt; glycogen synthase kinase-3
Dietary Restriction (DR) extends longevity in diverse species suggesting that there is a conserved mechanism for nutrient regulation and prosurvival responses1. We have discovered a role for the HECT E3 ubiquitin ligase WWP-1 as a positive regulator of lifespan in C. elegans in response to diet restriction. We find that overexpression of wwp-1 in worms extends lifespan up to 20% under conditions of ad libitum feeding. This extension is dependent upon the FoxA transcription factor pha-4, and independent of the FoxO transcription factor, daf-16. Reduction of wwp-1 completely suppresses the extended longevity of diet-restricted animals. However, loss of wwp-1 does not affect the long lifespan of animals with compromised mitochondrial function or reduced insulin/IGF-1 signaling. Overexpression of a mutant form of WWP-1 lacking catalytic activity suppresses the increased lifespan of diet-restricted animals, indicating that WWP-1 ubiquitin ligase activity is essential for longevity. Additionally, we find that the E2 ubiquitin conjugating enzyme, UBC-18, is essential and specific for DR induced longevity. UBC-18 interacts with WWP-1 and is required for the ubiquitin ligase activity of WWP-1 and the extended longevity of worms overexpressing wwp-1. Taken together, our results indicate that WWP-1 and UBC-18 function to ubiquitinate substrates that regulate DR induced longevity.
Mutations in the PARK6 gene coding for PTEN-induced kinase 1 (PINK1) cause recessive early-onset parkinsonism. Although PINK1 and Parkin promote the degradation of depolarized mitochondria in cultured cells, little is known about changes in signaling pathways that may additionally contribute to dopamine neuron loss in recessive parkinsonism. Accumulating evidence implicates impaired Akt cell survival signaling in sporadic and familial PD (PD). IGF-1/Akt signaling inhibits dopamine neuron loss in several animal models of PD and both IGF-1 and insulin are neuroprotective in various settings. Here, we tested whether PINK1 is required for insulin-like growth factor 1 (IGF-1) and insulin dependent phosphorylation of Akt and the regulation of downstream Akt target proteins. Our results show that embryonic fibroblasts from PINK1-deficient mice display significantly reduced Akt phosphorylation in response to both IGF-1 and insulin. Moreover, phosphorylation of glycogen synthase kinase-3β (GSK-3β) and nuclear exclusion of FoxO1 are decreased in IGF-1 treated PINK1-deficient cells. In addition, phosphorylation of ribosomal protein S6 is reduced indicating decreased activity of mitochondrial target of rapamycin (mTOR) in IGF-1 treated PINK1−/− cells. Importantly, the protection afforded by IGF-1 against staurosporine-induced metabolic dysfunction and apoptosis is abrogated in PINK1-deficient cells. Moreover, IGF-1-induced Akt phosphorylation is impaired in primary cortical neurons from PINK1-deficient mice. Inhibition of cellular Ser/Thr phosphatases did not increase the amount of phosphorylated Akt in PINK1−/− cells, suggesting that components upstream of Akt phosphorylation are compromised in PINK1-deficient cells. Our studies show that PINK1 is required for optimal IGF-1 and insulin dependent Akt signal transduction, and raise the possibility that impaired IGF-1/Akt signaling is involved in PINK1-related parkinsonism by increasing the vulnerability of dopaminergic neurons to stress-induced cell death.
PINK1; Parkinson’s disease; IGF-1; Akt signaling; GSK-3β; apoptosis
Insulin-like growth factor-1 (IGF-1) is a small protein that promotes cell survival and growth, often acting over long distances. Although for decades IGF-1 has been considered to have therapeutic potential, systemic side effects of IGF-1 are significant, and local delivery of IGF-1 for tissue repair has been a long-standing challenge. In this study, we designed and purified a novel protein, heparin-binding IGF-1 (Xp-HB-IGF-1), which is a fusion protein of native IGF-1 with the heparin-binding domain of heparin-binding epidermal growth factor-like growth factor. Xp-HB-IGF-1 bound selectively to heparin as well as the cell surfaces of 3T3 fibroblasts, neonatal cardiac myocytes and differentiating ES cells. Xp-HB-IGF-1 activated the IGF-1 receptor and Akt with identical kinetics and dose response, indicating no compromise of biological activity due to the heparin-binding domain. Because cartilage is a proteoglycan-rich environment and IGF-1 is a known stimulus for chondrocyte biosynthesis, we then studied the effectiveness of Xp-HB-IGF-1 in cartilage. Xp-HB-IGF-1 was selectively retained by cartilage explants and led to sustained chondrocyte proteoglycan biosynthesis compared to IGF-1. These data show that the strategy of engineering a “long-distance” growth factor like IGF-1 for local delivery may be useful for tissue repair and minimizing systemic effects.
heparin binding domain; tissue repair; biosynthesis
IGF-II plays a crucial role in fetal and cancer development by signaling through the IGF-I receptor. We have shown that inhibition of IGF-II by resveratrol (RSV) induced apoptosis and that proIGF-II (highly expressed in cancer) was more potent than mIGF-II in inhibiting this effect. Thus, we hypothesized that IGF-II differentially regulates the signaling cascade of the IGF-I receptor to stimulate the anti-apoptotic proteins Bcl-2 and Bcl-XL to prevent apoptosis. RSV treatment to breast cancer cells inhibited Bcl-2 and Bcl-XL expression and induced mitochondrial membrane depolarization. ProIGF-II was more potent than mIGF-II in: (1) activating the PI3/Akt pathway, (2) regulating Bcl-2 and Bcl-XL expression, and (3) inducing phosphorylation/nuclear translocation of Cyclic AMP-responsive element binding protein. Furthermore, IGF-II differentially regulated the intracellular translocation of Bcl-2 and Bcl-XL, a critical process in breast cancer progression to hormone-independence. Our study provides a novel mechanism of how proIGF-II promotes progression and chemoresistance in breast cancer development.
Insulin-like growth factor II; Bcl-2; Bcl-XL; MCF-7; Akt; CREB
The etiology of Graves’ orbitopathy (GO) remains enigmatic and thus controversy surrounds its pathogenesis. The role of the TSH receptor (TSHR) and the activating antibodies directed against it in the hyperthyroidism of Graves’ disease (GD) is firmly established. Less well elucidated is what part the TSHR pathway might play in the development of GO. Also uncertain is the participation of other cell surface receptors in the disease. Elevated levels of IGF-1 receptor (IGF-1R) have been found in orbital fibroblasts as well as B and T cells from patients with GD. These abnormal patterns of IGF-1R display are also found in rheumatoid arthritis and carry functional consequences. In addition, activating IgGs capable of displacing IGF-1 from IGF-1R have also been detected in patients with these diseases. IGF-1R forms a complex with TSHR which is necessary for at least some of the non-canonical signaling observed following TSHR activation. Functional TSHR and IGF-1R have also been found on fibrocytes, CD34+ bone marrow-derived cells from the monocyte lineage. Levels of TSHR on fibrocytes greatly exceed those found on orbital fibroblasts. When ligated by TSH or M22, a TSHR-activating monoclonal antibody, fibrocytes produce extremely high levels of several cytokines and chemokines. Moreover, fibrocytes infiltrate both the orbit and thyroid in GD. In sum, based on current evidence, IGF-1R and TSHR can be thought of as “partners in crime”. Involvement of the former probably transcends disease boundaries, while TSHR may not.
Graves’ ophthalmopathy; TSH receptor; IGF-1 receptor; orbital fibroblast; fibrocyte
Insulin/insulin-like growth factor-I (IGF-I) pathways are recognized as critical signaling pathways involved in the control of lifespans in lower organisms to mammals. Caloric restriction (CR) reduces plasma concentration of insulin, growth hormone (GH), and IGF-I. CR retards various age-dependent disorders such as nuerodegenerative diseases and extends lifespan in laboratory rodents. These beneficial effects of CR are partly mimicked in spontaneous or genetically engineered rodent models of reduced insulin and GH/IGF-I axis. Most of these long-living rodents show increased insulin sensitivity; however, recent study has revealed that some other rodents show normal or reduced insulin sensitivity. Thus, increased insulin sensitivity might be not prerequisite for lifespan extension in insulin/GH/IGF-I altered longevity rodent models. These results highlighted that, for lifespan extension, the intracellular signaling molecules of insulin/GH/IGF-I pathways might be more important than actual peripheral or systemic insulin action.
Insulin; GH; IGF-I; calorie restriction.
High levels of insulin-like growth factor-1 (IGF-1) have been associated with a significant increase in colon cancer risk. Additionally, IGF-1 inhibits apoptosis and stimulates proliferation of colonic epithelial cells in vitro. Unfortunately, IGF-1 knockout mice have severe developmental abnormalities and most do not survive, making it difficult to study how genetic ablation of IGF-1 affects colon tumorigenesis. To test the hypothesis that inhibition of IGF-1 prevents colon tumorigenesis, we utilized a preexisting mouse model containing a deletion of the igf1 gene in the liver through a Cre/loxP system. These liver-specific IGF-1 deficient (LID) mice display a 50–75% reduction in circulating IGF-1 levels. We conducted a pilot study to assess the impact of liver-specific IGF-1 deficiency on azoxymethane (AOM)-induced colon tumors. LID mice had a significant inhibition of colon tumor multiplicity in the proximal area of the colon compared to their wild-type littermates. We examined markers of proliferation and apoptosis in the colons of the LID and wild-type mice to see if these were consistent with tumorigenesis. We observed a decrease in proliferation in the colons of the LID mice and an increase in apoptosis. Finally, we examined cytokine levels to determine whether IGF-1 interacts with inflammatory pathways to affect colon tumorigenesis. We observed a significant reduction in the levels of 7 out of 10 cytokines that were measured in the LID mice as compared to wild-type littermates. Results from this pilot study support the hypothesis that reductions in circulating IGF-1 levels may prevent colon tumorigenesis and affect both proliferation and apoptosis. Future experiments will investigate downstream genes of the IGF-1 receptor.
IGF-1; colon; azoxymethane
Insulin-like growth factor-1 (IGF-1) is essential to hippocampal neurogenesis and the neuronal response to hypoxia/ischemia injury. IGF (IGF-1 and -2) signaling is mediated primarily by the type 1 IGF receptor (IGF-1R) and modulated by six high-affinity binding proteins (IGFBP) and the type 2 IGF receptor (IGF-2R), collectively termed IGF system proteins. Defining the precise cells that express each is essential to understanding their roles. With the exception of IGFBP-1, we found that mouse hippocampus expresses mRNA for each of these proteins during the first 2 weeks of postnatal life. Compared to postnatal day 14 (P14), mRNA abundance at P5 was higher for IGF-1, IGFBP-2, -3, and -5 (by 71%, 108%, 100%, and 98%, respectively), lower for IGF-2, IGF-2R, and IGFBP-6 (by 65%, 78%, and 44%, respectively), and unchanged for IGF-1R and IGFBP-4. Using laser capture microdissection (LCM), we found that granule neurons and pyramidal neurons exhibited identical patterns of expression of IGF-1, IGF-1R, IGF-2R, IGFBP-2, and -4, but did not express other IGF system genes. We then compared IGF system expression in mature granule neurons and their progenitors. Progenitors exhibited higher mRNA levels of IGF-1 and IGF-1R (by 130% and 86%, respectively), lower levels of IGF-2R (by 72%), and similar levels of IGFBP-4. Our data support a role for IGF in hippocampal neurogenesis and provide evidence that IGF actions are regulated within a defined in vivo milieu.
insulin-like growth factor (IGF); hippocampus; dentate gyrus; laser capture microdissection (LCM)
The IGF-IR is a multifunctional tyrosine kinase receptor involved in several biological processes including cell proliferation, differentiation, DNA repair, and cell survival. In the brain IGF-I plays a critical role during embryonic and early postnatal development. In the mature brain, IGF-I binding sites have been found in different regions of the brain, and multiple reports confirmed a strong neuroprotective action of the IGF-IR against different pro-apoptotic insults. When the IGF-IR signaling system is insufficiently deployed, either by low level of expression in elderly individuals, or by the inhibition associated with inflammatory cytokines, neuronal function and survival could be compromised. The examples of such CNS pathologies include HIV associated dementia, diabetic neuropathies, and Alzheimer’s disease. On the other hand, elevated expression activity of the IGF-IR may support uncontrolled cell proliferation and protection from apoptosis. Probably the best example of the IGF-IR involvement in brain tumors is medulloblastomas in which functional cooperation between viral oncoprotein, JC virus large T-antigen, and IGF-IR has been recently established. Therefore, better understanding of the beneficial and potentially harmful aspects of the IGF-IR can be critical for the development of new clinical regimens against neurodegenerative disorders and brain tumors.
IGF-IR; Neuroprotection; Brain Tumors; Review
Understanding of the interactions between estradiol (E2) and insulin-like growth factor-I (IGF-I) is still incomplete. Cell lines derived from the MCF-7 breast cancer cells were generated with suppressed expression of the IGF-I receptor (IGF-IR), termed IGF-IR.low cells, by stable transfection using small interfering RNA (siRNA) expression vector. Vector for control cells carried sequence generating non-interfering RNA. Concomitant with reduction in the IGF-IR levels, the IGF-IR.low cells also showed a reduction in estrogen receptor α (ERα) and progesterone receptor expressions and an elevation in the expression of ERβ. The number of the IGF-IR.low cells was reduced in response to IGF-I and human growth hormone plus epidermal growth factor, but E2 did not cause increase in the number of the IGF-IR.low cells compared to controls. Proliferation rate of IGF-IR.low cells was only reduced in response to E2 compared to controls, whereas their basal and hormone stimulated apoptosis rate was increased. Phosphorylation of p38 mitogen activated protein kinase (p38 MAPK) was increased in the IGF-IR.low cells after treatment with E2, without affecting control cells. Further, phosphorylation of the tumor suppressor protein p53 was elevated in the IGF-IR.low cells compared to the controls. Summary, suppressing the IGF-IR expression decreased the level of ERα but increased the level of ERβ. Overall growth rate of the IGF-IR.low cells was reduced mostly through an increase in apoptosis without affecting proliferation substantially. We hypothesize that a decreased ERα:ERβ ratio triggered a rapid phosphorylation of p38 MAPK which in turn phosphorylated the p53 tumor suppressor and accelerated apoptosis rate.
Breast Cancer; Estrogen receptors; IGF-I receptor; Apoptosis; Cell growth control
Previous in vivo studies have suggested a long term regulatory role for insulin in the exocrine pancreas. Furthermore, we reported that pancreatic acini have specific receptors for IGF I and II, and using different techniques (acid washing, trypsinisation, electron microscope autoradiography), that CCK8 reduces the internalisation of IGF II. To now directly study the long term role for IGF and insulin in the exocrine pancreas we used AR42J cells, a rat cell line that is derived from a transplantable tumour of the acinar pancreas. Hormone binding studies with 125I-labelled hormones indicated that those cells have insulin receptors, relatively fewer receptors for IGF II but in contrast with normal acini no detectable IGF I receptors. Insulin at concentrations as low as 1 nm stimulated the growth of AR42J cells, as measured by an increase in cell number, DNA and protein content. At 100 nM insulin had maximal effects stimulating the growth by about 50%. IGF I and II had only very weak growth promoting effects probably due to their interaction with the insulin receptor. Additionally insulin increased amylase synthesis over the same concentration range that it stimulated growth. But immunoprecipitation studies revealed that insulin induced a selective increase of amylase synthesis over general protein synthesis. These studies indicate, therefore, that insulin is a growth promoting hormone for AR42J cells and that additionally it seems to specifically regulate amylase synthesis. The role for the IGFs in the exocrine pancreas, however, still remains to be determined.
The type 1 insulin-like growth factor receptor (IGF1R) and its ligands (IGF1 and IGF2) have been implicated in a variety of physiological processes and in diseases such as cancer. In addition to IGF1R, IGF2 also activates the insulin receptor (IR) isoform A and therefore antibodies against IGF2 can inhibit cell proliferation mediated by the signaling through both IGF1R and IR triggered by IGF2. We identified a new human monoclonal antibody (mAb), m708.2, which bound to IGF1 and IGF2 but not to insulin. m708.2 potently inhibited signal transduction mediated by the interaction of IGF1 or IGF2 with the IGF1R and IGF2 with the IR. It also inhibited the growth of the breast cancer cell line MCF-7. An affinity-matured derivative of m708.2, m708.5, bound to IGF1 with equilibrium dissociation constant, KD = 200 pM and to IGF2 with KD = 60 pM. m708.5 inhibited signal transduction mediated by IGF1 and IGF2 and cancer cell growth more potently than m708.2. These results suggest that m708.5 could have potential as a candidate therapeutic for cancers driven by the IGF1,2 interactions with IGF1R and IR.
mAb; IGF1; IGF2; picomolar affinity; phage display; yeast display
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.
Three groups of rats were included in the experimental protocol young controls (17 weeks old); untreated old rats (103 weeks old); and aging rats (103 weeks old) treated with IGF-II (2 μg/100 g body weight and day) for 30 days.
Compared with young controls, untreated old rats showed an increase of oxidative damage in isolated mitochondria with a dysfunction characterized by: reduction of mitochondrial membrane potential (MMP) and ATP synthesis and increase of intramitochondrial free radicals production and proton leak rates. In addition, in untreated old rats mitochondrial respiration was not blocked by atractyloside. In accordance, old rats showed an overexpression of the active fragment of caspases 3 and 9 in liver homogenates. IGF-II therapy corrected all of these parameters of mitochondrial dysfunction and reduced activation of caspases.
The cytoprotective effects of IGF-II are related to mitochondrial protection leading to increased ATP production reducing free radical generation, oxidative damage and apoptosis.
Advanced or metastatic renal cell carcinoma (RCC) has a poor prognosis, because it is relatively resistant to conventional chemotherapy or radiotherapy. Treatments with human interferon-α2b alone or in combination with mammalian target of rapamycin (mTOR) inhibitors have led to only a modest improvement in clinical outcome. One observation made with mTOR inhibitors is that carcinomas can overcome these inhibitory effects by activating the insulin-like growth factor-I (IGF-I) signaling pathway. Clinically, there is an association of IGF-I receptor (IGF-IR) expression in RCC and poor long-term patient survival. We have developed a humanized anti-IGF-IR monoclonal antibody, hR1, which binds to RCC, resulting in effective down-regulation of IGF-IR and moderate inhibition of cell proliferation in vitro. In this work, we evaluate the anti-tumor activity of two novel IGF-1R-targeting agents against renal cell carcinoma given alone or in combination with an mTOR inhibitor.
hR1 was linked by the DOCK-AND-LOCK™ (DNL™) method to four Fabs of hR1, generating Hex-hR1, or to four molecules of interferon-α2b, generating 1R-2b. Eight human RCC cell lines were screened for IGF-1R expression and sensitivity to treatment with hR1 in vitro. Synergy with an mTOR inhibitor, temsirolimus, was tested in a cell line (ACHN) with low sensitivity to hR1.
Hex-hR1 induced the down-regulation of IGF-IR at 10-fold lower concentrations compared to the parental hR1. Sensitivity to growth inhibition mediated by hR1 and Hex-hR1 treatments correlated with IGF-1R expression (higher expression was more sensitive). The potency of 1R-2b to inhibit the in vitro growth of RCC was also demonstrated in two human cell lines, ACHN and 786-O, with EC50–values of 63 and 48 pM, respectively. When combined with temsirolimus, a synergistic growth-inhibition with hR1, Hex-hR1, and 1R-2b was observed in ACHN cells at concentrations as low as 10 nM for hR1, 1 nM for Hex-hR1, and 2.6 nM for 1R-2b.
Both Hex-hR1 and 1R-2b proved to be more potent than parental hR1 in inhibiting growth of RCC in vitro. Synergy was achieved when each of the three hR1-based agents was combined with temsirolimus, suggesting a new approach for treating RCC.
Dock-and-Lock; Renal cell carcinoma; Insulin-like growth factor-I receptor; Hex-hR1; 1R-2b; mTOR inhibitors